WO2024200613A1 - Compositions and methods using trigonelline and oleuropein for preventing or treating conditions or disorders in skeletal muscle in a pet animal - Google Patents

Abstract

The present invention relates to compositions and methods for preventing or treating skeletal muscle conditions or diseases. The present invention also relates to compositions and methods that help to increase NAD+ levels in skeletal muscle. Preferably, the present invention relates to compositions and methods that use trigonelline and oleuropein and/or metabolite thereof, for preventing or treating skeletal muscle conditions or diseases in pet animals. The recipient of the compositions of the invention can be, for example, an elderly, a pet animal with sarcopenia or in need of the compositions and methods of the invention for recovery of skeletal muscles, for example, after exercise, muscle injury or surgery.

Description

COMPOSITIONS AND METHODS USING TRIGONELLINE AND OLEUROPEIN FOR PREVENTING OR TREATING CONDITIONS OR DISORDERS IN SKELETAL MUSCLE IN A PET ANIMAL

BACKGROUND OF THE INVENTION

Age-related loss of muscle mass and function is inevitable in all individuals; however its progression largely depends on genetic and environmental factors such as physical activity and nutritional intake, including adequate intake of vitamins. Sarcopenia has been defined as the point where the age-related loss of muscle mass and function gets debilitating and impacts quality of life. In contrast, frailty is another classification of age-related physical function decline that features low muscle strength and functionality, but not muscle mass. Sarcopenia is defined clinically according to low muscle mass and function, using cutoffs which stratify the elderly population for individuals in a state of pathological mobility. Muscle loss and related diseases such as cachexia or sarcopenia have important clinical implications because they are associated with increased morbidity and mortality.

Whereas cachexia and sarcopenia are important areas of research for development in humans, they are beginning to be recognized in veterinary medicine (L M Freeman, 2012, J Vet Intern Med. 26(1) 3-17).

Trigonelline is an important NAD+ precursor which feeds into the NAD+ pathway. NAD+ is an enzyme co-factor that is essential for the function of several enzymes related to reductionoxidation reactions and energy metabolism. NAD+ functions as an electron carrier in cell metabolism of amino acids, fatty acids, and carbohydrates. NAD+ serves as an activator and substrate for sirtuins, a family of protein deacetylases that have been implicated in metabolic function and extended lifespan in lower organisms. The co-enzymatic activity of NAD+, together with the tight regulation of its biosynthesis and bioavailability, makes it an important metabolic monitoring system that is clearly involved in the aging process and important for production of energy to allow skeletal muscle to properly function.

Moreover, Mitochondria are the primary source of aerobic energy production in mammalian cells and also maintain a large Ca2+ gradient across their inner membrane, providing a signaling potential for this molecule. Furthermore, mitochondrial Ca2+ plays a role in the mitochondria in the regulation of ATP generation and potentially contributes to the orchestration of cellular metabolic homeostasis. Specifically, activation of mitochondrial Ca2+ import increases cellular energy metabolism (Glancy, B. and R. S. Balaban (2012). "Role of mitochondrial Ca2+ in the regulation of cellular energetics." Biochemistry 51 (14): 2959-2973).

The present inventors noted that advancing age includes a gradual decrease in muscle function, capacity and reactivity, associated with a decline in muscle mitochondrial function. Age-related decrease in muscle mass is responsible for almost all loss of strength and power in older individuals, with an increase in fatigue. This decrease is due to inter-related factors: lifestyle, structural changes of the muscle, and metabolic changes.

The present inventors recognized this problem and addressed it by the surprising discovery that oleuropein and/or metabolites thereof are bioactives that activate mitochondrial calcium in combination with trigonelline. Calcium is essential for skeletal muscle contraction, but there are very limited solutions to increase mitochondrial calcium uptake through natural bioactives in order to influence bioenergetics. Therefore, without being bound by theory, the present inventors believe that a combination of trigonelline and at least one of oleuropein or metabolite thereof increases muscle energy metabolism and mitochondrial function, which in turn can increase muscle mass, functionality and performance.

SUMMARY OF THE INVENTION

Accordingly, in a general embodiment, the present disclosure provides a method of achieving at least one result selected from the group consisting of (i) improved mitochondrial calcium uptake in muscle cells, (ii) improved utilization of calcium in muscle cells, (iii) increased mitochondrial energy in muscle cells, (iv) improvement in at least one of muscle functionality, muscle performance, lean muscle mass or muscle strength, (v) decreased muscle fatigue, (vi) increased mobility and (vii) treatment or prevention of a muscle disorder linked to calcium depletion or deficiency (e.g., reduction in incidence and/or severity) (viii) increasing NAD+ levels in skeletal muscle to prevent and/or treat skeletal muscle diseases and/or conditions. The method comprises orally administering to a pet animal an effective amount of a combination of trigonelline and at least one of oleuropein and/or metabolite thereof.

In an embodiment, the present invention relates to a method of decreasing muscle fatigue in a pet animal who participates in exercise, the exercise comprising at least one of 1) resistance exercise, 2) anaerobic or repeated sprint-type exercise, or 3) endurance exercise, the method comprising orally administering to the pet animal an effective amount of a combination of trigonelline and at least one of oleuropein or metabolite thereof.

In another embodiment, the composition increases NAD⁺ biosynthesis and energy production in skeletal muscle.

In some embodiments, at least a portion of the trigonelline is provided from a plant source by a plant extract in the composition, such as one or more of a coffee extract, a hemp extract, pumpkin seed extract and/or a fenugreek extract, for example a plant extract enriched in trigonelline.

In an embodiment, the metabolite of oleuropein is selected from the group consisting of oleuropein aglycone, hydroxytyrosol, homovanillyl alcohol, isohomovanillyl alcohol, glucuronidated forms thereof, sulfated forms thereof, derivatives thereof, and mixtures thereof.

In an embodiment, the composition formulation is selected from the group consisting of: of a petfood product, a treat, a veterinary petfood or supplement, and combinations thereof.

