Compositions and methods for producing intracellular nicotinamide adenine dinucleotide (NAD+) using trigonelline for treating or preventing physiological disorders or conditions

Abstract

The composition essentially comprises or comprises trigonelline. The composition can be used in food or beverage applications, pharmaceutical preparations, or dietary supplements. The composition can be administered to a mammal to treat or prevent a mitochondrial-related disease or a condition associated with altered mitochondrial function in an individual in need thereof or at risk. The mitochondrial-related disease or condition is selected from the group consisting of adverse effects of aging, stress (e.g., oxidative stress), obesity, overweight, reduced metabolic rate, metabolic syndrome, diabetes mellitus, diabetic complications, hyperlipidemia, neurodegenerative disease, cognitive impairment, stress-induced or stress-related cognitive dysfunction, mood disorders, anxiety disorders, age-related neuronal cell death or dysfunction, chronic kidney disease, renal failure, trauma, infection, cancer, hearing loss, macular degeneration, myopathy and dystrophy, and combinations thereof.

Description

[Technical Field]

[0001] [Background technology]

[0001] The present disclosure generally relates to the use of trigonelline to inhibit intracellular NAD + Intracellular NAD+ levels in cells and tissues can be increased to improve cell and tissue survival and / or overall cell and tissue health.

[0002] Nicotinic acid and nicotinamide are compounds derived from nicotinamide adenine dinucleotide (NAD + ) is a vitamin form of tryptophan. Eukaryotes convert tryptophan to NAD via the kynurenine pathway. + Niacin can be synthesized de novo, and niacin supplementation prevents pellagra, which can occur in populations consuming a tryptophan-deficient diet. Nicotinic acid is phosphoribosylated to nicotinic acid mononucleotide (NaMN), which is then adenylated to form nicotinic acid adenine dinucleotide (NaAD), which is then amidated to form NAD. + Generate.

[0003]

[0003] NAD + NAD is an enzyme cofactor essential for the function of several enzymes involved in reduction-oxidation reactions and energy metabolism. + NAD functions as an electron carrier in the cellular metabolism of amino acids, fatty acids, and carbohydrates. + NAD functions as an activator and substrate for sirtuins, a family of protein deacetylases involved in metabolic function and lifespan extension in lower organisms. + The coenzyme activity of NAD, as well as the tight regulation of its biosynthesis and bioavailability, + is clearly involved in the aging process and has become an important system for monitoring metabolism. [Summary of the Invention]

[0004]

[0004] The present disclosure provides compositions that consist essentially of or comprise trigonelline. In some embodiments, at least a portion of the trigonelline is provided by a plant extract in the composition, such as one or more of coffee extract, hemp extract, pumpkin seed extract, and / or fenugreek seed extract, e.g., a plant extract enriched in trigonelline.

[0005]

[0005] In a preferred embodiment, at least a portion of the trigonelline is provided from a fenugreek extract.

[0006]

[0006] In some embodiments, at least a portion of the trigonelline is provided from an algae source, such as a kelp (Laminariaceae) extract.

[0007] In one embodiment, the composition is selected from the group consisting of a food product, a dietary supplement, an oral nutritional supplement (ONS), a medical food, and combinations thereof.

[0008] In another embodiment, the present disclosure provides a method for treating or preventing (e.g., reducing the incidence and / or severity of) a mitochondrial-associated disease or a condition associated with altered mitochondrial function in an individual in need or at risk. The method comprises: + The method includes orally administering to an individual a composition consisting essentially of or comprising trigonelline in an amount effective to increase the biosynthesis of trigonelline.

[0009]

[0009] The mitochondrial-related disease or condition may be selected from the group consisting of adverse effects of aging, stress (e.g., oxidative stress), obesity, overweight, slowed metabolic rate, metabolic syndrome, diabetes mellitus, diabetic complications, hyperlipidemia, neurodegenerative disease, cognitive impairment, stress-induced or stress-related cognitive dysfunction, mood disorders, anxiety disorders, age-related neuronal cell death or dysfunction, chronic kidney disease, renal failure, trauma, infection, cancer, hearing loss, macular degeneration, myopathy and dystrophies, and combinations thereof.

[0010]

[0010] NAD + Increasing the biosynthesis of NAD can provide one or more benefits to an individual, e.g., a human (e.g., a human undergoing treatment), a pet or horse (e.g., a pet or horse undergoing treatment), or a livestock or poultry (e.g., a livestock or poultry used in agriculture). Preferably, NAD + The biosynthesis of is increased in one or more cells of a mammal, for example, one or more cells that are part of at least one body part selected from the group consisting of the liver, kidney, brain, and skeletal muscle.

[0011] In one embodiment, the composition is administered enterally.

[0012] In one embodiment, the composition is selected from the group consisting of a food product, a dietary supplement, an oral nutritional supplement (ONS), a medical food, and combinations thereof.

[0013] In another embodiment, the present disclosure provides a unit dosage form of a composition consisting essentially of or comprising trigonelline, the unit dosage form containing trigonelline in an amount effective to treat or prevent (e.g., reduce the incidence and / or severity of) a mitochondrial-associated disease or a condition associated with altered mitochondrial function in an individual in need or at risk. The composition can be selected from the group consisting of a food product, a dietary supplement, an oral nutritional supplement (ONS), a medical food, and combinations thereof.

[0014] In another embodiment, the present disclosure provides a method for promoting neurite outgrowth, comprising administering a composition consisting essentially of or comprising trigonelline to one or more cells (e.g., one or more cells that are part of at least one body part selected from the group consisting of the liver, kidney, brain, and skeletal muscle) to promote NADPH expression. +The method includes orally administering to an individual an amount effective to increase the biosynthesis of, for example, an amount effective to treat or prevent (e.g., reduce the incidence and / or severity of) a mitochondrial-associated disease or a condition associated with altered mitochondrial function in an individual in need thereof or at risk thereof.

[0015] An advantage of one or more embodiments provided by the present disclosure is the enhanced effect on oxidative metabolism and the prevention of DNA damage.

[0016] Another advantage of one or more embodiments provided by the present disclosure is that NAD decreases with age. + The goal is to replenish the pool.

[0017] Yet another advantage of one or more embodiments provided by the present disclosure is that they help compensate for the metabolic slowdown that accompanies aging.

[0018] Another advantage of one or more embodiments provided by the present disclosure is that they help increase fatty acid metabolism.

[0019] Yet another advantage of one or more embodiments provided by the present disclosure is that they help the body metabolize fat and increase lean body mass.

[0020] Another advantage of one or more embodiments provided by the present disclosure is that it helps maintain cardiac health.

[0021] Yet another advantage of one or more embodiments provided by the present disclosure is that they help support healthy LDL cholesterol and fatty acid levels in the blood.

[0022] Another advantage of one or more embodiments provided by the present disclosure is treating or preventing neurodegeneration, such as age-related neurodegeneration, and / or promoting neurite outgrowth.

[0023]

[0023] Additional features and advantages are described in, and will be apparent from, the following detailed description and drawings. [Brief explanation of the drawings]

[0024]

