Nicotinamide riboside trioleate chloride, compositions containing the compound, and methods of making and using the compound

Abstract

Nicotinamide riboside trioleate chloride (NRTOCl) is a novel hydrophobic derivative of nicotinamide riboside chloride. It can be used in compositions formulated for oral administration, preferably in beverages, such as ready-to-drink (RTD) beverages sealed in a container, or in powders formulated for reconstitution with a diluent to form a reconstituted beverage.

Description

[Technical field]

[0001] [Background technology]

[0001] This disclosure relates generally to a novel hydrophobic derivative of nicotinamide riboside chloride, i.e., nicotinamide riboside trioleate chloride (NRTOCl). This disclosure also relates to methods of making and using the compound, and to compositions, such as liquid compositions, including beverages, that contain the compound.

[0002]

[0002] Nicotinamide adenine dinucleotide (NAD + ) is a key coenzyme in redox-mediated energy metabolism and mitochondrial function 1,2 In non-oxidation and reduction reactions, NAD + is a key cofactor for regulating the activity of two essential protein families, sirtuins (SIRTs) and poly(ADP-ribose) polymerases (PARPs) 3~5 Sirtuins have several important roles in maintaining nuclear, mitochondrial, cytoplasmic, or metabolic homeostasis. The most important role of PARP is to repair DNA and maintain chromatin structure and function. 3~5 .

[0003]

[0003] NAD + Levels decrease during aging, and this decrease leads to defects in nuclear and mitochondrial function, resulting in many age-related symptoms. 6~10 . NAD + Replenishing precursors to NAD + This can restore levels of ATP and prevent many age-related diseases, including neurodegenerative and cardiovascular diseases, as well as metabolic disorders. 11~17 Recent studies have shown that NAD + It has been shown that boosting the immune system may be useful in preventing and treating liver cancer. 18 .

[0004]

[0004] Nicotinamide riboside (NR) is the most important NAD +It is a precursor of NAD, which can be administered orally and is a potent NAD inhibitor in mammalian cells. + You can boost the level of 17、19 NR converts NAD to NAD in fewer steps. + It is metabolized to other NADs such as niacin and nicotinamide. + It is more efficient than the precursor. (Figure 1) 19 .

[0005]

[0005] Several studies have confirmed that NR supplementation shows numerous health benefits in many animals and humans, especially in middle-aged and elderly people. For example, NR supplementation reduces DNA and mitochondrial damage. 20 , Alzheimer's disease 21 ,obesity 22~24 , diabetes 24、25 , muscle degeneration 7 , and aging 26 NR supplementation not only improves lactation and breastfeeding behavior in new mothers, but also improves the quality of breast milk by increasing the transfer of nutrients from the mother to the breast milk. 13 Studies have also demonstrated that NR exhibits antiviral effects against HIV and Hepatitis B. 26 .

[0006]

[0006] Infectious diseases caused by pathogens, particularly SARS-CoV-2 and COVID-19, are characterized by the activation of NAD + Dramatically reduces levels, resulting in abnormal immune responses 26 The use of NR is similar to that of NAD. + Maintaining stable levels helps fight COVID-19 infection and activates the innate immune response to fight infection 26、27 .

[0007] [Summary of the invention] Nicotinamide riboside chloride (NRCl) is the chloride salt of NR, which is commercially available in capsule form as Niagen™. NRCl is a compound that is capable of inhibiting NAD +Used as a safe dietary supplement approved by the U.S. Food and Drug Administration (FDA) to boost levels 28 One of the challenges in using and storing NR is its inherent instability to hydrolysis. NR is a quaternary ammonium salt that contains a reactive N-glycosidic bond that can be spontaneously cleaved in aqueous solution to yield nicotinamide and D-ribose degradation products. As a result, it is difficult to develop ready-to-drink (RTD) beverages containing NR (Figure 2).

[0008]

[0008] The experimental examples described herein report the synthesis of nicotinamide riboside trioleate chloride (NRTOCl) as a novel hydrophobic NRCl derivative by the reaction of NRCl with oleoyl chloride (Figure 3). In contrast to NRCl, this novel compound is not soluble in water, but is readily dissolved in canola oil, corn oil, and medium chain triglyceride (MCT) oil at room temperature. The aqueous stability of NRCl and NRTOCl was tested at 35°C, and the results confirmed that NRTOCl was 88 times more stable than NRCl.

[0009]

[0009] As NRTOCl was readily soluble in canola oil, an oil-in-water emulsion was made by dissolving NRTOCl in canola oil in the presence of sodium caseinate as an edible emulsifier. The stability of NRTOCl in this emulsion was greatly increased, and NRTOCl was 213 times more stable in the emulsion compared to NRCl under the same conditions at a temperature of 35°C. Finally, the bioavailability of NRTOCl was investigated by testing its digestibility in a simulated intestinal phase. The results demonstrate that NRTOCl is digestible (e.g., 1-10% or even 1-20%) and liberates NR in the presence of porcine pancreatin in a simulated intestinal phase. These results obtained indicate that NRTOCl may be promising for use as a NR booster in beverages, such as ready-to-drink (RTD) beverages.

[0010] Furthermore, as shown in additional experimental examples, the specific use of long chain fatty acids in NRTOCl is advantageous over short chain fatty acids in nicotinamide riboside tributyrate chloride (NRTBCl), which did not exhibit good solubility.

[0011]

[0011] Additional features and advantages are described herein, and will be apparent from the drawings and detailed description that follow. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic outlining the conversion of NR to NAD+ in mammalian cells. [Diagram 2] FIG. 1 is a schematic diagram outlining the hydrolysis of NR. [Diagram 3] FIG. 1 is a schematic outlining the synthesis of NRTOCl using oleoyl chloride as disclosed herein. [Figure 4] 1 is a graph showing the results of FT-IR of an experimental example disclosed herein, particularly NRTOCl. [Diagram 5] 1 is a graph showing the results of 1H NMR of an experimental example disclosed herein, specifically NRTOCl in CDCl3. [Figure 6] 1 is a graph showing an expanded view of the 1H NMR results of an experimental example disclosed herein, in particular NRTOCl. [Figure 7] 1 is a graph showing 13C NMR results for an experimental example disclosed herein, specifically NRTOCl in CDCl3. [Figure 8] 1 is a graph showing an enlarged view of 13C NMR results for an experimental example disclosed herein, in particular NRTOCl. [Figure 9A] 9A and 9B are graphs showing SRM LC-MS results for the experimental examples disclosed herein, specifically NRTOCl: Figure 9A is the SRM LC of NRTOCl, and Figure 9B is the mass spectrum of NRTOCl. [Figure 9B]9A and 9B are graphs showing SRM LC-MS results for the experimental examples disclosed herein, specifically NRTOCl: Figure 9A is the SRM LC of NRTOCl, and Figure 9B is the mass spectrum of NRTOCl. [Figure 10A] 10A-10C are photographs showing results from an example experiment disclosed herein: Figure 10A is NRTOCl dispersed in water, and Figures 10B-10C are transmission electron microscope (TEM) images of NRTOCl dispersed in water. [Figure 10B] 10A-10C are photographs showing results from an example experiment disclosed herein: Figure 10A is NRTOCl dispersed in water, and Figures 10B-10C are transmission electron microscope (TEM) images of NRTOCl dispersed in water. [Figure 10C] 10A-10C are photographs showing results from an example experiment disclosed herein: Figure 10A is NRTOCl dispersed in water, and Figures 10B-10C are transmission electron microscope (TEM) images of NRTOCl dispersed in water. [Figure 11] 1 is a graph showing results of the experimental examples disclosed herein, in particular the stability of NRTOCl and NRCl in DI water at 35° C. [Figure 12] FIG. 1 is a schematic outlining the hydrolysis of NRTO+ and NR+ from the N-glycosidic bond. [Figure 13] FIG. 1 is a schematic outlining the hydrolysis of NRTOCl nanoparticles in the outer layer in contact with water when NRTOCl is dispersed in water. [Figure 14] 1 is a graph showing the results of the first experimental example disclosed herein, specifically, the hydrolytic stability over 26 days at 35° C. of an NRTOCl emulsion compared to an NRCl emulsion or NRCl in water. [Figure 15] 1 is a graph showing the results of the first experimental example disclosed herein, specifically, the comparison of hydrolytic stability over 42 days at 25° C. between an NRTOCl emulsion and NRCl in water. [Figure 16] FIG. 1 is a schematic outlining the digestion of NRTO+ in a simulated intestinal phase. [Figure 17A] 17A and 17B are graphs showing the SRM LC-MS results of the first example experiment disclosed herein, specifically, the SRM LC of free NRCl, and FIG. 17B is the mass spectrum of free NRCl. [Figure 17B] 17A and 17B are graphs showing the SRM LC-MS results of the first example experiment disclosed herein, specifically, the SRM LC of free NRCl, and FIG. 17B is the mass spectrum of free NRCl. [Figure 18] 1 shows an FT-IR of nicotinamide riboside tributyrate chloride (NRTBCl) from the second example disclosed herein. [Figure 19] 1 shows the 1H NMR of NRTBCl in CDCl3 from the second example disclosed herein. [Figure 20] 13C NMR of NRTBCl in CDCl3 from the second example disclosed herein. [Figure 21A] 21A and 21B show SRM LC-MS of NRTBCl from the second example experiment disclosed herein: Figure 21A shows the SRM LC of NRTBCl, and Figure 21B shows the mass spectrum of NRTBCl. [Figure 21B] 21A and 21B show SRM LC-MS of NRTBCl from the second example experiment disclosed herein: Figure 21A shows the SRM LC of NRTBCl, and Figure 21B shows the mass spectrum of NRTBCl. [Figure 22A] 1 shows size measurements of NRTBCl in water from the second example disclosed herein. [Figure 22B] 1 shows size measurements of NRTBCl in water from the second example disclosed herein. [Figure 22C] 1 shows size measurements of NRTBCl in water from the second example disclosed herein. [Figure 23] The second experimental example disclosed herein shows the stability of NRTBCl in MilliQ (MQ) water at 35° C. for 6 days.