In an embodiment, the pet animal is a selected from the group consisting of an aging subject; an elderly subject; a subject with muscle fatigue or muscle weakness; a subject with impaired mobility; a frail subject; a pre-frail subject; a sarcopenic subject; a subject recovering from prefrailty, frailty, sarcopenia or impaired mobility; a subject undergoing physical rehabilitation (e.g., from an injury to one or more of a muscle, a bone, a ligament, or the nervous system); and an athlete pet.

In another embodiment, the present disclosure provides a method of treating in a pet animal in need thereof or preventing in a pet animal at risk thereof (e.g., reducing incidence and/or severity) at least one condition selected from the group consisting of (i) impairment in at least one of muscle functionality, muscle performance, lean muscle mass or muscle strength, (ii) muscle fatigue or muscle weakness, (iii) pre-frailty, frailty, sarcopenia or impaired mobility, (iv) a muscle disorder linked to calcium depletion or deficiency, (v) conditions of restrictions of NAD⁺ bioavailability and (vi) skeletal muscle diseases and/or conditions, comprising orally administering to the pet animal in need thereof or at risk thereof an effective amount of a combination of trigonelline and at least one of oleuropein or metabolite thereof.

One advantage of one or more embodiments provided by the present invention is to replenish NAD⁺ pools, which decline with age. Another advantage of one or more embodiments provided by the present invention is to help offset slowing of the metabolism associated with aging.

An advantage of one or more embodiments provided by the present invention is to potentiate benefits on oxidative metabolism and prevent DNA damage.

Yet another advantage of one or more embodiments provided by the present invention is to help the body to metabolize fat and increase lean body mass.

Another advantage of one or more embodiments provided by the present invention is to maintain or increase skeletal muscle function in a subject.

Another advantage of one or embodiments provided by the present invention is maintenance of muscle function, for example, as measured by skeletal muscle contraction and relaxation without pain, cramping and muscle spasm.

Another advantage of one or more embodiments provided by the present invention is to maintain or increase skeletal muscle mass in a subject.

Another advantage of one or more embodiments provided by the present invention is to prevent or reduce skeletal muscle wasting in a subject.

Another advantage of one or more embodiments provided by the present invention is to enhance recovery of skeletal muscle after intense exercise.

Another advantage of one or more embodiments provided by the present invention is to enhance recovery of skeletal muscle after injury.

Another advantage of one or more embodiments provided by the present invention is to enhance recovery of skeletal muscle after trauma or surgery.

Yet another advantage of one or more embodiments provided by the present invention is to support improvements, as mentioned above, in the skeletal muscle after diseases and conditions such as: cachexia or precachexia; sarcopenia, myopathy, dystrophy, and/or recovery after intense exercise, muscle injury or surgery. In particular, cachexia is associated with cancer, chronic heart failure, renal failure, chronic obstructive pulmonary disease, AIDS, autoimmune disorders, chronic inflammatory disorders, cirrhosis of the liver, anorexia, chronic pancreatitis, metabolic acidosis and/or neurodegenerative disease. Additional features and advantages are described herein and will be apparent from the following Figures and Detailed Description.

DESCRIPTION OF FIGURES

FIG. 1 is a graph showing the beneficial effect of the combination of 3pM Oleuropein aglycone plus 500pM Trigonelline iodine on mitochondria activation, via mitochondrial Ca2+, in C2C12- derived myotubes. The graph shows that Oleuropein aglycone alone (3pM) slightly enhances mitochondrial Ca2+ by 17%, compared with buffer (white). Conversely, the combination of Oleuropein aglycone (3pM) plus Trigonelline (500 pM) enhances mitochondrial Ca2+ by 76%, compared with Trigonelline alone (buffer, black). The data are plotted as a percentage of the integrated mitochondrial calcium response, obtained in presence of the buffer. Mitochondrial Ca2+ was stimulated in all the conditions with 5 mM caffeine. Results are expressed as mean +/- SEM, from n = 16 biological replicates, from 2 independent experiments. * indicates statistically significant difference between the conditions, at P < 0.05 (unpaired t-test).

FIG. 2 is a graph showing the beneficial effect of the combination of 10pM Oleuropein aglycone plus 500pM Trigonelline iodine on mitochondria activation, via mitochondrial Ca2+, in C2C12-derived myotubes. The graph shows that Oleuropein aglycone alone (10pM) slightly enhance mitochondrial Ca2+ by 28%, compared with buffer (white). Conversely, the combination of Oleuropein aglycone (10pM) plus Trigonelline (500 pM) enhances mitochondrial Ca2+ by 67%, compared with Trigonelline alone (buffer black). The data are plotted as a percentage of integrated mitochondrial calcium response obtained in presence of the buffer. Mitochondrial Ca2+ was stimulated in all the conditions with 5 mM caffeine. Results are expressed as mean +/- SEM, from n = 16 biological replicates, from 2 independent experiments. * indicates statistically significant difference between the conditions, at P < 0.05 (unpaired t- test).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

All percentages are by weight of the total weight of the composition unless expressed otherwise. Similarly, all ratios are by weight unless expressed otherwise. When reference is made to the pH, values correspond to pH measured at 25 °C with standard equipment. As used herein, “about,” “approximately” and “substantially” are understood to refer to numbers in a range of numerals, for example the range of -10% to +10% of the referenced number, preferably -5% to +5% of the referenced number, more preferably -1 % to +1 % of the referenced number, most preferably -0.1 % to +0.1 % of the referenced number.

Furthermore, all numerical ranges herein should be understood to include all integers, whole or fractions, within the range. Moreover, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

As used herein and in the appended claims, the singular form of a word includes the plural, unless the context clearly dictates otherwise. Thus, the references “a,” “an” and “the” are generally inclusive of the plurals of the respective terms. For example, reference to “an ingredient” or “a method” includes a plurality of such “ingredients” or “methods.” 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 “both X and Y.”

Similarly, the words “comprise,” “comprises,” and “comprising” are to be interpreted inclusively rather than exclusively. Likewise, the terms “include,” “including” and “or” should all be construed to be inclusive, unless such a construction is clearly prohibited from the context. However, the embodiments provided by the present disclosure may lack any element that is not specifically disclosed herein. Thus, a disclosure of an embodiment defined using the term “comprising” is also a disclosure of embodiments “consisting essentially of’ and “consisting of’ the disclosed components. “Consisting essentially of’ means that the embodiment comprises more than 50 wt.% of the identified components, preferably at least 75 wt.% of the identified components, more preferably at least 85 wt.% of the identified components, most preferably at least 95 wt.% of the identified components, for example at least 99 wt.% of the identified components.

