WO2024086312A1 - Compositions de nad+ améliorées, leurs procédés de fabrication et leurs méthodes d'utilisation - Google Patents

Compositions de nad+ améliorées, leurs procédés de fabrication et leurs méthodes d'utilisation Download PDF

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WO2024086312A1
WO2024086312A1 PCT/US2023/035557 US2023035557W WO2024086312A1 WO 2024086312 A1 WO2024086312 A1 WO 2024086312A1 US 2023035557 W US2023035557 W US 2023035557W WO 2024086312 A1 WO2024086312 A1 WO 2024086312A1
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nad
subject
composition
enhanced
tga
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PCT/US2023/035557
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English (en)
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Keith R. Latham
Regina L. GARLAND
Wendy Komac
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Bionadrx Holdings, Inc. Dba Bryleos
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Publication of WO2024086312A1 publication Critical patent/WO2024086312A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide

Definitions

  • the present disclosure relates to the field of chemical compound compositions.
  • the disclosure relates to chemical compound compositions and pharmaceutical formulations for treating conditions, diseases, and disorders.
  • Nicotinic acid and nicotinamide are the vitamin forms of nicotinamide adenine dinucleotide (NAD+) found in all living cells.
  • NAD+ plays an essential role in the synthesis of adenosine triphosphate (ATP), an organic compound that provides energy for many processes in living cells, such as muscle contraction, nerve impulse propagation, condensate dissolution, and chemical synthesis, and as such NAD+ is a crucial nutrient for animal health.
  • ATP adenosine triphosphate
  • Eukaryotes synthesize NAD+ de novo from tryptophan via the kynurenine pathway (Krehl, et al.
  • Nicotinic acid is phosphoribosylated to nicotinic acid mononucleotide (NMN), which is then adenylated to form nicotinic acid adenine dinucleotide (NAAD), which in turn is amidated to form NAD+ (Preiss and Handler (1958) J. Biol. Chem. 233:488-492; Preiss and Handler (1958b) J. Biol. Chem. 233:493-50).
  • NPN nicotinic acid mononucleotide
  • NAAD nicotinic acid adenine dinucleotide
  • NAD+ depleted levels of NAD+ are known to lead to a number of conditions and disorders in humans. For example, drug and alcohol abuse has been shown to deplete NAD+ levels, causing recovery from addiction to be a physically and psychologically brutal process.
  • NAD+ has application in numerous diseases and conditions. For example, niacin supplementation prevents the pellagra that can occur in populations with a tryptophan-poor diet.
  • U.S. Pat. No. 3,412,190 to O'Holleran teaches the use of NADPH for the lipids in the blood stream. NADPH has also been used to treat alcoholism (see, e.g., Canadian Patent 670,909).
  • currently available commercially produced NAD+ is chemically and biologically unstable. It has a limited shelf life, and requires special storage condition requirements (such as, for example, cooling or dry storage) to manufacture and produce it.
  • NAD is metabolized in the body rapidly, and thus a higher dosage is required for efficacy.
  • NAD+ supplementation has been by IV which has been reported to be painful, and the dosage required to be therapeutic has been high due to instability.
  • the risks associated with the administration of IV NAD+ include fluid overload in patients with compromised renal, hepatic or cardiac function, and the side effect profile has included pain in the back of the neck, paresthesias at the base of the tongue, dizziness, and nausea (Pritsos et al. (1999) US 5,888,532).
  • NAD+ can have enhanced stability and have a higher bioavailability as compared to commercially available NAD+ supplements when prepared by vacuumization. This discovery has been exploited to provide the instant disclosure, which provides enhanced NAD+ compositions, pharmaceutical formulations, and methods of producing and using the same.
  • the disclosure provides an enhanced NAD+ composition with increased shelf life, stability, and bioavailability relative to NAD+ (e.g., NAD+ monohydrate), such as commercially obtainable and other known forms of NAD.
  • NAD+ e.g., NAD+ monohydrate
  • the enhanced composition comprises vacuumized NAD+ and phosphorus.
  • the composition consists essentially of vacuumized NAD+, and phosphorus.
  • the enhanced composition comprises vacuumized NAD+ and polyethylene glycol.
  • the enhanced composition comprises vacuumized NAD+.
  • the unit dose of NAD+ in the composition described herein is about 5 mg to about 4000 mg. In some embodiments, the unit dose of NAD+ is about 500 mg.
  • 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.
  • the composition comprises from about 5 mg to about 4000 mg NAD+.
  • the composition comprises about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2000 mg, or about 4000 mg NAD+.
  • One aspect of the instant disclosure relates to a composition
  • a composition comprising nicotinamide adenine dinucleotide (NAD+), polyethylene glycol (PEG), and a phosphorus content of about 3.0% to about 11.0%.
  • NAD+ nicotinamide adenine dinucleotide
  • PEG polyethylene glycol
  • 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%.
  • the concentration of NAD+ in the composition described herein is about 200 mg/ml to about 1000 mg/ml. In some embodiments, the concentration of NAD+ is about 500 mg/ml.
  • the enhanced NAD+ composition is in powder form.
  • the composition has a longer shelf life relative to a commercially obtained NAD+, e g. NAD+ monohydrate.
  • the disclosure provides a pharmaceutical formulation comprising the enhanced NAD+ composition described supra, and at least one ingredient which facilitates administration to a subject and/or which can further treat the subject’s disease or condition.
  • the pharmaceutical formulation comprises polyethylene glycol (PEG ) of any molecular weight and/or combinations thereof, such as, but not limited to, PEG 300, PEG 400, PEG monomethyl ether (MME) 550, PEG 600, PEG 1000, PEG MME 2000, PEG 3350, and/or PEG 4000.
  • PEG polyethylene glycol
  • MME monomethyl ether
  • the composition is a powder.
  • compositions comprising Liothyronine, Polyethylene glycol, micro-porous glucose, and a pharmaceutically acceptable excipient.
  • compositions comprising LevaDopa and Polyethylene glycol and a pharmaceutically acceptable excipient.
  • 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.
  • the PEG is PEG 3350.
  • the PEG has a molecular mass over 4000 g/mol.
  • the PEG is a higher molecular weight PEG than is commercially available. Illustrative methods of PEG synthesis are described in Khanal, A., Fang,S. Chemistry.2017 Oct.26; 23(60): 15133-15142, incorporated herein by reference in its entirety.
  • the composition further comprises traces of an inert gas.
  • the inert gas comprises Nitrogen, Helium, Xenon, or Argon.
  • the inert gas comprises Nitrogen.
  • the inert gas comprises Helium.
  • the inert gas comprises Xenon.
  • the inert gas comprises Argon.
  • NAD+ composition such as an enhanced NAD+ composition
  • the process comprises blending NAD+ and PEG and vacuumizing the blended mixture of NAD+ and PEG under vacuum and inert gas conditions.
  • vacuumizing comprises placing the mixture of NAD+ and PEG under reduced pressure under vacuum at a pressure between about 24 inches of Hg and about 30.25 inches of Hg. Units of measurements of pressure may be readily interconverted by one skilled in the art.
  • vacuumizing further comprises replacing the vacuum with an inert gas into the blended mixture to bring the pressure in the blender to about 1 atm.
  • the process further comprises pre-treating the NAD+ by passing the NAD+ through a 0.25 mm sieve. In some embodiments, the process further comprises pre-treating the NAD+ to produce a particle that is 0.152 mm in size.
  • the co-formulation of the NAD+ with PEG provides increased product stability. For example, such pre-treating may be performed prior to mixing NAD+ and PEG. In some embodiments, such pre-treating is performed after mixing NAD+ and PEG (e.g., pre-formulating the NAD+/PEG mixture). In some embodiments, a measured portion of PEG is pre-vacuumized before adding NAD+ to the composition.
  • 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.
  • the PEG is PEG 3350.
  • the PEG has a molecular mass over 4000 g/mol.
  • the blending of NAD+ (e.g., the pre-formulated NAD+) and PEG (e.g., the pre-formulated PEG) is performed in a blender.
  • the vacuumizing comprises placing the mixture of NAD+ and PEG under a stable vacuum.
  • the vacuumizing further comprises pumping an inert gas into the blended mixture under vacuum to bring the pressure in the blender back to about 1 atmosphere (atm).
  • the inert gas comprises Helium, Nitrogen, Xenon, and/or Argon.
  • the inert gas comprises Nitrogen.
  • the inert gas comprises Helium.
  • the inert gas comprises Xenon.
  • the inert gas comprises Argon.
  • the vacuumizing is performed at least twice.
  • a further aspect of the instant disclosure relates to NAD+ compositions made by the process described herein.
  • the process comprises blending NAD+ and PEG and vacuumizing the blended mixture of NAD+ and PEG under vacuum and inert gas conditions.
  • a NAD+ composition described herein comprises water in an amount less than about 0.2%.
  • the dose of the NAD+ composition described herein is about 5 mg to 4000 mg. In some embodiments, the dose of the NAD+ composition is about 500 mg per gram of composition.
  • 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.
  • MME monomethyl ether
  • the composition further comprises traces of an inert gas.
  • the inert gas comprises Nitrogen, Helium, Xenon, or Argon.
  • the inert gas comprises Nitrogen.
  • the inert gas comprises Helium.
  • the inert gas comprises Xenon.
  • the inert gas comprises Argon.
  • 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).
  • 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).
  • 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).
  • the ratio of NAD+ to PEG in a composition of the present disclosure is 2:1 (wt/wt). In some embodiments, the ratio of NAD+ to PEG in a composition of the present disclosure is 3: 1 (wt/wt). In some embodiments, the ratio of NAD+ to PEG in a composition of the present disclosure is 4: 1 (wt/wt). In some embodiments, the ratio of NAD+ to PEG in a composition of the present disclosure is 5: 1 (wt/wt). In some embodiments, the ratio of NAD+ to PEG in a composition of the present disclosure is 6: 1 (wt/wt).
  • the ratio of NAD+ to PEG in a composition of the present disclosure is 7: 1 (wt/wt). In some embodiments, the ratio of NAD+ to PEG in a composition of the present disclosure is 8:1 (wt/wt). In some embodiments, the ratio of NAD+ to PEG in a composition of the present disclosure is 9: 1 (wt/wt). In some embodiments, the ratio of NAD+ to PEG in a composition of the present disclosure is 10:1 (wt/wt). In some embodiments, the ratio of NAD+ to PEG in a composition of the present disclosure is 20: 1 (wt/wt).
  • the composition comprises phosphorus at about 3.0% to about 11.0% (wt/wt).
  • the ratio of NAD+ to PEG is about 1 : 1 (wt/wt).
  • the ratio of NAD+ to PEG 3350 is about 1: 1 (wt/wt).
  • the enhanced NAD+ composition is in powder form in some embodiments.
  • the pharmaceutical formulation is suitable for oral administration to a subject.
  • the pharmaceutical formulation is in powder, liquid, aqueous, tablet, suppository, or capsule form.
  • the formulation is suitable for subcutaneous implant and may be formulated as a nasal spray, cream, toothpaste, or shampoo.
  • the enhanced NAD+ pharmaceutical formulation when administered to a subject, increases NAD levels in the subject by at least about 10% relative to a baseline level of intracellular NAD (icNAD) in the subject.
  • the pharmaceutical is capable of increasing NAD+ levels in the subject by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%>, about 52%, about 55%, about 60%, about 65%, about 70%, about 75%, 80%, about 85%, about 90%, about 95%, or about 100%, relative to a baseline level of intracellular NAD in the subject.
  • the pharmaceutical formulation is capable of increasing a subject’s intracellular levels of NAD, and in certain embodiments the formulation is capable of increasing intravascular levels of NAD in the subject. In some embodiments, the formulation does not significantly change extracellular NAD levels in the subject when administered to the subject.
  • the subject to which the present enhanced NAD+ pharmaceutical formulation is administered has, or has a risk of having, a disease or disorder correlated with decreased NAD levels.
  • the subject has a cardiovascular disorder, a neurodegenerative disorder, a neuropsychiatric disorder, a liver disorder [e.g., non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH)], a kidney disorder, a disorder of oxidative stress, a disorder of lipid metabolism, an addition to a substance use, a digestive disorder, a pancreatic disorder, an endocrine disorder, an immune system disorder, an autoimmune disorder, a reproductive system disorder, a metabolic system disorder, obesity, a skin disorder, and/or a bone disorder.
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • the present pharmaceutical formulation which, when administered to a subject, is capable of: improving liver and/or kidney function in the subject, optionally by decreasing serum levels of gamma-glutamyl transferase (GGT), bilirubin, alkaline phosphatase (ALP), and/or albumin; decreasing oxidative stress in the subject, optionally by decreasing serum GGT, increasing plasma gamma-glutamyl glutamine (L-Glu-L-Gln), decreasing circulating glutamate, decreasing at least one of histidine metabolites, upregulating glutamate utilization, increasing plasma abundance of glutathione S-transferase alpha- 1 (GSTA1), increasing mitochondrial carbonic anhydrase 5 (CA5A), decreasing plasma mitochondrial superoxide dismutase 2 (SOD2), and/or increasing plasma sirtuin 1 (SIRT1) and/or alcohol dehydrogenase 4 (ADH4); decreasing nitrosative stress and/or improves am
  • GGT gamma-
  • the pharmaceutical formulation is capable of modulating expression of at least one biomarker in a subject when administered to the subject.
  • the at least one biomarker is a protein or metabolite that indexes endothelial, liver, and/or overall mitochondrial functioning and/or integrity, one of upregulated fatty acid-binding proteins, or one of downregulated proteins associated with endothelial, liver, and/or pancreatic injuries.
  • at least one biomarker is a marker for an opiate in the blood of a subject.
  • the biomarker marker is an increased level of circulating serotonin receptor 1A (5-HT1AR), dopamine release at the nucleus accumbens, and/or at least one liver enzyme.
  • the presence of the at least one biomarker is related to an addiction to a substance use, such as opiates and other drugs, including, but not limited to, e.g., increased circulating levels of serotonin receptor 1 A (5-HT1AR), increased dopamine release at the nucleus accumbens, and/or increased levels of at least one liver enzyme.
  • the presence of the biomarker may also or alternatively be related to a neurodegenerative and or neuropsychiatric disease, including, e.g., increased levels of proteins with neurological effects, improved levels associated with neurodegenerative diseases, depression, anxiety, or chronic pain, oxidative stress, and/or nitrosative stress.
  • the presence of the biomarker may also or alternatively be related to a cardiovascular disease, including, e.g., improved LDL/HDL ratio, vascular and/or endothelial health, injury resistance, and/or oxidative stress.
  • the presence of the biomarker may also or alternatively be related to an exocrine pancreas and/or digestion function, including, e g., decreased circulate levels of pancreatic enzymes.
  • the presence of the biomarker may also or alternatively be related to an inflammation and/or immune function, including, e.g., improved adaptive or innate immune function or inflammation.
  • the presence of the biomarker may also or alternatively be related to: a preproduction function, including, e.g., INSL3; a metabolic disease and/or obesity, including, e.g., GALNT2, FABP1, and/or RBP2; and/or skin and/or bone health, such as collagen synthesis, skin inflammation in psoriasis and/or atopic dermatitis, and/or bone development, including, e.g., PPIB, WFDC12, INSL4, and/or MATN3.
  • a preproduction function including, e.g., INSL3
  • a metabolic disease and/or obesity including, e.g., GALNT2, FABP1, and/or RBP2
  • skin and/or bone health such as collagen synthesis, skin inflammation in psoriasis and/or
  • the pharmaceutical formulation provided herein does not increase any anxiety, stress, depression, fatigue, or any combination thereof in a subject.
  • the pharmaceutical formulation provided herein does not elicit unfavorable change(s) of vital signs, does not elicit unfavorable change(s) to body compositions, does not elicit unfavorable change(s) to a clinical laboratory test result, or combination thereof.
  • the disclosure provides an enhanced NAD+ pharmaceutical formulation for use in a method of: mitigating chemical addiction withdrawal symptoms in a subject in need thereof; mitigating opioid withdrawal symptoms in a subject in need thereof; increasing nicotinamide adenine dinucleotide (NAD+) levels in a subject; and/or preventing or treating a disease or disorder correlated to decreased levels of nicotinamide adenine dinucleotide (NAD+) in a subject in need thereof.
  • NAD+ nicotinamide adenine dinucleotide
  • the disclosure provides a kit comprising the enhanced NAD+ composition or the enhanced NAD+ pharmaceutical formulation provided herein.
  • the disclosure provides a method of making the enhanced NAD+ composition as described herein.
  • the method comprises vacuumizing NAD+ (e.g., NAD+ monohydrate) in a container under vacuum; and replacing the vacuum in the container with an inert gas or CO2.
  • vacuumizing the NAD+ comprises: placing the NAD+ under a vacuum or under a reduced pressure of between about 24 inches of Hg and about 30.25 inches of Hg.; and replacing the vacuum with an inert gas to increase the pressure in the container to about 1 atmosphere (atm).
  • vacuumization is performed at least twice.
  • the inert gas which replaces the vacuum comprises Argon, Helium, Nitrogen, and/or Xenon.
  • the NAD+ is vacuumized more than once, more than twice, or more than three times.
  • the NAD is pretreated before being vacuumized.
  • pretreatment comprises processing the NAD+ to obtain a preselected average NAD+ particle size.
  • processing comprises passing the NAD+ through a sieve, and/or grinding, milling, or jet-milling the NAD+.
  • the present disclosure also provides an enhanced NAD+ composition made by a method as described herein.
  • the disclosure provides a method of making an enhanced NAD+ pharmaceutical formulation as described herein, comprising mixing the enhanced NAD+ composition of the disclosure, such as that described above, with a compound, component, and/or ingredient which to form aa blended mixture.
  • the secondary compound, component or ingredient is one that facilitates administration to a subject and/or which also has therapeutic efficacy.
  • the ingredient comprises PEG of any molecular weight or mixtures of PEG having different molecular weights.
  • the NAD+ is pretreated as described above to provide NAD+ particles of a preselected particle size for homogeneous mixing with the secondary compound, component, or ingredient.
  • the NAD+ is pretreated before being mixed with PEG in a blender. Pretreatment may also or alternatively comprise vacuumization of either the NAD+ or PEG before they are mixed.
  • the blended mixture of NATH and PEG is then vacuumized in the blender under vacuum or under a reduced pressure, in some embodiments, of between about 24 inches of Hg and about 30.25 inches of Hg.
  • the vacuum is then replaced with an inert gas or CO2 to increase the pressure in the blender to about 1 atmosphere (atm).
  • vacuumization before or after mixing is performed at least twice.
  • the inert gas which replaces the vacuum comprises Argon, Helium, Nitrogen, and/or Xenon.
  • the disclosure provides a method of mitigating chemical addiction withdrawal symptoms in a subject in need thereof.
  • the method comprises: administering a therapeutically effective amount of the present enhanced NAD+ pharmaceutical formulation to the subject, the amount being effective to mitigate at least one chemical addiction withdrawal symptom in the subject.
  • administration of the formulation increases the level of NAD in the subject by at least 5%. In other embodiments, administration of the formulation increases the level of NAD in the subject by at least 5% to 10%, at least about 6%, at least about 7%, at least about 8%, at least about 9% or at least about 10%. In certain embodiments, the increased NAD amount is an intracellular level, and in particular embodiments, an intracellular level of NAD is increased to greater than about 35 pM.
  • intracellular level of NAD is increased to greater than about 35 gM, greater than about 36 gM, greater than about 37 gM, greater than about 38 gM, greater than about 39 gM, greater than about 40 gM, greater than about 41 gM, greater than about 42 gM, greater than about 43 gM, greater than about 44 gM, or greater than about 45 gM.
  • the method is effective to increase a plasma level of N-methyl-nicotinamide (MeNAM) or N-methyl-2-pyridone-5-carboxamide (2PY) by at least 5% in the subject.
  • extracellular level of NAD alternatively or also is increased.
  • the amount administered is effective to increase a level of icNAD to a level within a selected physiological range.
  • a minimum value of the selected physiological range is from at least 5% to at least 50% higher than a baseline level of intracellular NAD in the subject, or at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher than a baseline level of intracellular NAD in the subject.