[0013] [Mode for carrying out the invention]

[0035] definition

[0036] Some definitions are provided below. However, definitions may be found in the "Embodiments" section below, and the heading "Definitions" above does not imply that such disclosure in the "Embodiments" section is not a definition.

[0014]

[0037] All percentages stated herein are by total weight of the composition unless otherwise indicated. As used herein, "about," "approximately," and "substantially" are understood to refer to numbers within a certain range of numerical values, for example, within the range of -10% to +10% of the referenced number, preferably within the range of -5% to +5% of the referenced number, more preferably within the range of -1% to +1% of the referenced number, and most preferably within the range of -0.1% to +0.1% of the referenced number. All numerical ranges herein should be understood to include all integers or fractions within the range. Furthermore, these numerical ranges should be interpreted as supporting claims directed to any number or subset of numbers within the range. For example, a disclosure of 1 to 10 should be interpreted as supporting ranges such as 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0015]

[0038] As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise. Thus, for example, reference to "a vitamin" or "the vitamin" includes both embodiments having a single vitamin and embodiments having two or more vitamins.

[0016]

[0039] The terms "comprise", "comprises", and "comprising" should be interpreted as inclusive rather than exclusive. Similarly, the terms "include", "including", and "or" should all be interpreted as inclusive unless such interpretation is clearly prevented by the context. However, the compositions disclosed herein may not include elements not specifically disclosed herein. Thus, disclosure of an embodiment using the term "comprising" includes disclosure of an embodiment "consisting essentially of" the specified components, and an embodiment "consisting of" the specified components.

[0017]

[0040] The terms "at least one of" and "and / or" used in the context of "at least one of X or Y" and "X and / or Y", respectively, should be interpreted as "X" or "Y" or "X and Y". For example, "at least one of sodium caseinate or lecithin" and "sodium caseinate and / or lecithin" should be interpreted as "sodium caseinate without lecithin" or "lecithin without sodium caseinate", or "both sodium caseinate and lecithin".

[0018]

[0041] As used herein, the terms "example" and "such as," especially when followed by a list of terms, are merely exemplary and illustrative and should not be considered exclusive or inclusive. As used herein, a state "associated with" or "linked with" another state means that the states occur simultaneously.

[0019]

[0042] "Prevention" includes reducing the risk, incidence and / or severity of a condition or disorder. The terms "treatment" and "treating" also include treatments that delay the onset of the pathological condition or disorder of interest, as well as curative, therapeutic or disease-modifying treatments, including, for example, therapeutic measures to cure, delay, reduce symptoms and / or halt the progression of a diagnosed pathological condition or disorder, and treatment of patients at risk of or suspected of having a disease, and patients who are unwell or diagnosed with a disease or medical condition. The terms "treatment" and "treating" do not necessarily mean treating until the subject is cured. The terms "treatment" and "treating" are also intended to include synergistic or otherwise potentiating one or more primary preventative or therapeutic measures. As a non-limiting example, treatment can be performed by the patient, a caregiver, a doctor, a nurse, or another medical professional.

[0020]

[0043] As used herein, a prophylactically or therapeutically "effective amount" is an amount that prevents a deficiency, treats a disease or medical condition in an individual, or, more generally, reduces symptoms, manages the progression of a disease, or provides a nutritional, physiological, or medical benefit to an individual. The relative terms "promote," "improve," "increase," "enhance," and the like, refer to the superiority of the compositions disclosed herein (including NRTOCl) and their properties and effects compared to the properties and effects of a similarly formulated composition, except that nicotinamide riboside chloride is substituted for NRTOCl.

[0021]

[0044] As used herein, the terms "food," "food product," and "food composition" refer to a product or composition intended for oral ingestion by humans or other mammals and containing at least one nutrient for humans or other mammals.

[0022]

[0045] As used herein, "nutritional composition" and "nutritional product" include any number of food ingredients and possibly optional additional ingredients based on the functional needs of the product and in full compliance with all applicable regulations. Optional ingredients may include, but are not limited to, conventional food additives such as one or more acidulants, additional thickeners, pH adjusting buffers or pH adjusting agents, chelating agents, colorants, emulsifiers, excipients, flavorings, minerals, osmotic agents, pharma-ceutically acceptable carriers, preservatives, stabilizers, sugars, sweeteners, modifiers and / or vitamins. Optional ingredients may be added in any suitable amount.

[0023]

[0046] As used herein, the term "unit dosage form" refers to physically discrete units suitable as dosage units for human and animal subjects, each unit containing a predetermined amount of a composition disclosed herein, optionally with a pharma- ceutically acceptable diluent, carrier, or vehicle, sufficient to produce the desired effect. The specifications for the unit dosage form depend on the particular compound used, the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0024]

[0047] A "subject" or "individual" is a mammal, preferably a human. The term "elderly", in relation to humans, means an age of at least 60 years, preferably greater than 63 years, more preferably greater than 65 years, and most preferably greater than 70 years. The term "older adult" in the human context means a postnatal age of 45 years or more, preferably greater than 50 years, more preferably greater than 55 years, and includes elderly people.

[0025]

[0048] Embodiment

[0049] Aspects of the present disclosure include nicotinamide riboside trioleate chloride (NRTOCl) and compositions comprising NRTOCl, e.g., liquid compositions such as beverages. The compositions may be food products or other nutritional compositions formulated for oral administration. The compositions may include emulsions in which NRTOCl is dispersed. For example, the emulsions may include an oil phase in which at least a portion of the NRTOCl is dispersed. In some embodiments, the oil includes at least one of canola oil, corn oil, or MCT oil. In some embodiments, the oil phase further includes an emulsifier, e.g., at least one of sodium caseinate or lecithin (preferably both in particularly preferred embodiments).

[0026]

[0050] Another aspect relates to the administration of NRTOCl (e.g., an effective amount thereof), or a composition comprising NRTOCl (e.g., an effective amount thereof), to an individual to increase nicotinamide adenine dinucleotide (NAD) levels in cells and tissues, e.g., to improve cell and tissue survival. + The method of the present invention is to promote an increase in intracellular levels of .

[0027]

[0051] Yet another embodiment is a method of reducing at least one of DNA damage or mitochondrial damage and / or treating or preventing at least one condition selected from the group consisting of: (a) a neurodegenerative condition; (b) overweight or obesity; (c) a cardiovascular disease, such as heart disease; (d) one or more of diabetes, hyperinsulinemia, insulin resistance, or insulin insensitivity; (d) muscle degeneration; (e) an age-related disease or disorder; (f) a viral infection, such as HIV, Hepatitis B, SARS-CoV-2, or COVID-19; (g) stress; (h) a blood clotting disorder; (i) inflammation; (j) cancer; (k) an eye disorder; and (l) flushing. The method comprises administering NRTOCl (e.g., an effective amount thereof) or a composition comprising NRTOCl (e.g., an effective amount thereof) to a subject in need of or at risk.