Where used herein, the term “example,” particularly when followed by a listing of terms, is merely exemplary and illustrative, and should not be deemed to be exclusive or comprehensive. Any embodiment disclosed herein can be combined with any other embodiment disclosed herein unless explicitly indicated otherwise. Accordingly, the terms “subject”, “individual” and “patient” refer to any pet animal that can benefit from the methods and compositions disclosed herein. “Pet animal” includes, but is not limited to, mammals, which includes but is not limited to rodents; aquatic mammals; domestic animals such as dogs, cats and other pets; farm animals such as sheep, pigs, cows and horses;

For animals, such as a companion animal (i.e. , cat or dog), an “older adult” or “ageing individual” has exceeded 50% of the average lifespan for its particular species and/or breed within a species.

The term “elderly” means a pet animal subject that has reached 60% of its likely lifespan, in some embodiments at least 70%, at least 80% or at least 90% of its likely lifespan. A determination of lifespan may be based on actuarial tables, calculations, or estimates, and may consider past, present, and future influences or factors that are known to positively or negatively affect lifespan. Consideration of species, gender, size, genetic factors, environmental factors and stressors, present and past health status, past and present nutritional status, and stressors may be taken into consideration when determining lifespan. An ageing cat or dog has an age from birth of at least about 5 years. An elderly or senior cat or dog has an age from birth of at least about 7 years.

“Muscle fatigue” means a reduced contractile force in one or more muscles due to a shortage of substrates within the muscle fiber and/or an accumulation of metabolites within the muscle fiber which interfere either with the release of calcium or with the ability of calcium to stimulate muscle contraction.

“Muscle weakness” is a condition where the force exerted by the muscles is less than would be expected. The U.S. Medical Research Council’s grading system for muscle strength is widely used to identify muscle weakness and the severity thereof. Specifically, the examiner assesses the patient’s ability to move the muscle against resistance provided by the examiner who, through experience, has developed a sense of the expected range of normal. This will vary from patient-to-patient depending upon the underlying size and conditioning of the subject; the fully trained athlete can be expected to perform differently from a small, sedentary, or deconditioned pet animal. The expected strength should also be adjusted for degree of atrophy in individualss with wasting illnesses. As used herein, a “athlete pet animal” is an individual who participates in at least one of 1) resistance exercise, 2) anaerobic or repeated sprint-type exercise, or 3) endurance exercise.

Sarcopenia

“Sarcopenia” is defined as the age-associated loss of muscle mass and functionality (including muscle strength and gait speed). Sarcopenia can be characterized by one or more of low muscle mass, low muscle strength and low physical performance.

Few studies investigating sarcopenia have been conducted in dogs and cats, but available information suggests that dogs and cats also lose LBM during aging. (Harper EJ. J Nutr 1998;128:2627S-2631S.). These data support the existence of a similar age-related loss of LBM as is seen in people. Although sarcopenia could be identified in healthy geriatric dogs by clinically relevant techniques, differences in pathophysiological mechanisms were not found, and additional research will be needed to identify both mechanisms and potential therapeutic targets (L M Freeman, 2012, J Vet Intern Med. 26(1) 3-17).

As used herein, “frailty” is defined as a clinically recognizable state of increased vulnerability resulting from aging-associated decline in reserve and function across multiple physiologic systems such that the ability to cope with everyday or acute stressors is compromised.

Cachexia and related diseases

Cachexia is the loss of lean body mass (LBM) that affects a large proportion of dogs and cats with congestive heart failure (CHF), chronic kidney disease (CKD), cancer, and a variety of other chronic diseases (L M Freeman, 2012, J Vet Intern Med. 26(1) 3-17).

Myopathy and related conditions

Myopathies are neuromuscular disorders in which the primary symptom is muscle weakness due to dysfunction of muscle fiber. Other symptoms of myopathy can include muscle cramps, stiffness, and spasm. Myopathies can be inherited (such as the muscular dystrophies) or acquired (such as common muscle cramps). Recovery after Muscle Injury from Surgery and Muscle Traumas

Muscle injuries can be caused by bruising, stretching or laceration causing acute or chronic soft tissue injury that occurs to a muscle, tendon, or both. It may occur as a result of fatigue, overuse, or improper use of a muscle. It may occur after physical trauma such as a fall, fracture or overuse during physical activity. Muscle injuries may also occur after surgery such as joint replacement arthroscopic surgery.

The terms “treatment” and “treating” include any effect that results in the improvement of the condition or disorder, for example lessening, reducing, modulating, or eliminating the condition or disorder. The term does not necessarily imply that a subject is treated until total recovery. Non-limiting examples of “treating” or “treatment of’ a condition or disorder include: (1) inhibiting the condition or disorder, i.e., arresting the development of the condition or disorder or its clinical symptoms and (2) relieving the condition or disorder, i.e., causing the temporary or permanent regression of the condition or disorder or its clinical symptoms. A treatment can be patient- or doctor-related.

The terms “prevention” or “preventing” mean causing the clinical symptoms of the referenced condition or disorder to not develop in an individual that may be exposed or predisposed to the condition or disorder but does not yet experience or display symptoms of the condition or disorder. The terms “condition” and “disorder” mean any disease, condition, symptom, or indication.

The relative terms “improved,” “increased,” “enhanced” and the like refer to the effects of the composition comprising a combination of trigonelline and high protein (disclosed herein) relative to a composition with less protein but otherwise identical.

The terms “food,” “food product” and “food composition” mean a product or composition that is intended for ingestion by an individual such as a pet animal and provides at least one nutrient to the individual. The compositions of the present disclosure, including the many embodiments described herein, can comprise, consist of, or consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise useful in a diet.

The term “beverage”, “beverage product” and “beverage composition” mean a product or composition for ingestion by an individual such as a pet animal and provides at least one nutrient to the individual. The compositions of the present disclosure, including the many embodiments described herein, can comprise, consist of, or consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise useful in a diet.