  • administration of the pharmaceutical formulation results in at least a 5% increase in the subject’s urinary excretion of nicotinamide or its metabolites.
  • the chemical addiction withdrawal symptoms suffered by the subject before administration of the formulation are related to detoxification from tobacco, heroin, opium, morphine, dihydromorphine, meperidine, codeine, cocaine, amphetamines, barbiturates, alcohol, tranquilizers, and/or opioids.
  • the subject to whom the pharmaceutical formulation is administered had scored at least about 3 or greater on the Drug Abuse Screening Test (DAST) before administration. In other embodiments, the subject to whom the pharmaceutical formulation is administered had scored about 6 or greater on the Drug Abuse Screening Test - 10 (DAST-10) before administration.
  • the amount of the pharmaceutical formulation administered is effective to mitigate opioid withdrawal symptoms such that the subject has a Clinical Opioid Withdrawal Scale (COWS) score of 12, 11, 10, 9, 8, 7, or 6 or lower. In some embodiments, the amount of formulation administered is effective to mitigate opioid withdrawal symptoms such that the client maintains a Clinical Opioid Withdrawal Scale (COWS) score of 12 or lower over the first about four days of acute detox.
  • COWS Clinical Opioid Withdrawal Scale
  • the pharmaceutical formulation is administered to the subject as a hydrated, dissolved, or suspended powder.
  • the formulation is administered via an oral, buccal, intramuscular, systemic, anal, vaginal, or sublingual route.
  • the formulation is administered via a multi-sip oral swish and swallow protocol.
  • the amount of NAD+ in the pharmaceutical formulation administered to the subject is from about 5 mg to about 4000 mg.
  • the amount of the enhanced NAD+ composition in the formulation comprises about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2000 mg, about 2200 mg, about 2400
  • the amount of the pharmaceutical formulation administered comprises a daily dose of about 1 mg per kg to about to about 100 mg/kg body weight of the subject. In some specific embodiments, the amount of the pharmaceutical formulation administered comprises a maximum daily dose of about 1 mg per kg, about 2 mg per kg, about 3 mg per kg, about 4 mg per kg, about 5 mg per kg, about 6 mg per kg, about 7 mg per kg, about 8 mg per kg, about 9 mg per kg, about 10 mg per kg, about 11 mg per kg, about 12 mg per kg, about 13 mg per kg, about 14 mg per kg, about 15 mg per kg, about 16 mg per kg, about 17 mg per kg, about 18 mg per kg, about 19 mg per kg, about 20 mg per kg, about 22 mg per kg, about 24 mg per kg, about 26 mg per kg, about 27 mg per kg, about 28 mg per kg, about 30 mg per kg, about 32 mg per kg, about 34 mg per kg, about 36 mg per kg, about 38 mg per kg, about 40 mg per kg, about 42 mg per kg, about 44
  • the method further comprises administering to the subject a pharmaceutically effective amount of an analgesic or a non-steroidal anti-inflammatory drug (NSAID).
  • the pharmaceutical formulation further comprises a pharmaceutically effective amount of an analgesic or a non-steroidal anti-inflammatory drug (NSAID).
  • 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.
  • the method further comprising administering a pharmaceutically effective amount of: an anti-diarrhea medication, such as, but not limited to, loperamide; an anti-nausea medication such as, but not limited to, ondansetron; an antispasmodic medication such as, but not limited to, tizanidine; and/or a sedative such as, but not limited to, promethazine.
  • an anti-diarrhea medication such as, but not limited to, loperamide
  • an anti-nausea medication such as, but not limited to, ondansetron
  • an antispasmodic medication such as, but not limited to, tizanidine
  • a sedative such as, but not limited to, promethazine.
  • the method further comprises monitoring a plurality of vital signs of the subject after administration of the formulation.
  • the method further comprises measuring an intracellular NAD or extracellular concentration of NAD in the subject before and/or after administration of the formulation, and the dosage of the pharmaceutical formulation to be administered may be selected based on the measured intracellular NAD or extracellular concentration of NAD in the subject.
  • the pharmaceutical formulation is administered over a period of at least two days, and may be administered over about two, three, four, or five or more days.
  • the pharmaceutical formulation is administered multiple doses within an about a 10 hour to about 16 hour period.
  • about one to about four doses are administered within a 10-16 hour period.
  • the formulation is administered in about one to about five daily doses of about 500 mg each.
  • the final daily dose of the formulation is administered immediately before the subject’s bedtime.
  • a first daily dose of the formulation is administered following about 12 hours of fasting by the subject.
  • the method may further comprise: monitoring at least one vital signs of the subject before and after administration of the composition: and/or measuring an intracellular NAD or extracellular concentration of NAD in the subject before administration and after administration of the pharmaceutical formulation; and/or selecting a dosage of the composition to be administered to the subject based on the measured baseline intracellular NAD or extracellular concentration of NAD in the subject.
  • the disclosure also provides a method of increasing NAD levels in a subject, the method comprising administering to the subject an amount of the enhanced NAD+ pharmaceutical formulation as provided herein, which is effective to increase NAD levels in the subject.
  • the subject is a healthy human subject.
  • the subject suffers from or is at risk for a disease or disorder, and administration of the formulation decreases a risk of and/or ameliorates at least one symptom of the disease or disorder.
  • the method may further comprise: monitoring at least one vital signs of the subject before and after administration of the pharmaceutical formulation: and/or measuring an intracellular concentration of NAD or extracellular concentration of NAD in the subject before administration and after administration of the pharmaceutical formulation; and/or selecting a dosage of the composition to be administered to the subject based on the measured baseline intracellular concentration of NAD or extracellular concentration of NAD in the subject.
  • the disclosure provides a method of preventing or treating a disease or disorder correlated with a decreased level of NAD in a subject in need thereof, the method comprising: administering an amount of the enhanced NAD+ pharmaceutical formulation as described herein effective to prevent or treat at least one symptom of the disease or disorder.
  • the disease or disorder is a cardiovascular disorder, a neurodegenerative disorder, a neuropsychiatric disorder, a liver disorder [e.g., non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH)], a kidney disorder, a disorder of oxidative stress, a disorder of lipid metabolism, an addition to a substance use, a digestive disorder, a pancreatic disorder, an endocrine disorder, an immune system disorder, an autoimmune disorder, a reproductive system disorder, a metabolic system disorder, obesity, a skin disorder, and/or a bone disorder.
  • a cardiovascular disorder e.g., non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH)
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • administering the formulation improves liver and/or kidney function in the subject, optionally by decreasing serum levels of gammaglutamyltransferase (GGT), bilirubin, alkaline phosphatase (ALP), and/or albumin; decreases oxidative stress in the subject, optionally by decreasing serum GGT, increasing plasma gammaglutamyl glutamine (L-Glu-L-Gkn), decreasing circulating glutamate, decreasing at least one of histidine metabolites, upregulating glutamate utilization, increasing plasma abundance of glutathione S-transferase alpha-1 (GSTA1), increasing mitochondrial carbonic anhydrase 5 (CA5A), decreasing plasma mitochondrial superoxide dismutase 2 (SOD2), and/or increasing plasma sirtuin 1 (SIRT1) and/or alcohol dehydrogenase 4 (ADH4); decreases nitrosative stress and/or improves ammonia clearance, optionally by upregulating mitochondrial CA5A and/or decreasing plasma
  • GGT gam
  • administering the enhanced NAD+ formulation to a subject modulates expression of at least one biomarker in the subject.
  • the at least one biomarker is a protein or metabolite associated with endothelial, liver, and/or overall mitochondrial functioning and/or integrity, or is an upregulated fatty acid-binding protein or a downregulated protein associated with endothelial, liver, and/or pancreatic injuries.
  • the at least one biomarker is related to: a substance use, such as opiates and other drugs, including, e.g., increased circulating levels of serotonin receptor 1A (5-HT1AR), increased dopamine release at the nucleus accumbens, and/or increased levels of at least one liver enzyme; a neurodegenerative and or neuropsychiatric disease, including, e.g., increased levels of proteins with neurological effects, improved levels associated with neurodegenerative diseases, depression, anxiety, or chronic pain, oxidative stress, and/or nitrosative stress.
  • the at least one biomarker is related to: a cardiovascular disease, including, e g., improved LDL/HDL ratio, vascular and/or endothelial health, injury resistance, and/or oxidative stress; an exocrine pancreas and/or digestion function, including, e.g., decreased circulate levels of pancreatic enzymes; an inflammation and/or immune function, including, e.g., improved adaptive or innate immune function or inflammation; a preproduction function, including, e.g., INSL3; a metabolic disease and/or obesity, including, e.g., GALNT2, FABP1, and/or RBP2; and/or skin and/or bone health, such as collagen synthesis, skin inflammation in psoriasis and/or atopic dermatitis, and/or bone development, including, e g., PPIB, WFDC12, INSL4, and/or MATN3.
  • a cardiovascular disease including, e g., improved LDL/HDL ratio
  • administering the enhanced NAD+ pharmaceutical formulation does not significantly increase at least one adverse symptom, such as, but not limited to, anxiety, stress, depression, fatigue, and/or a significant unfavorable change of vital signs, body compositions, and/or a clinical laboratory test result.
  • adverse symptom such as, but not limited to, anxiety, stress, depression, fatigue, and/or a significant unfavorable change of vital signs, body compositions, and/or a clinical laboratory test result.
  • 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 a baseline level of intracellular NAD in the subject.
  • the method may further comprise: monitoring at least one vital signs of the subject before and after administration of the pharmaceutical formulation: and/or measuring an intracellular concentration of NAD or extracellular concentration of NAD in the subject before administration and after administration of the pharmaceutical formulation; and/or selecting a dosage of the composition to be administered to the subject based on the measured baseline intracellular concentration of NAD or extracellular concentration of NAD in the subject.
  • 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 the intracellular NAD level.
  • Another aspect of the present disclosure relates to a method of treating a subject suffering from, or preventing in a subject, an age-related disease comprising administering to the subject, a therapeutically effective amount of an NAD+ composition disclosed herein.
  • the disclosure also provides NAD+ compositions for use in the manufacture of a medicament or supplement for treating age related diseases.
  • the present disclosure relates to a method for treating or preventing central nervous system disorders or diseases, comprising administering to a subject in need thereof a therapeutically effective amount of an NAD+ composition described herein.
  • Another aspect of the present disclosure relates to a method for treating a subject for (e.g., reducing) chemical addiction or substance abuse, comprising administering to the subject a therapeutically effective amount of an NAD+ composition described herein.
  • 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 SIRTI in the subject.
  • 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 PLBI in the subject.
  • PLB1 cryptococcal phospholipase
  • 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.
  • MME membrane metalloendopeptidase
  • Another aspect of the present disclosure relates to a method for treating a subject suffering from a disease or disorder modulated by SIRTI, comprising administering to the subject a therapeutically effective amount of an NAD+ composition described herein.
  • Another aspect of the present disclosure relates to a method for treating a subject suffering from a disease or disorder modulated by cryptococcal phospholipase (PLBI), comprising administering to the subject a therapeutically effective amount of an NAD+ composition described herein.
  • PLBI cryptococcal phospholipase
  • Another aspect of the present disclosure relates to a method for treating a subject suffering from a disease or disorder modulated by membrane metalloendopeptidase (MME), comprising administering to the subject a therapeutically effective amount of an NAD+ composition described herein.
  • MME membrane metalloendopeptidase
  • Another aspect of the present disclosure relates to a method for increasing a 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.
  • NAD+ compositions are provided as a component in solution for use in the treatment of a subject suffering from an age-related disorder, and/or for use in the treatment of a subject suffering from of a CNS disease or disorder.
  • the CNS disorder is chemical addiction.
  • the CNS disorder is substance abuse.
  • NAD+ compositions are provided as a component in solution for use in the enhancement of energy for autophagy, a process in which cellular debris is removed.
  • NAD+ compositions are provided as a component in solution for use in increasing longevity and prevention of disease in humans, animals, and/or plants.
  • Another aspect of the present disclosure relates to a method of promoting cellular NAD+ metabolism or NAD+ cellular homeostasis in a subject, comprising administering to the subject an amount of the composition described herein effective to promote NAD+ metabolism or NAD+ cellular homeostasis.
  • Another aspect of the present disclosure relates to a method of treating a patient suffering from at least one condition of metabolic syndrome, comprising administering to the subject a therapeutically effective amount of the composition described herein, wherein the at least one condition is increased blood pressure, high blood sugar, excess body fat around the waist, abnormal cholesterol or triglyceride levels, or any combination thereof.
  • Another aspect of the present disclosure relates to a method of increasing the amount of functional polypeptide of at least one of SIRT1, PLB1, NPL, ENPP5, GLSTA, GALNT3, FABP1, NRCAM, NLGN2, ARTN, UPB1, MME, CTSB, CTSL, or HRAS polypeptide in a subject in need thereof, comprising administering to the subject an amount of the composition described herein effective to increase the amount of functional polypeptide of at least one of SIRT1, PLB1, NPL, ENPP5, GLSTA, GALNT3, FABP1, NRCAM, NLGN2, ARTN, UPB1, MME, CTSB, CTSL, or HRAS polypeptide.
  • Certain APIs like thyroid hormones contain bound water that can be removed by azeotroping solvents to provide product stability.
  • the thyroid hormone is dissolved in a solvent and combined with a pharmaceutically acceptable excipient in a mixture and the solvent is removed under vacuum to result in a powder that is suitable for further processing.
  • FIG. 1 is a graph demonstrating the intracellular increase of NAD following treatment with an illustrative NAD+ composition (“A”) as compared to placebo treatment (“B”);
  • FIG. 2 is a graph depicting the impact of treatment with an illustrative NAD+ composition (“A”) on total bilirubin (prnol/L) as compared to placebo treatment (“B”) over time;
  • A illustrative NAD+ composition
  • B placebo treatment
  • FIG. 3 is a graph depicting the impact of treatment with an illustrative NAD+ composition (“A”) on total bilirubin (pmol/L) as compared to placebo treatment (“B”) over time;
  • A illustrative NAD+ composition
  • B placebo treatment
  • FIG. 4 is a graph depicting the impact of treatment with an illustrative NAD+ composition (“A”) on total bilirubin (pmol/L) as compared to placebo treatment (“B”) over a series of time points (days);
  • A illustrative NAD+ composition
  • B placebo treatment
  • FIG. 5 is a graph depicting the impact of treatment with an illustrative NAD+ composition (“A”) on liver and alkaline phosphatase measured by enzyme activity in Units/Liter as compared to placebo treatment (“B”) over time;
  • A illustrative NAD+ composition
  • B placebo treatment
  • FIG. 6 is a graph depicting the impact of treatment with an illustrative NAD+ composition (“A”) on liver and alkaline phosphatase measured by enzyme activity in Units/Liter as compared to placebo treatment (“B”) over time;
  • A illustrative NAD+ composition
  • B placebo treatment
  • FIG. 7 is a graph depicting the impact of treatment with an illustrative NAD+ composition (“A”) on liver and alkaline phosphatase measured by enzyme activity in Units/Liter as compared to placebo treatment (“B”) over a series of time points;
  • A illustrative NAD+ composition
  • B placebo treatment
  • FIG. 8 is a graph depicting the impact of treatment with an illustrative NAD+ composition (“A”) on gamma-glutamyl transferase (GGT) as compared to placebo treatment (“B”) over time;
  • FIG. 9 is a graph depicting the impact of treatment with an illustrative NAD+ composition (“A”) on gamma-glutamyl transferase (GGT) as compared to placebo treatment (“B”) over time;
  • FIG. 10 is a graph depicting the impact of treatment with an illustrative NAD+ composition (“A”) on gamma-glutamyl transferase (GGT) as compared to placebo treatment (“B”) over a series of time points;
  • FIG. 11 is a graph depicting the impact of treatment with an illustrative NAD+ composition (“A”) on LDL/HDL ratio as compared to placebo treatment (“B”) over time;
  • FIG. 12 is a graph depicting the impact of treatment with an illustrative NAD+ composition (“A”) on LDL/HDL ratio as compared to placebo treatment (“B”) over time;
  • FIG. 13 is a graph depicting the impact of treatment with an illustrative NAD+ composition (“A”) on LDL/HDL ratio as compared to placebo treatment (“B”) over a series of time points relative to treatment;
  • A illustrative NAD+ composition
  • B placebo treatment
  • FIG. 14 is a schematic of NAD+ metabolism
  • FIG. 16 is a panel of graphs depicting the plasma concentration of hypoxanthine, 4- hydroxyphenylpyruvate, docosahexaenoylcholine, cortisone, gamma-glutamylglutamine, urate, indolelacetate, glycohyocholate, tetrahydrocortisone glucuronide, and glutamate measured in treatment (A) and placebo (B) participants at three time points during the Study described in Example 6. (P ⁇ 0.05 for all graphs);
  • FIG. 17 is a graph depicting the top 25 enriched metabolite sets observed in participants treated with NAD+.
  • Metabolite enrichment analysis (MetaboAnalyst 5.0) identified NAD+, histidine, aspartate/ glutamate metabolism, and arginine biosynthesis as the most affected (Kyoto Encyclopedia of Genes and Genomes) KEGG metabolic pathways;
  • FIG. 18 is a set of graphs demonstrating the intracellular increase of NAD following treatment with an illustrative NAD+ composition as compared to placebo treatment, and the changes relative to baseline (“BL”; prior to treatment) for individual participants within each treatment group;
  • FIG. 19 is a set of graphs depicting the plasma concentration of NAD metabolite 1- methylnicotinamide (MeNAM) measured in NAD- or placebo-treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group;
  • MeNAM NAD metabolite 1- methylnicotinamide
  • FIG. 20 is a set of graphs depicting the plasma concentration of NAD metabolite Nl- 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, and the change relative to baseline for individual participants within each treatment group;
  • FIG. 21 is a set of graphs depicting the plasma concentration of SIRT1 measured in NAD+- or placebo-treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group;
  • FIG. 22 is a set of graphs depicting the plasma concentration of methionine synthase (MTR) measured in NAD+- or placebo-treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group;
  • MTR methionine synthase
  • FIG. 23 is a set of graphs depicting the plasma concentration of glutathione S-transferase alpha-1 (GSTA1) measured in NAD+- or placebo-treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group;
  • GSTA1 glutathione S-transferase alpha-1
  • FIG. 24 is a set of graphs depicting the plasma concentration of mitochondrial superoxide dismutase 2 measured in NAD+- or placebo-treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group;
  • FIG. 25 is a set of graphs depicting the plasma concentration of hypoxanthine measured in NAD+- or placebo-treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group;
  • FIG. 26 is a set of graphs depicting the plasma concentration of gamma-glutamyl transferase (GGT) measured in NAD+- or placebo-treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group;
  • GTT gamma-glutamyl transferase
  • FIG. 27 is a set of graphs depicting the plasma concentration of total bilirubin (TBILI) measured in NAD+- or placebo-treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group;
  • TILI total bilirubin
  • FIG. 28 is a table summarizing changes in a number of proteins that modulate neuronal signaling pathways or neuron growth after treatment with NAD+ during the Study described in Examples 5 and 6;
  • FIG. 29 is a table summarizing changes in a number of immune and inflammatory related proteins after treatment with NAD+ during the Study described in Examples 5 and 6;
  • FIG. 30 is a set of graphs depicting the plasma concentration of fatty-acid binding protein 1 (FABP1) measured in NAD- or placebo-treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group; and
  • FBP1 fatty-acid binding protein 1
  • FIG. 31 is a set of graphs depicting the plasma concentration of retinol binding protein 2 (RBP2) measured in NAD- or placebo-treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group.
  • RBP2 retinol binding protein 2
  • FIG. 32A is a graph of the TGA thermogram for Sample 6, replicate 1 (Sample 6-1).
  • FIG. 32B is a graph of the TGA thermogram for Sample 6, replicate 2 (Sample 6-2).
  • FIG. 32C is an overlay of the TGA thermograms for Sample 6 replicates (Samples 6-1, 6-2).
  • FIG. 33A is a graph of the TGA thermogram for Sample 7, replicate 1 (Sample 7-1).