[0028]

[0052] The term "neurological condition" refers to a disorder of the nervous system. A neurological condition may be the result of damage to the brain, spinal column, or nerves caused by disease or injury. Non-limiting examples of symptoms of a neurological condition include paralysis, muscle weakness, poor coordination, loss of sensation, epilepsy / seizures, confusion, pain, and altered level of consciousness. Responses to touch, pressure, vibration, limb position, heat, cold, and pain, as well as reflexes, can be assessed to determine if a subject has a nervous system disorder.

[0029]

[0053] Some neurological conditions are lifelong and their onset can be experienced at any time. Other neurological conditions, such as cerebral palsy, are present from birth. Some neurological conditions, such as Duchenne muscular dystrophy, generally manifest in early childhood, while other neurological conditions, such as Alzheimer's disease and Parkinson's disease, primarily affect older adults. Some neurological conditions develop suddenly due to injury or disease, such as head injury or stroke, or cancer of the brain and spine.

[0030]

[0054] In one embodiment, the neurological condition is the result of a traumatic brain injury. Additionally, or alternatively, the neurological condition is the result of an energy deficiency in the brain or muscles.

[0031]

[0055] Examples of neurological conditions include migraine, memory impairment, age-related memory impairment, brain injury, neurorehabilitation, stroke and post-stroke, amyloid lateral sclerosis, multiple sclerosis, cognitive impairment, mild cognitive impairment (MCI), post-intensive care cognitive impairment, age-related cognitive impairment, Alzheimer's disease, Parkinson's disease, Huntington's disease, inborn errors of metabolism (such as glucose transporter 1 deficiency syndrome and pyruvate dehydrogenase complex deficiency), bipolar disorder, schizophrenia, and / or epilepsy.

[0032]

[0056] It will be appreciated that the compounds, compositions and methods of the invention may be beneficial in the prevention and / or treatment of the above neurological conditions, particularly in maintaining or improving brain or nervous system function.

[0033]

[0057] "Diabetes" refers to hyperglycemia or ketoacidosis, as well as chronic common metabolic abnormalities resulting from prolonged hyperglycemic states or impaired glucose tolerance. "Diabetes" includes both type I and type II (non-insulin-dependent diabetes mellitus or NIDDM) of the disease. Risk factors for diabetes may include, but are not limited to, the following: waist size greater than 40 inches for men or greater than 35 inches for women, blood pressure greater than 130 / 85 mmHg, triglycerides greater than 150 mg / dL, fasting blood glucose greater than 100 mg / dL, or high density lipoprotein less than 40 mg / dL for men or less than 50 mg / dL for women.

[0034]

[0058] The term "hyperinsulinemia" refers to a condition in which an individual has higher than normal levels of insulin in the blood.

[0035]

[0059] The term "insulin resistance" refers to a state in which normal amounts of insulin produce a subnormal biological response compared to the biological response in a subject who does not have insulin resistance.

[0036]

[0060] "Insulin resistance disorder" as described herein refers to any disease or condition caused by or contributed to by insulin resistance. Examples include diabetes, obesity, metabolic syndrome, insulin resistance syndrome, syndrome X, insulin resistance, elevated blood pressure, hypertension, high blood cholesterol, dyslipidemia, hyperlipidemia, dyslipidemia, atherosclerosis including stroke, coronary artery disease or myocardial infarction, hyperglycemia, hyperinsulinemia and / or hyperproinsulinemia, impaired glucose tolerance, delayed insulin release, diabetic complications such as coronary heart disease, angina pectoris, congestive heart failure, stroke, cognitive function in dementia, retinopathy, peripheral neuropathy, nephropathy. , glomerulonephritis, glomerulosclerosis, nephrotic syndrome, hypertensive nephrosclerosis, some types of cancer (e.g., endometrial, breast, prostate, and colon cancer), pregnancy complications, poor female reproductive health (e.g., menstrual irregularities, infertility, irregular ovulation, polycystic ovarian syndrome (PCOS)), lipodystrophy, cholesterol-related disorders (e.g., gallstones, cholecystitis, and cholelithiasis), gout, obstructive sleep apnea and breathing disorders, osteoarthritis, and bone loss (osteoporosis).

[0037]

[0061] An "overweight" individual has a body mass index (BMI) of at least 25, and an "obese" individual has a body mass index (BMI) of at least 30. Overweight and obesity may or may not be associated with insulin resistance.

[0038]

[0062] In some embodiments, a method of treating or preventing cancer includes administering NRTOCl (e.g., an effective amount thereof), or a composition comprising NRTOCl (e.g., an effective amount thereof), to a subject in need of or at risk of cancer, e.g., by inhibiting inosine 5' monophosphate dehydrogenase and / or reducing the amount of NAD+ in cells, including cancer.

[0039]

[0063] "Cancer" refers to any of a variety of cellular diseases involving malignant neoplasms characterized by the proliferation of undifferentiated cells. It is not intended that the diseased cells will actually invade surrounding tissues and metastasize to new body sites. Cancer may involve any tissue of the body and have many different forms in each body area. Most cancers are named after the type of cell or organ in which they originate.

[0040]

[0064] The cancer may be selected from the group consisting of pancreatic cancer, endometrial cancer, small cell and non-small cell carcinoma of the lung (including squamous cell carcinoma, adenocarcinoma, and large cell carcinoma), squamous cell carcinoma of the head and neck, bladder cancer, ovarian cancer, cervical cancer, breast cancer, kidney cancer, CNS cancer, and colon cancer, myeloid and lymphocytic leukemia, lymphoma, liver tumor, medullary thyroid cancer, multiple myeloma, melanoma, retinoblastoma, and soft tissue and bone sarcoma. Optionally, the NRTOCl is administered in combination with another chemotherapeutic agent, for example in the same composition.

[0041]

[0065] Another aspect is a method of reducing weight in a subject or preventing weight gain in a subject, the method comprising administering NRTOCl (e.g., an effective amount thereof) or a composition comprising NRTOCl (e.g., an effective amount thereof) to a subject in need of or at risk.

[0042]

[0066] Yet another aspect is a method of treating or preventing drug toxicity and / or adverse drug reactions, the method comprising administering NRTOCl (e.g., an effective amount thereof) or a composition comprising NRTOCl (e.g., an effective amount thereof) to a subject in need of or at risk, e.g., a subject who is concurrently administered a drug such as a statin.

[0043]

[0067] "Adverse drug reaction" means any reaction to a drug that is harmful, unintended, and occurs at a prophylactic, diagnostic, or therapeutic dose, and includes side effects, toxicity, hypersensitivity, drug interactions, complications, or other idiosyncrasies. Side effects are adverse symptoms, often resulting from the pharmacological effects of therapeutic serum concentrations of the drug on unintended organ systems (e.g., blurred vision from antihistamines with anticholinergic properties). Toxic side effects are adverse symptoms or other effects resulting from excessive or prolonged chemical exposure to the drug (e.g., digitalis toxicity, liver toxicity). Hypersensitivity is an immune-mediated adverse reaction (e.g., anaphylaxis, allergy). Drug interactions are adverse effects resulting from interactions with other drugs, foods, or disease states (e.g., warfarin and erythromycin, cisapride and grapefruit, loperamide and Clostridium difficile enteritis). Complications are diseases caused by drugs (e.g., gastric ulcers caused by NSAIDs, thrombosis caused by estrogen). Adverse drug reactions may be mediated by known or unknown mechanisms (e.g., agranulocytosis associated with chloramphenicol or clozapine). Such adverse drug reactions can be identified by subject observation, assays, or animal models well known in the art.

[0044]

[0068] In one embodiment, NRTOCl is used to decrease the level and / or activity of a sirtuin protein and is selected from the group consisting of the following compounds: nicotinamide (NAM), suranim; NF023 (G-protein antagonist); NF279 (purinergic receptor antagonist); trolox (6-hydroxy-2,5,7,8,tetramethylchroman-2-carboxylic acid); (-)-epigallocatechin (hydroxyl is at the 3,5,7,3',4',5' positions); (-)-epigallocatechin gallate (hydroxyl is at the 5,7,3',4',5' positions); and a gallate ester at the 3-position); cyanidin chloride (3,5,7,3',4'-pentahydroxyflavylium chloride); delphinidin chloride (3,5,7,3',4',5'-hexahydroxyflavylium chloride); myricetin (cannabiscetin; 3,5,7,3',4',5'-hexahydroxyflavone); 3,7,3',4',5'-pentahydroxyflavone; gossypetin (3,5,7,8,3',4'-hexahydroxyflavone), sirtinol; and splitomycin.