As used herein, “complete nutrition” contains sufficient types and levels of macronutrients (protein, fats and carbohydrates) and micronutrients to be sufficient to be a sole source of nutrition for the subject to which the composition is administered. Individuals can receive 100% of their nutritional requirements from such complete nutritional compositions.

The term “enterally administering” encompasses oral administration (including oral gavage administration), as well as rectal administration, although oral administration is preferred. The term "parenterally administering" refers to delivery of substances given by routes other than the digestive tract and covers administration routes such as intravenous, intra-arterial, intramuscular, intracerebroventricular, intraosseous, intradermal, intrathecal, and also intraperitoneal administration, intravesical infusion and intracavernosal injection.

Preferred parenteral administration is intravenous administration. A particular form of parenteral administration is delivery by intravenous administration of nutrition. Parenteral nutrition is “total parenteral nutrition” when no food is given by other routes. “Parenteral nutrition” is preferably a isotonic or hypertonic aqueous solution (or solid compositions to be dissolved, or liquid concentrates to be diluted to obtain an isotonic or hypertonic solution) comprising a saccharide such as glucose and further comprising one or more of lipids, amino acids, and vitamins.

Embodiments

An aspect of the present disclosure is a method of achieving at least one result selected from the group consisting of (i) improved mitochondrial calcium uptake in muscle cells, (ii) improved utilization of calcium in muscle cells, (iii) increased mitochondrial energy in muscle cells, (iv) improvement in at least one of muscle functionality, muscle performance, lean muscle mass or muscle strength, (v) decreased muscle fatigue or muscle weakness, (vi) increased mobility and (vii) treatment or prevention of a muscle disorder linked to calcium depletion or deficiency (e.g., reduction in incidence and/or severity) (viii) increasing NAD+ levels in skeletal muscle to prevent and/or treat skeletal muscle diseases and/or conditions. The method comprises orally administering to a pet animal an effective amount of a combination of trigonelline and at least one of oleuropein or metabolite thereof. Another aspect of the present disclosure is a method of treating in a pet animal in need thereof or preventing in an pet animal at risk thereof (e.g., reducing incidence and/or severity) at least one condition selected from the group consisting of (i) impairment in at least one of muscle functionality, muscle performance, lean muscle mass or muscle strength, (ii) muscle fatigue or muscle weakness, (iii) pre-frailty, frailty, sarcopenia or impaired mobility, and (iv) a muscle disorder linked to calcium depletion or deficiency. The method comprises orally administering to the pet animal in need thereof or at risk thereof an effective amount of a combination of trigonelline and at least one of oleuropein or metabolite thereof.

This also results in an improved vitality and/or energy in the pet animal.

In another embodiment, decreasing muscle fatigue is in a pet animal who participates in exercise, the exercise comprising at least one of 1) resistance exercise, 2) anaerobic or repeated sprint-type exercise, or 3) endurance exercise, the method comprising orally administering to the pet animal an effective amount of a combination of trigonelline and at least one of oleuropein or metabolite thereof. The combination is administered to the pet animal before the exercise, and/or during the exercise, and/or after the exercise, preferably less than two hours before the exercise, and/or during the exercise, and/or less than two hours after the exercise.

In a more preferred embodiment, the combination of trigonelline and at least one of oleuropein or metabolite thereof is administered to the pet animal less than one hour before the exercise.

In a preferred embodiment, the composition is administered to provide an amount of the combination that is effective to increase NAD+, for example, in muscle. The effective amount of the combination of trigonelline and at least one of oleuropein or metabolite thereof varies with the particular composition, the age and condition of the recipient, and the particular disorder or disease being treated.

Trigonelline

“Trigonelline” is here defined as any compound comprising 1-methylpyridin-1-ium-3-carboxylate, including, for example, any salt thereof (e.g., Chloride or Iodide salt) and/or a form in which the ring therein may be reduced.

In some embodiments, trigonelline is represented by the structure of formula 1 , being able to establish a salt with an anion (X-), such as a halogen, for example, 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, Pyridinium 3-carboxy-1 -methyl-.

1 In some embodiments, trigonelline is represented by the structure of formula 2 in its inner salt form. The structure of formula 2 is also known as Caffearine, Gynesine, N-Methylnicotinate, Trigenolline, Coffearine, Trigonellin, Coffearin, Betain nicotinate, Betaine nicotinate, 1- methylpyridinium-3-carboxylate, Nicotinic acid N-methylbetaine, 1 -Methylpyridinio-3- carboxylate, 1-Methyl-3-pyridiniumcarboxylate, N-Methylnicotinic acid, Trigenelline, Caffearin, 3- Carboxy-1 -methylpyridinium hydroxide inner salt, N'-Methylnicotinate, 1-methylpyridin-1-ium-3- carboxylate, 3-Carboxy-1 -methylpyridinium hydroxide inner salt, Pyridinium 3-carboxy-1 -methylhydroxide inner salt, 1-methylpyridine-3-carboxylic acid, 1-methylpyridin-1-ium-3-carboxylic acid, 1 -methylnicotinate, Trigonelline (S), N-methyl-nicotinate, Pyridinium 3-carboxy-1 -methylhydroxide inner salt (8CI), N'-Methylnicotinic acid, N-Methylnicotinic acid betaine, Nicotinic acid N- methylbetaine, 1 -Methyl-Nicotinic Acid Anion, Pyridinium 3-carboxy-1 -methyl- inner salt, 1- Methyl-5-(oxylatocarbonyl)pyridinium-3-ide, Pyridinium 3-carboxy-1 -methyl- inner salt, 3- carboxy-1-methyl-Pyridinium hydroxide inner salt).

2 In some embodiments, optionally “trigonelline” can include metabolites and pyrolysis products thereof, such as nicotinamide, nicotinamide riboside, 1 -methylnicotinamide, 1-methyl-2- pyridone-5-carboxamide (Me2PY), 1-methyl-4-pyridone-5-carboxamide (Me4PY), and alkylpyridiniums, such as 1-methyl-pyridinium (NMP) and 1 ,4-dimethylpyridinium; although as noted later herein, some embodiments exclude one or more of these metabolites and pyrolysis products of trigonelline.