  • FIG. 33B is a graph of the TGA thermogram for Sample 7, replicate 2 (Sample 7-2).
  • FIG. 33C is an overlay of the TGA thermograms for Sample 7 replicates (Samples 7-1, 7-2).
  • FIG. 34A is a graph of the TGA thermogram for Sample 8, replicate 1 (Sample 8-1).
  • FIG. 34B is a graph of the TGA thermogram for Sample 8, replicate 2 (Sample 8-2).
  • FIG. 34C is an overlay of the TGA thermograms for Sample 8 replicates (Samples 8-1, 8-2).
  • FIG. 35A is a graph of the TGA thermogram for Sample 9, replicate 1 (Sample 9-1).
  • FIG. 35B is a graph of the TGA thermogram for Sample 9, replicate 2 (Sample 9-2).
  • FIG. 35C is an overlay of the TGA thermograms for Sample 9 replicates (Samples 9-1, 9-2).
  • FIG. 36 is an overlay of the TGA thermograms for Samples 6, 7, 8 and 9.
  • the present disclosure relates to enhanced NAD+ compositions useful in the treatment of diseases and disorders regulated by intracellular levels of NAD.
  • the present disclosure further relates to methods of manufacturing of NAD+ drug products that are stable, safer for use and that have a longer shelf life.
  • the present disclosure also provides new methods of increasing intracellular NAD and methods of treatments of central nervous system diseases and disorders including chemical addiction and substance abuse.
  • the present disclosure provides for enhanced delivery of NAD+ to target cells to treat substance abuse including, but not limited to, drug or alcohol addiction.
  • NAD+ is known to be chemically and biologically unstable.
  • the present disclosure provides herein NAD+ compositions having an enhanced stability compared to traditional NAD+ compositions.
  • the illustrative enhanced NAD+ compositions may have a slower degradation rate and/or a longer half-life (in vitro and/or in vivo), a longer shelf-life, and/or a less stringent storage condition requirement (e.g., cooling or dry storage), compared to a control NAD+ composition without the property or not produced by the conditions and processes described herein (e.g., a NAD+ composition from a commercial source).
  • NAD+ is a crucial nutrient for animal health.
  • the present disclosure provides herein NAD+ compositions with enhanced properties, which may be administered to an individual (e.g., a human) as a nutrient and/or a pharmaceutical agent to prevent, ameliorate, and/or treat a disease or disorder related to insufficient icNAD in the body.
  • the enhanced NAD+ compositions described herein increases the icNAD concentration and/or NAD+ degradation in the individual.
  • the individual is having tryptophan- poor diet.
  • the individual has a genetic mutation and/or other mechanisms (e.g., living or environmental conditions, food or drug effects, etc.) resulting in a reduction or inhibition of NAD production, a reduction of NAD stability, and/or an increase of NAD degradation or turnover in the individual.
  • the individual may have a genetic mutation in the kynurenine pathway, thus reducing or inhibiting the production chain from nicotinic acid to NMN to NAD and then to NAD.
  • the individual may have another genetic mutation affecting the production, transportation, storage, and/or utilization of NAD in the individual.
  • the enhanced NAD+ compositions described herein may also enhance NAD-related biochemical and biological reactions in an individual in need thereof.
  • the enhanced NAD+ compositions described herein may enhance the bio-functions of oxidoreductases in the individual, e.g., enhancing synthesizing enzymes such as poly(ADPribose) polymerases and cADPribose synthases.
  • the enhanced NAD+ compositions described herein may be administered to the individual to prevent, ameliorate, and/or treat the disease or disorder.
  • the enhanced NAD+ compositions described herein may be a substitute of current commercially available NAD+ compositions, or other NAD+ compositions without the property or not produced by the current conditions or process, for its enhanced stability.
  • the enhanced NAD+ compositions described herein may be used as a supplement and/or a pharmaceutical agent with improved bioavailability for oral administration (or through other administration routes) to an individual.
  • the enhanced NAD+ compositions described herein may be more stable in the direct interaction with water, radiation, oxygen, and/or UV light, and may be resistant to hydrolysis in the small intestine by brush border cells.
  • the articles “a” and “an” refer to one or to more than one i.e., at least one) of the grammatical object of the article.
  • an element means one element or more than one element.
  • use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.
  • the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ⁇ 20% or ⁇ 10%, including ⁇ 5%, ⁇ 1%, and ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
  • NAD refers to “nicotinamide adenine dinucleotide” and encompasses NAD ⁇ and/or NADH.
  • NAD ⁇ as used in this disclosure describes compositions consisting of NAD ⁇ .
  • compositions comprising NAD ⁇ that has been prepared according to the present disclosure.
  • ‘vacuumizing” or “vacuumization” is a procedure that purifies active primary ingredients, components (i.e., excipients), intermediates, secondary compounds, ingredients, or components, and final products (such as, but not limited to, powder and tablets).
  • Vacuumizing consists of subjecting a composition or mixture of components to decreased pressure or vacuum, followed by the replacement of the vacuum with an inert gas or CO2.
  • one vacuumization cycle comprises subjecting a composition or mixture of components to decreased pressure or vacuum, followed by the replacement of the vacuum with an inert gas or CO2. The cycles are subsequent and sequential starting with vacuum under specific pressure and followed with replacement of the air with an inert gas to compensate or re-saturate the pressure created by the vacuum at similar pressure.
  • NAD ⁇ and combinations of NAD ⁇ and PEG are vacuumized to provide the compositions and pharmaceutical formulations described herein.
  • ‘NAD+ monohydrate” or “P-NAD [P-Nicotinamide Adenine Dinucleotide, Oxidized free acid hydrate]” is a compound which is commercially available, and which may include phosphorus.
  • the term “blended” refers to mixed components or ingredients, for example, NAD+ is mixed with a secondary component such as one or more excipients of various weight (e.g., PEG, including but not limited to, pre-formulated PEG) to achieve a homogeneous distribution of the active ingredient in the final drug product.
  • a secondary component such as one or more excipients of various weight (e.g., PEG, including but not limited to, pre-formulated PEG) to achieve a homogeneous distribution of the active ingredient in the final drug product.
  • PEG including but not limited to, pre-formulated PEG
  • substrate or “excipient substrate” refers to a therapeutically suitable excipient that may be used to bind or immobilize an active agent or drug to its surface through non- covalent intermolecular forces or interactions according to methods of the present disclosure.
  • the optimal choice for a substrate may depend on the solvent and the active agent or drug, such that the active agent or drug may interact with and become immobilized to the substrate.
  • the substrate may also be chosen depending on the final dosage form of the product.
  • any classic therapeutically acceptable excipient(s), such as micro-crystalline cellulose, etc., may be used as a substrate according to the present disclosure because they are considered (i) GRAS compound because they have been shown to be non-toxic, (ii) compatible with a wide range of solvents, and (iii) present a surface topology (e.g., but not limited to, porosity) and surface chemistry suitable for drug immobilization without covalent chemical interactions.
  • Compounds or drugs immobilized to micro-crystalline cellulose are compatible with other excipients and may be blended with other excipients to make formulations that may be formed into tablets by direct compression.
  • a micro-crystalline cellulose powder flow having the active agent or drug immobilized to it may also be used for fdling capsules.
  • degrade means to chemically decompose. Degradation is distinct from melting in that it involves the breaking of chemical bonds or the formation of new chemical bonds. Methods of degradation can include hydrolysis, solventolysis, thermolysis, or oxidation.
  • a “subject” or “patient” can be a mammal, e.g., but not limited to, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, chordate, or non-human primate, such as, but not limited to, a monkey, chimpanzee, or baboon.
  • an “effective amount” when used in connection with a pharmaceutical formulation encompasses an amount that causes a particular result, such as reducing or eliminating at least one symptom or negative effect of ae disorder or disease, or which prevents the manifestation of the disease or disorder in a subject as described herein.
  • An effective amount also refers to that amount which can cause a predetermined or preselected result, change, or activity.
  • carrier encompasses carriers, excipients, and diluents and means a material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting a pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body of a subject.
  • the “carrier” can be used to manipulate the release rate of the API (e.g., NAD+) to include rapid or sustained release.
  • the terms “treat” and “treatment” are synonymous with the term “prevent” and are meant to indicate a postponement of development of diseases, preventing the development of diseases, and/or reducing severity of such symptoms that will or are expected to develop.
  • these terms include ameliorating existing disease symptoms, preventing additional symptoms, ameliorating, or preventing the underlying causes of symptoms, inhibiting the disorder or disease, e.g., arresting the development of the disorder or disease, relieving the disorder or disease, causing regression of the disorder or disease, relieving a condition caused by the disease or disorder, or stopping or alleviating the symptoms of the disease or disorder.
  • disorder is used in this disclosure to mean, and is used interchangeably with, the terms disease, condition, or illness, unless otherwise indicated.
  • administered refers to either directly administering a pharmaceutical formulation to a subject.
  • container encompasses any vessel, dish, or other structure into which NAD+ or a mixture of NAD+ and any other chemical component is placed and which can withstand decreased pressure or vacuum.
  • a container may also be a blender or a container that enables mixing of components.
  • administer refers to either directly administering a disclosed salt or a composition to a subject, or administering a prodrug derivative or analog of the salt or composition to the subject, which can form an equivalent amount of active salt within the subject's body.
  • the present disclosure relates to enhanced NAD+ compositions and pharmaceutical formulations containing the same useful in the treatment of diseases, disorders, and conditions related to decreased levels of intracellular NAD.
  • the present disclosure further relates to methods of manufacturing enhanced NAD+ compositions and pharmaceutical formulations or “drug products” that are stable, therapeutic, and bioavailable.
  • the present disclosure also provides new methods of increasing intracellular levels of NAD and of treating various disorders associated with a decrease in intracellular NAD.
  • the present disclosure provides for enhanced delivery of NAD+ to target cells to treat substance abuse including, but not limited to, drug or alcohol addiction. Also disclosed are methods of detecting biomarkers associated with decreased intracellular levels of NAD.
  • NAD+ is known to be chemically and biologically unstable.
  • the present disclosure provides herein NAD+ compositions and formulations having an enhanced stability compared to traditional NAD+ compositions.
  • the illustrative enhanced NAD+ compositions may have a slower degradation rate and/or a longer half-life (in vitro and/or in vivo), a longer shelf-life, and/or a less stringent storage condition requirement (e.g., cooling or dry storage), compared to a control NAD+ composition without the property or not produced by the conditions and processes described herein (e.g., a NAD+ composition from a commercial source).
  • the present disclosure provides NAD+ compositions having an enhanced stability and bioavailability compared to NAD+ (e.g., NAD+ monohydrate)-containing compositions.
  • NAD+ e.g., NAD+ monohydrate
  • the present enhanced NAD+ compositions have a slower degradation rate and/or a longer half-life (in vitro and/or in vivo), a longer shelf-life, and/or a less stringent storage condition requirement (e.g., but not limited to, cooling or dry storage), compared to a control NAD+ not produced by the conditions and processes described herein.
  • the chemical name of Formula I is: [[(2R,3S,4R,5R)-5-(6- aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2R,3S,4R,5R)-5-(3- carbamoylpyridin-l-ium-l-yl)-3,4-dihydroxyoxolan-2-yl]methyl hydrogen phosphate.
  • the chemical formula of Formula I is C21H2XN7O14P2T
  • the molecular weight of protonated NAD+ positively charged, anhydrate
  • the CASRN of Formula I is 53-84-9 and the source is PubChem (CID 5893).
  • the molecular weight of NAD+ (zwitterion, anhydrate) is 663.43 g/mol.
  • the enhanced NAD+ compositions described herein may be a substitute of current commercially available NAD+ compositions, or other NAD+ compositions without the property or not produced by the current conditions or process, for its enhanced stability.
  • the enhanced NAD+ compositions described herein may be used as a supplement and/or a pharmaceutical agent with improved bioavailability for oral administration (or through other administration routes) to an individual.
  • the enhanced NAD+ compositions described herein may be more stable in the direct interaction with water, radiation, oxygen, and/or UV light,
  • This compound is an active component of various enhanced compositions and pharmaceutical formulations of the present disclosure (described below), where it is present from about 5 mg to about 4000 mg.
  • 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 dose of NAD+ is about 50 mg to about 2000 mg. In some embodiments of the present disclosure, the amount of NAD+ is about 100 mg to about 1500 mg. In some embodiments of the present disclosure, the amount of NAD+ is about 200 mg to about 1000 mg. In further embodiments, the amount of NAD+ is about 300 mg to about 900 mg.
  • 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.
  • Enhanced NAD+ compositions according to the disclosure may also include phosphorus as a minor component originally from the commercially obtained NAD+ (e.g., NAD+ monohydrate) which remains after the vacuumization process (described below), or which may be added.
  • the content of phosphorus is from about 0.5% to about 9.33%.
  • an enhanced NAD+ composition may comprise from about 0.5% (wt/wt) to about 5.5% (wt/wt).
  • the content of phosphorus is about 1.0% to about 5.0%.
  • the content of phosphorus is about 4.5% to about 5.0%.
  • the content of phosphorus is about 3.5% to about 6.0%.
  • the content of phosphorus is about 3.0% to about 5.5%. In some embodiments, the content of phosphorus is about 4.0% to about 5.5%. In some embodiments, the content of phosphorus is about 3.5% to about 5.5%. In certain embodiments, the content of phosphorus is about 4.5% to about 4.7%. In other embodiments, the content of phosphorus is about 4.5% to about 5.5%. In some embodiments, the content of phosphorus is about 3.0%, about 3.5%, about 4.5%, about 4.58%, 4.6%, about 4.67%, about 4.7%, about 4.8%, about 4.9%.
  • the content of phosphorus is about 5.0%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 5.6%, about 5.6%, about 5.8%, about 5.9%, about 6.0% about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, or about 9.33%.
  • the NAD+ composition comprises 3.0% (wt/wt) NAD+ and about to 9.33% (wt/wt) phosphorus.
  • the enhanced NAD+ composition further contains traces of an inert gas.
  • the NAD+ composition may include s traces of Argon, Nitrogen, Xenon, or Helium, and may also include anhydrous CO2.
  • the enhanced composition of the present disclosure comprises an amount of water, moisture, and/or solvent that is less than about 0.2% (weight/ weight) measured by standard methods (e.g., Karl Fischer titration).
  • the enhanced NAD+ compositions of the present disclosure have a density of about 0.51g/cm 3 . In some embodiments, the NAD+ compositions of the present disclosure have a density of about 0.4g/cm 3 . In some embodiments, the enhanced NAD+ compositions of the present disclosure have a density from about 0.27 g/cm 3 to about 0.51 g/cm 3 . In some embodiments, the enhanced NAD+ compositions of the present disclosure have a density of about 0.52 g/cm 3 . In some embodiments, the enhanced NAD+ compositions of the present disclosure have a density of about 0.53 g/cm 3 .
  • 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 0.5 6 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 .
  • 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 from about 0.51 g/cm 3 to about 0.75 g/cm 3 .
  • control NAD+ refers to NAD+ (e.g., NAD+ monohydrate) which may be synthesized or which is commercially available.
  • enhanced NAD+ compositions described herein are at least 10%, at least 20%, at least 30%, at least 40%, 50%, at least 60%, at least 70%, 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, or at least 900% more stable than control NAD+
  • Other exemplary enhanced NAD+ compositions according to the disclosure are 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more, stable than a control NAD+.
  • Such stability may be represented by the degradation rate, half-life
  • the enhanced NAD+ compositions of the instant disclosure are also stable under physiological conditions.
  • the instant NAD+ compositions have a higher bioavailability in cells and organs as compared to commercially available NAD+ supplements.
  • the instant NAD+ compositions have a higher bioavailability in target cells (e.g., liver, kidney, immune cells) as compared to commercially available NAD+ supplements.
  • the NAD+ compositions described herein display a low rate of degradation.
  • the instant enhanced NAD+ compositions can be stored without signs of degradation for 36 months at room temperature (RT) (20 °C to 22 °C) and/or under suitable storage conditions, including, but not limited to, refrigeration (0 °C to 4 °C) or freezing (0 °C to - 20 °C).
  • the enhanced NAD+ composition has enhanced solid form stability.
  • the enhanced NAD+ composition of the instant disclosure shows greater stability than commercially available NAD+ supplements.
  • the instant enhanced NAD+ compositions also show increased stability in aqueous solution.
  • no significant degradation of the instant NAD+ compositions in aqueous solution phase occurs for up to 72 hours.
  • degradation of the enhanced NAD+ composition is less than 5% after 4 days in an aqueous solution.
  • the compositions remain stable in aqueous solution after 5 days with 10% degradation or less.
  • the enhanced NAD+ composition has enhanced aqueous stability.
  • the enhanced NAD+ compositions of the present disclosure have a density 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 from about 0.27 g/cnT to about 0.51 g/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 .
  • 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 from about O.51g/cm 3 to about 0.75 g/cm 3 .
  • the enhanced NAD+ compositions of the present disclosure comprise a crystalline component comprising polyethylene glycol, and an amorphous component comprising amorphous NAD+.
  • the enhanced NAD+ composition further comprises an amorphous polyethylene glycol.
  • the ratio of NAD+ to polyethylene glycol in the composition is about 1 : 1 (wt/wt).
  • the ratio of NAD+ to polyethylene glycol in the composition is 1 : 1 (wt/wt).
  • the polyethylene glycol has an average molecular weight of between about 300 and about 4000.
  • the polyethylene glycol is PEG335O.
  • the composition is characterized by having a first onset temperature of between 179.39 °C ( ⁇ 0.10°C) and 182.61 °C ( ⁇ 0.10°C) by thermogravimetric analysis (TGA) thermogram.
  • TGA thermogravimetric analysis
  • the composition is characterized by having a weight loss from 14.39 wt% to 16.98 wt% in the range from about 170 °C to about 300 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 300 °C under nitrogen gas at a ramp rate of 10 °C/min.
  • TGA thermogravimetric analysis
  • the composition is characterized by having a weight loss from 57.83 wt% to 58.55 wt% in the range from about 300 °C to about 800 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 800 °C under nitrogen gas at a ramp rate of 10 °C/min.
  • TGA thermogravimetric analysis
  • the composition is characterized by having a TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with any one of FIG. 33A, FIG. 33B, FIG. 34A, FIG. 34B, FIG. 35A, or FIG. 35B.
  • TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with FIG. 33 A or FIG. 33B.
  • compositions of the present disclosure comprising a crystalline component comprising polyethylene glycol and an amorphous component comprising amorphous NAD+
  • TGA thermogravimetric analysis
  • thermogravimetric analysis TGA thermogram collected from ambient temperature to at least 170 °C under nitrogen gas at a ramp rate of 10 °C/min
  • thermogravimetric analysis TGA thermogram collected from ambient temperature to at about 800 °C under nitrogen gas at a ramp rate of 10 °C/min and from about 800 °C to about 900 °C under air at a ramp rate of 10 °C/min, and/or
  • a first onset temperature from 179.39 °C to 180.15 °C, and/or
  • the composition is characterized by having one characteristic selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having two characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having three characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having four characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having five characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having all six characteristics selected from (1), (2), (3), (4), (5), and (6).
  • compositions of the present disclosure comprising a crystalline component comprising polyethylene glycol and an amorphous component comprising amorphous NAD+
  • the composition is characterized by having a TGA thermogravimetric analysis (TGA) thermogram having:
  • thermogravimetric analysis TGA thermogram collected from ambient temperature to at least 170 °C under nitrogen gas at a ramp rate of 10 °C/min
  • thermogravimetric analysis TGA thermogram collected from ambient temperature to at least 300 °C under nitrogen gas at a ramp rate of 10 °C/min
  • thermogravimetric analysis TGA thermogram collected from ambient temperature to at about 800 °C under nitrogen gas at a ramp rate of 10 °C/min and from about 800 °C to about 900°C under air at a ramp rate of 10 °C/min, and/or
  • a first onset temperature from 178.62 °C to 180.11 °C, and/or
  • the composition is characterized by having one characteristic selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having two characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having three characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having four characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having five characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having all six characteristics selected from (1), (2), (3), (4), (5), and (6).