[0024] [Figure 1] Enzymatic quantification of NAD+ concentrations in humans and zebrafish upon treatment with trigonelline. Figure 1A shows enzymatic quantification of NAD+ concentrations in human skeletal muscle myotubes (HSMMs) treated with trigonelline at doses of 5 μM, 50 μM, 500 μM, and 1 mM for 6 hours. Figure 1B shows enzymatic quantification of NAD+ concentrations in zebrafish larvae (DPF4) treated with trigonelline at doses of 500 μM and 1 mM for 16 hours. # and * indicate differences from control (one-way ANOVA, p<0.1, p<0.05). Data are presented as mean ± SEM. [Figure 2-1]Liquid chromatography-mass spectrometry measurements of NAD+ concentrations and stable isotope-labeled NAD+ incorporation in myotubes upon treatment with isotope-labeled trigonelline. Figure 2A shows the relative NAD+ concentrations measured by liquid chromatography-mass spectrometry (LC-MS) in human skeletal muscle myotubes (HSMMs) from two different donors after treatment with a 500 μM dose of trigonelline for 6 hours compared to control. Figure 2B shows the fractional labeling of NAD+ (13C-carbonyl) after treatment with 500 μM isotope-labeled trigonelline (13C-carbonyl, CH3) for 6 hours (Σi.mi / (n.Σmi), where i = isotopologue and m = isotopologue abundance). Values ​​are corrected for natural abundance, normalized to maximum incorporation, and expressed as percentages compared to control. Measured by LC-MS. Figure 2C shows the structure of the trigonelline stable isotope tracer (C-carbonyl, CH) used to assess label incorporation into NAD (C-carbonyl). In both structures, the isotopically labeled atom (D corresponds to deuterium or H, and C corresponds to carbon-13) is highlighted. ** and **** indicate differences from the respective controls (unpaired t-test, p<0.01 and p<0.0001, respectively). Data are presented as mean ± SEM (n=3). [Figure 2-2]Liquid chromatography-mass spectrometry measurements of NAD+ concentrations and stable isotope-labeled NAD+ incorporation in myotubes upon treatment with isotope-labeled trigonelline. Figure 2A shows the relative NAD+ concentrations measured by liquid chromatography-mass spectrometry (LC-MS) in human skeletal muscle myotubes (HSMMs) from two different donors after treatment with a 500 μM dose of trigonelline for 6 hours compared to control. Figure 2B shows the fractional labeling of NAD+ (13C-carbonyl) after treatment with 500 μM isotope-labeled trigonelline (13C-carbonyl, CH3) for 6 hours (Σi.mi / (n.Σmi), where i = isotopologue and m = isotopologue abundance). Values ​​are corrected for natural abundance, normalized to maximum incorporation, and expressed as percentages compared to control. Measured by LC-MS. Figure 2C shows the structure of the trigonelline stable isotope tracer (C-carbonyl, CH) used to assess label incorporation into NAD (C-carbonyl). In both structures, the isotopically labeled atom (D corresponds to deuterium or H, and C corresponds to carbon-13) is highlighted. ** and **** indicate differences from the respective controls (unpaired t-test, p<0.01 and p<0.0001, respectively). Data are presented as mean ± SEM (n=3). [Figure 3] Enzymatic quantification of NAD+ uptake in liver and muscle upon trigonelline (Trig) treatment. Enzymatic quantification of NAD+ in mice 120 minutes after receiving 250 mg / kg trigonelline by oral gavage (Figure 3A, Figure 3C) or intraperitoneal administration (Figure 3B, Figure 3D). * indicates difference from control (unpaired t-test, p<0.05). Data are expressed as mean ± SEM. [Figure 4]NAD+ measured in human primary myoblasts after treatment with chemically synthesized trigonelline or trigonelline-rich fenugreek seed extract. Figure 4A shows human skeletal muscle myotubes (HSMMs) treated with different doses of synthetic trigonelline monohydrate for 16 hours and quantification of NAD+. Figure 4B shows human skeletal muscle myotubes (HSMMs) treated with different doses of trigonelline-rich fenugreek seed extract (40.45% trigonelline) for 16 hours and quantification of NAD+ (one-way ANOVA, p<0.05, p<0.01, p<0.001, respectively). Data are expressed as mean ± SD. [Figure 5] Liver NAD+ levels in C57BL / 6JRj mice measured 120 minutes after oral gavage of 300 mg / kg trigonelline chloride (Trig) or an equimolar amount of fenugreek seed extract. *, **, and **** indicate differences from control (one-way ANOVA, p<0.05, p<0.01, and p<0.001, respectively). Data are expressed as mean ± SD. [Figure 6] NAD+ levels in C. elegans total lysates measured in day 1 adult animals and day 8 aged worms treated with 1 mM trigonelline chloride compared to age-matched controls. *, **, **** indicate difference from control (one-way ANOVA, p<0.05, p<0.01, p<0.001, respectively). Data are expressed as mean ± SD. [Figure 7-1]C. elegans survival rate, mean speed, distance, and motility. Figure 7A shows the survival curve of worms treated with 1 mM trigonelline chloride (Trig), which increased lifespan by 21%. Figure 7B shows the mean speed measured in spontaneous mobility assays performed on adult day 1 of worms treated with 1 mM trigonelline chloride compared to controls. Figure 7C shows the distance traveled during spontaneous mobility assays at older ages. Figure 7D shows the percentage of worms that responded to physical stimulation by post-stimulus motility scores assessed in aged worms on days 8 and 11. * and ** indicate differences from the respective controls (Student's test, p<0.05 and p<0.01, respectively). In Figures 7A and 7D, data are presented as mean ± SD. In Figures 7B and 7C, data are presented as mean ± SEM. [Figure 7-2] C. elegans survival rate, mean speed, distance, and motility. Figure 7A shows the survival curve of worms treated with 1 mM trigonelline chloride (Trig), which increased lifespan by 21%. Figure 7B shows the mean speed measured in spontaneous mobility assays performed on adult day 1 of worms treated with 1 mM trigonelline chloride compared to controls. Figure 7C shows the distance traveled during spontaneous mobility assays at older ages. Figure 7D shows the percentage of worms that responded to physical stimulation by post-stimulus motility scores assessed in aged worms on days 8 and 11. * and ** indicate differences from the respective controls (Student's test, p<0.05 and p<0.01, respectively). In Figures 7A and 7D, data are presented as mean ± SD. In Figures 7B and 7C, data are presented as mean ± SEM. [Figure 8] Mitochondrial DNA to nuclear DNA ratio (mt / nDNA) in C. elegans. Figure 8 shows the ratio of mitochondrial-encoded genes (nduo-1) to nuclear-encoded genes (act-1) in aged worms at day 8. * indicates difference from control (Student's test, p<0.05). Data are presented as mean ± SD. DETAILED DESCRIPTION OF THE INVENTION

[0025]

[0046] definition

[0047] 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.

[0026]

[0048] All percentages stated herein are by weight of the total composition unless otherwise specified. As used herein, "about," "approximately," and "substantially" are understood to refer to numbers within a numerical range, for example, within -10% to +10% of the referenced number, preferably within -5% to +5%, more preferably within -1% to +1%, and most preferably within -0.1% to +0.1% of the referenced number.

[0027]

[0049] All numerical ranges herein should be understood to include all integers or fractions within that range. Furthermore, these numerical ranges should be construed to support claims directed to any number or subset of numbers within that range. For example, a disclosure of 1 to 10 should be construed to support ranges of 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0028]

[0050] As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a component" or "the component" includes two or more components.

[0029]

[0051] 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 embodiments "consisting essentially of," as well as embodiments "comprising of," the specified components. Any embodiment disclosed herein can be combined with any other embodiment disclosed herein.

[0030]

[0052] The term "and / or" used in the context of "X and / or Y" should be interpreted as "X" or "Y" or "X and Y". Similarly, "at least one of X or Y" should be interpreted as "X" or "Y" or "X and Y". For example, "at least one of obesity or diabetes" should be interpreted as "obesity" or "diabetes", or "both obesity and diabetes".

[0031]

[0053] As used herein, the terms "examples" and "such as," particularly when followed by a list of terms, are merely exemplary and illustrative and should not be considered exclusive or exhaustive. As used herein, a condition "associated with" or "linked with" another condition means that the conditions occur simultaneously, preferably that the conditions are caused by the same underlying condition, and most preferably that one of the specified conditions is caused by the other specified condition.

[0032]

[0054] The terms "food," "food product," and "food composition" refer to a product or composition intended for consumption by an individual, such as a human, and providing at least one nutrient to such an individual. Food products typically include at least one of protein, lipid, carbohydrate, and optionally one or more vitamins and minerals. The compositions of the present disclosure, including many of the embodiments described herein, can include, consist of, or essentially consist of the elements disclosed herein as well as any additional or optional ingredients, components, or elements described or not described herein that are useful in a diet.

[0033]

[0055] As used herein, the term "isolated" means separated from one or more other compounds or components with which the compound may otherwise be found, e.g., in nature. Preferably, for example, "isolated" means that the specified compound is separated from at least a portion of the cellular material with which it is typically found in nature. In one embodiment, an isolated compound is free of any other compounds.