The composition can comprise a pharmacologically effective amount of trigonelline in a pharmaceutically suitable carrier. In aqueous liquid compositions, the trigonelline concentration preferably ranges from about 0.05 wt.% to about 4 wt.%, or from about 0.5 wt.% to about 2 wt.% or from about 1.0 wt.% to about 1.5 wt.% of the aqueous liquid composition.

In particular embodiments, the method can comprise administering daily trigonelline in the weight range of 0.05 mg - 1 g per kg body weight of the pet animal, preferably 1 mg -200 mg per kg body weight, more preferably 5 mg - 150 mg per kg body weight, even more preferably 5 mg - 80 mg per kg body weight, or most preferably 5 mg - 20 mg per kg body weight.

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

In one embodiment, the composition comprises trigonelline which is chemically synthesized which is at least about 90% trigonelline, preferably at least about 98% trigonelline.

In a preferred embodiment, at least a portion of the trigonelline is provided by a plant extract, for example an extract from one or more of coffee bean (e.g., a green coffee extract), Japanese radish, fenugreek seed, garden pea, hemp seed, pumpkin seed, oats, potato, dahlia, Stachys species, Strophanthus species, Laminariaceae species (especially Laminaria and Saccharina), Postelsia palmaeformis, Pseudochorda nagaii, Akkesiphycus or Dichapetalum cymosum. The plant extract is preferably enriched in trigonelline, i.e. , the starting plant material comprises one or more other compounds in addition to the trigonelline, and the enriched plant material has a ratio of the trigonelline relative to at least one of the one or more other compounds that is higher than the ratio in the starting plant material.

Therefore, some embodiments of the composition comprise plant sources and/or enriched plant sources that provide at least a portion of the trigonelline in the composition.

In a preferred embodiment, the composition comprises enriched fenugreek extract which provides at least about 25 - 50% trigonelline in the composition. As used herein, a “composition consisting essentially of trigonelline” contains trigonelline and is substantially free or completely free of any additional compound that affects NAD+ production other than the trigonelline. In a particular non-limiting embodiment, the composition consists of the trigonelline and one or more excipients.

In some embodiments, the composition consisting essentially of trigonelline is optionally substantially free or completely free of other NAD+ precursors, such as one or more of trigonelline derivatives; metabolites and pyrolysis products of trigonelline, 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 and 1 ,4-dimethylpyridinium; nicotinic acid (“niacin”); or L-tryptophan.

As used herein, “substantially free” means that any of the other compound present in the composition is no greater than 1.0 wt.% relative to the amount of trigonelline, preferably no greater than 0.1 wt.% relative to the amount of trigonelline, more preferably no greater than 0.01 wt.% relative to the amount of trigonelline, most preferably no greater than 0.001 wt.% relative to the amount of trigonelline.

Oleuropein and metabolites

In an embodiment, at least a portion of the oleuropein is obtained by extraction, e.g., by extraction from a plant such as a plant belonging to the Oleaceae family, preferably one or more of the stems, the leaves, the fruits or the stones of a plant belonging to the Oleaceae family such as Olea europaea (olive tree), a plant of genus Ligustrum, a plant of genus Syringa, a plant of genus Fraximus, a plant of genus Jasminum and a plant of genus Osmanthus. Additionally or alternatively, at least a portion of the oleuropein can be obtained by argan oil, produced from kernels of the argan tree (Argania spinosa) or by chemical synthesis.

Non-limiting examples of suitable metabolites of oleuropein include oleuropein aglycone, hydroxytyrosol, homovanillyl alcohol, isohomovanillyl alcohol, and mixtures thereof.

In some embodiments, the at least one of oleuropein or metabolite thereof is the only polyphenol in the composition and/or the only polyphenol administered to the pet animal.

The effective amount of the combination of oleuropein or metabolite thereof varies with the particular composition, the age and condition of the recipient, and the particular disorder or disease being treated. Nevertheless, in a general embodiment, 0.001 mg to 1.0 g of the at least one of oleuropein or metabolite thereof can be administered to the individual per day, preferably from 0.01 mg to 0.9 g of the at least one of oleuropein or metabolite thereof per day, more preferably from 0.1 mg to 750 mg of the at least one of oleuropein or metabolite thereof per day, more preferably from 0.5 mg to 500 mg of the at least one of oleuropein or metabolite thereof per day, and most preferably from 1.0 mg to 200 mg of the at least one of oleuropein or metabolite thereof per day.

Composition formulation

The composition includes petfood compositions to supply the necessary dietary requirements for an animal, animal treats (e.g., biscuits), dietary supplements, veterinary petfood or supplement, and combinations thereof. The compositions may be a dry composition (e.g., kibble), semi-moist composition, wet composition, or any mixture thereof. In one embodiment, the composition is a dietary supplement such as a gravy, drinking water, beverage, yogurt, powder, granule, paste, suspension, chew, morsel, treat, snack, pellet, pill, capsule, tablet, or any other suitable delivery form.

In some embodiments, in addition to trigonelline and vitamins, the composition may contain additional components such as proteins, carbohydrates and fats. The dietary supplement can comprise a high concentration of the UFA and NORC, and B vitamins and antioxidants. This permits the supplement to be administered to the animal in small amounts, or in the alternative, can be diluted before administration to an animal. The dietary supplement may require admixing, or can be admixed with water or other diluent prior to administration to the animal.

In various embodiments, pet food or pet treat compositions comprise from about 15% to about 50% crude protein. The crude protein material may comprise 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 protein useful herein include pork, lamb, equine, poultry, fish, and mixtures thereof.

In another embodiment, the food composition is a pet food composition such as a premium or super-premium pet food composition. In one embodiment, the pet food is formulated for canines and has a protein content of about 20-30%, preferably about 24-28%, and more preferably about 25-27%. In one embodiment, the protein content of a dog food composition is about 26% by weight. In another embodiment, the formulation is for felines and has a protein content of about 35-45%, preferably about 37-42%, and more preferably about 39-41 %. In one embodiment, the protein content of a cat food composition is about 40%. In a preferred embodiment, the composition is a food product comprising about 15% to about 50% protein, about 5% to about 40% fat, about 5% to about 10% ash content, and having a moisture content of about 5% to about 20%.