  • compositions of the present disclosure comprising a crystalline component comprising polyethylene glycol and an amorphous component comprising amorphous NAD+
  • the composition is characterized by having a TGA thermogravimetric analysis (TGA) thermogram having:
  • thermogravimetric analysis TGA
  • second weight loss 15.17 wt% to 16.98 wt% in the range from about 170 °C to about 300 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 300 °C under nitrogen gas at a ramp rate of 10 °C/min
  • TGA thermogravimetric analysis
  • thermogravimetric analysis TGA thermogram collected from ambient temperature to at about 800 °C under nitrogen gas at a ramp rate of 10 °C/min and from about 800 °C to about 900°C under air at a ramp rate of 10 °C/min, and/or
  • a first onset temperature from 179.83 °C to 182.61 °C, and/or
  • the composition is characterized by having one characteristic selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having two characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having three characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having four characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having five characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having all six characteristics selected from (1), (2), (3), (4), (5), and (6).
  • the present disclosure also provides pharmaceutical formulations comprising NAD compositions with enhanced properties, which may be administered to an individual (e.g., a human) as a nutrient and/or a pharmaceutical agent to prevent, ameliorate, and/or treat a disease or disorder related to insufficient NAD in the body.
  • an individual e.g., a human
  • the enhanced NAD+ pharmaceutical formulations described herein increases the NAD concentration and/or NAD degradation in the individual.
  • the individual is having tryptophan-poor diet.
  • the individual has a genetic mutation and/or other mechanisms (e.g., living or environmental conditions, food or drug effects, etc.) resulting in a reduction or inhibition of NAD production, a reduction of NAD stability, and/or an increase of NAD degradation or turnover in the individual.
  • the individual may have a genetic mutation in the kynurenine pathway, thus reducing or inhibiting the production chain from nicotinic acid to NMN to NAD and then to NAD.
  • the individual may have another genetic mutation affecting the production, transportation, storage, and/or utilization of NAD+ in the individual.
  • the enhanced NAD+ compositions described herein may also enhance NAD-related biochemical and biological reactions in an individual in need thereof.
  • the enhanced NAD+ pharmaceutical formulations described herein may enhance the bio-functions of oxidoreductases in the individual, e.g., enhancing synthesizing enzymes such as poly(ADPribose) polymerases and cADPribose synthases.
  • the enhanced NAD+ pharmaceutical formulations described herein may be administered to the individual to prevent, ameliorate, and/or treat the disease or disorder.
  • the enhanced NAD+ pharmaceutical formulations described herein may be a substitute of current commercially available NAD+ compositions, or other NAD+ compositions without the property or not produced by the current conditions or process, for its enhanced stability.
  • the enhanced NAD+ compositions described herein may be used as a supplement and/or a pharmaceutical agent with improved bioavailability for oral administration (or through other administration routes) to an individual.
  • the enhanced NAD+ compositions described herein may be more stable in the direct interaction with water, radiation, oxygen, and/or UV light, and if administered as part of a pharmaceutical formulation, is resistant to hydrolysis in the small intestine by brush border cells.
  • the enhanced NAD+ pharmaceutical formulations of the present disclosure comprise the enhanced NAD+ composition as described above, as well as at least one additional secondary component, compound, or ingredient that can aid in administration and/or treatment of a subject.
  • this ingredient is PEG, a polymer of ethylene glycol) which is “generally recognized as safe” (GRAS) under the US Federal Drug Administration (FDA).
  • PEG has various sizes depending on its molecular weight which follows the term “PEG.”
  • the enhanced NAD+ composition comprises PEG300, PEG400 PEGMME (Polyethylene glycol monomethyl ether) 550, PEG600, PEG1000, PEGMME 2000, PEG3350, and/or PEG4000.
  • composition of the present disclosure comprises PEG of any molecular weight at a concentration of about 10% to about 75%
  • 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%.
  • the ratio of NAD+ to PEG of any molecular weight in an NAD+ composition 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).
  • the ratio of NAD+ to PEG of any molecular weight in a composition of the present disclosure is 2: 1 (wt/wt), 3: 1 (wt/wt), 4: 1 (wt/wt), 5: 1 (wt/wt), 6: 1 (wt/wt), 7: 1 (wt/wt), 8: 1 (wt/wt), 9: 1 (wt/wt), 10:1 (wt/wt), 1 1 : 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).
  • the enhanced NAD+ pharmaceutical formulations of the instant disclosure are also stable under physiological conditions.
  • these formulations have a higher bioavailability in cells and organs as compared to commercially available NAD+ supplements.
  • nicotinamide mononucleotide NNN
  • NR nicotinamide riboside
  • NAM nicotinamide
  • the instant NAD+ formulations have a higher bioavailability in target cells (e.g, liver, kidney, immune cells) as compared to commercially available NAD+ supplements.
  • formulations can be in solid as well as liquid dosage forms, such as tablets, capsules, powders, lozenges, suppositories , syrups, elixirs, sterile solutions, suspensions or emulsions, pastes, ointments, jellies, waxes, oils, lipids, encapsulation in lipid (cationic or anionic) containing vesicles (such as liposomes, microparticles, microcapsules, or LIPOFECTINTM), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax, semi-solid gels, and semi-solid mixtures containing carbowax.
  • lipid (cationic or anionic) containing vesicles such as liposomes, microparticles, microcapsules, or LIPOFECTINTM
  • DNA conjugates anhydrous absorption pastes, oil-in-water and water-in-oil emulsion
  • the amount of pharmaceutical formulation to be administered depends on the concentration or amount of NAD+ in the formulation, the mode of administration, the disorder to be treated, the severity of the disorder being treated, the weight of the patient, and the like, as known by the professional treating a subject.
  • These pharmaceutical formulations may include carriers, excipients, flavorings, dyes, and/or other agents that provide suitable delivery, tolerance, and the like to the subject.
  • Such useful and appropriate agents can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
  • the pharmaceutical formulation comprises about 0.5% (wt/wt) magnesium stearate. In some embodiments, the pharmaceutical formulation comprises about 0.1% (wt/wt), about 0.2% (wt/wt), about 0.3% (wt/wt), about 0.4% (wt/wt), about 0.5% (wt/wt), about 0.6% (wt/wt), about 0.7% (wt/wt), about 0.8% (wt/wt), about 0.9% (wt/wt), or about 1% (wt/wt) magnesium stearate.
  • the tablet form may further comprise glucose, e.g., at from about 10% to about 75% glucose.
  • glucose e.g., at from about 10% to about 75% glucose.
  • Some non-limiting embodiments of the NAD+ formulations comprise glucose at a concentration of about 1 10%, about 20%, about 30%, about 40%, about 41%, about 41.42%, about 45%, about 50%, about 60%, about 70%, or about 75%.
  • the composition of the present disclosure further comprises polyethylene glycol at a concentration of about 50%.
  • 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%.
  • Useful dyes include, but are not limited to, Allura Red AC (Red 40) or Blue #1 .
  • the compositions may comprise Allura Red AC at a concentration of from about 0.1% to about 3%.
  • the composition comprises Allura Red AC at a concentration of about 0.1%, about 0.5%, about 1%, about 1.25%, about 1.5%, about 1.75%, about 2%, about 2.5%, or about 3%.
  • the enhanced NAD+ pharmaceutical formulation of the present disclosure further comprises a flavoring such as, but not limited to, methyl salicylate.
  • a flavoring such as, but not limited to, methyl salicylate.
  • methyl salicylate can be present at a concentration of about 0.1 ml/kg to about 2.0 ml/kg.
  • methyl salicylate can be present in the composition at a concentration of about 0.1 ml/kg, about 0.2 ml/kg, about 0.3 ml/kg, about 0.4 ml/kg, about 0.5 ml/kg, about 0.6 ml/kg, about 0.7 ml/kg, about 0.8 ml/kg, about 0.9 ml/kg, about 1 ml/kg, about 1.5 ml/kg, about 1.7 ml/kg, or about 2 ml/kg.
  • the enhanced NAD+ pharmaceutical formulations may additionally include other therapeutic compounds or drugs, such as, but not limited to analgesic agents.
  • a powder form of the formulation may include acetylsalicylic acid at a concentration of about 0.1% to about 0.7%.
  • the NAD+ formulations comprises acetylsalicylic acid at a concentration of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, or about 0.7%.
  • the pharmaceutical composition may include, but is not limited to, for example, magnesium stearate, glucose, polyethylene glycol, Red 40, salicylate methyl, Blue #1, or any combination thereof.
  • compositions described herein may be administered orally, anally, systemically, intramuscularly, optically, vaginally, buccally, subcutaneously, etc.
  • the route and delivery system for administration are known by those with medical skill in the art.
  • liquid forms of the NAD+ formulation may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), for example, by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intra-tracheal, epidural, and oral routes, and may be administered together with other biologically active.
  • epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.
  • intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intra-tracheal, epidural, and oral routes for example, by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intra-tracheal, epidural, and oral routes, and may be administered together with other biologically active.
  • composition described herein is in a powder form.
  • the NAD+ is placed in a container under reduced pressure of vacuum or under a reduced pressure of between about 24 inches of Hg and about 30.25 inches of Hg; and replacing the vacuum with an inert gas to increase the pressure in the container to about 1 atmosphere (atm).
  • the inert gas which replaces the vacuum comprises Argon, Helium, Nitrogen, and/or Xenon. Vacuumization can be performed more than once and at least twice. Vacuumization can be performed more than once, e.g., once, twice, or at least twice.
  • the inert gas used for vacuumization comprises Argon. In another embodiment, the inert gas used for vacuumization comprises Xenon. In another embodiment, the inert gas used for vacuumization comprises anhydrous, high purity Nitrogen. In another embodiment, the inert gas used for vacuumization comprises Helium.
  • the NAD+ may be pretreated before being vacuumized.
  • the NAD+ can be processing to obtain a preselected average NAD+ particle size. Processing can include, but it not limited to, passing the NAD+ through a sieve, and/or grinding, milling, or jet-milling the NAD+ to obtain the preselected particle size.
  • the NAD+ is ground or milled into a particle about 0.05 mm to about 0.4 mm in size in size.
  • the NAD+ particle is about 0.05 mm, about 0.07 mm, about 0.09 mm, about 0.1 mm, about 0.110 mm, about 0.120 mm, about 0.140 mm, about 0.150 mm, about 0.153 mm, about 0.155 mm, about 0. 160 mm, about 0.180 mm, about 0.2 mm, about 0.220 mm, about 0.250 mm, about 0.270 mm, about 0.300 mm, about 0.320 mm, about 0.340 mm, about 0.350 mm, about 0.370 mm, or about 0.4 mm in size.
  • the enhanced NAD+ pharmaceutical formulations according to the present disclosure can be prepared by mixing NAD+ with any other components, to obtain a blended mixture, which is then vacuumized under vacuum and inert gas conditions.
  • the vacuumized mixture may contain traces of an inert gas.
  • the resulting vacuumized mixture contains traces of Argon, Nitrogen, Xenon, or Helium.
  • APIs such as those according to the disclosure may be initiated by mixing vacuumized or un-vacuumized NAD+ with other components in a container, e ., in a blender, to form a blended mixture.
  • a container e.g., in a blender
  • the term “blended mixture” refers to mixed components of the composition, e.g., various amounts of one or more ingredient with another or others.
  • NAD+ is mixed with PEG of any molecular weight, which may be commercially-obtained, and which may be pretreated as described herein to obtain a preselected particle size, to achieve a homogeneous distribution of NAD+ and PEG in the final pharmaceutical formulation (or “drug product”).
  • PEG of any molecular weight which may be commercially-obtained, and which may be pretreated as described herein to obtain a preselected particle size, to achieve a homogeneous distribution of NAD+ and PEG in the final pharmaceutical formulation (or “drug product”).
  • the commercially obtained PEG of any particular molecular weight or weights, as described above, is used in the preparation is GRAS under the FDA.
  • the process of preparation comprises preformulating or pretreating commercially obtained NAD+ and/ or PEG.
  • the pretreated NAD+ and/or PEG is vacuumized prior to mixing.
  • the NAD+ or PEG is vacuumized prior to being pretreated and mixed.
  • both NAD+ and PEG are separately vacuumized before mixing, before or after being sized, and/or before and/or before mixing.
  • Processing or pretreatment of NAD+ is performed to allow for homogeneous distribution with excipients in the NAD+ formulation of the present disclosure.
  • the NAD+ is ground or milled into a particle about 0.05 mm to about 0.4 mm in size.
  • Another aspect of the disclosure relates to a process of making an enhanced NAD+ composition, said process comprising blending NAD+ and polyethylene glycol and vacuumizing the blended mixture of NAD+ and PEG under vacuum and/or inert gas conditions.
  • the process of preparation comprises preformulating NAD+.
  • pre-formulating the NAD+ involves grinding or milling the NAD+.
  • 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+ formulation of the present disclosure.
  • the process of pre-formulation of NAD+ involves grinding or milling the NAD+ and then vacuumizing the ground or milled NAD+ product by itself prior to blending.
  • the process of preparation further comprises preformulating commercial PEG.
  • pre-formulating the PEG comprises grinding or milling the commercial PEG.
  • grinding and/or milling the commercial PEG is performed to reduce the commercial PEG into particles of super fine size to allow for homogeneous distribution with excipients and the active ingredient (NAD+) in the NAD+ formulation of the present disclosure.
  • pre-formulation of PEG comprises vacuumizing the commercial PEG and then grinding or milling the vacuumized PEG product by itself prior to blending.
  • the pretreated NAD+ and/or PEG is vacuumized prior to mixing.
  • the NAD+ or PEG is vacuumized prior to being pretreated and mixed.
  • both NAD+ and PEG are separately vacuumized before mixing, before or after being sized, and/or before and/or before mixing.
  • the process of mixing comprises mixing the NAD+ and PEG or the pretreated NAD+ and/or pretreated PEG.
  • Mixing of NAD+ with PEG enables, e.g., non- covalent coating or immobilization of NAD+ to PEG, results in stabilization of NAD+ and prevents its degradation.
  • Mixing can be carried out, e.g., for about 20 minutes to about an hour at room temperature (RT). For example, the mixture is blended for at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 1 hour, at RT.
  • the blended mixture of NAD+ and PEG is then vacuumized by being placed under a cycle of vacuum or reduced pressure and may be followed by inert gas re-saturation.
  • the blended mixture of NAD+ and PEG is placed under a reduced pressured of about 22 inches of Hg to about 29 inches of Hg. In some embodiments of process of preparation, the blended mixture of NAD+ and PEG is placed under a reduced pressured of about 26 inches of Hg to about 30 inches of Hg. In some embodiments, the blended mixture of NAD+ and PEG is placed under a reduced pressure of about 24 inches of Hg to about 29.92 inches of Hg. In some embodiments, the blended mixture of NAD+ and PEG is placed under a reduced pressured of about 24 inches of Hg to about 28 inches of Hg.
  • the blended mixture of NAD+ and PEG is placed under a reduced pressured of about 24 inches of Hg. In some embodiments, the blended mixture of NAD+ and PEG is placed under a reduced pressured of about 26 inches of Hg. In some embodiments, the blended mixture of NAD+ and PEG is placed under a reduced pressured of about 28 inches of Hg. In some embodiments, the blended mixture of NAD+ and PEG is placed under a reduced pressured of about 30 inches of Hg. In some embodiments, the blended mixture of NAD+ and PEG is placed under a reduced pressured of about 30.25 inches of Hg.
  • the removal of solvents also results in the azeotropic and/or evaporative removal of contaminant components, such as, but not limited to, water, air, oxygen, etc. which accelerates the degradation of the NAD+ product.
  • contaminant components such as, but not limited to, water, air, oxygen, etc. which accelerates the degradation of the NAD+ product.
  • the process of vacuumizing may also include placing the blended mixture of NAD+ and PEG under a cycle of vacuum or reduced pressure may be followed by inert gas re-saturation.
  • non-covalent coating or immobilization of an active agent e.g., NAD+
  • an active agent e.g., NAD+
  • further stabilization of the product is also believed to occur by azeotropic and/or evaporative removal of contaminant components, such as, but not limited to, water and oxygen, during solvent evaporation, and the vacuum may be replaced with an inert, anhydrous gas such as, but not limited to, Nitrogen (N2), Argon (Ar), or Xenon (Xe).
  • the blended mixture under vacuum is re-saturated with an inert gas to restore the pressure in the blender to 1 atm.
  • the inert gas used in the resaturation during the vacuumization process comprises Nitrogen, Argon, Xenon, or Helium, any of which may be of high purity.
  • Other displacement gases may also be used to achieve further advantages.
  • anhydrous carbon dioxide (CO2) may be used as an inert replacement gas that may also serve an additional purpose of acidifying the local environment of a substrate for active agents or drugs that are more stable in acidic environments.
  • Vacuumization of the blended mixture is performed for at least one cycle. In other embodiments, vacuumization of the blended mixture is done in at least two cycles. In other embodiments, vacuumization of the blended mixture is done in at least three, four or more cycles.
  • the commercial PEG used in the preparation is GRAS under the FDA.
  • the commercial PEG used in the preparation is PEG300.
  • the commercial PEG used in the preparation is PEG400.
  • the commercial PEG used in the preparation is PEGMME 550.
  • the commercial PEG used in the preparation is PEG600.
  • the commercial PEG used in the preparation is PEG1000.
  • the commercial PEG used in the preparation is PEGMME 2000.
  • the commercial PEG used in the preparation is PEG335O.
  • the commercial PEG used in the preparation is PEG4000.
  • the commercial PEG used in the preparation is PEG300, PEG400, PEGMME 550, PEG600, PEG1000, PEGMME 2000, PEG3350, PEG4000, or any combination thereof.
  • the process of preparation comprises blending the pre-formulated NAD+ and pre-formulated PEG.
  • compositions according to the disclosure can be made from the enhanced NAD+ compositions according to the process described above, mixed with PEG, and may also include the addition of other components such as, but not limited to, excipients, flavorings, sugars dyes, magnesium stearate, etc.
  • Non-limiting embodiments of such secondary ingredients include magnesium stearate, glucose, Red40, Blue#l, methyl salicylate, and water.
  • a further aspect of the present disclosure is to provide an enhanced NAD+ composition made by the process of the instant disclosure which comprises nicotinamide adenine dinucleotide (NAD+) and polyethylene glycol (PEG) with a phosphorus content of about 1.0% (wt/wt) to about 9.33% (wt/wt).
  • a further aspect of the present disclosure is to provide an enhanced NAD+ composition made by the process of the instant disclosure which comprises nicotinamide adenine dinucleotide (NAD+) and polyethylene glycol (PEG) with a phosphorus content of about 1.0% to about 4.67%.
  • a further aspect of the present disclosure is to provide an enhanced NAD+ composition made by the process of the instant disclosure which comprises nicotinamide adenine dinucleotide (NAD+) and polyethylene glycol (PEG) with a phosphorus content of about 0.5% to about 9.33%.
  • NAD+ nicotinamide adenine dinucleotide
  • PEG polyethylene glycol
  • the enhanced NAD+ pharmaceutical formulations of the present disclosure can be used in the treatment of a variety of conditions, diseases and disorders. For example, they can be used to preventing or ameliorating anti-aging processes, in treating or preventing age-related disorders, CNS disorders, and in increase intracellular NAD levels
  • the present enhanced NAD+ pharmaceutical formulations are potent and are efficacious at clinically achievable doses, are stable in a variety of potential dosage forms, possess acceptable solubility, bioavailability, acceptable pH, and have a reduced propensity to absorb and/or degrade in the presence of water, oxygen, UV light water, display ease of handling and consumption, all of which are consistent with the development, manufacture, and use of a medicament.
  • the NAD+ composition comprises an amorphous component.
  • the enhanced NAD+ pharmaceutical formulations disclosed herein offer increased biological activity toward increased cellular NAD levels, increased stability, and more physiologically acceptable pH.
  • PEG poly(ethylene glycol)
  • the inclusion of PEG in the formulations herein provide a novel “molecular shuttle” for the delivery of NAD+, for example, but not limited to, across the buccal membranes into the blood.
  • This formulation provides multiple advantages that are useful to assist in NAD+-dependent cellular activities.