[0034]

[0056] "Prevention" includes reducing the risk, incidence, and / or severity of a condition or disorder. The terms "treatment," "treat," and "to alleviate" include both prophylactic or preventive treatment (which prevent and / or delay the onset of the targeted condition or disorder) and curative, therapeutic, or disease-modifying treatment, including therapeutic measures that cure, delay, attenuate the symptoms, and / or halt the progression of a diagnosed condition or disease; and include treatment of patients who are ill or diagnosed as suffering from a disease or medical condition, as well as treatment of patients at risk of or suspected of having the disease. The term does not necessarily imply that a subject is treated until cured. The terms "treatment" and "treating" also refer to maintaining and / or promoting the health of individuals who are not afflicted with the disease but who may be susceptible to an ill health condition. The terms "treatment / therapy," "treat / treating," and "alleviating" are also intended to include synergistic or otherwise potentiating action of one or more primary preventative or therapeutic measures. The terms "treatment / therapy," "treat / treating," and "alleviating" are further intended to include dietary management of a disease or condition, or dietary management for the prophylaxis or prevention of a disease or condition. Treatment may be patient-related or physician-related.

[0035]

[0057] As used herein, the term "unit dosage form" refers to a physically discrete unit suitable as a dosage unit for human and animal subjects, each unit containing a predetermined amount of a composition disclosed herein, in an amount sufficient to produce a desired effect, together with a pharmaceutically acceptable diluent, carrier, or vehicle. 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.

[0036]

[0058] As used herein, an "effective amount" is an amount that prevents a deficiency, treats a disease or medical condition in an individual, or more generally, alleviates symptoms, manages the progression of a disease, or provides a nutritional, physiological, or medical benefit to an individual. The relative terms "improved," "increased," "promoted," and the like refer to the effect of a composition disclosed herein, i.e., a composition comprising trigonelline, compared to an identical composition but without trigonelline. As used herein, "promoting" refers to enhancing or inducing compared to a value prior to administration of a composition disclosed herein.

[0037]

[0059] A "subject" or "individual" is a mammal, preferably a human. The term "elderly," in reference 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 older, preferably greater than 50 years, and more preferably greater than 55 years, and includes elderly individuals.

[0038]

[0060] "Overweight" is defined in humans as a body mass index (BMI) of 25-30 kg / m 2 "Obesity" is defined in humans as a BMI of at least 30 kg / m 2 , e.g., 30 to 39.9 kg / m 2 "Weight loss" is defined as a loss of total body weight. Weight loss can refer to a loss of total body weight to improve one or more of, for example, health, fitness, or appearance.

[0039]

[0061] "Diabetes" includes both types I and II of the disease. Non-limiting examples of risk factors for diabetes include a waistline greater than 40 inches for men or 35 inches for women, blood pressure equal to or 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.

[0040]

[0062] As used herein, the term "metabolic syndrome" refers to a combination of medical abnormalities that, when occurring together, increase the risk of developing cardiovascular disease and diabetes. Metabolic syndrome affects one in five people in the United States, with prevalence increasing with age. Some studies estimate the prevalence in the United States to be 25% of the population. According to the International Diabetes Foundation's consensus worldwide definition (2006), metabolic syndrome is central obesity in combination with any two of the following:

[0063] Elevated triglycerides: greater than 150 mg / dL (1.7 mmol / L) or treatment directed at this lipid abnormality;

[0064] Lowering HDL cholesterol: less than 40 mg / dL (1.03 mmol / L) in men and less than 50 mg / dL (1.29 mmol / L) in women, or treatment directed at this lipid abnormality;

[0065] Elevated blood pressure: systolic BP > 130 or diastolic BP > 85 mmHg, or treatment of previously diagnosed hypertension; and

[0066] Elevated fasting plasma glucose (FPG) >100 mg / dL (5.6 mmol / L) or previously diagnosed type 2 diabetes.

[0041]

[0067] As used herein, "neurodegenerative disease" or "neurodegenerative disorder" refers to any condition in the central nervous system that results in a gradual loss of functional neurons. In one embodiment, the neurodegenerative disease is associated with age-related cell death. Non-limiting examples of neurodegenerative diseases include mild cognitive impairment, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (also known as ALS and Lou Gehrig's disease), peripheral neuropathy, AIDS dementia complex, adrenoleukodystrophy, Alexander disease, Alpers disease, ataxia-telangiectasia, Batten disease, bovine spongiform encephalopathy (BSE), Canavan disease, corticobasal degeneration, Creutzfeldt-Jakob disease, and dementia with Lewy bodies. Neurodegenerative diseases include, but are not limited to, fatal familial insomnia, frontotemporal lobar degeneration, Kennedy's disease, Krabbe's disease, Lyme disease, Machado-Joseph's disease, multiple sclerosis, multiple system atrophy, neuroacanthocytosis, Niemann-Pick disease, Pick's disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum's disease, Sandhoff's disease, diffuse myelinating sclerosis, spinocerebellar ataxia, subacute combined spinal degeneration, tabes dorsalis, Tay-Sachs disease, toxic encephalopathy, transmissible spongiform encephalopathy, and wobbly hedgehog syndrome. The present disclosure is not limited to a particular embodiment of a neurodegenerative disease, and the neurodegenerative disease can be any neurologically related condition known to one of skill in the art.

[0068] As used herein, "cognitive function" refers to any mental process that involves symbolic activity, such as perception, memory, attention, speech comprehension, speech production, reading, imagery creation, learning, and reasoning, preferably at least memory.

[0042]

[0069] Methods for measuring cognitive function are well known and can include, for example, individual tests or battery tests for any aspect of cognitive function. One such test is the Prudhoe Cognitive Function Test by Margallo-Lana et al. (2003) J. Intellect. Disability Res. 47:488-492. Another such test is the Mini Mental State Exam (MMSE), which is designed to assess orientation to time and place, memory, attention and calculation, recall, language use and comprehension, repetition, and complex commands. Folstein et al. (1975) J. Psych. Res. 12:189-198. Such tests can be used to objectively assess cognitive function, allowing changes in cognitive function to be measured and compared, for example, in response to treatment using the methods disclosed herein.

[0043]

[0070] As used herein, "cognitive disorder" refers to any condition that impairs cognitive function. Non-limiting examples of cognitive disorders include delirium, dementia, learning disabilities, attention deficit disorder (ADD), and attention deficit hyperactivity disorder (ADHD).

[0044]

[0071] As used herein, "neurite" refers to any projection from the cell body of a neuron, such as an axon or dendrite. Because it can be difficult to distinguish between axons and dendrites until differentiation is complete, the term is frequently used in reference to immature or developing neurons, particularly cells in culture. Neurites are often packed with microtubule bundles, and their growth is stimulated by nerve growth factor (NGF), as well as tau protein, microtubule-associated protein 1 (MAP1), and microtubule-associated protein 2 (MAP2). The neural cell adhesion molecule N-CAM simultaneously binds to another N-CAM and the fibroblast growth factor receptor, stimulating the tyrosine kinase activity of the receptor and inducing neurite outgrowth.

[0045]

[0072] As used herein, "trigonelline" refers to any compound containing 1-methylpyridin-1-ium-3-carboxylate, including, for example, any salt thereof (e.g., chloride or iodide salt) and / or forms in which the ring may be reduced.

[0046]

[0073] In some embodiments, trigonelline can be represented by the structure of Formula 1 and can be formed into a salt with an anion (X-), such as a halogen (e.g., iodide or chloride). The structure of Formula 1 is also known as 3-carboxy-1-methylpyridinium, N-methylnicotinic acid, 1-methylpyridine-3-carboxylic acid, 1-methylpyridin-1-ium-3-carboxylic acid, pyridinium 3-carboxy-1-methyl-hydroxide inner salt (8CI), 1-methylnicotinic acid, and 3-carboxy-1-methylpyridinium.