In an embodiment, at least a portion of the protein is selected from the group consisting of (i) protein from an animal source, (ii) protein from a plant source and (iii) a mixture thereof.

In an embodiment, at least a portion of the protein is selected from the group consisting of (i) free form amino acids, (ii) unhydrolyzed protein, (iii) partially hydrolyzed protein, (iv) extensively hydrolyzed protein, and (v) mixtures thereof. The protein can comprise essential amino acids and/or conditionally essential amino acids, e.g., such amino acids that may be insufficiently delivered due to low caloric intake or illness. For example, the protein can comprise one or more essential amino acids selected from the group consisting of histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine; and each of these amino acids (if present) may be administered in the composition in a daily dose from about 0.0476 to about 47.6 mg amino acid/kg body weight. Notably, lower intake of methionine leads to lower levels of protein translation and ultimately muscle synthesis. The protein can comprise one or more conditionally essential amino acids (e.g., amino acids conditionally essential in illness or stress) selected from the group consisting of arginine, cysteine, glutamine, glycine, proline, ornithine, serine and tyrosine; and each of these amino acids (if present) may be administered in the composition in a daily dose from about 0.0476 to about 47.6 mg amino acid/kg body weight.

In an embodiment, the composition comprises branched chain amino acids in at least one form selected from the group consisting of (i) free form, (ii) bound to at least one additional amino acid, and (iii) mixtures thereof. The branched chain amino acids can comprise leucine, isoleucine and/or valine in an amount effective to activate mTOR in the individual.

In an embodiment, at least a portion of the protein is 5 to 95% hydrolyzed.

In an embodiment, the protein has a formulation selected from the group consisting of (i) at least 50% of the protein has a molecular weight of 1-5 kDa, (ii) at least 50% of the protein has a molecular weight of 5-10 kDa and (iii) at least 50% of the protein has a molecular weight of 10- 20 kDa.

In one embodiment, the composition may include a source of carbohydrates. Any suitable carbohydrate may be used in the composition including, but not limited to, starch (e.g., modified starch, amylose starch, tapioca starch, corn starch), sucrose, lactose, glucose, fructose, corn syrup solids, maltodextrin, xylitol, sorbitol or combinations thereof.

The source of carbohydrates is preferably not greater than 50 energy % of the composition, more preferably not greater than 36 energy % of the composition, and most preferably not greater than 30 energy % of the composition. The composition can have a high protein carbohydrate energy ratio, for example greater than 0.66, preferably greater than 0.9 and more preferably greater than 1.2.

In an embodiment, the composition may include a source of fat. The source of fat may include any suitable fat or fat mixture. Non-limiting examples of suitable fat sources include vegetable fat, such as olive oil, corn oil, sunflower oil, high-oleic sunflower, rapeseed oil, canola oil, hazelnut oil, soy oil, palm oil, coconut oil, blackcurrant seed oil, borage oil, lecithins, and the like, animal fats such as milk fat; or combinations thereof.

In some embodiments, the food composition is a wet food, such as a canned food, frozen food, or fresh food product. In some embodiments, the food composition is shelf stable. In other embodiments, it must be refrigerated. In other embodiments, the food composition is an intermediate moisture product or a dry food product as described above.

In some embodiments, the composition is administered to the animal in conjunction with one or more drugs. In a preferred embodiment, the composition is administered to the animal on a daily basis, preferably in a single dose.

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

Methods and Uses of the Composition

A composition of the invention can be administered to a pet animal in need of preventing and/or treating skeletal muscle diseases and conditions. For example, to increase NAD+ in skeletal muscle. Non-limiting examples of such muscle include one or more of the following: vastus lateralis, gastrocnemius, tibialis, soleus, extensor, digitorum longus (EDL), biceps femoris, semitendinosus, semimembranosus, gluteus maximus, extra-ocular muscles, face muscles or diaphragm. In some embodiments, the combination of trigonelline and at least one of oleuropein or metabolite thereof is administered to a pet animal selected from the group consisting of an aging subject; an elderly subject; a subject with muscle fatigue or muscle weakness; a subject with impaired mobility; a frail subject; a pre-frail subject; a sarcopenic subject; a subject recovering from pre-frailty, frailty, sarcopenia or impaired mobility; a subject undergoing physical rehabilitation (e.g., from an injury to one or more of a muscle, a bone, a ligament, or the nervous system) and an athlete pet.

In some embodiments, the pet animal is healthy.

In some embodiments, the pet animal has sarcopenia, frailty, muscle fatigue or muscle weakness, or impairment in one or more of muscle functionality, muscle performance, lean muscle mass or muscle strength, but optionally is otherwise healthy.

For example, the combination of trigonelline and at least one of oleuropein or metabolite thereof can be administered to an athlete pet before, during and/or after exercise, for example less than two hours before the exercise or less than one hour before the exercise and less than two hours after the exercise or less than one hour after the exercise.

The composition of the invention can be administered to a pet animal, e.g., an ageing pet animal or a critically ill pet animal, or a pet animal recovering from surgery or injury of the skeletal muscle; in a therapeutically effective dose. The therapeutically effective dose can be determined by the person skilled in the art and will depend on a number of factors known to those of skill in the art, such as the severity of the condition and the weight and general state of the pet animal.

The composition is preferably administered to the pet animal at least two days per week, more preferably at least three days per week, most preferably all seven days of the week; for at least one week, at least one month, at least two months, at least three months, at least six months, or even longer. In some embodiments, the composition is administered to the pet animal consecutively for a number of days, for example at least until a therapeutic effect is achieved. In an embodiment, the composition can be administered to the pet animal daily for at least 30, 60 or 90 consecutive days.

The above examples of administration do not require continuous daily administration with no interruptions. Instead, there may be some short breaks in the administration, such as a break of two to four days during the period of administration. The ideal duration of the administration of the composition can be determined by those of skill in the art.