  • the enhanced NAD+ pharmaceutical formulations disclosure herein may be administered to a subject (e g., but not limited to, a human) as a nutrient and/or a therapeutic agent to prevent, ameliorate, and/or treat a disease or disorder related to insufficient NAD+ in the body.
  • a subject e g., but not limited to, a human
  • a therapeutic agent to prevent, ameliorate, and/or treat a disease or disorder related to insufficient NAD+ in the body.
  • the enhanced NAD+ pharmaceutical formulations are administered to increase the NAD concentration and/or NAD stability (i.e., half-life) in the individual.
  • the individual is having tryptophan-poor diet, a genetic mutation and/or other characteristics (e ., but not limited to, living or environmental conditions, food or drug effects), resulting in a reduction or inhibition of NAD production, a reduction of NAD stability, and/or an increase of NAD degradation or turnover in the individual.
  • the individual has a genetic mutation in the kynurenine pathway which results in the reduction or inhibition of the production chain from nicotinic acid to NMN to NAAD and then to NAD.
  • the individual has another genetic mutation affecting the production, transportation, storage, and/or utilization of NAD+ in the individual.
  • the enhanced NAD+ pharmaceutical formulations described herein may also enhance NAD+-related biochemical and biological reactions in an individual in need thereof.
  • NAD+ is a co-factor of oxidoreductases
  • the enhanced NAD+ compositions described herein enhances the bio-functions of oxidoreductases in the individual, e.g., but not limited to, enhancing synthesizing enzymes such as poly(ADPribose) polymerases and cADPribose synthases.
  • an enhanced NAD+ pharmaceutical formulation is administered to the individual to prevent, ameliorate, and/or treat the disease or disorder.
  • Another aspect of the present disclosure relates to a method of treating or preventing, an age-related disease, in a subject, comprising administering to the subject, a therapeutically effective amount of an NAD+ enhanced pharmaceutical formulation disclosed herein.
  • Such formulations can be manufactured as a medicament or supplement for treating many NAD+ deficiency-related diseases and disorders as described herein, such as, but not limited to, age- related diseases, central nervous system disorders, chemical addiction or substance abuse, such as, but not limited to, abuse related to tobacco, heroin, opium, morphine, dihydromorphine, meperidine, codeine, cocaine, amphetamines, barbiturates, alcohol, or tranquilizer use.
  • the present disclosure relates to a method for treating a subject suffering from, or preventing in a subject, a central nervous system disorder or disease, comprising administering to the subject a therapeutically acceptable amount of a composition of NAD+ described herein.
  • a method for treating a subject suffering from chemical addiction or substance abuse comprising administering to the subject a therapeutically effective amount of an NAD+ composition described herein.
  • the substance abused is tobacco, heroin, opium, morphine, dihydromorphine, meperidine, codeine, cocaine, amphetamines, barbiturates, alcohol, or tranquilizers.
  • Other methods of treatment include methods for increasing NAD-dependent deacetylase sirtuin-1 (SIRT1), increasing cryptococcal phospholipase (PLB1), and for increasing membrane metalloendopeptidase (MME) in a subject in need thereof, comprising administering to the subject an amount of an enhanced NAD+ pharmaceutical formulation the deficiency.
  • SIRT1 NAD-dependent deacetylase sirtuin-1
  • PLA1 cryptococcal phospholipase
  • MME membrane metalloendopeptidase
  • the present disclosure also provides a method for treating a subject suffering from a disease or disorder modulated by SIRT1, comprising administering to the subject an amount of an NAD+ composition described herein effective to modulate SIRT1.
  • PLB1 cryptococcal phospholipase
  • a method of treating a subject suffering from a disease or disorder modulated by membrane metalloendopeptidase comprises administering to the subject a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein.
  • the present disclosure also relates to a method for increasing a plasma proteome in a subject in need thereof, comprising administering to the subject in need thereof an amount of an enhanced NAD+ pharmaceutical formulation described herein effective to increase the plasma proteome of the subject.
  • the CNS disorder is chemical addiction.
  • the CNS disorder is substance abuse.
  • the method of treating is related to the treatment of a subject suffering from alcoholism and comprises administering to the subject a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation.
  • the disclosure relates to treatment of a subject suffering from alcohol -induced organ damage and comprises administering to the subject a therapeutically effective amount of the enhanced NAD+.
  • the disclosure relates to a method of treating a subject suffering from schizophrenia, comprising administering to the subject a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the disclosure relates to a method of treating a subject suffering from ischemia, inflammation, sepsis, free radical damage following myocardial infarction, and/or oxidative stress, comprising administering to the subject a therapeutically effective amount of the enhanced NAD+ composition described herein.
  • the disclosure relates to a method of treating a subject suffering from Alzheimer’s disease, comprising administering to the subject a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the disclosure relates to a method of treating a subject suffering from excitotoxicity, ischemia, and/or oxidative stress, comprising administering to the subject an effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the disclosure relates to a method of treating a subject suffering from pellagra, comprising administering to the subject a therapeutically effective amount of the enhanced NAD pharmaceutical formulation described herein.
  • the disclosure relates to a method of treating a subject suffering from pseudohypoxia, comprising administering to the subject a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the disclosure provides a method of treating a subject suffering from hypertrophic cardiomyopathy (HCM), comprising administering to the subject a therapeutically effective amount of the enhanced NAD+ + pharmaceutical formulation described herein.
  • HCM hypertrophic cardiomyopathy
  • the disclosure provides a method of treating a subject suffering from cellular senescence in brain aging, comprising administering to the subject a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein. In some embodiments, the disclosure provides a method of treating a subject suffering from ischemic brain damage, comprising administering to the subject a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein. In some embodiments, the disclosure provides a method of treating a subject suffering from Parkinson’s disease, comprising administering to the subject a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein. In some embodiments, the disclosure provides a method of treating a subject suffering from a prion disease, comprising administering to the subject a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the present disclosure also provides a method of treating a subject suffering from Parkinson’s disease, comprising administering the enhanced NAD+ pharmaceutical formulation described herein.
  • the disclosure provides a method of treating a subject suffering from a prion disease, comprising administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the disclosure provides a method of treating a subject suffering from bupivacaine-induced neurotoxicity, comprising administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the disclosure also provides a method of treating a subject suffering from arrhythmia, comprising administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the disclosure provides a method of treating a subject suffering from nicotinamide-induced mitophagy, comprising administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the also disclosure provides a method of treating a subject suffering from ischemic reperfusion, comprising administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the disclosure provides a method of treating a subject suffering from traumatic brain injury, comprising administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the disclosure provides a method of treating a subject suffering from synchrotron radiation X-ray- induced tissue injury, comprising administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the disclosure provides a method of treating a subject suffering from autoimmune diseases, comprising administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the disclosure provides a method of treating a subject suffering from diabetes, comprising administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the disclosure provides a method of treating a subject suffering from cancer, comprising administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the disclosure provides a method of treating a subject suffering from viral infection, comprising administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the viral infection is the coronavirus disease 2019 (COVID-19) and variants thereof.
  • the disclosure provides a method of promoting sobriety and/or satiety in a subject, comprising administering to the subject an amount of the enhanced NAD+ pharmaceutical formulation described herein effective to promote sobriety and/or satiety.
  • the disclosure provides a method of promoting cellular NAD+ metabolism and NAD+ cellular homeostasis in a subject, comprising administering to the subject an amount of the enhanced NAD+ pharmaceutical formulation described herein effective to promote cellular NAD+ metabolism.
  • the disclosure provides a method of preventing or treating a subject suffering from or in a risk of having at least one of cardiovascular diseases, comprising administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • cardiovascular diseases may include, for example, diseases or disorders related to an abnormality in any one of lipid metabolism, vascular effects/hypertension, homocysterine and the folate cycle, oxidative stress, inflammation, resting heart rate, metabolite levels, or any combinations thereof.
  • the disclosure provides a method of preventing or treating a subject suffering from or in a risk of having at least one of liver diseases, comprising administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the enhanced NAD+ composition described herein is capable of improving liver functions, resulting in, for example, a decrease in serum GGT, bilirubin, ALP, and/or albumin.
  • the enhanced NAD+ composition described herein is capable of reducing at least one of metabolites such as l-palmitoyl-2- arachidonoyl-GPC.
  • the enhanced NAD+ pharmaceutical formulation described herein is capable of reducing oxidative stress.
  • the disclosure also provides a method of preventing or treating a subject suffering from or in a risk of having at least one of diseases or disorders related to an abnormality in exocrine pancreas function and/or digestion, comprising administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the enhanced NAD+ pharmaceutical formulation pharmaceutical formulation described herein is capable of decreasing levels and/or functions of at least one of pancreatic enzymes [e.g., pancreatic lipase-related protein 1 (PNLIPRP1), pancreatic ribonuclease (RNASE 1), carboxypeptidase Al (CPA1), carboxypeptidase Bl (CPB1), etc.].
  • pancreatic enzymes e.g., pancreatic lipase-related protein 1 (PNLIPRP1), pancreatic ribonuclease (RNASE 1), carboxypeptidase Al (CPA1), carboxypeptidase Bl (CPB1), etc.
  • the disclosure provides a method of preventing or treating a subject suffering from or in a risk of having at least one of neurodegenerative and/or neuropsychiatric diseases, comprising administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • neurodegenerative diseases may include, for example, Alzheimer’s disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and Huntington’s disease.
  • neuropsychiatric diseases may include, for example, depression, anxiety, bipolar disorder, and schizophrenia.
  • diseases or disorders may be correlated with abnormal expression levels and/or functions of at least one of proteins listed in the table in FIG. 28.
  • the disclosure provides a method of preventing or treating a subject suffering from or in a risk of having at least one of diseases or disorders related to an abnormality in inflammation and/or immune modulation, comprising administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • diseases or disorders may include, for example, autoimmune diseases, including multiple sclerosis (MS), inflammatory bowel disease (IBD), and rheumatoid arthritis (RA).
  • diseases or disorders may be correlated with abnormal expression levels and/or functions of at least one of proteins listed in the table in FIG. 29.
  • the disclosure provides a method of preventing or treating a subject suffering from or in a risk of having at least one of diseases or disorders related to an abnormality in reproductive function and/or fertility, comprising administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the disclosure provides a method of preventing or treating a subject suffering from or in a risk of having at least one of metabolic diseases (such as obesity and insulin resistance syndromes), comprising administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the enhanced NAD+ composition described herein is capable of increasing expression levels and/or functions of at least one of N-acetyl-galactosaminyl-transferase 2 (GALNT2), fatty-acid binding protein 1 (FABP1), retinol binding protein 2 (RBP2), Glycohyocholic acid (GHCA), and Glyco- beta-muricholate.
  • GALNT2 N-acetyl-galactosaminyl-transferase 2
  • FBP1 fatty-acid binding protein 1
  • RBP2 retinol binding protein 2
  • GHCA Glycohyocholic acid
  • the enhanced NAD+ pharmaceutical formulation described herein is capable of decreasing expression levels and/or functions of at least one of pyrraline, urea, glutamate, acisoga, nonadecanoate, and l-palmitoyl-2-arachidonoyl-GPC.
  • the disclosure provides a method of preventing or treating a subject suffering from or in a risk of having at least one of skin and/or bone diseases, comprising administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation described herein.
  • the enhanced NAD+ composition described herein is capable of increasing expression levels and/or functions of SIRT1 and/or Matrilin-3 (MATN3).
  • the enhanced NAD+ pharmaceutical formulation described herein decreases expression levels and/or functions of at least one of Peptidyl-prolyl cis-trans isomerase B (PPIB), whey-acidic protein 4-di sulfide-core 12 (WFDC12), and insulin-like peptide 3 (INSL3).
  • PPIB Peptidyl-prolyl cis-trans isomerase B
  • WFDC12 whey-acidic protein 4-di sulfide-core 12
  • INSL3 insulin-like peptide 3
  • the disclosure provides a method of preventing or treating a subject suffering from post-viral long-term symptoms such as Long Covid. In other embodiments, the disclosure provides a method of preventing post-viral long-term symptoms. In some embodiments, the method comprises identifying a subject suffering from, or having a risk of suffering from, post-viral long-term symptoms such as Long Covid; and administering, e.g., orally, to the subject an amount of an enhanced NAD+ pharmaceutical formulation to the subject, wherein the amount is effective to increase a level of icNAD by at least 5% in the subject, thereby mitigating or preventing post-viral long-term symptoms in the subject.
  • the composition is be administered to the subject after a period of acute illness due to a viral disease such as Covid- 19.
  • the subject is diagnosed based on a positive test result for SARS-CoV-2 and persistence of one or more Long Covid symptoms over a period of months.
  • Long Covid symptoms include fatigue, post- exertional malaise, fever, difficulty breathing, shortness of breath, coughing, chest pain, heart palpitations, difficulty concentrating, headache, sleep disorder, dizziness, lightheadedness, pins- and-needles feeling, change in smell or taste, depression, anxiety, diarrhea, stomach pain, joint or muscle pain, rash, and changes in menstrual cycle.
  • compositions comprising Liothyronine, Polyethylene glycol, micro-porous glucose, and a pharmaceutically acceptable excipient.
  • compositions comprising LevaDopa and Polyethylene glycol and a pharmaceutically acceptable excipient.
  • the process of blending comprises mixing the pre-formulated NAD+ and PEG for at least 20 minutes at room temperature. In another embodiment, the process of blending involves mixing the pre-formulated NAD+ and PEG for at least 30 minutes at room temperature. In yet another embodiment, the process of blending involves mixing the pre-formulated NAD+ and PEG for at least 40 minutes at room temperature. In still another embodiment, the process of blending comprises mixing the pre-formulated NAD+ and PEG for at least 50 minutes at room temperature. In another embodiment, the process of blending comprises mixing the pre-formulated NAD+ and PEG for at least 1 hour at room temperature.
  • the process of preparation further comprises pre-formulating commercial PEG.
  • pre-formulating the PEG comprises grinding, sieving, or milling the commercial PEG.
  • grinding and/or milling the commercial PEG is performed to reduce the commercial PEG into particles of super fine size to allow for homogeneous distribution with excipients and the active ingredient.
  • the mixture is processed through a sieve of 2.5 mm or other size as best to allow for homogeneous distribution and compatibility with excipients.
  • the process of pre-formulation of PEG comprises grinding, sieving, or milling the commercial PEG and then vacuumizing the ground, sieved, or milled PEG product by itself prior to blending.
  • pre-formulation of PEG comprises vacuumizing the commercial PEG and then grinding, sieving, or milling the vacuumized PEG product by itself prior to blending.
  • the commercial PEG used in the preparation is GRAS under the FDA.
  • the commercial PEG used in the preparation is PEG300.
  • the commercial PEG used in the preparation is PEG400.
  • the commercial PEG used in the preparation is PEGMME 550.
  • the commercial PEG used in the preparation is PEG600.
  • the commercial PEG used in the preparation is PEG1000.
  • the commercial PEG used in the preparation is PEGMME 2000.
  • the commercial PEG used in the preparation is PEG3350.
  • the commercial PEG used in the preparation is PEG4000.
  • the commercial PEG used in the preparation is PEG300, PEG400, PEGMME 550, PEG600, PEG1000, PEGMME 2000, PEG335O, PEG4000, or any combination thereof.
  • Embodiment 1 An enhanced nicotinamide adenine dinucleotide (NAD+) composition, comprising a crystalline component comprising crystalline polyethylene glycol, and an amorphous component comprising amorphous NAD+, wherein the composition is characterized by having a first onset temperature of between 179.39 °C ( ⁇ 0.10°C) and 182.61 °C ( ⁇ 0.10°C) by therm ogravi metric analysis (TGA) thermogram.
  • TGA therm ogravi metric analysis
  • Embodiment 2 An enhanced nicotinamide adenine dinucleotide (NAD+) composition, comprising a crystalline component comprising crystalline polyethylene glycol, and an amorphous component comprising amorphous NAD+, wherein the composition is characterized by having a weight loss from 14.39 wt% to 16.98 wt% in the range from about 170 °C to about 300 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 300 °C under nitrogen gas at a ramp rate of 10 °C/min.
  • TGA thermogravimetric analysis
  • An enhanced nicotinamide adenine dinucleotide (NAD+) composition comprising a crystalline component comprising crystalline polyethylene glycol, and an amorphous component comprising amorphous NAD+, wherein the composition is characterized by having a weight loss from 57.83 wt% to 58.55 wt% in the range from about 300 °C to about 800 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 800 °C under nitrogen gas at a ramp rate of 10 °C/min.
  • TGA thermogravimetric analysis
  • Embodiment 4 The composition of any one of the preceding embodiments, further comprising an amorphous polyethylene glycol.
  • Embodiment 5 An enhanced nicotinamide adenine dinucleotide (NAD+) composition, comprising a crystalline component comprising crystalline polyethylene glycol, and an amorphous component comprising amorphous NAD+, wherein the composition is characterized by having a TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with any one of FIG. 33A, FIG. 33B, FIG. 34A, FIG. 34B, FIG. 35A, or FIG. 35B.
  • TGA thermogravimetric analysis
  • Embodiment 6 The composition of embodiment 5, which is characterized by having a TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with FIG. 33A or FIG. 33B.
  • TGA thermogravimetric analysis
  • Embodiment 7 The composition of embodiment 5, which is characterized by having a TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with FIG. 34A or FIG. 34B
  • Embodiment 8 The composition of embodiment 5, which is characterized by having a TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with FIG. 35A or FIG. 35B.
  • Embodiment 9 An enhanced nicotinamide adenine dinucleotide (NAD+) composition, comprising a crystalline component comprising crystalline polyethylene glycol, and an amorphous component comprising amorphous NAD+, wherein the composition is characterized by having a TGA thermogravimetric analysis (TGA) thermogram having: a first weight loss from 2.42 wt% to 3.25 wt% in the range from ambient temperature to about 170 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 170 °C under nitrogen gas at a ramp rate of 10 °C/min, and/or a second weight loss from 13.66 wt% to 14.61 wt% in the range from about 170 °C to about 300 °C by thermogravimetric analysis (
  • Embodiment 10 An enhanced nicotinamide adenine dinucleotide (NAD+) composition, comprising a crystalline component comprising crystalline polyethylene glycol, and an amorphous component comprising amorphous NAD+, wherein the composition is characterized by having a TGA thermogravimetric analysis (TGA) thermogram having: a first weight loss from 2.32 wt% to 3.55 wt% in the range from ambient temperature to about 170 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 170 °C under nitrogen gas at a ramp rate of 10 °C/min, and/or a second weight loss from 14.39 wt% to 16.22 wt% in the range from about 170 °C to about 300 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 300 °C under nitrogen gas at a ramp rate of 10 °C/min, and/or a third weight loss from 57.83 wt
  • Embodiment 11 An enhanced nicotinamide adenine dinucleotide (NAD+) composition, comprising a crystalline component comprising crystalline polyethylene glycol, and an amorphous component comprising amorphous NAD+, wherein the composition is characterized by having a TGA thermogravimetric analysis (TGA) thermogram having: a first weight loss from 3.62 wt% to 3.96 wt% in the range from ambient temperature to about 170 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 170 °C under nitrogen gas at a ramp rate of 10 °C/min, and/or a second weight loss from 15.17 wt% to 16.98 wt% in the range from about 170 °C to about 300 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 300 °C under nitrogen gas at a ramp rate of 10 °C/min, and/or a third weight loss from 58.05 wt
  • Embodiment 12 The composition of any of the preceding embodiments, wherein the ratio of NAD+ to polyethylene glycol in the composition is 1 : 1 (wt/wt).
  • Embodiment 13 The composition of any of the preceding embodiments, wherein the polyethylene glycol has an average molecular weight of between about 300 and about 4000.
  • Embodiment 14 The composition of any of the preceding embodiments, wherein the polyethylene glycol is PEG3350.
  • Embodiment 15 The composition of any of the preceding embodiments, which is in powder form.
  • Embodiment 16 The composition of any of the preceding embodiments, which is suitable for oral administration to a subject.
  • Embodiment 17 The composition of any of the preceding embodiments, wherein the composition has a shelf life of at least 6 months at room temperature.