[0045]

[0069] NRTOCl can be administered to humans or animals, particularly companion animals, pets, or livestock. The composition has beneficial effects on all age groups. Preferably, the composition is formulated for administration to infants, juveniles, adults, or the elderly.

[0046]

[0070] Preferably, NRTOCl is orally administered to an individual in a beverage, and the unit dosage form is a predetermined amount of the beverage (eg, a predetermined amount of the beverage containing an effective amount of NRTOCl).

[0047]

[0071] In some embodiments, the supplement may be a ready-to-drink (RTD) beverage in a container, and the unit dosage form is a predetermined amount of the RTD beverage sealed in a container that is opened for oral administration. For example, the predetermined amount of the RTD beverage can include an effective amount of NRTOCl. The RTD beverage is a liquid that can be consumed orally without the addition of any further ingredients.

[0048]

[0072] In other embodiments, the method includes reconstituting a powder unit dosage form comprising NRTOCl with a diluent, such as water or milk, to form a beverage that is then orally administered to an individual (e.g., within about 10 minutes after reconstitution, within about 5 minutes after reconstitution, or within about 1 minute after reconstitution). The powder unit dosage form can be sealed in a sachet or other package that can be opened for reconstitution and subsequent oral administration.

[0049]

[0073] The unit dosage form of the supplement may contain excipients, emulsifiers, stabilizers, and mixtures thereof.

[0050]

[0074] NRTOCl can be administered at least 1 day per week, preferably at least 2 days per week, more preferably at least 3 or 4 days per week (e.g., every other day), and most preferably at least 5 days per week, 6 days per week, or 7 days per week. The administration period can be at least 1 week, preferably at least 1 month, more preferably at least 2 months, and most preferably at least 3 months, e.g., at least 4 months.

[0051]

[0075] In one embodiment, administration is at least daily, for example, a subject may receive administration one or more times a day. In some embodiments, administration continues for the remaining life of an individual. In other embodiments, administration is performed until there is no detectable symptom of a medical condition. In a specific embodiment, administration is performed until there is a detectable improvement in at least one symptom, and in further cases, administration is continued to maintain remission.

[0052]

[0076] In view of the foregoing disclosure, an embodiment provided herein is NRTOCl. Another embodiment is a composition comprising NRTOCl and, optionally, at least one of a protein, a lipid, a carbohydrate, a vitamin, or a mineral. In some embodiments, the composition is formulated for oral administration, preferably as a beverage, such as a ready-to-drink (RTD) beverage sealed in a container, or a powder formulated for reconstitution with a diluent to form a reconstituted beverage.

[0053]

[0077] Another embodiment is a unit dosage form of a composition comprising NRTOCl, the unit dosage form comprising a therapeutically or prophylactically effective amount of NRTOCl for the individual to whom the unit dosage form is administered.

[0054]

[0078] Yet another embodiment is a method of making a composition, the method comprising adding NRTOCl to at least one other ingredient, in some embodiments, the composition is formulated for oral administration and the at least one other ingredient is edible.

[0055]

[0079] In some embodiments of these methods, the composition is an emulsion, preferably an oil-in-water emulsion, comprising an oil phase in which at least a portion of the NRTOCl is dispersed, and optionally an emulsifier, such as at least one of sodium caseinate or lecithin. The oil phase can comprise at least one of canola oil, corn oil, or medium chain triglyceride (MCT) oil, in which at least a portion of the NRTOCl is dispersed.

[0056]

[0080] In some embodiments of these methods, the composition is administered to the individual daily for at least one week. In some embodiments of these methods, the individual is selected from the group consisting of human infants, human children, human adolescents, human adults, and human geriatrics, and animals, such as companion animals.

[0057]

[0081] In some embodiments of these methods, the composition is preferably administered orally as a beverage, more preferably as a ready-to-drink (RTD) beverage sealed in a container that is opened prior to administration, or as a powder formulated to be reconstituted with a diluent to form a reconstituted beverage prior to administration.

[0058]

[0082] [Example]

[0083] The following non-limiting examples generally illustrate the concepts underlying the embodiments disclosed herein.

[0059]

[0084] Example 1

[0085] 1. Experimental Section

[0086] 1.1. Overview

[0087] Materials: Nicotinamide riboside chloride (beta form) was donated by ChromaDex Company. Oleoyl chloride with 89% purity was purchased from Aldrich, silica gel (P60, 40-63 μm, 60 Å) was purchased from SiliCycle, and Silica Gel 60 F254 Coated Aluminum-Backed TLC Sheets were purchased from EMD Millipore (Billerica, MA, USA). Bovine bile (B3883) and pancreatin from porcine pancreas (P7545, 8x USP) were purchased from Aldrich.

[0060]

[0088] Characterization: Using a Bruker INOVA 500 NMR spectrometer. 1 H-NMR and 13 C-NMR spectra were obtained in CDCl3. Fourier transform infrared (FTIR) spectra were obtained on a Shimadzu IRAffinity-1S spectrophotometer using an 8 cm -1The measurements were recorded by collecting 128 scans at a resolution of 100 nm. Ultraviolet-visible (UV-vis) spectroscopy was recorded on a Shimadzu UV-2600 spectrophotometer. High-performance liquid chromatography (HPLC) measurements were performed using an Agilent 1200 LC System equipped with a Binary SL Pump & Diode Array Detector and a Shodex RI-501 Refractive Index Detector (single channel).

[0061]

[0089] The HPLC was equipped with an ultraviolet detector (HPLCUV). Reversed-phase HPLC was performed on a Luna 100Å (150 mm×4.6 mm). The column temperature was set at 25° C. The injection volume was 10.0 μL, ammonium acetate (20 mM) was used as the mobile phase, and the flow rate was 0.7 mL / min for 45 or 60 min. All samples were filtered using a 13 mm nylon syringe filter with a pore size of 0.22 μm before measurement.

[0062]

[0090] For LC-MS analysis, an LC (Agilent 1100 series) coupled to a mass spectrometer was used. For reversed-phase chromatography, a Phenomenex Luna Omega (Phenomenex) LC column with the following specifications was used: 100 x 4.6 mm, 3 μm, polar C18, pore size 100 Å, with a flow rate of 0.3 mL / min. The LC eluent contained MilliQ water (solvent A) and acetonitrile (solvent B), with gradient elution (time course of composition of solution A:solution B: 95:5 at 0 min, 95:5 at 3 min, 85:15 at 15 min, 90:10 at 17 min, and 95:5 at 20 min).

[0063]

[0091] The mass spectrometer (Finnigan LTQ mass spectrometer) was equipped with an electrospray interface (ESI) set in positive mode for electrospray ionization to analyze NRTOCl, NRCl, and nicotinamide. The optimized parameters were a sheath gas flow rate of 20 arbitrary units, spray voltage set at 4.00 kV, capillary temperature of 350° C., capillary voltage of 41.0 V, and tube lens set at 125.0 V.

[0064]

[0092] The particle size distribution, mean particle size (zeta average size), and zeta potential of NRTOCl in DI water were measured using a commercial dynamic light scattering instrument (Nano-ZS, Malvern Instruments, Worcestershire, UK). The structure and morphology of the prepared NRTOCl nanoparticles were evaluated using a Tecnai F20 TEM / STEM transmission electron microscope (200 kV).

[0065]

[0093] 1.2. General Procedure for the Synthesis of NRTOCl

[0094] In a round bottom flask in an ice bath, 200 mg of NRCl, 0.55 mL of pyridine, and 4.75 mL of DMF were added. Then, 2.0 mL of oleoyl chloride was added dropwise and the reaction mixture was stirred under a nitrogen blanket for 3 h. The progress of the reaction was followed by TLC. After 3 h, 5 mL of methanol was added to the reaction mixture to neutralize the excess amount of oleoyl chloride, and then the solvent was evaporated under reduced pressure using a rotary evaporator. The crude product was extracted into hexane and finally purified using column chromatography on SiO2. The eluent was a mixture of CH3OH (12%) and EtOAc (88%). The purified NRTOCl was obtained as a pale cream-colored fatty product in 64.3% (479.2 mg) (lambda in methanol). max was 267 nm).

[0066]

[0095] 1.3. Preparation of 15 wt% NRTOCl in Canola Oil as Stock for Making Oil-in-Water Emulsions

[0096] 2154 mg of canola oil was added to a 15 mL Falcon tube containing 380 mg of NRTOCl. The tube was then placed in a water bath and shaken at approximately 35°C until the NRTOCl was completely dissolved in the canola oil. After the NRTOCl was dissolved in the canola oil, the sample was kept at room temperature for further testing. During the storage of NRTOCl in canola oil at room temperature, the solution was stable and clear with no sediment.