In a preferred embodiment, the composition is administered to the pet animal orally or enterally (e.g. tube feeding). For example, the composition can be administered to the pet animal as a beverage, a capsule, a tablet, a powder or a suspension.

The composition can be any kind of composition that is suitable for pet animal consumption. For example, the composition may be selected from the group consisting of food compositions, dietary supplements, nutritional compositions, nutraceuticals, powdered nutritional products to be reconstituted in water or milk before consumption, food additives, medicaments, beverages and drinks. In an embodiment, the composition is an oral nutritional supplement (ONS), a complete nutritional formula, a pharmaceutical, a medical or a food product. In a preferred embodiment, the composition is administered to the pet animal as a beverage. The composition may be stored in a sachet as a powder and then suspended in a liquid such as water for use.

In some instances where oral or enteral administration is not possible or not advised, the composition may also be administered parenterally.

In some embodiments, the composition is administered to the pet animal in a single dosage form, i.e. all compounds are present in one product to be given to an pet animal in combination with a meal. In other embodiments, the composition is co-administered in separate dosage forms, for example at least one component separately from one or more of the other components of the composition.

These methods can consist essentially of administering the composition consisting essentially of trigonelline and oleuropein or metabolite thereof.

Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition. Similarly, unit dosage forms for injection or intravenous administration may comprise the compounds in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier, wherein each dosage unit, for example, mL or L, contains a predetermined amount of the composition containing one or more of the compounds.

The above examples of administration do not require continuous daily administration with no interruptions. Instead, there may be some short breaks in the administration, such as a break of two to four days during the period of administration. The ideal duration of the administration of the composition can be determined by those of skill in the art.

In an embodiment, the trigonelline and the at least one of oleuropein or metabolite thereof can be administered in the same composition, for example a unit dosage form containing both the trigonelline and the at least one of oleuropein or metabolite thereof.

In an alternative embodiment, the trigonelline and the at least one of oleuropein or metabolite thereof can be administered sequentially in separate compositions. The term “sequentially” means that the trigonelline and the at least one of oleuropein or metabolite thereof are administered in a successive manner such that the at least one of oleuropein or metabolite thereof is administered at a first time without the trigonelline, and the trigonelline is administered at a second time (before or subsequent to the first time) without the at least one of oleuropein or metabolite thereof. The time between sequential administrations may be, for example, one or several seconds, minutes or hours in the same day; one or several days or weeks in the same month; or one or several months in the same year.

Skeletal Muscle diseases or conditions

Method and uses of the composition are provided for increasing NAD+ in a subject by administering an effective amount of a composition in an effect unit dose form to prevent and/or treat skeletal muscle diseases or conditions such as: sarcopenia, cachexia or precachexia, myopathy, dystrophy, and/or recovery after intense exercise, muscle injury or surgery.

In another embodiment, muscle functionality that can be improved by the methods disclosed herein comprises a characteristic selected from the group consisting of muscle strength, gait speed, and combinations thereof. Muscle function is typically defined as strength per unit of appendicular skeletal muscle mass or per muscle volume.

Non-limiting examples of a muscle disorder linked to calcium depletion or deficiency that can be treated by the methods disclosed herein include muscular dystrophies, congenital core myopathies and mitochondrial myopathies. Particular non-limiting examples include Barth syndrome; chronic progressive external ophthalmoplegia (cPEO); Kearns-Sayre syndrome (KSS); Leigh syndrome; mitochondrial DNA depletion syndromes (MDDS); mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS); mitochondrial neurogastrointestinal encephalomyopathy (MNGIE); myoclonus epilepsy with ragged red fibers (MERRF); neuropathy, ataxia, and retinitis pigmentosa (NARP); and Pearson syndrome.

The pet animal can be at risk of a disorder or condition (e.g., sarcopenia, frailty, muscle fatigue or muscle weakness, or impairment in one or more of muscle functionality, muscle performance, lean muscle mass or muscle strength), in which case the effective amount of the composition is a prophylactically effective dose; or the pet animal can have a disorder or condition, in which case the effective amount of the composition is a therapeutically effective dose. In some embodiments, the methods comprise identifying the pet animal as having the condition or being at risk of the condition before the administration.

In another embodiment, the present disclosure provides a method of treating or preventing impaired mobility in an older adult. The method comprises orally administering to the older adult an effective amount of a combination of trigonelline and at least one of oleuropein or metabolite thereof. The older adult can be an elderly pet animal. In some embodiments, the older adult has a condition selected from the group consisting of frailty, pre-frailty, sarcopenia, recovering from sarcopenia, osteoporosis, osteoarthritis, malnutrition, at risk of malnutrition, undergoing rehabilitation, scheduled to undergo rehabilitation within the next year, and combinations thereof.

EXAMPLE

Comparative effect of combination of oleuropein and Trigonelline vs single compounds on mitochondrial activation in muscle cells

Materials and Methods

To test the effect of Oleuropein aglycone (Oea), Trigonelline and their combination on mitochondrial activation in living cells, the inventors measured mitochondrial calcium elevation in myotubes differentiated from C2C12 cells.

C2C12 cells were purchased from ATCC. C2C12 cells were seeded in 384-well plates at a density of 4500 cells per well in DMEM medium, high glucose (Gibco) + 10% fetal calf serum. Myotubes were differentiated from C2C12 cells by growing the cells in DMEM containing 2% horse serum, for 6 days. Mitochondrial calcium measurements were carried out using myotubes infected with the adenovirus (from Sirion biotech) expressing the luminescent mitochondrially-targeted calcium sensor mitochondrial mutated aequorin (Montero et al., 2000). For aequorin reconstitution, 48 hours after infection, myotubes were incubated for 2h at room temperature (22 ±°C) in standard Aequorin buffer (145 mM NaCI, 5 mM KCI, 1 mM MgCI2, 1 mM CaCI2, 10 mM glucose and 10 mM Hepes, pH 7.4) with 1 pM wild type coelenterazine. For treatment, Trigonelline Iodine (0 pM, buffer, or 500 pM, as indicated in the figures) and Oleuropein aglycone (3 pM or 10 pM, as indicated in the figures) were incubated for 2h in Aequorin buffer. Myotubes were stimulated with 5 mM of caffeine and the total calcium transiting during stimulation was calculated as the area under the curve, during caffeine stimulation. Luminescence was measured at the FLIPR cell imaging reader (Molecular devices). Calibration of the luminescence data into Calcium concentration was carried out using an algorithm as described previously (Bonora et al., 2013). Custom module analysis based on Excel (Microsoft) and GraphPad Prism 7.02 (GraphPad) software was used for quantification. Results

As shown in FIG. 1 and FIG.2, the combination of Trigonelline and Oleuropein promotes the beneficial effects of the 2 compounds by boosting the calcium uptake into the mitochondria.