  • Embodiment 18 The composition of any of the preceding embodiments, wherein the composition has a shelflife of at least 1 year at room temperature.
  • Embodiment 19 A pharmaceutical formulation comprising a composition of any one of embodiments 1-18.
  • Embodiment 20 The formulation of embodiment 19, which, when administered to a subject, increases NAD levels in the subject by at least about 10% relative to a baseline level of intracellular NAD in the subject.
  • Embodiment 21 The formulation of embodiment 19 or 20, which, when administered to a subject, increases intracellular levels of NAD in the subject.
  • Embodiment 22 The formulation of any one of embodiments 19-21, which, when administered to a subject, modulates expression of at least one biomarker in the subject.
  • Embodiment 23 The formulation of any one of embodiments 19-22, for use in a method of mitigating chemical addiction withdrawal symptoms in a subject in need thereof; mitigating opioid withdrawal symptoms in a subject in need thereof; increasing nicotinamide adenine dinucleotide (NAD) levels in a subject; and/or preventing or treating a disease or disorder correlated to decreased levels of NAD in a subject in need thereof.
  • NAD nicotinamide adenine dinucleotide
  • Embodiment 24 A kit comprising the composition of any one of embodiments 1-18.
  • Embodiment 25 A method of preparing the enhanced NAD+ composition of any one of embodiments 1-18, comprising
  • Embodiment 26 The method of embodiment 25, wherein the inert gas comprises argon or nitrogen.
  • Embodiment 27 The method of embodiment 25 or 26 wherein step (1) comprises subjecting NAD+ to at least three vacuumization cycles.
  • Embodiment 28 The method of any one of embodiments 25-27, wherein the polyethylene glycol has an average molecular weight of between about 300 and about 4000.
  • Embodiment 29 The method of any one of embodiments 25-28, wherein the polyethylene glycol is PEG3350.
  • Embodiment 30 The method of any one of embodiments 25-29, further comprising subjecting the blended mixture to at least one additional vacuumization cycle.
  • Embodiment 31 The method of any one of embodiments 25-29, further comprising subjecting the blended mixture to at least three additional vacuumization cycles.
  • Embodiment 32 The method of any one of embodiments 25-29, further comprising subjecting the blended mixture to at least six additional vacuumization cycles.
  • Embodiment 33 The method of any one of embodiments 25-32, further comprising pretreating the NAD+ prior to step (1).
  • Embodiment 34 The method of embodiment 33, wherein pretreating the NAD+ comprises passing the NAD+ through a sieve, and/or grinding , milling, or jet-milling the NAD+.
  • Embodiment 35 An enhanced NAD+ composition prepared by the method of any one of embodiments 25-34.
  • Embodiment 36 A method of making an enhanced NAD+ pharmaceutical formulation, comprising mixing the enhanced NAD+ composition of any of embodiments 1-18 with and at least one ingredient or excipient that facilitates administration to a subject and/or which can further treat the subject’s disease or condition.
  • Embodiment 37 A method of mitigating chemical addiction withdrawal symptoms in a subject in need thereof, the method comprising: administering a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation of any one of embodiments 19-23 to the subject, the amount being effective to mitigate chemical addiction withdrawal symptoms in the subject.
  • Embodiment 38 The method of embodiment 36 or 37, wherein the therapeutically effective amount increases the level of NAD in the subject by at least 5%.
  • Embodiment 39 The method of any one of embodiments 36-38, wherein the amount of the pharmaceutical formulation administered is effective to mitigate opioid withdrawal symptoms such that the client maintains a Clinical Opioid Withdrawal Scale (COWS) score of 12 or lower.
  • COWS Clinical Opioid Withdrawal Scale
  • Embodiment 40 The method of any one of embodiments 37-39, wherein the level of NAD increased in the subject is an intracellular level of NAD.
  • Embodiment 41 The method of any one of embodiments 37-40, wherein an intracellular level of NAD is increased to greater than about 35 pM.
  • Embodiment 42 The method of any one of embodiments 37-41, wherein the level of NAD increased in the subject is an extracellular level of NAD.
  • Embodiment 43 The method of any one of embodiments 37-42, wherein the administration of the enhanced NAD+ pharmaceutical formulation is effective to increase a plasma level of N-methyl-nicotinamide (MeNAM) or N-methyl-2-pyridone-5-carboxamide (2PY) by at least 5% in the subject.
  • MeNAM N-methyl-nicotinamide
  • 2PY N-methyl-2-pyridone-5-carboxamide
  • Embodiment 44 The method of any one of embodiments 37-43, wherein the amount of NAD+ in the pharmaceutical formulation administered to the subject is from about 5 mg to about 4000 mg.
  • Embodiment 45 The method of any one of embodiments 37-44, wherein the amount of the pharmaceutical formulation administered comprises a maximum daily dose of about 1 mg per kg to about to about 100 mg/kg body weight of the subject.
  • Embodiment 46 The method of any one of embodiments 37-45, further comprising administering to the subject a pharmaceutically effective amount of an analgesic or a non-steroidal anti-inflammatory drug (NS AID).
  • AID non-steroidal anti-inflammatory drug
  • Embodiment 47 The method of any one of embodiments 37-45, wherein the pharmaceutical formulation further comprises a pharmaceutically effective amount of an analgesic or a non-steroidal anti-inflammatory drug (NSAID).
  • NSAID non-steroidal anti-inflammatory drug
  • Embodiment 48 The method of any one of embodiments 37-47 further comprising monitoring at least one vital sign of the subject before and after administration of the pharmaceutical formulation.
  • Embodiment 49 The method of any one of embodiments 37-48, further comprising measuring an intracellular NAD or extracellular concentration of NAD in the subject before administration and after administration of the pharmaceutical formulation.
  • Embodiment 50 The method of embodiment 49, further comprising selecting a dosage of the composition to be administered to the subject based on the measured baseline intracellular concentration of NAD or extracellular concentration of NAD in the subject.
  • Embodiment 51 A method of increasing nicotinamide adenine dinucleotide (NAD) levels in a subject, the method comprising: administering an amount of the enhanced NAD+ pharmaceutical formulation of any one of embodiments 19-23 effective to increase NAD levels in the subject.
  • NAD nicotinamide adenine dinucleotide
  • Embodiment 52 The method of embodiment 51, wherein the subject is a healthy adult.
  • Embodiment 53 A method of preventing or treating a disease or disorder correlated to decreased levels of NAD in a subject in need thereof, the method comprising: administering an amount of the enhanced NAD+ pharmaceutical formulation of any one of embodiments 19-23 effective to prevent or treat the disease or disorder in the subject.
  • Embodiment 54 A method of treating a subject suffering from a chemical addiction, comprising administering to the subject a pharmaceutically effective amount of the enhanced NAD+ pharmaceutical formulation of any one of embodiments 19-23.
  • Embodiment 55 The method of embodiment 54, wherein the chemical is tobacco, heroin, opium, morphine, dihydromorphine, meperidine, codeine, cocaine, amphetamines, barbiturates, alcohol, tranquilizers, or opioids.
  • Embodiment 56 A method of promoting cellular NAD metabolism or NAD cellular homeostasis in a subject, comprising administering to the subject an amount of the enhanced NAD+ pharmaceutical formulation of any one of embodiments 19-23 effective to promote NAD metabolism or NAD cellular homeostasis.
  • Embodiment 57 A method of treating a patient suffering from at least one condition of metabolic syndrome, comprising administering to the subject a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation of any one of embodiments 19-23, wherein the at least one condition is increased blood pressure, high blood sugar, excess body fat around the waist, abnormal cholesterol or triglyceride levels, or any combination thereof.
  • Embodiment 58 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 the enhanced NAD+ pharmaceutical formulation of any one of embodiments 19-23 effective to increase SIRT1, PLB1, NPL, ENPP5, GLSTA, GALNT3, FABP1, NRCAM, NLGN2, ARTN, UPB1, MME, CTSB, CTSL, or HRAS polypeptide.
  • Embodiment 59 A method of preparing the enhanced NAD+ composition of any one of embodiments 1-18, comprising
  • each vacuumization cycle comprises placement in a container under vacuum and back-fdling the container with an inert gas or CO2.
  • Embodiment 60 A method of preparing an enhanced NAD+ composition comprising a crystalline component comprising crystalline polyethylene glycol, and an amorphous component comprising amorphous NAD+, the method comprising
  • each vacuumization cycle comprises placement in a container under vacuum and back-fdling the container with an inert gas or CO2.
  • Embodiment 61 The method of embodiment 59 or 60, wherein the inert gas comprises argon or nitrogen.
  • Embodiment 62 The method of one of embodiments 59-61 wherein step (2) comprises at least three vacuumization cycles.
  • Embodiment 63 The method of any one of embodiments 59-61, wherein step (2) comprises at least six vacuumization cycles.
  • Embodiment 64 The method of any one of embodiments 59-63, wherein the polyethylene glycol has an average molecular weight of between about 300 and about 4000.
  • Embodiment 65 The method of any one of embodiments 59-64, wherein the polyethylene glycol is PEG3350.
  • Embodiment 66 The method of any one of embodiments 59-65, further comprising pretreating the NAD+ prior to step (1).
  • Embodiment 67 The method of embodiment 66, wherein pretreating the NAD+ comprises passing the NAD+ through a sieve, and/or grinding, milling, or jet-milling the NAD+.
  • Embodiment 68 The method of any one of embodiments 59-67, wherein the NAD+ is subjected to at least one vacuumization cycle prior to step (1).
  • Embodiment 69 An enhanced NAD+ composition prepared by the method of any one of embodiments 59-68.
  • Example 1 Process for the Preparation of Enhanced NAD+ Composition
  • NAD+ and PEG including particle sizing and “vacuumizing” were weighed and combined into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids.
  • the Blender was tightly sealed. The mixture was blended for a minimum of 20 minutes.
  • the blended NAD+ was then vacuumized for 3 cycles, which each cycle lasting at least 20 minutes and comprising placing the blended NAD+ under vacuum and replacing the vacuum with Argon
  • the blended, enhanced vacuumized product was then dispensed into a bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which was size-matched to the product volume.
  • the liner bag was then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.
  • Example 1 A Process for the Preparation of Enhanced NAD+ Composition
  • NAD+ and PEG including particle sizing and “vacuumizing” were weighed and combined into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids.
  • the Blender was tightly sealed.
  • the mixture was blended for a minimum of 20 minutes.
  • the blended NAD+ was then vacuumized for 3 cycles, which each cycle lasting at least 20 minutes and comprising placing the blended NAD+ under vacuum and replacing the vacuum with Nitrogen
  • the blended, enhanced vacuumized product was then dispensed into a bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which was size-matched to the product volume.
  • the liner bag was then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.
  • Example IB Process for the Preparation of Enhanced NAD+ Composition
  • NAD+ and PEG including particle sizing and “vacuumizing” are weighed and combined into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids.
  • the Blender is tightly sealed.
  • the mixture is blended for a minimum of 20 minutes.
  • the blended NAD+ is then vacuumized for 2 cycles, which each cycle lasting at least 20 minutes and comprising placing the blended NAD+ under vacuum and replacing the vacuum with Argon.
  • the blended, enhanced vacuumized product is then dispensed into a bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which is size-matched to the product volume.
  • the liner bag is then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.
  • Example 2 Process for Preparation of Vacuumized NAD+
  • NAD+ was placed into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids.
  • the Blender was tightly sealed.
  • the NAD+ was blended for a minimum of 20 minutes.
  • the blended NAD+ was then vacuumized for 3 cycles, which each cycle lasting at least 20 minutes and comprising placing the blended NAD+ under vacuum and replacing the vacuum with Argon.
  • the blended, enhanced vacuumized NAD+ product was then dispensed into a bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which was size-matched to the NAD+ product volume.
  • the liner bag was then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.
  • Example 2A Process for Preparation of Vacuumized NAD+
  • NAD+ was placed into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids.
  • the Blender was tightly sealed.
  • the NAD+ was blended for a minimum of 20 minutes.
  • the blended NAD+ was then vacuumized for 3 cycles, which each cycle lasting at least 20 minutes and comprising placing the blended NAD+ under vacuum and replacing the vacuum with Nitrogen.
  • the blended, enhanced vacuumized NAD+ product was then dispensed into a bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which was size-matched to the NAD+ product volume.
  • the liner bag was then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.
  • Example 2B Process for Preparation of Vacuumized NAD+
  • NAD+ is placed into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids.
  • the Blender is tightly sealed.
  • the NAD+ is blended for a minimum of 20 minutes.
  • the blended NAD+ is then vacuumized for 2 cycles, which each cycle lasting at least 20 minutes and comprising placing the blended NAD+ under vacuum and replacing the vacuum with Argon.
  • the blended, enhanced vacuumized NAD+ product is then dispensed into a bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which is size-matched to the NAD+ product volume.
  • the liner bag is then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.
  • Example 3 Process for Preparation of Vacuumized PEG
  • Pre-qualified commercial PEG was placed into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids.
  • the Blender was tightly sealed.
  • the preparation was blended for a minimum of 20 minutes.
  • the blended NAD+ was then vacuumized for 3 cycles, which each cycle lasting at least 20 minutes and comprising placing the blended NAD+ under vacuum and replacing the vacuum with Argon.
  • the blended, enhanced vacuumized product was then dispensed into the bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which was size-matched to the product volume.
  • the liner bag was then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.
  • Example 3B Process for Preparation of Vacuumized PEG
  • Pre-qualified commercial PEG was placed into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids.
  • the Blender was tightly sealed.
  • the preparation was blended for a minimum of 20 minutes.
  • the blended NATH was then vacuumized for 3 cycles, which each cycle lasting at least 20 minutes and comprising placing the blended NAD+ under vacuum and replacing the vacuum with Nitrogen
  • the blended, enhanced vacuumized product was then dispensed into the bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which was size-matched to the product volume.
  • the liner bag was then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.
  • Example 3B Process for Preparation of Vacuumized PEG
  • Pre-qualified commercial PEG is placed into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids.
  • the Blender is tightly sealed.
  • the preparation is blended for a minimum of 20 minutes.
  • the blended NAD+ is then vacuumized for 2 cycles, which each cycle lasting at least 20 minutes and comprising placing the blended NAD+ under vacuum and replacing the vacuum with Argon.
  • the blended, enhanced vacuumized product was then dispensed into the bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which is size-matched to the product volume.
  • the liner bag is then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.
  • Example 4 Process for the Preparation of Enhanced NAD+ Composition
  • Pre-treated (ground, milled or jet-milled) NAD+ at 50% NAD+ G/100 g and PEG including particle sizing and “vacuumizing” were weighed and combined into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids.
  • the Blender was tightly sealed. The mixture was blended for a minimum of 20 minutes. The blended mixture was placed under vacuum and the vacuum was replaced with Argon and the mixture was then vacuumized for 3 cycles lasting at least 20 minutes each.
  • the blended, enhanced vacuumized product was then dispensed into a bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which was size-matched to the product volume.
  • the liner bag was then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.
  • Example 4A Process for the Preparation of Enhanced NAD+ Composition
  • Pre-treated (ground, milled or jet-milled) NAD+ at 50% NAD+ G/100 g and PEG including particle sizing and “vacuumizing” were weighed and combined into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids.
  • the Blender was tightly sealed. The mixture was blended for a minimum of 20 minutes. The blended mixture was placed under vacuum and the vacuum was replaced with Nitrogen and the mixture was then vacuumized for 3 cycles lasting at least 20 minutes each.
  • the blended, enhanced vacuumized product was then dispensed into a bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which was size-matched to the product volume.
  • the liner bag was then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.
  • Example 4B Process for the Preparation of Enhanced NAD+ Composition
  • Pre-treated (ground, milled or jet-milled) NAD+ at 50% NAD+ G/100 g and PEG including particle sizing and “vacuumizing” are weighed and combined into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids.
  • the Blender is tightly sealed.
  • the mixture is blended for a minimum of 20 minutes.
  • the blended mixture is placed under vacuum and the vacuum is replaced with Argon and the mixture is then vacuumized for 2 cycles lasting at least 20 minutes each.
  • the blended, enhanced vacuumized product is then dispensed into a bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which is size-matched to the product volume.
  • the liner bag is then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.
  • a colorimetric assay that measures NAD+ and NADH present in biological samples was performed.
  • NAD+ a cell lysate sample was added to a microcentrifuge tube, combined with 0.1N HC1, and mixed thoroughly. The tube was incubated at 80 °C and protected from light. Assay Buffer was added to the tube to shift the pH of the sample back to neutral. Sample pH was adjusted to between 6.0 and 8.0, accordingly with acid or base.
  • Example 6 Multi-OMIC Characterization of the Effects of Oral Enhanced NAD+ in a Wellness Cohort
  • the present disclosure provides for delivery of an enhanced NAD+ pharmaceutical formulation to target cells to treat substance abuse including, but not limited to, drug or alcohol addiction.
  • Enhanced NAD+ refers to an enhanced NAD+ pharmaceutical formulation according to the instant disclosure.
  • the NAD+ is delivered as an oral NAD+ pharmaceutical formulation that is chemically stable, bioavailable and pharmacologically active.
  • a Research Study was conducted to investigate the multi-omic effects of oral enhanced Nicotinamide adenine dinucleotide (NAD+) in a constrained “wellness cohort” composed of healthy individuals.
  • NAD+ Nicotinamide adenine dinucleotide
  • the study followed a double-blind placebo-controlled design with repeated measures. Enrolled 60 participants randomized approximately equally to the enhanced NAD+ or the placebo group (i.e., 30 enhanced NAD+, 30 placebo).
  • the study sample size was determined by a preliminary analysis of statistical power carried out in G*Power 3.0 (Faul, Erdfelder, Lang, & Buchner, 2007) taking the number of blood draws and anticipated effect sizes into account, and allowing the study to attain 80% power to detect (at a nominal 5% significance level) large as well as medium-size effects.
  • Study Days 1-7 wash-out period; Study Days 8-12: treatment dose administration; Study Day 13: Post-Study.
  • Each dose of enhanced NAD+ was delivered by oral administration as a swish and swallow.
  • the first dose on Day 8 was administered following 12h of fasting. Participants were asked to record the date and time of each administration. Participants also agreed not to significantly change their diet or to discontinue the use of supplements while participating in the study.
  • a designated study coordinator actively monitored dose administration and blood draw compliance with the assistance of technology that allows the participant to receive reminders, and provide quick responses to questions pertaining to the ongoing study protocol.
  • Doses were administered with 3-4 hours between doses. If a dose was missed, it was taken within 2 hours of the scheduled dose; another 2 hours were allowed to pass prior to resuming the dose schedule such that all the doses for that day were completed. On Days 9 and 10 there was a 5th dose to be taken at least 2 hours after the 4th dose (see Table 1 for daily dosing schedule on days 8-12).
  • BMI Body Mass Index
  • Type I juvenile or Type 2 diabetes mellitus [0290] Type I juvenile or Type 2 diabetes mellitus
  • Inflammatory bowel disease (ulcerative colitis, Crohn’s disease);
  • Venous blood (up to 30ml) was collected using venipuncture. Blood was collected twice at baseline (Day 3 or 4 of the washout period and before administration of study dose, and Day 7 of the study before administration of the treatment dose) to establish an estimate of baseline intracellular NAD levels. Upon commencement of dose administration on Day 8, blood was collected on Day 10 and on Day 13.
  • Plasma or serum were isolated using standard centrifugation protocols, frozen, and stored until use at -80° C. Whole blood or isolated fractions were processed for further assaying using the manufacturers’ recommended practices for sample handling, randomization, and plating, where applicable. Assays
  • Extracellular serum NAD and intracellular NAD concentrations were measured by Jinfiniti Precision Medicine (“Jinfiniti”) per manufacturer’s protocols.
  • CBC Complete Blood Count
  • CMP Comprehensive Metabolic Panel
  • hs-CRP hs-CRP
  • PEA Proximity Extension Assays
  • Metabolomics were performed using Metabolon’s ultra-high-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) Global Platform, detecting approximately 1,000 small metabolites.
  • the metabolites represent super-pathways: Lipids (449), Amino Acid (183), Xenobiotics (91), Nucleotide (35), Peptide (31), Cofactors/Vitamins (23), Carbohydrate (22), Partially Characterized (6), with >200 metabolites unassigned.