[0047] [ka]

[0048]

[0074] In some embodiments, trigonelline is represented by its inner salt structure of Formula 2. The structure of Formula 2 also includes caffearine, ginesin, N-methylnicotinate, trigenolin, coffearine, trigonelline, coffearin, betaine nicotinate, nicotinic acid betaine, 1-methylpyridinium-3-carboxylate, nicotinic acid N-methylbetaine, 1-methylpyridinio-3-carboxylate, 1-methyl-3-pyridinium carboxylate, N-methylnicotinic acid, trigenelin, caffearin, 3-carboxy-1-methylpyridinium hydroxide inner salt, N'-methylnicotinate, 1-methylpyridin-1-ium-3-carboxylate, 3-carboxy-1-methylpyridinium hydroxide Also known as pyridinium 3-carboxy-1-methyl-hydroxide inner salt, pyridinium 3-carboxy-1-methyl-hydroxide inner salt, 1-methylpyridine-3-carboxylic acid, 1-methylpyridin-1-ium-3-carboxylic acid, 1-methylnicotinate, trigonelline (S), N-methyl-nicotinate, pyridine-3-carboxy-1-methyl-hydroxide inner salt (8CI), N'-methylnicotinic acid, N-methylnicotinic acid betaine, N-methylbetaine nicotinate, 1-methyl-nicotinic acid anion, pyridinium 3-carboxy-1-methyl-hydroxide inner salt, 1-methyl-5-(oxylatocarbonyl)pyridinium-3-ide, pyridinium 3-carboxy-1-methyl-hydroxide inner salt, 3-carboxy-1-methyl-pyridinium hydroxide inner salt.

[0049] [ka]

[0050]

[0075] In some embodiments, "trigonelline" can optionally include its metabolites and thermal decomposition products, such as nicotinamide, nicotinamide riboside, 1-methylnicotinamide, 1-methyl-2-pyridone-5-carboxamide (Me2PY), 1-methyl-4-pyridone-5-carboxamide (Me4PY), and alkyl-pyridiniums such as 1-methyl-pyridinium (NMP) and 1,4-dimethylpyridinium; however, as described herein below, some embodiments exclude one or more of these metabolites and thermal decomposition products of trigonelline.

[0051]

[0076] Embodiment

[0077] The present disclosure provides compositions essentially comprising and consisting of trigonelline. Another aspect of the present disclosure is a unit dosage form of a composition essentially comprising or comprising trigonelline, the unit dosage form containing an amount of trigonelline effective to treat or prevent (e.g., reduce the incidence and / or severity of) a mitochondrial-associated disease or a condition associated with altered mitochondrial function in an individual in need thereof or at risk thereof. The method comprises: + The method includes orally administering to an individual a composition consisting essentially of or comprising trigonelline in an amount effective to increase the biosynthesis of trigonelline.

[0052]

[0078] The mitochondrial-related disease or condition may be selected from the group consisting of adverse effects of aging, stress, obesity, overweight, slowed metabolic rate, metabolic syndrome, diabetes mellitus, diabetic complications, hyperlipidemia, neurodegenerative disease, cognitive impairment, stress-induced or stress-related cognitive dysfunction, mood disorders, anxiety disorders, age-related neuronal cell death or dysfunction, chronic kidney disease, renal failure, trauma, infection, cancer, hearing loss, macular degeneration, myopathy and dystrophies, and combinations thereof.

[0053]

[0079] NAD +Increasing the biosynthesis of NAD can provide one or more benefits to an individual, e.g., a human (e.g., a human undergoing treatment), a pet or horse (e.g., a pet or horse undergoing treatment), or a livestock or poultry (e.g., a livestock or poultry used in agriculture). Preferably, NAD + In some embodiments, the composition is administered to an elderly adult or an aged individual.

[0054]

[0080] The composition can contain a pharmacologically effective amount of trigonelline in a pharmaceutically suitable carrier. In the aqueous liquid composition, the trigonelline concentration is preferably in the range of about 0.05% to about 4% by weight, about 0.5% to about 2% by weight, or about 1.0% to about 1.5% by weight of the aqueous liquid composition.

[0055]

[0081] In a specific embodiment, the method is a treatment to increase plasma trigonelline to a level in the range of, for example, 50 to 6000 nmol, preferably 100 to 6000 nmol per L of plasma. The method may comprise administering trigonelline daily in a weight range of 0.05 mg to 1 g, preferably 1 mg to 200 mg, more preferably 5 mg to 150 mg, even more preferably 10 mg to 120 mg, or most preferably 40 mg to 80 mg per kg of body weight.

[0056]

[0082] For non-human mammals such as rodents, some embodiments include administering an amount of the composition to provide 1.0 mg to 1.0 g of trigonelline per kg of body weight of the non-human mammal, preferably 10 mg to 500 mg of trigonelline, more preferably 25 mg to 400 mg of trigonelline, and most preferably 50 mg to 300 mg of trigonelline per kg of body weight of the non-human mammal.

[0057]

[0083] For humans, some embodiments involve administering an amount of the composition to provide 1.0 mg to 10.0 g of trigonelline per kg of human body weight, preferably 10 mg to 5.0 g of trigonelline, more preferably 50 mg to 2.0 g of trigonelline, and most preferably 100 mg to 1.0 g of trigonelline per kg of human body weight.

[0058]

[0084] In some embodiments, at least a portion of the trigonelline is isolated. Additionally or alternatively, at least a portion of the trigonelline can be chemically synthesized.

[0059]

[0085] In one embodiment, the composition comprises chemically synthesized trigonelline that is at least about 90% trigonelline, preferably at least about 98% trigonelline.

[0060]

[0086] In a preferred embodiment, at least a portion of the trigonelline is provided by a plant or algae extract, such as an extract from one or more of coffee beans (e.g., green coffee bean extract), radish, fenugreek seeds, peas, hemp seeds, oats, potatoes, dahlias, perilla species (Stachys), oleander species (Strophanthus), Laminariaceae (particularly the genera Laminaria and Saccharina), sea palm (Postelsia palmaeformis), false kelp (Pseudochrada nagaii), false kelp (Akkesiphycus), or Dichapetalum cymosum. The plant extract is preferably enriched in trigonelline. That is, the starting plant material contains one or more other compounds in addition to trigonelline, and the enriched plant material has a higher ratio of trigonelline to at least one of the one or more other compounds than the ratio in the starting plant material.

[0061]

[0087] Thus, some embodiments of the composition include a plant source and / or a concentrated plant source that provides at least a portion of the trigonelline in the composition.

[0062]

[0088] In a preferred embodiment, the composition comprises a concentrated fenugreek extract providing at least about 25-50% trigonelline in the composition, and in a more preferred embodiment, the composition comprises a concentrated fenugreek extract providing at least about 28-40% trigonelline.

[0063]

[0089] As used herein, a "composition essentially comprising trigonelline" refers to a composition that contains trigonelline and is substantially free of, or completely free of, any additional compounds other than trigonelline that affect NAD production. In certain non-limiting embodiments, the composition consists of trigonelline and one or more excipients.

[0064]

[0090] In some embodiments, a composition essentially comprising trigonelline is optionally substantially free of, or completely free of, one or more of: other NAD+ precursors, e.g., trigonelline derivatives; metabolites and thermal degradation products of trigonelline, e.g., nicotinamide, nicotinamide riboside, 1-methylnicotinamide, 1-methyl-2-pyridone-5-carboxamide (Me2PY), 1-methyl-4-pyridone-5-carboxamide (Me4PY), and alkyl-pyridiniums such as 1-methyl-pyridinium and 1,4-dimethylpyridinium; nicotinic acid (“niacin”); or L-tryptophan.

[0065]

[0091] In some embodiments, a composition essentially comprising trigonelline optionally is substantially free of, or completely free of, glycine; functional derivatives of glycine; N-acetylcysteine; or functional derivatives of N-acetylcysteine.

[0066]

[0092] In some embodiments, a composition essentially comprising trigonelline optionally is substantially free of, or completely free of, one or more of: chlorogenic acid; anthocyanins; 25-hydroxyvitamin D3; poly(ADP-ribose) polymerase (PARP-1) inhibitor compounds; pipecolic acid; myo-inositol; piperidine-2-carboxylic acid; tartaric acid; mannitol; renieraten; adenine; uronic acid (UA); adenine; uracil; friideline; nicotinamide riboside; or α-amyrin.