[0067]

[0097] 1.4. Preparation of Aqueous Phase for Making Oil-in-Water Emulsions Containing NRTOCl Using Sodium Caseinate (2 wt%), KCl (0.3 wt%), NaCl (0.1 wt%), CaCl2 (0.2 wt%), and NaN3 (0.01 wt%)

[0098] 2 g of sodium caseinate was slowly added to 100 mL of DI water at 70° C. and the mixture was stirred for 5 minutes. The temperature was then increased to 75° C. for 10 minutes. After the solution was cooled to 50° C., 0.3 g of KCl, 0.1 g of NaCl, and 0.01 g of NaN3 were added to the mixture and stirred for 2 minutes. Then, 0.2 g of CaCl2 was slowly added to the solution and stirred for an additional 5 minutes. After the temperature was reduced to 25° C., the mixture was diluted to 100 mL by adding DI water and homogenized at 10,000 rpm for 2 minutes.

[0068]

[0099] 1.5. Preparation of Oil-in-Water Emulsions of NRTOCl and NRCl for Stability Studies

[0100] Three different NRTOCl oil-in-water emulsions were made according to the following process.

[0069]

[0101] 1.5.1.Cas emulsion:

[0102] An emulsion was prepared using 480 mg of oil stock containing 15 wt% NRTOCl in canola oil and an aqueous phase (14.52 g) of Na caseinate (2 wt%), KCl (0.3 wt%), NaCl (0.1 wt%), CaCl2 (0.2 wt%), and NaN3 (0.01 wt%). The oil phase was added to the aqueous phase and homogenized at 16,800 rpm for 150 seconds at room temperature.

[0070]

[0103] 1.5.2.Cas-Lec Emulsion:

[0104] An emulsion was prepared using 480 mg of oil stock containing 15 wt% NRTOCl in canola oil and 0.025 g of lecithin. The aqueous phase (14.5 g) contained Na caseinate (2 wt%), KCl (0.3 wt%), NaCl (0.1 wt%), CaCl2 (0.2 wt%), and NaN3 (0.01 wt%). The oil phase was added to the aqueous phase and homogenized at 16,800 rpm for 150 seconds at room temperature.

[0071]

[0105] Tween Emulsions:

[0106] An emulsion was made using 480 mg of oil stock containing 15 wt% NRTOCl in canola oil and 14.52 g of an aqueous phase containing 2 wt% Polysorbate 80 (Tween 80). The oil phase was added to the aqueous phase and homogenized at 16,800 rpm for 150 seconds at room temperature.

[0072]

[0107] 1.5.4.NR Emulsion:

[0108] An emulsion was prepared using approximately 480 mg of canola oil in an aqueous phase (14.52 g) of NRCl (19 mg), Na caseinate (2 wt%), KCl (0.3 wt%), NaCl (0.1 wt%), CaCl2 (0.2 wt%), and NaN3 (0.01 wt%). The oil phase was added to the aqueous phase and homogenized at 16,800 rpm for 150 seconds at room temperature.

[0073]

[0109] 1.6. Sample preparation of NRTOCl and NRCl for measurement of nicotinamide released during stability studies

[0110] After stabilization for a specific time for NRTOCl and NRCl emulsions, 1.5 mL of each emulsion was centrifuged (14,000 rpm) at room temperature for 20 min, and then the aqueous phase was separated and filtered through a 0.22 μm filter for the determination of released nicotinamide by HPLC analysis.

[0074]

[0111] 1.7. In vitro digestion test

[0112] The in vitro digestion of NRTOCl in pure form and NRTOCl dissolved in MCT oil was investigated as follows.

[0075]

[0113] 1.7.1. In vitro digestion study of pure NRTOCl dispersed in simulated intestinal phase

[0114] The buffer for the simulated intestinal phase was prepared according to the following protocol: 29 Specifically, 60 mg of NRTOCl was dissolved in 0.3 mL of ethanol and added to 10 g of buffer containing 400 mg of bovine bile. 0.75 mL of CaCl2 solution (0.3 M) was added to the mixture, and the pH was adjusted to about 7 using HCl (1 M). 400 mg of fresh porcine pancreatin was then dispersed in 4 mL of buffer solution and added to the mixture. The sample was placed in a 37°C incubator (250 rpm) for 30 min. The sample was then removed from the incubator, the pH was adjusted to about 7, and returned to the incubator for another 30 min. The incubation step and pH adjustment were repeated every 30 min until 2 h had elapsed. After placing the sample in an ice bath to inactivate the enzyme, 1.5 mL of the sample was centrifuged at 14,000 rpm for 10 min. Finally, the aqueous phase was separated and filtered through a 0.22 μm filter and the released NR and nicotinamide were measured.

[0076]

[0115] 1.7.2. In vitro digestion study of NRTOCl in MCT oil in a simulated intestinal phase

[0116] NRTO 29% w / w in MCT oil was prepared by dissolving 60 mg NRTOCl in 150 mg MCT oil. The aqueous phase was 10 g of buffer solution containing 400 mg of bovine bile. The oil phase was added to the aqueous phase and homogenized at 15,000 rpm for 150 seconds at room temperature. 0.75 mL of CaCl2 solution (0.3 M) was added to the mixture and the pH was adjusted to about 7 using HCl (1 M). 400 mg of fresh porcine pancreatin was then dispersed in 5 mL of buffer solution and added to the mixture. The sample was placed in a 37°C incubator (250 rpm) for 30 minutes. The sample was then removed from the incubator, the pH was adjusted to about 7 (with NaOH 1 M) and returned to the incubator for another 30 minutes. The incubation and pH adjustment steps were repeated every 30 minutes until 2 hours had elapsed. After placing the samples in an ice bath to inactivate the enzyme, 1.5 mL samples were centrifuged at 14,000 rpm for 10 min. Finally, the aqueous phase was separated and filtered through a 0.22 μm filter to measure the released NR and nicotinamide.

[0077]

[0117] 2. Results and Discussion

[0118] In this study, chemical modification was performed as a fatty ester derivative to increase the hydrolytic stability of NR, thereby increasing its hydrophobicity. For this purpose, a new compound, NRTOCl, was synthesized by the reaction between NRCl and oleoyl chloride. The best results were obtained when the reaction was carried out using DMF as the solvent and pyridine as the base. After purification of NRTOCl by column chromatography on SiO2, the pure product was observed by FTIR, 1 H NMR, 13 It was characterized by C NMR and LC-MS.

[0078]

[0119] First, to determine the functional groups in the structure of NRTOCl, the FTIR spectrum of this compound was examined (Figure 4). -1 and 3123 cm -1 The two peaks at 3005 cm indicate the asymmetric contraction and symmetric stretching of the NH2 bond in the amide functional group. The stretching vibrations of alkenes and aromatic CH bonds are at about 3005 cm-1 Appears in. 2922cm -1 and 2853 cm -1 The two peaks at 1744 cm are due to the asymmetric and symmetric stretching vibrations of aliphatic CH. -1 The strong peak at 1689 cm indicates the carbonyl of the ester group. The carbonyl of the amide functional group is at 1689 cm -1 The presence of a C=C bond is seen at 1622 cm -1 This is indicated by the peak at 1458cm -1 and 1379 cm -1 The two peaks indicate the deformation vibrations of the methylene group and the methyl group, respectively. -1 The broad peak between 677 cm is due to the stretching of the C-O bond of the ester group. -1 , 721cm -1 , and 915 cm -1 The bands indicate bending vibrations of alkenes and aromatic CH bonds. 30 .