  • Non-negligible pathway activation and enzymatic reaction enhancement was observed as evidence favoring bioavailability of administered enhanced NAD+ dose.
  • NAD+ supplementation in the cohort resulted in amplification of the urea cycle (likely via the xanthine component given that hypoxanthine is an intermediate metabolite), facilitation of pyruvate «- ⁇ lactate conversion contributing to glycolysis, increased lipid oxidation (observed in lipid fractions decreasing while bile acid metabolites increase), as well as increase in glutamate ⁇ -> glutamine interconversion (FIGs. 8-10).
  • Example 6 Further study was performed following Example 6 to show that an illustrative oral enhanced NAD+ pharmaceutical formulation modulates levels of various biomarkers related in several therapeutic areas, where statistically significant differences between treatment and placebo group were observed, including, but not limited to, cardiovascular diseases, liver diseases, exocrine pancreas/digestion, neurodegenerative/neuropsychiatric disease, inflammation/immune function, reproduction, metabolic disease and obesity, and skin and bone health.
  • NAFLD non-alcoholic fatty liver disease
  • Exocrine pancreas/digestion - the enhanced NAD+ composition differentially decreased circulating levels of four pancreatic enzymes.
  • Neurodegenerative/neuropsychiatric diseases - the enhanced NAD+ composition differentially increased levels of 6 proteins with known neurological effects and decreased levels of 6 other proteins.
  • Several of the proteins altered by the enhanced NAD+ composition treatment have been previously associated with neurodegenerative diseases, depression, anxiety, or chronic pain. The observed improvements in oxidative and nitrosative stress also have relevance in neurodegenerative disease.
  • Inflammation/immune function There were no differences seen between groups in clinical inflammatory markers such as hsCRP. However, there were significant increases or decreases in the oral enhanced NAD+ treatment group in 15 immune proteins that play a role in regulating adaptive or innate immune function and inflammation.
  • Metabolic disease and obesity - the enhanced NAD+ composition differentially upregulated 3 proteins associated with metabolic diseases and obesity.
  • the study also examined the effects of the illustrative oral formulation versus placebo on a comprehensive range of clinical laboratory tests and multi-omic peripheral biomarkers associated with inflammation; lipid metabolism; oxidative stress/antioxidant capacity; liver, kidney, and mitochondrial function, as well as patient-reported outcomes, including mental wellbeing and daily reports of physical symptoms.
  • ROS daily Review of Systems
  • the study examined a comprehensive array of about 4,000 circulating biomarkers utilizing blood samples from baseline, Day 11 (treatment Day 4), and Day 13 (immediate posttreatment day). Samples were biobanked and preserved in liquid nitrogen at -80 °C for batch processing and assaying of longitudinal samples from the same participant was performed simultaneously.
  • Metabolon Global Platform used for untargeted metabolomics of plasma samples from the study; these metabolites include amino acids, carbohydrates, lipids, nucleotides, peptides, as well as partially characterized molecules; -3,000 proteins whose abundance in plasma was assayed using Olink’s Explore 3,072 PEA proteomics platform with NGS readout (Illumina’s HiSeq 2000); a wide range of interrogated proteins targeted a host of biological pathways, including cellular membrane maintenance, nutrient transport, oxygen species formation, lipid transport and degradation, inflammation, immune signaling, and mitochondrial functioning.
  • the illustrative enhanced NAD+ pharmaceutical formulation used for treatment was an oral NAD+ formulation consisting of 500 mg NAD+ and 500 mg PEG 3350. The composition was taken four times daily (q.i.d.) for 5 consecutive days on Day 8 through Day 12. On each of Days 9 and 10, one additional dose was taken. For each dose, a measured amount of the composition powder was dissolved in water and participants took the dose orally using a “swish and swallow” procedure. The supplement meets the FDA criteria for administration in humans (including compliance with GMP regulations).
  • One endpoint of the study was the change in intracellular NAD (icNAD) concentrations after treatment.
  • SIRT1 is an NAD-dependent enzyme that typically increases in plasma when levels of NAD+ are increased. SIRT1 is actively involved in the regulation of energy sensing and homeostasis, oxidative stress response, and anti-inflammatory cascades. It also relates to a number of chronic diseases, including cardiometabolic and neurodegen erative diseases.
  • proteomic and metabolomic assays used generate results that are relative, rather than absolute (quantitative) values. This is indicated in the X-axes of the figures which are labeled either “Relative abundance” (for metabolites) or “NPX” (for proteins).
  • NPX stands for Normalized Protein expression, which is Olink’s arbitrary unit (in Log2 scale) for proteomics reports.
  • Enhanced NAD+ differentially impacted several proteins that have vascular effects and are known to play a role in hypertension or stroke.
  • the orally administered enhanced NAD+ pharmaceutical formulation significantly increased the levels of FGF12, EDIL3, FLT1 (VEGFR1), and FLRT2.
  • Fibroblast growth factor 12 is a potent inhibitor of the vascular Smooth Muscle Cell (SMC) phenotype switch, resulting in healthier blood vessels. FGF12 strongly induces a healthier phenotype of human aortic SMCs and results in a more favorable response to arterial injury in rats. Conversely, lower FGF12 levels have been implicated in pulmonary arterial hypertension and associated mechanistically with vascular damage in this disease in animal models.
  • SMC Smooth Muscle Cell
  • EGF-like repeat and discoidin I-like domain-containing protein 3 (EDIL3) is an extracellular matrix protein that acts as a pro-angiogenic factor, mediator of the immune and antiinflammatory response, and a regulator of endothelial cell adhesion and migration. Overall, EDIL3 plays an important role in mediating angiogenesis and may be important in vessel wall remodeling and development. The effect of the modest increase in circulating levels in healthy patients is unknown.
  • Vascular endothelial growth factor receptor 1 (VEGFR1) protein binds to VEGFR-A, VEGFR-B and placental growth factor and plays an important role in angiogenesis and vasculogenesis. Expression of this receptor is found in vascular endothelial cells, placental trophoblast cells and peripheral blood monocytes. It is involved in the regulation of angiogenesis by regulating endothelial cell proliferation and senescence. In mice, VEGFR1 levels are increased in ischemic muscle tissue compared to non-ischemic muscle and VEGFR1 appears to play a role in vascular recovery from ischemia.
  • VEGFR1 Vascular endothelial growth factor receptor 1
  • 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.
  • FLRT2 is essential for development of the epicardium and loss of this protein impairs expansion of the ventricular myocardium and reduces endocardial volume. Its role in cardiac function in adults is unclear.
  • the illustrative enhanced NAD+ pharmaceutical formulation differentially decreased circulating levels of the protein SERPINI1 (neuroserpin).
  • Neuroserpin inhibits the activity of an enzyme called tissue plasminogen activator (tPA), which plays a role in cell migration, blood clotting, and inflammation.
  • tPA tissue plasminogen activator
  • administration of neuroserpin following aortic allograft transplant exerted an anti-inflammatory effect, reducing plaque growth and CD3+ T cell invasion.
  • neuroserpin levels in the brain increase in the region surrounding the lesion, providing a neuroprotective action.
  • Increased levels of the amino acid homocysteine are an independent risk factor for atherosclerosis, as there is a correlation between serum homocysteine and incidence of coronary, carotid and peripheral vascular disease, likely mediated by homocysteine’s adverse effects on vascular endothelium and arterial structure.
  • the enzyme methionine synthase (MTR) converts homocysteine to methionine, using methylated vitamin B12 (methylcobalamin) thus decreasing levels of homocysteine.
  • Oxidative stress is considered a major risk factor for many diseases including cardiovascular disease, with increased oxidative stress associated with arterial hypertension, arrhythmia, and atherosclerotic plaque formation.
  • administration of the illustrative oral NAD+ formulation was associated with beneficial changes in a variety of metabolites associated with glutathione (GSH), a key cellular antioxidant.
  • GSH glutathione
  • GSTA1 is an enzyme that utilizes GSH to bind electrophilic compounds and ROS, thus reducing oxidative stress, while SOD2 is another player in cellular response to oxidative stress that binds superoxide anions to convert them to H2O2 and 02. Importantly, SOD2 is under transcriptional regulation by NAD+-dependent sirtuins. Decreasing levels of plasma SOD2 may indicate relaxing demand for its antioxidant capacity.
  • clinical inflammatory biomarkers e g., cardiac CRP, hsCRP, TNF or IL-6.
  • Metabolites related to cardiovascular health that were differentially upregulated by the illustrative oral enhanced NAD+ formulation compared to placebo include homoarginine and glyco-beta-muricholate, while metabolites which were significantly decreased in the enhanced NAD+ group included docosahexaenoyl choline, urea, hypoxanthine, glutamate, acisoga, and 1- palmitoyl-2-arachidonoyl-GPC. Details on these metabolites in relation to CVD are shown below:
  • Homoarginine is an amino acid that appears to have a protective effect against cardiovascular disease. Homoarginine appears to act as a weak substrate for endothelial nitric oxide synthase, which improves vascular function.
  • Glyco-beta-muricholate is a type of bile acid.
  • Mouse studies show muri cholic acids promote resistance to hypercholesterolemia.
  • Docosahexaenoyl choline (choline docosahexaenoate), is a type of acyl choline, a class of molecules involved in fatty acid metabolism. Research has also shown association between this metabolite and serum lipids in Caucasian and African-American adults.
  • Urea cycle metabolites Both urea and hypoxanthine are involved in the urea cycle which breaks down toxic ammonia derived from the metabolism of proteins and gut bacterial waste. Urea cycle metabolites are a statistically over-represented cluster in relation to cardiometabolic disease. Excess levels of urea are associated with oxidative stress, which plays a significant role in CVD.
  • Glutamate (glutamic acid) is an amino acid and an excitatory neurotransmitter. In addition to its neurological role, higher plasma glutamate has been associated with heart failure.
  • - Acisoga is a polyamine metabolite. It has been shown to be predictive of atrial fibrillation. Recently, acisoga has also been suggested as a novel biomarker in heart failure, correlating with reduced left ventricular function.
  • 1 -palmitoyl -2-arachidonoyl-GPC is a type of phosphatidylcholine, functionally related to arachidonic acid and hexadecenoic acid. This compound is predictive of ischemic stroke risk in women, but not men.
  • Non-alcoholic fatty liver disease and non-alcoholic steatohepatitis (NASH) are common, chronic liver diseases.
  • NAFLD and NASH are strongly associated with both obesity and Type 2 diabetes, thus as those conditions have increased in prevalence globally, so has the prevalence of fatty liver diseases. In the US, it’s estimated that 1 in 4 people have NAFLD.
  • liver enzymes AST or ALT No effects were detected for the liver enzymes AST or ALT, or for BUN or Cr (all P’s >0.05).
  • oxidative stress has been recognized as a significant risk factor for both onset and progression of various liver diseases, including alcoholic liver disease as well as NASH.
  • liver diseases including alcoholic liver disease as well as NASH.
  • human evidence for use of antioxidant compounds in established liver disease is limited, there is evidence in animal models and for preventive benefits in humans.
  • the antioxidant benefits of enhanced NAD+ administration discussed above regarding cardiovascular disease are relevant for liver diseases. This is especially important given the central role of the liver in overall metabolism, including in relation to development of Type 2 diabetes.
  • GGT an enzyme responsible for the breakdown and freeing of glutathione, may not only be a sensitive marker of liver damage and cholestasis but an overall sensitive marker of oxidative stress. Circulating GGT is a good biomarker of cellular redox status. Additionally, serum concentrations of GGT inversely correlate with serum antioxidants.
  • PNLIPRP1 pancreatic lipase-related protein 1
  • RNASE1 pancreatic ribonuclease
  • CPA1 carboxypeptidase Al
  • CPB1 carboxypeptidase Bl
  • PNLIPRP1 is a lipolytic enzyme secreted in the gastrointestinal tract. The function of this protein is unclear.
  • RNASE 1 belongs to the Ribonuclease A superfamily, which consists of 8 described enzymes. Pancreatic ribonuclease is a pancreatic enzyme that catalyzes the breakdown of RNA and plays a role in the digestion of RNA in vertebrate species. RNase 1 was historically considered as a digestive enzyme but is now thought to have additional functions regarding host defense similar to other members of this enzyme superfamily.
  • CPA1 is a member of the carboxypeptidase A family of zinc metalloproteases. This enzyme is produced in the pancreas and preferentially cleaves C-terminal branched-chain and aromatic amino acids from dietary proteins. Elevated protein levels (i.e., opposite of what was observed here) have been associated with pancreatic cancer.
  • CPB1 is another enzyme produced in the pancreas. Very elevated levels may be a serum marker for pancreatitis.
  • ACOX1 acyl-CoA oxidase 1
  • PLXNA4 Plexin A-4
  • ASAHI acid ceramidase
  • MTR Methionine synthase
  • GRN progranulin
  • SERPINI1 neuropeptide
  • ACOX1 is the first enzyme of the fatty acid beta-oxidation pathway, which catalyzes the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs. It donates electrons directly to molecular oxygen, thereby producing hydrogen peroxide, which has a variety of adverse effects in the body. Decreasing this protein suggests improved oxidative stress, which may be regulated by SIRT enzymes. ACOX1 seems to play a major role in neuronal function.
  • Plexin A4 binds to neuropilin 1 and 2 and transduces signals from Sema3A, Sema6A, and Sema6B. These Nrp-plexin and semaphorin complexes initiate cascades that regulate diverse processes in the nervous system such as axon pruning and repulsion, dendritic attraction and branching, regulation of cell migration, and vascular remodeling. Both upregulation and downregulation of Plexin A4 has been observed following neural injury suggesting a dynamic role for Plexin A4 in neural maintenance and regeneration. Plexin A-4 has been implicated in the pathology of Alzheimer’s disease.
  • the acid ceramidase enzyme is found in lysosomes, cell compartments that digest and recycle materials. Within lysosomes, acid ceramidase breaks down lipids called ceramides. Ceramides are typically found within the membranes that surround cells and play a role in regulating cell maturation, growth and division of cells (proliferation), and controlled cell death (apoptosis). Additionally, ceramides are a component of a fatty substance called myelin that insulates and protects nerve cells.
  • Methionine synthase is responsible for the regeneration of methionine from homocysteine and is the enzyme responsible for linking the SAMe cycle to one-carbon metabolism via the folate cycle. Both homocysteine and SAMe-dependent reactions have important implications for neurological health. Increased levels of homocysteine have been associated with increased levels of amyloid-beta in the brains of Alzheimer’s patients and may be an independent risk factor for neurodegenerative diseases. Methionine synthase is also an important regulator of brain anti-oxidant status and has been described as having a broad and dynamic role in coordinating metabolism in the brain during development and aging.
  • Progranulin is found in tissues throughout the body, but it is most active in cells that are dividing rapidly, such as skin cells (fibroblasts), immune system cells, and certain brain cells. This protein helps regulate the growth, division, and survival of these cells. Progranulin is active in several types of brain cells, however, little is known about this protein's role in the brain. It appears to be critical for the survival of neurons.
  • Neuroserpin inhibits the activity of an enzyme called tissue plasminogen activator (tPA), which plays a role in cell migration, blood clotting, and inflammation.
  • tissue plasminogen activator tPA
  • neuroserpin is active in the nervous system where it helps control the growth of neurons, especially axons which are required for the transmission of nerve impulses.
  • Neuroserpin also plays a role in the development of synapses, and helps regulate synaptic plasticity, which suggests that it may be important for learning and memory.
  • Neuroserpin has been shown to have a protective effect on the brain following stroke, and has been reported to be upregulated in Alzheimer’s disease.
  • Proteins related to neurologic function that significantly and preferentially increased in the NAD+ group compared to placebo include: NPL (N-acetylneuraminate lyase), 5-HT1AR (5- HT [serotonin] receptor 1A), FOLH1 (Glutamate carboxypeptidase 2), HRAS (GTPase Hras), CRIP2 (Cysteine-rich protein 2), and FLRT2 (Fibronectin Leucine-rich Repeat Transmembrane protein 2).
  • NPL is one of a family of lyase enzymes and it catalyzes the reversible cleavage and synthesis of sialic acids (monosaccharide sugars).
  • Sialic acids have a variety of potential medical uses including anti-viral, anti-microbial and neural substrate uses.
  • addition (sialylation) or removal (desialylation) of sialic acid in microglia cells has been associated with neurodegenerative diseases.
  • a specific type of sialic-acid modified molecule, polysialic acid has also been associated with psychiatric disorders.
  • 5 -HT1AR is involved in neuromodulation and impacts a variety of conditions.
  • 5-HT1AR agonists e.g., buspirone
  • 5-HT1AR also seems to play a role in modulating chronic pain.
  • the potential for a role of NAD+ in depression is suggested by the importance of the SIRT1 pathway, which is strongly influenced by NAD, in bipolar depression and related neurological conditions. As described below, administration of enhanced NAD+ significantly increased plasma SIRT1 levels.
  • 5 -HT1A receptor activation has also been shown to increase dopamine release in the medial prefrontal cortex, striatum, and hippocampus, and so may be useful for improving the symptoms of schizophrenia and Parkinson's disease.
  • the potential for an impact on Parkinson’s disease is particularly interesting given that several older clinical studies found significant benefits of administering IV NADH to patients with Parkinson’s disease.
  • GCPII is a zinc metalloenzyme found in prostate epithelium, kidney, small intestine, and the nervous system. In the intestine, this enzyme catalyzes a reaction that results in the release of folic acid, which can then be used in the body as a vitamin. In the brain, GCPII has been shown to both indirectly and directly increase the concentration of glutamate. Potent and selective GCPII inhibitors have been shown to decrease brain glutamate and provide neuroprotection in preclinical models of stroke, amyotrophic lateral sclerosis, and neuropathic pain.
  • HRAS is an important regulator of signal transduction and cell cycle.
  • the RAS signaling pathway is involved in the regulation of developmental processes, including cell growth, proliferation, and differentiation, in the central nervous system.
  • Germline mutations in the RAS signaling pathway genes are associated with a group of neurodevelopmental disorders, collectively called RASopathies, which include neurofibromatosis type 1, Noonan syndrome, cardio-facio-cutaneous syndrome, and Costello syndrome.
  • RASopathies include neurofibromatosis type 1, Noonan syndrome, cardio-facio-cutaneous syndrome, and Costello syndrome.
  • HRAS activation in neurons attenuates the generation of precursor cells in the hippocampus, which has the potential to impact spatial short-term memory and object recognition.
  • CRIP2 is found in many different tissues and is believed to play a role in the differentiation of smooth muscle tissue. CRIP2 is found in the spinal cord and may play an inhibitory role in inflammatory pain.
  • 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.
  • Glutamate is an amino acid and an excitatory neurotransmitter. Plasma glutamate levels have been found to be higher in patients with migraine and the glutamine/glutamate ratio is altered in patients with schizophrenia. Higher plasma glutamate levels have also been found in individuals who develop post-stroke depression.
  • Cortisone is a metabolite of the steroid stress hormone cortisol. Both cortisol and cortisone are considered markers of stress, and some research suggests that plasma cortisone may be an even more sensitive marker of stress. Cortisone is increased in patients with depression.
  • 1,2-dipalmitoyl-GPC (16:0/16:0) is a type of phosphatidylcholine glycerophospholipid containing two chains of palmitic acid. 1,2-dipalmitoyl-GPC may correlate with levels of multiple Alzheimer’s disease biomarkers in the cerebrospinal fluid of older adults and is one of the top 3 metabolite predictors of Alzheimer’s disease progression in a cohort with mild cognitive impairment.
  • both oxidative stress and nitrosative stress have implications for neurodegenerative disease.
  • Administration of enhanced NAD+ had a number of beneficial effects on markers of antioxidant status.
  • Increased oxidative stress is observed both peripherally and in the brain of patients with Alzheimer’s disease and plays an important role in the degeneration of dopamine neurons in Parkinson’s disease.
  • Nitrosative stress refers to the joint biochemical reactions of nitric oxide (NO) and superoxide (Ch') when an oxygen metabolism disorder occurs in the body.