[0067]

[0093] In some embodiments, the composition essentially comprising trigonelline optionally contains ketones and ketone precursors, such as medium-chain triglycerides (MCT); MCT derivatives; ketone esters, such as monoesters (e.g., (R)-3-hydroxybutyl-(R)-3-hydroxybutyrate) and acetoacetate diesters (e.g., R,S-1,3-butanediol acetoacetate diester); ketone salts; BHB (β-hydroxybutyrate) and salts thereof, such as sodium salts, magnesium salts, potassium salts, calcium salts, and combinations thereof; D-BHB and salts thereof, such as sodium salts, magnesium salts, potassium salts, calcium salts, and combinations thereof; β-hydroxypentanoate and salts thereof, such as sodium salts, magnesium salts, potassium salts, calcium salts, and combinations thereof; The MCT is substantially free of or completely free of: sodium salts, magnesium salts, potassium salts, calcium salts, and combinations thereof; D-β-hydroxypentanoate and its salts, such as sodium salts, magnesium salts, potassium salts, calcium salts, and combinations thereof; β-ketopentanoate and its salts, such as sodium salts, magnesium salts, potassium salts, calcium salts, and combinations thereof; hexanoylethyl β-hydroxybutyrate; octanoylethyl β-hydroxybutyrate; hexanoylhexyl β-hydroxybutyrate; acetoacetate (AcA) and its salts, such as sodium salts, magnesium salts, potassium salts, calcium salts, and combinations thereof; and mixtures thereof. MCT contains three fatty acid moieties, each fatty acid moiety independently having 6 to 12, 6 to 11, 6 to 10, 7 to 12, 7 to 11, 7 to 10, 8 to 12, 8 to 11, or 8 to 10 carbon atoms.

[0068]

[0094] In some embodiments, the composition consisting essentially of trigonelline optionally includes 4-hydroxyisoleucine; acetylcholine; 25-α-spirosta-3,5-diene; 3,4,7-trimethylcoumarin; 3-hydroxy-4,5-dimethyl-2-furanone; 4-hydroxyisoleucine lactone; 4-methyl-7-acetoxycoumarin; 7-acetoxy-4-methylcoumarin; α-galactosidase; α-mannosidase; aluminum; arabinose; arachidic acid; behenic acid; β-carotene; β-mannan; β-sitosterol; biotin; calpain; choline; coumarin; cyanocobalamin; d-mannose; digalactosylmyo-inositol; dihydroactinidiolide, dihydrobenzofuran; diosin; diosgenin; elemene; endo-β-mannanase; fenugreek; folacin; galactinol; galactomannan; gentianin; gytoge. Nin; Glaecnin-h; Glaecnin-n; Homoorientin; Isovitexin; Kaempferol; Lecithin; Lignin; Luteolin; Muurolein; Myo-inositol; Neotigogenin; Niacin; Nicotinic acid; Oleic acid; Orientin; Orientin-arabinoside; p-Coumaric acid; Palmitic acid; Protopectin; Pyridoxine; Quercetin; Raffinose; Riboflavin; Rutin; Saponin; Selenin; Stachyose; Stearic acid Substantially free of, or completely free of, one or more of: phosphate; thiamine; threonine; tigogenin; trigofenoside; trigoforin; trigoneroside; trilin; verbascose; vicenin-1; vicenin-2; vitexin; vitexin-2'-op-coumarate; vitexin-7-glucoside; xanthophylls; yamogenin; yamogenin-3,26-biglycoside; and yamogenin-tetroside.

[0069]

[0095] As used herein, "substantially free" means that any other compounds present in the composition are less than 1.0% by weight relative to the amount of trigonelline, preferably less than 0.1% by weight relative to the amount of trigonelline, more preferably less than 0.01% by weight relative to the amount of trigonelline, and most preferably less than 0.001% by weight relative to the amount of trigonelline.

[0070]

[0096] Yet another aspect of the present disclosure is a method of treating or preventing (e.g., reducing the incidence and / or severity of) a mitochondrial-associated disease or a condition associated with altered mitochondrial function in an individual in need thereof or at risk thereof. + The method includes orally administering to an individual a composition consisting essentially of or comprising trigonelline in an amount effective to increase the biosynthesis of trigonelline.

[0071]

[0097] The mitochondrial-related disease or condition may be selected from the group consisting of adverse effects of aging, stress (e.g., oxidative stress), obesity, overweight, slowed metabolic rate, metabolic syndrome, diabetes mellitus, diabetic complications, hyperlipidemia, neurodegenerative disease, cognitive impairment, stress-induced or stress-related cognitive dysfunction, mood disorders, anxiety disorders, age-related neuronal cell death or dysfunction, chronic kidney disease, renal failure, trauma, infection, cancer, hearing loss, macular degeneration, myopathy and dystrophies, and combinations thereof.

[0072]

[0098] For example, aging is associated with oxidative stress, declining glutathione levels, and a decrease in the redox ratio NAD + The compositions disclosed herein can treat or prevent these adverse effects of aging. For example, trigonelline can reduce NAD + and the inventors found that increased NAD + However, we believe that this can lead to increased glutathione production through redox recycling.

[0073]

[0099] As another example, depression is associated with low glutathione, and anxiety is associated with oxidative stress. The compositions disclosed herein can treat or prevent these conditions.

[0074]

[0100] These methods may essentially comprise administering a composition that essentially comprises or comprises trigonelline. As used herein, "a method that essentially comprises or comprises administering a composition that comprises trigonelline" refers to a method that essentially comprises administering a composition that comprises NAD + This means that any additional compound other than trigonelline that affects the production of is not administered within 1 hour of administration of trigonelline, preferably not within 2 hours of administration of trigonelline, more preferably not within 3 hours of administration of trigonelline, and most preferably not on the same day as administration of trigonelline. Non-limiting examples of compounds that may optionally be excluded from the method include those described above with respect to exclusion from the composition itself.

[0075]

[0101] Another aspect of the present disclosure is a unit dosage form of a composition consisting essentially of or comprising trigonelline, the composition being effective in treating at least one of the following conditions: diabetes (type I or type II), diabetic complications (e.g., diabetic dyslipidemia and / or diabetic microvascular complications, such as nephropathy, retinopathy, and / or neuropathy), insulin resistance, metabolic syndrome, dyslipidemia, overweight, obesity, overweight, elevated cholesterol levels, elevated triglyceride levels, elevated fatty acid levels, fatty liver disease (e.g., non-alcoholic fatty liver disease with or without inflammation), cardiovascular disease (e.g., heart failure and / or cardiac systolic dysfunction), neurodegenerative diseases (e.g., due to aging), depression, anxiety, loss of motivation / decreased motivation, cognitive impairment, myopathy such as statin-induced myopathy, non-alcoholic steatohepatitis, tinnitus, dizziness, alcohol hangover, hearing loss, osteoporosis, high blood pressure, atherosclerosis / coronary artery disease, myocardial damage after stress (e.g., due to burns or trauma), traumatic brain injury (including concussion), cystic fibrosis, inflammation, cancer, HIV infection, stroke, migraine, and cerebral ischemia.

[0076]

[0102] For example, aging is associated with oxidative stress, declining glutathione levels, and a decrease in the redox ratio NAD +The compositions disclosed herein can treat or prevent these adverse effects of aging. For example, trigonelline can reduce NAD + and the inventors found that increased NAD + However, we believe that this can lead to increased glutathione production through redox recycling.

[0077]

[0103] As another example, depression is associated with low glutathione, and anxiety is associated with oxidative stress. The compositions disclosed herein can treat or prevent these conditions.

[0078]

[0104] Another aspect of the present disclosure is a method for treating at least one of the above conditions, comprising administering a therapeutically effective amount of a composition consisting essentially of or comprising trigonelline to an individual having the condition. Another aspect of the present disclosure is a method for preventing at least one of the above conditions, comprising administering a prophylactically effective amount of a composition consisting essentially of or comprising trigonelline to an individual at risk of at least one of the conditions.

[0079]

[0105] Compositions consisting essentially of or comprising trigonelline can treat or prevent ocular conditions that result directly or indirectly from reduced GSH levels, including reduced levels in the eye lens, which is known to be rich in glutathione. Non-limiting examples of such conditions include cataracts and / or glaucoma, presbyopia (age-related loss of near vision requiring magnifying glasses), and presbycusis (age-related hearing loss requiring hearing aids).