[0079]

[0120] Furthermore, NRTOCl 1 H NMR was performed in CDCl3 at room temperature. Integration showed the presence of 111 hydrogens consistent with the structure of this compound (Figure 5). 1 H NMR clearly shows that the most unshielded proton (H1) at 10.34 ppm is due to the hydrogen located on the pyridinium ring between the positive nitrogen and the amide group (Figure 6). 30In NRTOCl, the chemical shifts of the NH2 protons are not equivalent. In this compound, one of the NH2 protons appears at 9.86 ppm and another at 6.28 ppm. The double peak at 9.44 ppm (J = 10 Hz) is due to H5, which is located in the ortho position of the positive nitrogen on the pyridinium ring. The chemical shift of H3, in the para position of the positive nitrogen, appears as a doublet peak at 9.34 ppm (J = 10 Hz). The last hydrogen on the pyridinium ring is H4, which appears as a triplet peak at 8.20 ppm (J = 10 Hz). In the structure of NRTO, there are four hydrogens on the ribose ring. The anomeric hydrogen (H1') is further influenced by the oxygen atom in the ribose ring and the positive nitrogen of the pyridinium ring, so this hydrogen appears as a doublet peak at 6.75 ppm (J = 5 Hz). H2' and H3' are adjacent groups and appear as two triplet peaks (J=5 Hz) with chemical shifts of 5.57 ppm and 5.43 ppm, respectively. H2' is closer to the anomeric center than H3', so its chemical shift is less shielded than that of H3'. A multiplet peak at 5.35 ppm with an integral of 6 confirms the presence of three HC=CH groups in the structure of NRTO. H4' in the ribose ring and one of the hydrogens of the methylene group attached to one oxygen of the ester group (H5') overlap each other and appear as a multiplet peak at 4.70 ppm with an integral of 2. The hydrogen of this methylene group is diastereotopic, so another hydrogen of this methylene group appears as a doublet of doublet peaks at 4.50 ppm (J1=14 Hz, J2=4 Hz). In the three fatty acid ester chains of NRTO, the three CH2 groups in the vicinity of the ester carbonyl group appear as a multiplet peak between 2.37 and 2.55 ppm (Figure 6). Furthermore, six methylene groups in the vicinity of the HC=CH group appear as a multiplet peak at 2.02 ppm. Three other methylene groups in the vicinity of the CH2 group attached to the carbonyl group appear as a multiplet peak at 1.63 ppm. The broad peak at 1.30 ppm with an integral value of 60 is due to the remainder of the 30 methylene groups. Finally, a triplet peak at 0.89 ppm (J=7.0 Hz) with an integral value of 9 confirms the presence of three methyl groups in the structure of NRTO.

[0080]

[0121] NRTOCl in CDCl3 13 The C NMR of this compound was also investigated at room temperature (Figure 7). 13 C NMR reveals three peaks at 173.1 ppm, 172.9 ppm, and 172.3 ppm that are attributed to three different carbonyls of the ester functional groups in the structure of NRTO ( FIG. 8 ).

[0081]

[0122] The peak at 162.5 ppm confirms the carbonyl of the amide group in this compound. There are five distinct peaks at 146.7 ppm, 142.5 ppm, 141.8 ppm, 134.6 ppm, and 127.9 ppm, indicating the presence of carbons in the pyridinium ring. The chemical shifts of the six carbons of the three -C=C groups are very close, appearing at 130.07 ppm, 130.06 ppm, 130.04 ppm, 129.67 ppm, and 129.62 ppm. Due to the close chemical shifts of these carbons, two carbons overlap each other (possibly at 129.62 ppm with higher intensity), resulting in the observation of five peaks of these alkene groups. The four peaks at 98.0 ppm, 82.9 ppm, 75.8 ppm, and 69.1 ppm completely indicated the presence of a ribose ring in the structure of NRTO. The chemical shift of the methylene bonded to the single oxygen of the ester group appears at 62.2 ppm. Three distinct peaks at 33.9 ppm, 33.8 ppm, and 33.7 ppm in the three fatty ester chains of NRTO are due to the three CH2 groups in the carbonyls near the ester groups (Figure 8). The rest of the methylene groups in these chains appear between 22.7 ppm and 31.9 ppm, and most of them overlap together because the chemical shifts of these carbons are close to each other. The strong peak at 14.1 ppm is due to the three methyl groups stacking together.

[0082]

[0123] For further confirmation, LC-MS measurements of this compound were performed to identify its molecular weight (Figures 9A and 9B). Selected reaction monitoring (SRM) results show a single peak at 1047.52 m / z (M-Cl) that is in exact agreement with the structure of NRTO. The fragment at 925.70 m / z is due to ribose-trioleate formed by the elimination of a nicotinamide molecule from NRTO. Overall, FTIR, 1 H NMR, 13 Spectral data obtained by C NMR and LC-MS fully confirm the structure of NRTOCl synthesized by the procedure of this example.

[0083]

[0124] Although NRTOCl is a quaternary ammonium salt, it was not soluble in water due to the presence of three oleic acid fatty acid ester groups in the structure. Therefore, for stability testing of NRTOCl, the compound was dispersed in DI water using 1% ethanol as a cosolvent. For this purpose, 15 mg of NRTOCl was dissolved in 0.15 mL of ethanol (1%), then 14.85 mL of DI water was added to the mixture and gently shaken. The NRTOCl concentration in this mixture was 1,000 ppm, the particle size was 192 nm, and the zeta potential was +65 mV. The average size of NRTOCl in this sample was 192 nm, but the TEM images disclosed smaller particles that were about 50 nm and spherical with an elliptical shape and an aspect ratio close to 1 (Figures 10A-C). In the nanoparticles, the NRTOCl structures are stacked on top of each other layer-by-laye. The high positive charge of NRTO makes the nanoparticles stable in the aqueous phase.

[0084]

[0125] After dispersing NRTOCl in DI water, NRCl was dissolved in DI water and the stability of these samples was investigated at 35° C. for 28 days (FIG. 11). The concentration of each sample was 1000 ppm.

[0085]

[0126] Similar to the hydrolysis reaction of NRTO and NR, both of these compounds released nicotinamide (Figure 12). The amount of nicotinamide released in each sample was measured by HPLC to calculate the remaining amount of NRTO and NR in each sample. No release of NR was detected from the NRTO sample during this test. This means that the heavy ester group in the NRTO sample was not hydrolyzed during the stability test. Since this test was performed in DI water under mild conditions, NRTO was hydrolyzed from the N-glycosidic bond.

[0086]

[0127] As shown in Figure 11, the results indicate that NRTO is more stable than NR in DI water at 35 °C, with 53.3% of NRTO remaining after 28 days, while 0.6% of NR remains. The trifunctionalization of NR with long-chain ester groups increases the hydrophobicity of this cation, resulting in a decrease in the accessibility of water to the N-glycosidic bond. After approximately 42% of NRTO is hydrolyzed, the slope of the hydrolysis profile decreases. After 28 days, 53.3% of NRTO remains.

[0087]

[0128] As shown in Figure 12, ribose trioleate was formed during the hydrolysis process to the same extent as hydrolyzed NRTO. Ribose trioleate is structurally more hydrophobic than NRTO. As the hydrolysis process of dispersed NRTO particles occurs from the outer layers, these layers are gradually converted to ribose trioleate, which acts as a superhydrophobic shell to minimize water penetration into the inner layers (Figure 13).

[0088]

[0129] Since NRTOCl is a very hydrophobic compound, the stability of NRTOCl was studied as a function of time using an emulsion system to assess its stability in the oil phase. For this purpose, a 15% w / w solution of NRTOCl in canola oil was used to prepare various emulsions at room temperature containing sodium caseinate, Tween 80, and lecithin as emulsifiers (2 wt.%).

[0089]

[0130] When Na-caseinate was used as the emulsifier, the negatively charged caseinate anion and the positively charged NRTO immediately aggregated in DI water, so the aqueous phase had to be a solution of NaCl (0.1 wt%), CaCl2 (0.2 wt%), and KCl (0.3 wt%). The total oil phase in these emulsions was 3.2 wt%, and the concentration of NRTOCl in the total volume (15 mL) of each emulsion was 4.4 mM. In the emulsions using Na-caseinate as the emulsifier, NaN3 (0.01 wt%) was added to prevent bacterial growth. When the NRTOCl emulsion was made with 2 wt% Na-caseinate, the average size and zeta potential in this emulsion (Cas emulsion) were 1020 nm and -14.4 mV, respectively. These results confirmed that the NRTO droplets in the oil phase were completely surrounded by caseinate anions, as the positive charge of NRTO on the droplet surface became negative.

[0090]

[0131] Using 2 wt% sodium caseinate and 2 wt% lecithin simultaneously as emulsifiers, the average size and zeta potential of the NRTO emulsion (Cas-Lac emulsion) were 1012 nm and -13.3 mV. NRTO was also prepared in an oil-in-water emulsion of canola oil using 2 wt% Tween 80 as an emulsifier in DI water (Tween emulsion). The corresponding average size and zeta potential of this emulsion were 531 nm and +49.2 mV, respectively. As expected from the use of Tween 80 as a neutral emulsifier, the positive charge of the NRTO droplets remained largely intact in the emulsion.