  • NO nitric oxide
  • Ch' superoxide
  • oxidative and nitrosative stress can exert parallel actions leading to cell damage and death and nitrosative stress has been implicated in a variety of diseases, including neurodegenerative diseases.
  • the instant study found a coordinated signal for the enhanced NAD+’s effectiveness in enhancing protection nitrosative stress.
  • NAD has also been implicated in autoimmune diseases, including multiple sclerosis (MS), inflammatory bowel disease (IBD), and rheumatoid arthritis (RA).
  • MS multiple sclerosis
  • IBD inflammatory bowel disease
  • RA rheumatoid arthritis
  • SIRT1 -deficient mice developed a severe form of experimental autoimmune encephalomyelitis (considered an animal model for MS) and spontaneous autoimmunity.
  • the homeostatic chemokine CXCL12 is an important factor in many physiological and pathological processes because it activates and/or induces migration of hematopoietic progenitor and stem cells, endothelial cells and most leukocytes.
  • the CXCL12-CXCL4 signaling axis is implicated in tumor biology and reductions in CXCL12 may be beneficial in this regard.
  • CXCL12 is also thought to be a major player in interactions between the immune system and the nervous system.
  • CCL8 is a chemokine belonging to the CC chemokine family that acts as a macrophage chemotactant. CCL8 activates many different immune cells, including mast cells, eosinophils and basophils, which are implicated in allergic responses, and monocytes, T cells, and NK cells that are involved in the inflammatory response.
  • Interleukin-2 Receptor subunit beta 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.
  • Interleukin-31 (IL-31) is produced mainly by activated CD4 + T-cells and interacts with a receptor expressed on epithelial cells and keratinocytes. IL-31 acts on a broad range of immune- and non-immune cells and therefore possesses potential pleiotropic physiological functions, including regulating hematopoiesis and immune response. IL-31 has been implicated in inflammatory bowel disease, airway hypersensitivity (asthma) and allergic dermatitis.
  • Interleukin-34 is a cytokine that promotes the differentiation and viability of monocytes and macrophages through the colony-stimulating factor-1 receptor. The highest levels of IL-34 are found in the brain and the skin. IL-34 has been associated with several autoimmune disease, including rheumatoid arthritis and systemic lupus erythematosus.
  • SIRT1 The NAD+-dependent enzyme SIRT1 is involved in regulation of sex hormone levels which are key to reproductive function. SIRT1 knockout mice have been shown to have decreased production of gonadotropin-releasing hormone, and subsequently decreased LH and FSH levels, which contributes to the observed effect of decreased SIRT1 on male fertility. In men, SIRT1 also regulates spermatogenesis and sperm maturation.
  • INSL3 insulin-like peptide 3
  • INSL3 is a protein hormone produced by gonadal tissues in both males and females. Circulating levels are higher in males than females. In males, INSL3 controls testicular descent during embryogenesis and declines with aging, presumably reflecting aging effects on the testes.
  • INSL3 may be involved in regulating female fertility.
  • the protein varies over the course of the menstrual cycle and decreases after menopause. (Note: Blood draws were not timed to menstrual cycle phase during the study).
  • INSL3 may also play a role in polycystic ovarian syndrome (PCOS).
  • PCOS polycystic ovarian syndrome
  • SIRT1 levels are reduced in diabetic patients with poor glycemic control compared to those with good glycemic control.
  • GALNT2 N-acetyl-galactosaminyl-transferase 2
  • FABP1 fatty-acid binding protein 1; FIG. 30
  • RBP2 retinol binding protein 2; FIG. 31
  • GALNT2 is a member of the glycosyltransferase 2 protein family. Recently, GALNT2 has attracted attention as a possible player in many clinical conditions that share the common ground of insulin resistance, including atherogenic dyslipidemia, type 2 diabetes, and obesity.
  • FABP1 is critical for fatty acid uptake and intracellular transport and has an important role in regulating lipid metabolism and cellular signaling pathways. It is primarily found in the liver. FABP1 may exert a protective effect against lipotoxicity by facilitating fatty acid oxidation or incorporation into triglycerides, binding these otherwise cytotoxic compounds. The ability to bind heme is another cytoprotective property. The role of FABP1 in substrate availability and in protection from oxidative stress suggests that FABP1 plays a pivotal role during intracellular bacterial/viral infections by reducing inflammation. Altered expression of the protein has been linked to metabolic conditions including obesity: higher circulating levels have been reported in Chinese adults with high BMI and insulin resistance.
  • Glycohyocholic acid is a glycine-conjugated form of the primary bile acid hyocholic acid.
  • Plasma GHCA has been found to be lower in individuals with obesity and those with Type 2 diabetes. Plasma levels of GHCA increase in patients who are no longer diabetic following gastric bypass surgery and can predict diabetes remission.
  • Muricholic acids are a type of bile acid. In obese humans, glyco-beta-muricholate has been found to correlate with insulin resistance.
  • Pyrraline is a type of Advanced Glycation Endproduct (AGE), harmful compounds formed in food and in vivo. AGEs are associated with diseases of aging and particularly Type 2 diabetes. Urinary pyrraline levels are higher in patients with Type 2 diabetes and are associated with glycemic control.
  • AGE Advanced Glycation Endproduct
  • Glutamate is an amino acid and an excitatory neurotransmitter. In addition to its neurological role, higher plasma glutamate has been associated with Type 2 diabetes.
  • Acisoga is a polyamine metabolite that is increased in children with obesity._Acisoga has also been found to be one of the metabolites significantly associated with new onset diabetes in a large longitudinal cohort in a model that controlled for other diabetes risk factors.
  • Nonadecanoate is a saturated fatty acid. Plasma levels of nonadecanoate were found to be significantly associated with glucose tolerance in a metabolomics study of adults with normal or impaired glucose tolerance. Nonadecanoate levels are also elevated in obese diabetic rodent models.
  • 1 -palmitoyl -2-arachidonoyl-GPC is a type of phosphatidylcholine, functionally related to arachidonic acid and hexadecenoic acid. This compound has been shown to be significantly higher in patients with obesity and metabolic syndrome compared to healthy obese or non-obese individuals.
  • PPIB Peptidyl-prolyl cis-trans isomerase B
  • ER endoplasmic reticulum
  • PPIB interacts with various other proteins to facilitate protein folding, especially for type I collagen.
  • PPIB is essential for collagen biosynthesis and post-translational modification and affects fibril assembly, matrix cross-linking, and bone mineralization.
  • WFDC12 (whey-acidic protein 4-di sulfide-core 12) - WFDC12 belongs to a family of proteins that are involved in innate immune defense including inhibition of neutrophil serine proteases and inhibition of the inflammatory response to lipopolysaccharide (LPS). WFDC12 is produced in several tissues but primarily the lung and the skin. Elevated levels of WFDC12 have been found in the affected (inflamed) skin of patients with psoriasis and atopic dermatitis.
  • PPIB plays a role in both skin and bone health, and significant changes were observed in two other proteins specifically associated with bone - decreased INSL3 and increased MATN3.
  • INSL3 insulin-like peptide 3
  • INSL3 is a protein hormone produced by gonadal tissues in both males and females. In addition to its important roles in the reproductive system, INSL3 plays a role in bone and musculoskeletal function and may impact other organs. INSL3 regulates the expression of genes involved in differentiation and maturation of primary human osteoblasts, such as ALP, COL1A1, COL6A1, and Osteonectin. Treatment of primary human osteoblasts with INSL3 improved mineralization of the bone matrix. Additionally, decreased INSL3 levels in Klinefelter’s syndrome patients are correlated with increased levels of serum sclerostin, which is involved in bone catabolism by inhibiting osteoblast differentiation and stimulating osteoclast activation.
  • MATN3 (Matrilin-3) - matrilin-3 is found in the extracellular matrix surrounding the cells that make up ligaments and tendons, and near 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 implicated in osteoarthritis.
  • Example 8 Mitigation of Opioid Withdrawal Symptoms by Orally Administration of Enhanced NAD+ Pharmaceutical Formulations
  • This Example illustrates using the oral formulation of the enhanced NAD+ composition described herein) to mitigate opioid withdrawal symptoms, thus facilitating abrupt opioid discontinuation in a human adult subject.
  • 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. The abnormalities in the brain-reward system that result from opioid use cause a need to keep taking drugs to avoid an opioid withdrawal syndrome (OWS). Opioid withdrawal is not life-threatening, but it serves as a barrier for entering into treatment and stopping opioid use; concern about withdrawal prevents some patients from seeking treatment, and symptoms of withdrawal contribute to relapse in patients attempting to recover.
  • Current treatment options for abrupt withdrawal include methadone, buprenorphine, and clonidine. In the clinical situations in which it is possible, a slow taper with the opioid on which the patient is physically dependent is preferred to abrupt withdrawal.
  • OWS The severity and duration of OWS varies as a function of the half-life of the opioid, the duration of opioid use, and patient-specific characteristics including health status.
  • Abrupt cessation of short-acting opioids e.g., fentanyl, heroin, hydrocodone, and oxycodone
  • severe OWS typically begin within 12 hours after a missed dose, peaks at 36-72 hours, and gradually tapers off over the following 4-7 days.
  • buprenorphine a long-acting opioid, can be less severe than withdrawal from short-acting opioids and of similar duration.
  • Withdrawal from methadone also produces milder symptoms; however, they may last 2 weeks or more.
  • OWS ulcerative colitis
  • aches/pains aches/pains, muscle spasms/twitching/tension, tremors, abdominal cramps, nausea/vomiting/diarrhea, anxiety/restlessness, irritability, and insomnia.
  • OWS will generally resolve after 5-14 days (depending on the half-life of the opioid), the distress in the first few days after discontinuation is severe. Without adequate treatment, many patients are unable to complete opioid discontinuation. While pain relief, relaxation, self-medication of depression and anxiety, or pleasure-seeking may be the initial reason for use of opioids, with prolonged use, avoidance of OW S often becomes the most powerful force driving continued use.
  • OUD opioid use disorder
  • OUD is characterized by a chronic and sustained manifestation of several symptoms within a 12-month period, including withdrawal symptoms, tolerance development, and an uncontrollable desire to seek and use drugs despite negative consequences on the patient’s daily life.
  • the time course of this neuropsychiatric disorder is characterized by cycling periods of exacerbated use and abstinence over years, separated by periods of treatment and remission, during which the relapse vulnerability remains high due to sustained neuroadaptations to the brain’s reward circuitry following chronic exposure to opioids.
  • Effective treatment of OWS may stabilize patients and set the stage for motivating patients to discontinue opioid use and enter longer-term treatment.
  • OUD OUD or opioid physical dependence
  • a long-term treatment strategy must be defined.
  • OUD is a chronic disorder and often requires long-term or even life-long treatment.
  • the treatment goal guides OWS treatment strategies.
  • Dependence on opioids occurs in a variety of patient types and clinical situations, which leads to different patient preferences and treatment strategies.
  • management of acute OWS is the first step during opioid discontinuation or dose reduction.
  • opioid withdrawal is typically managed by slowly tapering the analgesic (referred to as detoxification or withdrawal management).
  • medication-tapering and withdrawal management can be supplemented with pharmaceutical intervention such as clonidine, lofexidine, naltrexone, or opioid medications such as buprenorphine or methadone.
  • clonidine With the use of clonidine, the time to opioid lapse, while longer than placebo, has been reported to not be statistically significant (duration of abstinence 34.8 ⁇ 3.7 days with clonidine compared to 25.5 ⁇ 2.7 days with placebo), and lofexidine results in only modest reductions of OWS symptoms by 12-14%, depending on the doses used. Both clonidine and lofexidine are associated with bradycardia, hypotension, orthostasis, somnolence, sedation, dry mouth, and serious effects on the cardiovascular system, which are an impediment to patient compliance and treatment success.
  • the present study illustrates use of orally administered enhanced NAD+ pharmaceutical formulations for mitigation of opioid withdrawal symptoms to facilitate abrupt opioid discontinuation in adults.
  • the formulation was designed to provide a less invasive route of administration compared with NAD+ administered intravenously. Swishing the formulation prior to swallowing increases the duration of exposure to the oral mucosa, which can allow for greater bioavailability compared to orally ingested NAD+.
  • an active component in the present formulations, NAD+ is considered safe (and even protective) on these organ systems.
  • an oral enhanced NAD+ formulation containing 50% nicotinamide adenine dinucleotide and 50% polyethylene glycol is used for human consumption.
  • a Phase 1 randomized, 2-part, open-label study is performed in healthy volunteers (e.g., of 18 to 55 years of age (inclusive)), including bioavailability (BA) and dose escalation study using an illustrative oral enhanced NAD+ formulation (via a swish-and-swallow administration), compared to NAD+ IV administration.
  • Exemplary doses include 500 mg, 1 g, 2 g, 3 g and 4 g.
  • a PK study in healthy adults is to assess the absolute BA (rate [C ma x] and extent [AUC] of systemic exposure) for the proposed enhanced NAD+ oral solution product compared to NAD+ delivered via IV administration (e.g., 750 mg infusion over 6 hours), as sufficient elevation of circulating NAD+ is requisite to the product’s therapeutic effect.
  • Swishing the enhanced NAD+ solution in the mouth may last as long as possible ( ⁇ 30-60 seconds) prior to swallowing the solution.
  • a fasted-fed period for the highest proposed dose can be used to assess the effects of food on the BA of the proposed enhanced NAD+ product.
  • In vitro drug-drug interaction (DDI) studies with NAD+ can be done to evaluate potential metabolism-mediated and transporter mediated drug interactions (as a substrate, inhibitor, or inducer).
  • a randomized and placebo-controlled Phase 2 study is performed to patients undergoing abrupt opioid discontinuation (with or without other drug use).
  • the highest safe dose of the illustrative enhanced NAD+ composition is determined in the Phase 1, such as at 500 mg/day, 1 g/day, 2 g/day, 3 g/day, or 4 g/day.
  • enhanced NAD+ oral solution 500 mg is administered 4 times per day (every 3-4 hours) for 5 days (on days 2 and 3, an additional dose will be given just before bedtime). On days 6 and 7 (tapering period), the same dose of NAD+ solution will be administered twice per day.
  • Placebo (all components of the oral solution, in the same volume, without API) is administered following the same schedule. After such 7-day dosing duration, an additional 7-day follow-up is applied for safety evaluation.
  • exemplary efficacy endpoints may include: change in Clinical Opiate Withdrawal Scale (COWS) compared to placebo evaluated on day 3 and day 5 of treatment, as well as the average COWS score over the course of treatment compared to placebo, change in Subjective Opiate Withdrawal Scale (SOWS) compared to placebo evaluated on day 3 and day 5 of treatment, as well as the average SOWS score over the course of treatment compared to placebo, and/or other exploratory endpoints.
  • COWS Clinical Opiate Withdrawal Scale
  • SOWS Subjective Opiate Withdrawal Scale
  • Exemplary inclusion criteria to enroll patients include those currently dependent on any other addictive or psychoactive substance in addition to opioids.
  • This Example illustrates using the enhanced NAD+ pharmaceutical formulation to mitigate symptoms related to viral infection, such as by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • SARS-CoV-2 has caused the global epidemic of coronavirus disease 2019 (COVID-19). COVID-19 patients usually develop severe respiratory distress, which may lead to death. According to the US Centers for Disease Control and Prevention (CDC), some people who have been infected with the SARS-CoV-2 virus that causes COVID-19 can experience long-term effects from their infection, known as Post-COVID Conditions (PCC) or Long CO VID. People call Post-COVID Conditions by many names, including: Long COVID, long-haul COVID, postacute COVID-19, post-acute sequelae of SARS CoV-2 infection (PASC), long-term effects of COVID, and chronic COVID.
  • PCC Post-COVID Conditions
  • PASC post-acute sequelae of SARS CoV-2 infection
  • Post-COVID Conditions can include a wide range of ongoing health problems; these conditions can last weeks, months, or years. Post-COVID Conditions are found more often in people who had severe COVID-19 illness, but anyone who has been infected with the virus that causes COVID- 19 can experience Post-COVID Conditions. People not vaccinated against COVID-19 and who become infected may have a higher risk of developing Post-COVID Conditions compared to people previously vaccinated. While most people with Post-COVID Conditions have evidence of infection or COVID-19 illness, in some cases, a person with Post-COVID Conditions may not have tested positive for the virus or known they were infected.
  • Post-COVID conditions may include a wide range of new, returning, or ongoing health problems that people experience after being infected with the virus that causes COVID- 19. Most people with COVID- 19 get better within a few days to a few weeks after infection, so at least four weeks after infection is the start of when Post-COVID Conditions could first be identified.
  • Post-COVID Conditions An important component of the virus that causes COVID- 19.
  • Most people with COVID- 19 get better within a few days to a few weeks after infection, so at least four weeks after infection is the start of when Post-COVID Conditions could first be identified.
  • An infected can experience Post-COVID Conditions. Most people with Post-COVID Conditions experienced symptoms days after first learning they had COVID-19, but some people who later experienced Post-COVID Conditions did not know when they got infected.
  • the enhanced NAD+ pharmaceutical formulation described herein is used to prevent or treat at least one of post-viral longterm symptoms.
  • a subject is identified as suffering from, or having a risk of suffering from, post-viral long-term symptoms such as Long Covid.
  • An amount of a composition comprising nicotinamide adenine dinucleotide (NAD+), such as the enhanced NAD+ composition described herein, is then orally administered to the subject, wherein the amount is effective to increase a level of NAD by at least 5% in the subject, thereby mitigating or preventing post- viral long-term symptoms in the subject.
  • the amount of NAD+ for administration can be designed based on the results of Phase I/II/III studies described in Example 7, or the other disclosures in the instant application.
  • Non-limiting mechanisms for using the enhanced NAD+ pharmaceutical formulations provided herein to prevent or treat COVID-19 infection, such as Long Covid symptoms, may include replenishment of the NAD+ levels in the host which has been diminished by viral infection, re-energize host cells, depress expression of inflammatory cytokines (thus suppress uncontrolled inflammation), etc.
  • Example 10 Thermogravimetric Analysis of Unenhanced vs. Enhanced NAD+ Powders
  • Sample 6 NAD+ and PEG3350 were combined in 1 : 1 wt ratio, stirred for 20 minutes at room temperature, and stored as Sample 6 (not enhanced).
  • Sample 7 NAD+ was subjected to three vacuumization cycles (each cycle comprising subjecting the solids to vacuum for about 20 minutes and then back-filling with argon).
  • Sample 8 A portion of Sample 7 was subjected to three further vacuumization cycles (each cycle comprising subjecting the solids to vacuum for about 20 minutes and then backfilling with argon). The solids were stirred for 20 minutes at room temperature, and stored as Sample 8 (an enhanced NAD+ composition).
  • Sample 9 A portion of Sample 8 was subjected to three further vacuumization cycles (each cycle comprising subjecting the solids to vacuum for about 20 minutes and then backfilling with argon). The solids were stirred for 20 minutes at room temperature, and stored as Sample 9 (an enhanced NAD+ composition).
  • the TGA was performed on the as-received powder samples. Sample analysis was duplicated. The samples were heated from ambient to 800°C under nitrogen gas at a rate of 10 °C/min and ramped to 900 °C at 10 °C/min under air.
  • thermograms are shown in FIG. 32A-C.
  • weight loss results are shown in Table 3.
  • thermograms are shown in FIG. 33A-C.
  • weight loss results are shown in Table 4.
  • thermograms are shown in FIG. 34A-C.
  • weight loss results are shown in Table 5.
  • thermograms are shown in FIG. 35A-C.
  • weight loss results are shown in Table 6.
  • FIG. 36 shows the overlay of thermograms of Samples 6, 7, 8, and 9.

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Abstract

La présente divulgation concerne des compositions de nicotinamide adénine dinucléotide (NAD+) et des formulations pharmaceutiques améliorées, utiles dans le traitement de troubles et de maladies régulés par des déficiences en NAD+, ainsi que des procédés de préparation et des méthodes d'utilisation de celles-ci.
PCT/US2023/035557 2022-10-19 2023-10-19 Compositions de nad+ améliorées, leurs procédés de fabrication et leurs méthodes d'utilisation WO2024086312A1 (fr)

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