[0080]

[0106] Yet another aspect of the present disclosure is a method for delaying the offset of metabolic decline, reducing oxidative stress, maintaining immune function, and / or maintaining cognitive function in a healthy middle-aged or elderly individual, comprising administering to the healthy middle-aged or elderly individual an effective amount of a composition consisting essentially of or comprising trigonelline.

[0081]

[0107] Yet another aspect of the present disclosure is a unit dosage form of a composition consisting essentially of or comprising trigonelline in an amount effective to promote neurite outgrowth, hi related embodiments, a method of promoting neurite outgrowth in an individual comprises administering to the individual an effective amount of a composition consisting essentially of or comprising trigonelline.

[0082]

[0108] The method of promoting neurite outgrowth can be an in vitro method or an in vivo method. In one embodiment, promoting neurite outgrowth includes inducing neurite outgrowth. For example, neurite outgrowth in a negative control sample may be negligible, while neurite outgrowth in an experimental or treatment sample may be substantial. In one embodiment, promoting neurite outgrowth includes enhancing neurite outgrowth. For example, neurite outgrowth in a negative control sample may be substantial, while neurite outgrowth in an experimental or treatment sample may be statistically significantly greater than the negative control. Of course, in some embodiments, promoting neurite outgrowth can encompass both enhancing and inducing.

[0083]

[0109] Yet another embodiment of the present disclosure is a unit dosage form of a composition essentially consisting of or comprising trigonelline, the composition containing trigonelline in an amount effective for weight management. "Weight management" for adults (e.g., at least 18 years of age) means that the individual has about the same body mass index (BMI) after ingesting the composition for one week, preferably after ingesting the composition for one month, and more preferably after ingesting the composition for one year, compared to the BMI at the time of starting to take the composition. "Weight management" for young individuals means that the BMI is about the same percentile for individuals of the corresponding age after ingesting the composition for one week, preferably after ingesting the composition for one month, and more preferably after ingesting the composition for one year, compared to the BMI percentile at the time of starting to take the composition.

[0084]

[0110] In a related embodiment, a method for weight management in an individual comprises administering to the individual an effective amount of a composition consisting essentially of or comprising trigonelline.

[0085]

[0111] In another aspect, the present disclosure provides a method for improving cognitive function. The method comprises administering to an individual an effective amount of a composition consisting essentially of or comprising trigonelline. The cognitive function can be selected from the group consisting of perception, memory, attention, language comprehension, language expression, reading comprehension, imagery, learning, reasoning, and combinations thereof. In one embodiment, the individual does not have a cognitive impairment, or the individual does have a cognitive impairment. The individual may be elderly and / or may have age-related cognitive decline.

[0086]

[0112] Yet another aspect of the present disclosure is a method for improving one or more of fetal metabolic programming to prevent future development of obesity, overweight, and / or diabetes, maternal and fetal health during gestational diabetes, motor skills and physical function, quality of life, lifespan, memory, cognition, recovery and survival after trauma (e.g., after surgery, after sepsis, after blunt or penetrating trauma from an accident or physical assault), or recovery from trauma and surgery, comprising administering to an individual at risk or in need thereof an effective amount of a composition consisting essentially of or comprising trigonelline.

[0087]

[0113] In some embodiments, trigonelline is administered directly to the infant. Additionally or alternatively, trigonelline can be administered to a pregnant mother to indirectly affect the fetus and / or to a nursing mother to indirectly affect the fetus.

[0088]

[0114] Yet another aspect of the present disclosure is a method for treating or preventing at least one physical condition selected from the group consisting of oxidative stress, conditions associated with oxidative stress (e.g., aging and its effects such as skin aging), decreased glutathione levels, and conditions associated with decreased glutathione levels, or (ii) a method for improving one or more of the following: fetal metabolic programming to prevent future onset of obesity, overweight, prediabetes, and / or diabetes, maternal and fetal health during gestational diabetes, athletic performance and physical function, quality of life, lifespan, memory, cognition, recovery and survival after trauma, or recovery from trauma and surgery. The method comprises administering to an individual at risk or in need thereof an effective amount of a composition consisting essentially of or comprising trigonelline. For example, trigonelline has been shown to inhibit NAD + and the inventors found that increased NAD + However, we believe that this can lead to increased glutathione production through redox recycling.

[0089]

[0115] In biology and psychology, the term "stress" refers to the failure of humans or other animals to respond appropriately to physiological, emotional, or physical threats, whether real or imagined. Psychobiologically, stress can be characterized as a manifestation of oxidative stress, i.e., an imbalance between the production and expression of reactive oxygen species and the ability of the living system to rapidly detoxify reactive intermediates or repair the resulting damage. Disruption of the normal redox state of tissues can cause toxic effects through the production of peroxides and free radicals, which damage all components of the cell, including proteins, lipids, and DNA. Some reactive oxidative species can also act as messengers through a phenomenon called "redox signaling."

[0090]

[0116] In humans, oxidative stress is involved in many diseases, including atherosclerosis, Parkinson's disease, heart failure, myocardial infarction, Alzheimer's disease, schizophrenia, bipolar disorder, fragile X syndrome, and chronic fatigue syndrome.

[0091]

[0117] One source of ROS under normal conditions in humans is the leakage of ROS from mitochondria during oxidative phosphorylation. Other enzymes that can produce superoxide (O2-) include xanthine oxidase, NADPH oxidase, and cytochrome P450. Another potent oxidant, hydrogen peroxide, is produced by a variety of enzymes, including several oxidases. Reactive oxygen species play an important role in cellular signaling processes known as redox signaling. Therefore, a balance between the production and consumption of ROS is necessary to maintain proper cellular homeostasis.

[0092]

[0118] Oxidative stress contributes to tissue damage after ultraviolet irradiation and hyperoxia and is suspected to be important in neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease.

[0093]

[0119] Oxidative stress is also thought to be associated with certain cardiovascular diseases, as oxidation of low-density lipoprotein (LDL) in the vascular endothelium is a precursor to plaque formation. Oxidative stress also plays a role in the ischemic cascade resulting from injury due to oxygen reperfusion after hypoxia, including both stroke and heart attack. Oxidative stress has also been implicated in chronic fatigue syndrome.

[0094]

[0120] Furthermore, the free radical theory of aging suggests that the biological process of aging is caused by increased oxidative stress in elderly individuals. The ability of cells to resist potential damage caused by oxidative stress depends on the balance between the production of oxidant free radicals and the availability of protective antioxidants. Among the various antioxidant defense systems, glutathione (GSH) is the most abundant intracellular component involved in comprehensive antioxidant defense. GSH is a tripeptide synthesized de novo in cells from the precursor amino acids glutamate, cysteine, and glycine in two steps catalyzed by glutamate cysteine ​​ligase (GCL, also known as γ-glutamylcysteine ​​synthetase, EC 6.3.2.2) and γ-L-glutamyl-L-cysteine:glycine ligase (also known as glutathione synthetase, EC 6.3.2.3).

[0095]

[0121] In each of the compositions and methods disclosed herein, the composition is preferably a food product such as a food additive, a food ingredient, a functional food, a dietary supplement, a medical food, a nutraceutical, an oral nutritional supplement (ONS) or a dietary supplement.

[0096]

[0122] The composition may be administered at least one day per week, preferably at least two days per week, more preferably at least three or four days per week (e.g., every other day), and most preferably at least five days per week, six days per week, or seven days per week. The administration period can be at least one week, preferably at least one month, more preferably at least two months, and most preferably at least three months, e.g., at least four months. In some embodiments, administration is at least daily; for example, the subject may receive one or more doses per day, and in one embodiment, multiple doses per day. In some embodiments, administration continues for the remainder of the individual's life. In another embodiment, administration is administered until there are no detectable symptoms of the medical condition. In a specific embodiment, administration is administered until there is a detectable improvement in at least one symptom, and in further cases, is continued to maintain remission.