Claims

1 . A method of achieving at least one result selected from the group consisting of (i) improved mitochondrial calcium uptake in muscle cells, (ii) improved utilization of calcium in muscle cells, (iii) increased mitochondrial energy in muscle cells, (iv) improvement in at least one of muscle functionality, muscle performance, lean muscle mass or muscle strength, (v) decreased muscle fatigue, (vi) increased mobility and (vii) treatment of a muscle disorder linked to calcium depletion or deficiency, (viii) increasing NAD+ levels in skeletal muscle to prevent and/or treat skeletal muscle diseases and/or conditions, the method comprising orally administering to a pet animal an effective amount of a combination of trigonelline and at least one of oleuropein or metabolite thereof.

2. The method of Claim 1 , wherein the pet animal is selected from the group consisting of an aging subject; an elderly subject; a subject with muscle fatigue or muscle weakness; a subject with impaired mobility; a frail subject; a pre-frail subject; a sarcopenic subject; a subject recovering from pre-frailty, frailty, sarcopenia or impaired mobility; a subject undergoing physical rehabilitation and an athlete pet.

3. The method of claim 1 , wherein decreasing muscle fatigue is in an pet animal who participates in exercise, the exercise comprising at least one of 1) resistance exercise, 2) anaerobic or repeated sprint-type exercise, or 3) endurance exercise, the method comprising orally administering to the pet animal an effective amount of a combination of trigonelline and at least one of oleuropein or metabolite thereof.

4. The method of Claim 3, wherein the combination of trigonelline and at least one of oleuropein or metabolite thereof is administered to the pet animal before the exercise, and/or during the exercise, and/or after the exercise.

5. The method of any of preceding claims, wherein at least a portion of the muscle cells are part of a skeletal muscle selected from the group consisting of gastrocnemius, tibialis, soleus, extensor digitorum longus (EDL), biceps femoris, semitendinosus, semimembranosus, gluteus maximus, and combinations thereof.

6. The method of any of preceding claims, wherein the combination of trigonelline and at least one of oleuropein or metabolite thereof is administered daily for at least one week.

7. The method of any preceding claim wherein the trigonelline is selected from an extract of coffee, fenugreek, hemp or algae.

8. The method of any preceding claim, wherein trigonelline is selected from an extract of fenugreek which contains at least about 25% - 50% trigonelline.

9. The method of any preceding claim, wherein trigonelline is chemically synthesized and contains at least about 90% trigonelline.

10. The method of any of preceding claims, wherein the metabolite of oleuropein is selected from the group consisting of oleuropein aglycone, hydroxytyrosol, homovanillyl alcohol, isohomovanillyl alcohol, glucuronidated forms thereof, sulfated forms thereof, derivatives thereof and mixtures thereof.

11 . The method of any of preceding claims, wherein the composition is selected from the group consisting: of a petfood product, a treat, a veterinary petfood or supplement, and combinations thereof.

12. The method of any of preceding claims, wherein the combination of trigonelline and at least one of oleuropein or metabolite thereof is administered daily for at least one week.

13. The method of any of preceding claims, wherein the trigonelline and the at least one of oleuropein or metabolite thereof are administered together in the same composition.

14. The method of any of preceding claims, wherein the trigonelline is administered separately in a different composition from the at least one of oleuropein or metabolite thereof.

15. The method of any of preceding claims, wherein the composition consists essentially of trigonelline and oleuropein or metabolite thereof.

16. The method of any of preceding claims, to maintain or increase skeletal muscle function.

17. The method of Claim 16 wherein maintenance of muscle function is measured by skeletal muscle contraction and relaxation without pain, cramping and muscle spasm.

18. The method of 16 wherein increased muscle function is measured by increase in the number of muscle stem cells and/or myoblasts and/or myotubes.

19. The method of Claim 16 to 18, to maintain or increase skeletal muscle mass in a subject.

20. The method of Claim 16 to 19, to prevent or reduce skeletal muscle wasting in a subject.

21 . The method of Claim 16 to 20, to enhance recovery of skeletal muscle after intense exercise.

22. The method of Claim 16 to 21 , to enhance recovery of skeletal muscle after injury, trauma or surgery.

23. The method of Claim 16 to 22, to enhance recovery of skeletal muscle after skeletal muscle disease and/or conditions.

24. The method of Claim 23, wherein skeletal muscle disease and/or condition is selected from the group consisting of: sarcopenia, cachexia or precachexia, myopathy, dystrophy, and/or recovery after intense exercise, muscle injury or surgery.

25. The method of Claim 24, wherein cachexia is associated with a disease selected from cancer, chronic heart failure, renal failure, chronic obstructive pulmonary disease, AIDS, autoimmune disorders, chronic inflammatory disorders, cirrhosis of the liver, anorexia, chronic pancreatitis, metabolic acidosis and/or neurodegenerative disease.

26. A method of treating in a pet animal in need thereof or preventing in a pet animal at risk thereof (e.g., reducing incidence and/or severity) at least one condition selected from the group consisting of (i) impairment in at least one of muscle functionality, muscle performance, lean muscle mass or muscle strength, (ii) muscle fatigue or muscle weakness, (iii) pre-frailty, frailty, sarcopenia or impaired mobility, (iv) a muscle disorder linked to calcium depletion or deficiency, (v) conditions of restrictions of NAD⁺ bioavailability and (vi) skeletal muscle diseases and/or conditions, comprising orally administering to the pet animal in need thereof or at risk thereof an effective amount of a combination of trigonelline and at least one of oleuropein or metabolite thereof.