[0091]

[0132] After making the NRTOCl emulsions, NRCl was also tried in a canola oil-in-water emulsion using 3.2% canola oil as a control. The aqueous phase was a solution of Na caseinate (2 wt%), NaCl (0.1 wt%), CaCl2 (0.2 wt%), KCl (0.3 wt%), and NaN3 (0.01 wt%). The concentration of NRCl in the total volume of this emulsion (15 mL) was 4.4 mM. Additionally, as another control, a 15 mL solution of NRCl in DI water was prepared at 4.4 mM. After preparing the triplicate NRTOCl emulsions and the two control samples, the hydrolytic stability of each sample was tested at 35°C for 26 days (Figure 14). The nicotinamide released from each sample was identified and the degradation rate was measured. After 26 days, the remaining amounts of NRTO in the Cas emulsion, Cas-Lec emulsion, and Tween emulsion were 93.7, 90.3, and 80.0%, respectively, whereas the remaining amounts in the NR emulsion and NR in DI water were 0.4% and 5.3%, respectively.

[0092]

[0133] The results showed that in all emulsions, the stability of NRTO was much better than that of NR in emulsions and water. The NR stability in emulsions was lower than that in DI water, indicating that NR cannot dissolve in the oil phase and is only present in the aqueous phase. Furthermore, the presence of some anions and nucleophiles in the aqueous phase of the emulsions increases the rate of NR hydrolysis. Cas and Cas-Lec emulsions showed better results in terms of stability compared to Tween emulsions, and in these samples, more than 90% of NRTO remained intact over 26 days. This result means that Na caseinate as an emulsifier may act to stabilize NRTO droplets in the aqueous phase better compared to Tween 80. As already explained, the caseinate anions can completely surround the surface of the NRTO droplets and neutralize the positive charge of NRTO on the outer surface of the droplets. This effect may result in a tendency for the lone pair of water to interphase NRTO to decrease. Another factor that may affect the stability of NRTO in these emulsions is the size of the droplets. In the Cas and Cas-Lec emulsions, the average size of the droplets is almost equal, about twice the average size of the droplets in the Tween emulsion. This result means that the surface area of ​​the droplets in the Cas and Cas-Lec emulsions was lower than that in the Tween emulsion. Therefore, the accessibility of water to the NRTO droplets in the Cas and Cas-Lec emulsions is lower than that in the Tween emulsion, so the rate of NRTO hydrolysis in these emulsions is lower than that in the Tween emulsion.

[0093]

[0134] Since NRTO in Cas and Cas-Lec emulsions showed better results for stability at 35 °C, the room temperature stability of NRTO in these emulsions was tested for a longer period (Figure 15). After 42 days, the remaining amount of NRTO was 95.0% in Cas emulsions and 93.7% in Cas-Lec emulsions. However, 52.0% of NR remained intact in water at room temperature during this period of stability testing. This result means that the hydrolysis rate of NRTO in the emulsions is negligible. During this period, these emulsions were stable and there was no visible phase separation. A slight increase in the average size of the NRTO droplets occurred in these emulsions: in Cas emulsions, the average size increased from 1020 nm to 1281 nm, and in Cas-Lec emulsions, this parameter increased from 1012 nm to 1106 nm. During all NRTO stability conditions, no NR was measured to be liberated from NRTO, confirming hydrolysis of the ester functionality. The overall stability results show that NRTO in canola oil-in-water emulsions is much more stable than NRTO dispersed in water and NR dispersed in water.

[0094]

[0135] After testing the stability of NRTOCl under different conditions, the compound was tested for digestibility to produce NR in simulated intestinal fluids. Porcine pancreatin, bovine bile, and a buffer solution around pH 7 were used for the enzymatic digestion. 29 Lipase enzymes in porcine pancreatin can hydrolyze fatty ester groups to produce NR, NRDO (nicotinamide riboside dioleate), NRMO (nicotinamide riboside monooleate), and oleic acid as the main products of digestion (Figure 16). Furthermore, nicotinamide (NAM) and ribose-trioleate (RTO) may be formed as products of hydrolysis but not digestion of NRTO. With this hypothesis in mind, NRTO digestion was tested in the simulated intestinal phase.

[0095]

[0136] First, following the digestion test, pure NRTOCl (60 mg) was dissolved in 0.3 mL of ethanol, and then this solution was added to 10 mL of buffer solution containing 400 mg of bovine bile to disperse NRTO in bovine bile solution. After that, 0.75 mL of CaCl2 solution (0.3 M) was added to the mixture, and HCl (1 M) was added to adjust the pH to 7.0. Finally, 400 mg of porcine pancreatin dispersed in 4 mL of buffer solution was added to the mixture, and the sample was placed in an incubator at 37°C for 2 hours. The use of 400 mg of bovine bile and 400 mg of porcine pancreatin was the optimal amount for the enzymatic digestion of NRTO. The NR and nicotinamide released from the sample were measured by LC-MS and HPLC analysis. The SRM LC-MS results show a single peak at 255.17 m / z (M-Cl), which is exactly consistent with the structure of NR (Figures 17A and 17B). In the mass spectrum, the fragment with m / z 123.04 is attributed to the nicotinamide molecule formed by the detachment of the ribose molecule from NR. The results showed that after 2 hours of NRTO digestion test, 27.5% of NR was released from this sample. This result means that at least 27.5% of NRTO is completely digested under the reaction conditions.

[0096]

[0137] Although 2.4% nicotinamide was measured as a side reaction in this sample, demonstrating that hydrolysis of N-glycosidic bonds occurred during the NRTO digestion process, the amount of this hydrolysis was low and almost negligible. Other products of NRTO hydrolysis included ribose trioleate, which was formed with the same amount of nicotinamide (2.4%) during the digestion process. This by-product was not dissolved in the aqueous phase and was measured directly. NRMO and NRDO could be other products of NRTO digestion. However, we did not have standards for either NRMO or NRDO, and therefore could not measure the liberation of these compounds. NRMO was detected in the aqueous phase at 519.35 m / z by LC-MS, but could not be measured because standards were not available.

[0097]

[0138] This study aimed to determine the digestibility and bioavailability of NRTO to liberate NR in a simulated intestinal phase. The results showed that 27.5% of NR was liberated, indicating the digestibility of NRTOCl as a new valuable compound.

[0098]

[0139] To develop the objectives of this study, the digestion of NRTO in the oil phase was studied. For this purpose and to increase the solubility of NRTO, MCT oil was used instead of canola oil. First, 60 mg of NRTOCl was dissolved in 150 mg of MCT oil to prepare about 29% (w / w) NRTOCl in MCT oil. The oil phase was then added to 10 mL of buffer containing 400 mg of bovine bile, and the mixture was homogenized at 15,000 rpm for 150 seconds at room temperature. Next, 0.75 mL of CaCl2 solution (0.3 M) was added to the mixture, and the pH was adjusted to 7.0 by adding HCl (1 M). Then, 400 mg of porcine pancreatin dispersed in 5 mL of buffer solution was added to the mixture, and the sample was placed in an incubator at 37°C for 2 hours. NR and nicotinamide released from the sample were measured by LC-MS and HPLC analysis. Compared to the pure digestion of NRTOCl, the liberation of NR from this sample was lower (11.3%). This result was expected because the majority of the oil phase was MCT oil, resulting in reduced accessibility of lipase molecules to NRTO. 3.9% nicotinamide was detected during the digestion of NRTO in MCT oil as a product of N-glycosidic bond hydrolysis. This porcine pancreatin contained multiple enzymes, including trypsin, chymotrypsin, α-amylase, lipase, and colipase. Thus, the porcine pancreatin lipase is not pure enough to have high activity. However, the use of porcine pancreatin and bovine bile at pH 7.0 is one of the best methods to simulate the digestion of lipids in the intestinal phase. 29 All the obtained results established the digestibility of NRTO (in pure form and in MTC oil) to produce NR in the simulated intestinal phase.