[0097]

[0123] The compositions disclosed herein may be administered to a subject enterally, for example orally, or parenterally. Non-limiting examples of parenteral administration include intravenous, intramuscular, intraperitoneal, subcutaneous, intra-articular, intrasynovial, intraocular, intrathecal, topical, and inhalation. Non-limiting examples of the composition forms include natural foods, processed foods, natural fruit juices, concentrates and extracts, injections, microcapsules, nanocapsules, liposomes, plasters, inhalation forms, nasal sprays, nasal drops, eye drops, sublingual tablets, and sustained-release formulations.

[0098]

[0124] The compositions disclosed herein can be administered in any of a variety of formulations for therapeutic purposes. More specifically, the pharmaceutical compositions can contain suitable pharmaceutically acceptable carriers or diluents and can be formulated as solid, semi-solid, liquid, or gaseous preparations, such as tablets, capsules, powders, granules, ointments, liquids, suppositories, injections, inhalants, gels, microspheres, and aerosols. Thus, administration of the compositions can be achieved in a variety of ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, and intratracheal administration. The active agent may be systemic after administration, or may be localized by the use of topical administration, intramural administration, or the use of an implant that acts to retain an effective dose at the implantation site.

[0099]

[0125] In pharmaceutical dosage forms, the compounds may be administered as pharmaceutically acceptable salts. They may also be used in appropriate association with other pharmaceutically active compounds. The following methods and additives are merely illustrative and in no way limiting.

[0100]

[0126] In oral formulations, the compounds can be used alone or in combination with suitable additives for producing tablets, powders, granules or capsules, for example, conventional additives such as lactose, mannitol, corn starch or potato starch; binders such as crystalline cellulose, functional cellulose derivatives, gum arabic, corn starch or gelatin; disintegrants such as corn starch, potato starch or sodium carboxymethylcellulose; lubricants such as talc or magnesium stearate; and, if desired, diluents, buffers, wetting agents, preservatives and flavoring substances.

[0101]

[0127] The compounds can be formulated as injectable preparations by dissolving, suspending or emulsifying them in aqueous or non-aqueous solvents, such as vegetable oils or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol, together with conventional additives such as solubilizers, isotonicity adjusting agents, suspending agents, emulsifying agents, stabilizers and preservatives, if desired.

[0102]

[0128] The compounds can be utilized in aerosol formulations to be administered via inhalation. For example, the compounds can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, and nitrogen.

[0103]

[0129] Furthermore, the compound can be prepared as a suppository by mixing with various bases, such as emulsifying bases or water-soluble bases.The compound can be administered rectally by suppository.Suppository can contain a vehicle such as cocoa butter, carbowax and polyethylene glycol, which melts at body temperature but solidifies at room temperature.

[0104]

[0130] Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, may be provided, with each dosage unit, e.g., a teaspoon, tablespoon, tablet, or suppository, containing a predetermined amount of the composition. Similarly, unit dosage forms for injection or intravenous administration may contain the compound in a composition as a solution in sterile water, saline, or another pharmaceutically acceptable carrier, with each dosage unit, e.g., mL or L, containing a predetermined amount of a composition containing one or more of the compounds.

[0105]

[0131] Compositions intended for non-human animals include food compositions, animal treats (e.g., biscuits), and / or dietary supplements that supplement the nutritional needs of animals. The compositions may be dry compositions (e.g., kibble), semi-moist compositions, wet compositions, or any mixture thereof. In one embodiment, the composition is a dietary supplement such as gravy, drinking water, beverage, yogurt, powder, granules, paste, suspension, chew, morsel, treat, snack, pellet, pill, capsule, tablet, or other suitable delivery form. The dietary supplement may contain high concentrations of UFAs and NORCs, as well as B vitamins and antioxidants. This allows such dietary supplements to be administered in small amounts to animals or can be diluted before administration to the animal. The dietary supplement may need to be or can be mixed with water or other diluents before administration to the animal.

[0106]

[0132] A "pet food" or "pet food composition" comprises about 15% to about 50% crude protein. The crude protein material may include vegetable proteins such as soybean meal, soy protein concentrate, corn gluten meal, wheat gluten, cottonseed, and peanut meal, or animal proteins such as casein, albumin, and meat protein. Examples of meat proteins useful herein include pork, lamb, horse, poultry, fish, and mixtures thereof. The composition may further comprise about 5% to about 40% fat. The composition may further comprise a carbohydrate source. The composition may comprise about 15% to about 60% carbohydrate. Examples of such carbohydrates include grains or cereals such as rice, corn, milo, sorghum, alfalfa, barley, soybeans, canola, oats, wheat, and mixtures thereof. The composition may also optionally contain other ingredients such as dried whey and other dairy by-products.

[0107]

[0133] In some embodiments, the ash content of the pet food composition ranges from less than 1% to about 15%, and in one aspect, from about 5% to about 10%.

[0108]

[0134] The moisture content can vary depending on the nature of the pet food composition. In one embodiment, the composition can be a complete and nutritionally balanced pet food. In this embodiment, the pet food can be a "wet food," a "dry food," or a food with an intermediate moisture content. "Wet food" refers to pet food typically sold in cans or foil bags and typically has a moisture content ranging from about 70% to about 90%. "Dry food" refers to pet food of a similar composition to wet food but containing a limited moisture content, typically ranging from about 5% to about 15% or 20%, and thus provided, for example, as small biscuit-like kibble. In one embodiment, the moisture content of the composition is about 5% to about 20%. Dry food products include a variety of foods with varying moisture contents that are relatively shelf-stable and resistant to microbial or fungal spoilage or contamination. Dry food product compositions that are extruded food products, such as pet foods or snack foods for companion animals, are also included.

[0109]

[0135] Example

[0136] The following non-limiting examples present scientific data that develop and support the concept of compositions consisting essentially of or including trigonelline for cellular nutrition.

[0110]

[0137] Example 1

[0138] Enzymatic quantification of NAD+ concentrations in humans and zebrafish after trigonelline treatment.

[0139] Human primary myoblasts were seeded at a density of 3,000 cells per well in 384-well plates in skeletal muscle cell medium (SKM-M, AMSbio). One day later, differentiation was induced by changing the medium for 4 days using differentiation medium (Gibco No. 31330-028). Cells were treated with trigonelline (Sigma #T5509) for 6 hours. NAD was measured using a bioluminescence assay (Promega NAD / NADH-Glo™ #G9071). This is shown in Figure 1A.

[0111]

[0140] Wild-type zebrafish embryos were cultured under standard laboratory conditions at 28°C in 6-well plates for 96 hours after fertilization (n=20–25). Larvae were treated with trigonelline (Sigma #T5509) for 16 hours. NAD was measured using a colorimetric NAD quantification assay (Biovision NAD / NADH Quantitation Colorimetric Kit #k337-100). This is shown in Figure 1B.

[0112]

[0141] Example 2

[0142] Enhancement of human myoblast differentiation by trigonelline.

[0143] Human primary myoblasts from two different donors were seeded in skeletal muscle cell medium (SKM-M, AMSbio) in 6-well plates at a density of 200,000 cells per well. After 1 day, differentiation was induced by changing the medium using differentiation medium (Gibco No. 31330-028) for 4 days. The cells were treated with isotope-labeled trigonelline ( 13 C carbonyl; 3 on methyl 2 H) for 6 hours.

[0113]

[0144] Cell extracts were separated on a Vanquish UHPLC + focusing LC system (Thermo Scientific) using a 150 × 2.1 mm, 5 μm hydrophilic liquid chromatography (HILIC) iHILIC-Fusion (P) column (Hilicon) and a front guard column (iHILIC-fusion (P), Hilicon). Metabolite separation was achieved by applying a linear solvent gradient to the normal phase at a flow rate of 0.25 mL / min and a temperature of 35 °C. Solvent A as the mobile phase was water (pH 9.3) containing 10 mM ammonium acetate and 0.04% (v / v) ammonium hydroxide, and solvent B was acetonitrile.

[0114]

[0145] Eluted metabolites were analyzed using an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) equipped with a heated electrospray ionization (H-ESI) source in positive and negative modes at a resolution of 60,000 at m / z 200. Instrument control and data analysis were performed using Xcalibur (Thermo Scientific).

[0115]

[0146] Figure 2A shows the enhancement of NAD+ levels in human myotubes upon treatment with 500 μM trigonelline. As shown in Figure 2B, trigonelline acts as an NAD+ precursor. In this human cell model, 500 μM labeled trigonelline ( 13 C-carbonyl, C 2 When treated with H3), the distribution of isotopes after 6 hours differs from the control. 13 C atom (trigonelline ( 13 C-carbonyl, C 2 H3)) NAD+(NAD+( 13 There is clear incorporation into the C-carbonyl. 13 When expressed as a percentage of C enrichment, NAD+ [ 13The [C] isotope enrichment is high (45%) compared to NAD+ in baseline conditions. Label incorporation was corrected for natural abundance using AccuCore (Su et al., Anal Chem 2017). The structure of the isotope-labeled tracer, trigonelline ( 13 C-carbonyl, C 2 H3), and the formed isotope-labeled NAD+ ( 13 C-carbonyl) is shown in Figure 2, C.

[0116]

[0147] Example 3

[0148] NAD+ concentrations in liver and muscle after oral or intraperitoneal administration of trigonelline.

[0149] Ten-week-old C57BL / 6JRj male mice were fed a diet (Safe 150) and then administered trigonelline (250 mg / kg, n = 5 / group) by oral gavage or intraperitoneal injection. 120 min after treatment, tissues were harvested and flash-frozen in liquid nitrogen. NAD was measured in the gastrocnemius muscle and liver using a colorimetric NAD quantitation assay (Biovision NAD / NADH Quantitation Colorimetric Kit #k337-100). Figure 3 shows enzymatic quantitation of NAD+ in mice 120 min after receiving 250 mg / kg trigonelline by oral gavage (Figure 3A, Figure 3C) or intraperitoneal administration (Figure 3B, Figure 3D).

[0117]

[0150] Example 4

[0151] NAD measured in human primary myoblasts after treatment with chemically synthesized trigonelline or with a trigonelline-rich fenugreek seed extract + .

[0152] Human primary myoblasts were seeded in 96-well plates at a density of 12,000 cells per well in skeletal muscle cell medium (SKM-M, AMSbio). After 1 day, the medium was changed for 4 days to induce differentiation. Cells were treated with various doses of synthetic trigonelline monohydrate (Figure 4A) or trigonelline-enriched fenugreek seed extract (Figure 4B) containing 40.45% trigonelline for 16 hours. Colorimetric NAD + Quantitative assay (Biovision NAD + / NADH Quantitation Colorimetric Kit #k337-100) + was measured.

[0118]

[0153] This study demonstrated that both chemically synthesized trigonelline and trigonelline derived from fenugreek seed extract significantly increased NAD levels compared to the control. + The results demonstrated that fenugreek seed extract showed a significant increase in trigonelline content, and was more potent than chemically synthesized trigonelline at lower doses.

[0119]

[0154] Example 5

[0155] NAD measured in mouse liver after treatment with chemically synthesized trigonelline or with a trigonelline-rich fenugreek seed extract + .

[0156] Ten-week-old C57BL / 6JRj male mice were orally gavaged with trigonelline (Sigma #T5509) or trigonelline-rich fenugreek seed extract (40.45% trigonelline) (equimolar to trigonelline at 300 mg / kg, n=8 / group). After 120 min of treatment, livers were harvested and flash-frozen in liquid nitrogen. An enzymatic method modified from that by Dall, M. et al. (Mol Cell Endocrinol, 2018.473:pp.245-256) was used to measure hepatic NAD. + was measured.

[0120]

[0157] This study demonstrated that both chemically synthesized trigonelline and trigonelline derived from fenugreek seed extract significantly increased NAD levels in the liver compared to controls. +It was demonstrated that the content of α-glucan was significantly increased.

[0121]

[0158] Example 6

[0159] C. elegans assay to measure viability, speed, motility, and post-stimulation movement.

[0160] Worm lifespan studies were conducted using approximately 100 worms per condition and were manually scored every other day. Trigonelline treatment and experimental measurements began on day 1 of adulthood for wild-type N2 worms in a long-term exposure regimen that continued until the end of the experiment. Figure 7A shows the mean worm survival (days) for control and trigonelline-treated worms. The survival curve for worms treated with 1 mM trigonelline chloride shows a 21% increase in lifespan.

[0122]

[0161] C. elegans motility tests were performed using Movement Tracker software (Mouchiroud, L. et al., Curr Protoc Neurosci 77, 8.37.1-8.37.21 (2016)). Experiments were repeated at least twice. Trigonelline treatment and experimental measurements began on day 1 of adulthood in wild-type N2 worms for the long-term exposure regimen that continued until the end of the experiment.

[0123]

[0162] Figure 7B shows the mean speed of worms treated with 1 mM trigonelline chloride in a spontaneous motility assay performed on day 1 of adulthood, compared with the control. The mean speed of worms treated with 1 mM trigonelline chloride was increased compared with the control.

[0124]

[0163] Figure 7C shows that worms treated with 1 mM trigonelline chloride significantly increased the distance traveled during spontaneous motility assays at late senescent stages compared to controls.

[0125]

[0164] Forty-five to sixty worms per condition were manually scored for motility after poking. Worms that failed to respond to any repeated stimulation were scored as dead. Results represent data obtained from at least two independent experiments. For the long-term exposure regimen, wild-type N2 worms were treated with trigonelline and experimental measurements were initiated on day 1 of adulthood.

[0126]

[0165] Figure 7D shows that post-stimulus locomotor scores assessed on aged worms at 8 and 11 days showed that worms treated with 1 mM trigonelline chloride were more responsive to physical stimuli than controls. * , ** indicates difference from the respective control (Student's test, p<0.05, p<0.01, respectively).

[0127]

[0166] Example 7

[0167] Improved structural integrity of myofibrils and myosin by treatment with trigonelline

[0168] Age-related changes in myosin structure are typically observed in myofibrils and myosin ATPase activity under high salt conditions, and myofibril structure becomes less organized with age.

[0128]

[0169] RW1596 (myo-3p:GFP) worms were harvested on day 1 (young adult) and day 11 (old adult) for muscle integrity assessment. Worms were fixed with tetramisole and analyzed by confocal microscopy to assess muscle fiber morphology as indicated by GFP fluorescence imaging. For the long-term exposure regimen, wild-type N2 worms were treated with 1 mM trigonelline chloride on day 1 of adulthood, and experimental measurements were performed.

[0129]

[0170] By observing the morphological structure of GFP-tagged myosin by fluorescence microscopy, we were able to see improved, more organized myofibrillar structure in 11-day-old trigonelline-treated worms compared to age-matched control worms.

[0130]

[0171] Example 8

[0172] Ratio of mitochondrial DNA to nuclear DNA in control and trigonelline-treated worms.

[0173] Absolute quantification of mtDNA copy number in wild-type N2 worms was performed by real-time PCR. The relative values ​​of nduo-1 and act-1 in each sample were compared to generate a ratio representing the relative levels of mitochondrial DNA per nuclear genome. For each biological data point, the average of at least two technical replicates was used. Each experiment was performed on at least 10 independent biological samples (separate worms). For the long-term exposure regimen, wild-type N2 worms were treated with 1 mM trigonelline chloride on day 1 of adulthood, and experimental measurements were initiated.

[0131]

[0174] Figure 8 shows the ratio of mitochondrial-encoded genes (nduo-1) to nuclear-encoded genes (act-1) in 8-day-old worms. * indicates difference from control (Student's test, p<0.05). Data are expressed as mean ± SD.

[0132]

[0175] In the trigonelline-treated group, mitochondrial expression relative to nuclear expression was higher than in the control group.

[0133]

[0176] 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. It is therefore intended that such changes and modifications be covered by the appended claims.

Claims

1. A composition for increasing NAD+ biosynthesis in one or more cells that are part of at least one body part selected from the group consisting of liver and skeletal muscle, comprising: A composition comprising a fenugreek seed extract containing at least 25% to 50% trigonelline.

2. 10. The composition of claim 1 formulated for enteral administration.

3. 3. The composition of claim 1 or 2, selected from the group consisting of a food product, a dietary supplement, an oral nutritional supplement (ONS), a medical food, and combinations thereof.

4. A unit dosage form of the composition of any one of claims 1 to 3.

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