[0099]

[0140] 3. Conclusion

[0141] A new hydrophobic NRCl derivative, NRTOCl, was synthesized. NRTOCl was synthesized by reacting NRCl with oleoyl chloride in the presence of pyridine. The pure product was obtained in 64.3% yield. 1 H NMR, 13 The structure of NRTOCl and its purity were also fully confirmed by the results of C NMR, FTIR, and LC-MS. NRTOCl was water insoluble due to the presence of three fatty acid esters in its structure. However, when EtOH was used as a cosolvent (1%), the molecule was dispersed in water as nanoparticles (average 192 nm) with a layer-by-layer structure. The stability of NRCl and NRTOCl in water for 28 days at 35°C was tested, and the results showed that NRTO was more than 88 times more stable than NRCl. In contrast to NRCl, NRTOCl was readily dissolved in canola oil, corn oil, and MCT oil at room temperature. This characteristic of NRTOCl was useful to evaluate its stability in the oil phase by making oil-in-water emulsions of canola oil in the presence of sodium caseinate, lecithin, and Tween 80 as emulsifiers. In canola oil-in-water emulsions, the stability of NRTO was greatly increased by using sodium caseinate (2 wt%) as the edible emulsifier, with 93.7% and 0.4% of NRTO and NR remaining unchanged in this system after 26 days and at 35°C, respectively. These findings demonstrated that NRTO is approximately 213 times more stable than NR in this emulsion system. The stability of NRTO was also tested in canola oil-in-water emulsions at room temperature for 42 days. The stability results confirmed that NRTO underwent negligible hydrolysis (5%) and remained almost unchanged during this period, while NR was 48% hydrolyzed. Finally, digestibility in a simulated intestinal phase was tested to investigate the bioavailability of the compound. The results demonstrated that NRTOCl was digestible and liberated NR in the presence of porcine pancreatin in a simulated intestinal phase. The obtained stability and digestibility results indicate that NRTOCl holds great promise for use as an NR booster in ready-to-drink (RTD) beverages.

[0100]

[0142] Example 2

[0143] General Procedure for the Synthesis of NR-Tributyrate Chloride (NRTBCl)

[0144] In a round-bottom flask in an ice bath, 300 mg of NRCl, 25 mg of 4-dimethylaminopyridine (DMAPY), 1.5 mL of butyric anhydride, and 9 mL of CH3CN were added and stirred under nitrogen atmosphere for 5 h. The progress of the reaction was followed by TLC. Then, the solvent was evaporated by rotary evaporator under reduced pressure, and the excess of butyric anhydride was washed with n-hexane. Finally, the crude product was purified by column chromatography on SiO2. The eluent was a mixture of CH3OH (35%) and EtOAc (65%). The purified NR-tributyrate chloride (NRTBCl) was obtained as a pale yellow viscous liquid in 71% (367 mg).

[0101]

[0145] Figure 18 shows the FT-IR of NR-tributyrate chloride. Figure 19 shows the 1H NMR of NRTBCl in CDCl3. Figure 20 shows the 1H NMR of NRTBCl in CDCl3. 13 C NMR is shown.

[0102]

[0146] Figures 21A and 21B show SRM LC-MS of NRTB. Figure 21A shows SRM LC of NRTB. Figure 21A shows the mass spectrum of NRTB.

[0103]

[0147] Solubility test of NRTBCl in long-chain triglycerides

[0148] To test the solubility of NRTBCl in long-chain triglycerides, we attempted to dissolve this compound in olive oil and corn oil. For this purpose, 15 mg of NRTBCl was added to 15 mL of olive oil and vigorously shaken with a vortex for 10 minutes at room temperature. The results showed that NRTBCl was not soluble in olive oil. This procedure was repeated using corn oil instead of olive oil. The results obtained revealed that NRTB is insoluble in corn oil.

[0104]

[0149] Solubility test of NRTBCl in medium chain triglycerides

[0150] To test the solubility of NRTBCl in medium-chain triglycerides, we attempted to dissolve the compound in coconut oil. For this purpose, 15 mg of NRTBCl was added to 15 mL of coconut oil and vigorously shaken using a vortex for 10 minutes at room temperature. The results showed that NRTBCl did not dissolve in coconut oil.

[0105]

[0151] Solubility test of NRTBCl in water at different pH

[0152] To test the solubility of NRTB in water, three aqueous solutions of NRTBCl were prepared at a concentration of 10,000 ppm at pH 5, pH 7, and pH 9. The results obtained by Zetasizer demonstrated that NR-tributyrate can be completely dissolved in water at various pHs without forming aggregates in the solution (Figures 22A, 22B, and 22C show the size measurements of NR-tributyrate chloride in water).

[0106]

[0153] Preliminary results of NRTBCl stability measurements at 35°C

[0154] The stability test of NRTBCl was carried out by dissolving NRTB in MQ water and keeping it at 35°C for 6 days. The concentration of each sample was 1000 ppm. The HPLC results showed that the peak of the by-product nicotinamide was overlapped with that of NRTBCl. The increase in the peak intensity at 33 min (flow rate: 0.75 mL / min) is because the by-product of the decomposition is nicotinamide (NA), which has a retention time of 33 min (precisely as NRTBCl). As shown in Figure 23, the peak intensity after 6 days was obviously higher than that after 0 h. This means that NRTBCl can be easily hydrolyzed at 35°C.

[0107]

[0155] It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. Accordingly, such changes and modifications are intended to be encompassed by the appended claims.

[0108]

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Claims

1. Nicotinamide riboside trioleate chloride (NRTOCl).

2. 1. A composition comprising nicotinamide riboside trioleate chloride (NRTOCl) and, optionally, at least one of a protein, a lipid, a carbohydrate, a vitamin, or a mineral.

3. The composition of claim 2 , wherein the composition is formulated for oral administration.

4. The composition of claim 3, wherein the composition is a beverage or a powder formulated to be reconstituted with a diluent to form a reconstituted beverage.

5. The composition described in claim 3, wherein the composition is a ready-to-drink (RTD) beverage sealed in a container.

6. 1. A unit dosage form of a composition comprising nicotinamide riboside trioleate chloride (NRTOCl), said unit dosage form comprising a therapeutically or prophylactically effective amount of NRTOCl for an individual to whom said unit dosage form is administered.

7. 1. A method of making a composition, said method comprising adding nicotinamide riboside trioleate chloride (NRTOCl) to at least one other ingredient.

8. 8. The method of claim 7, wherein the composition is formulated for oral administration and the at least one other ingredient is edible.

9. 1. A composition for promoting an increase in intracellular levels of nicotinamide adenine dinucleotide (NAD+) in cells and tissues, the composition comprising nicotinamide riboside trioleate chloride (NRTOCl), the composition being for administration to a subject.

10. The composition described in claim 9, wherein the subject is a middle-aged or elderly person or an elderly human.

11. (i) inflammation; (j) cancer; (k) eye disorders; and (l) hot flashes. A composition for reducing at least one of DNA damage or mitochondrial damage and / or treating or preventing at least one condition selected from the group consisting of: (a) a neurodegenerative condition; (b) overweight or obesity; (c) cardiovascular disease; (d) one or more of diabetes, hyperinsulinemia, insulin resistance, or insulin insensitivity; (d) muscle degeneration; (e) an age-related disease or disorder; (f) a viral infection; (g) stress; (h) a blood clotting disorder; (i) inflammation; (j) cancer; (k) an eye disorder; and (l) hot flashes, the composition comprising nicotinamide riboside trioleate chloride (NRTOCl), and the composition is for administration to a subject in need or at risk.

12. 1. A composition for reducing weight and / or preventing weight gain in a subject, said composition comprising nicotinamide riboside trioleate chloride (NRTOCl), said composition being for administration to a subject in need or at risk.

13. 1. A composition for treating or preventing drug toxicity and / or adverse drug reactions, said composition comprising nicotinamide riboside trioleate chloride (NRTOCl) and for administration to a subject in need or at risk.

14. The composition of any one of claims 9 to 13, wherein the composition is an oil-in-water emulsion comprising an oil phase in which at least a portion of the NRTOCl is dispersed.

15. The composition described in Claim 14, wherein an emulsifier selected from sodium caseinate and lecithin is further dispersed in the oil phase.

16. 15. The composition of claim 14, wherein the oil phase comprises at least one of canola oil, corn oil, or medium chain triglyceride (MCT) oil in which at least a portion of the NRTOCl is dispersed.

17. The composition of any one of claims 9 to 13, wherein the composition is administered to the subject daily for at least one week.

18. 14. The composition of any one of claims 9 to 13, wherein the subject is selected from the group consisting of a human infant, a human child, a human adolescent, a human adult, and an elderly human, and an animal.

19. The composition according to any one of claims 9 to 13, wherein the composition is for oral administration.

20. The composition of claim 19, wherein the composition is a beverage or a powder formulated to be reconstituted with a diluent to form a reconstituted beverage prior to administration.

21. The composition of claim 19, wherein the composition is a ready-to-drink (RTD) beverage sealed in a container that is opened prior to administration.

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