Compositions comprising combination of caffeine and oleuropein or metabolites thereof and uses thereof for improving muscle function

Abstract

The present invention discloses a combination of caffeine and at least one of oleuropein or a metabolite thereof, which can be used for oral administration to a subject in an effective amount to achieve at least one of (i) improve mitochondrial calcium uptake in muscle cells, (ii) improve calcium utilization in muscle cells, (iii) increase energy of mitochondrial in muscle cells, in some embodiments, the methods may include (i) at least one of: (i) at least one of muscle function, muscle performance, or muscle strength, (v) reduce muscle fatigue, (vi) enhance mobility, and / or (vii) treat muscle diseases associated with calcium depletion or deficiency. The individual may be at least one of: an aged individual; elderly individuals; individuals with muscle fatigue or myasthenia; individuals of which mobility is impaired; weak individuals; weak early-stage individuals; a sarcopenia individual; individuals that recover from early stage of weakness, weakness, sarcopenia, or impaired mobility; individuals undergoing physical rehabilitation; an athlete; or a pet.

Description

Background Art

[0001] The present disclosure generally relates to compositions and methods using a combination of caffeine and oleuropein or at least one of its metabolites. More specifically, the present disclosure relates to compositions and methods for enhancing bioenergetics and mitochondrial function by a combination of caffeine and oleuropein or at least one of its metabolites to promote mitochondrial calcium import, which in turn can enhance muscle contraction and muscle performance, thereby improving, maintaining muscle function, or reducing muscle function loss.

[0002] Sarcopenia is defined as the age-related loss of muscle mass and function, including muscle strength and walking speed. Muscle function and physical ability decline with the loss of muscle mass. Impaired muscle function is highly predictive of the incidence of loss of mobility, disability, and death in advanced age. Sarcopenia is becoming more and more common with the growing elderly population, with 45% of the elderly population in the United States having moderate to severe symptoms. The direct and indirect costs attributed to sarcopenia in U.S. healthcare have reached nearly $19 billion. Therefore, preventing and / or treating sarcopenia will have a huge impact on the health and quality of life of our society, and thus on the healthcare-related economy. Unfortunately, the etiology and pathophysiological mechanisms of sarcopenia remain poorly understood, making it difficult to take effective preventive or therapeutic measures. Summary of the invention

[0003] Mitochondria are the main source of aerobic energy production in mammalian cells and also maintain a large Ca2+ gradient on their inner membrane, providing a signaling potential for the molecule. In addition, mitochondrial Ca2+ plays a role in the regulation of ATP production in mitochondria and potentially contributes to the coordination of cellular metabolic homeostasis. (Glancy, B. and RS Balaban (2012), "Role of mitochondrial Ca2+ in the regulation of cellular energetics", Biochemistry, Vol. 14, No. 51: pp. 2959-2973).

[0004] The inventors have noted that age-related increases include a gradual decline in muscle function, muscle capacity, and muscle responsiveness. For example, a 50-year-old loses about 10% of muscle area, and muscle strength decreases by 15% per decade in the 60s and 70s and by about 30% thereafter. Age-related reductions in muscle mass are responsible for nearly all of the loss of strength and power in the elderly, and are accompanied by increased fatigue. This reduction is attributed to interrelated factors: lifestyle, structural changes in muscle, and metabolic changes.

[0005] The inventors recognized this problem and solved it by the surprising discovery that oleuropein and its metabolites are bioactive substances that activate mitochondrial calcium in combination with caffeine. Calcium is necessary for skeletal muscle contraction, but solutions to affect bioenergetics by increasing mitochondrial calcium uptake through natural bioactive substances are very limited. Therefore, without being bound by theory, the inventors believe that the combination of caffeine with at least one of oleuropein or its metabolites enhances bioenergetics and mitochondrial function to promote mitochondrial calcium import, which in turn can enhance muscle contraction and muscle performance, thereby improving, maintaining muscle function or reducing muscle function loss.

[0006] Thus, in general embodiments, the present disclosure provides a method of achieving at least one result selected from the group consisting of: (i) improving mitochondrial calcium uptake in muscle cells, (ii) improving calcium utilization in muscle cells, (iii) increasing mitochondrial energy in muscle cells, (iv) improving at least one of muscle function, muscle performance, or muscle strength, (v) reducing muscle fatigue, (vi) enhancing mobility, and (vii) treating or preventing muscle diseases associated with calcium depletion or deficiency (e.g., reducing incidence and / or severity). The method comprises orally administering to an individual an effective amount of a combination of caffeine and at least one of oleuropein or a metabolite thereof.

[0007] In one embodiment, the present invention is directed to a method of reducing muscle fatigue in an individual participating in exercise comprising at least one of 1) resistance exercise, 2) anaerobic or repetitive sprint-type exercise, or 3) endurance exercise, the method comprising orally administering to the individual an effective amount of a combination of caffeine and at least one of oleuropein or a metabolite thereof.

[0008] In one embodiment, the subject is selected from the group consisting of: an aging subject; an elderly subject; a subject with muscle fatigue or 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., recovering from an injury to one or more of the muscle, bone, ligament, or nervous system); an athlete; and a pet.

[0009] In one embodiment, at least a portion of the muscle cells are part of a skeletal muscle selected from the group consisting of the gastrocnemius, tibialis, soleus, extensor digitorum longus (EDL), biceps femoris, semitendinosus, semimembranosus, gluteus maximus, and combinations thereof.

[0010] In one embodiment, the combination of caffeine and at least one of oleuropein or its metabolites is administered orally daily for at least one week, preferably for at least one month.

[0011] In one 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.

[0012] In one embodiment, the combination of caffeine and at least one of oleuropein or its metabolites is administered in the form of a composition selected from the group consisting of a food composition, a dietary supplement, a nutritional composition, a beverage, a nutraceutical, a powdered nutritional product to be reconstituted with water or milk before consumption, a food additive, a medicament, a drink, a pet food, and combinations thereof.

[0013] In one embodiment, caffeine is administered with oleuropein or at least one of its metabolites in the same composition.

[0014] In one embodiment, caffeine and at least one of oleuropein or a metabolite thereof are administered separately in different compositions.

[0015] In one embodiment, caffeine is administered together with oleuropein or at least one of its metabolites in the form of a food product further comprising a component selected from the group consisting of protein, carbohydrate, fat, and mixtures thereof.

[0016] In another embodiment, the present disclosure provides a method of treating in an individual with relevant needs or preventing (e.g., reducing incidence and / or severity) at least one condition selected from the following in an individual at risk: (i) impairment of at least one of muscle function, muscle performance, or muscle strength, (ii) muscle fatigue or weakness, (iii) pre-frailty, weakness, sarcopenia, or impaired mobility, and (iv) muscle diseases associated with calcium depletion or deficiency. The method comprises orally administering to an individual with relevant needs or at relevant risk an effective amount of a combination of caffeine and at least one of oleuropein or its metabolites.

[0017] In another embodiment, the present disclosure provides a unit dosage form comprising a combination of caffeine and at least one of oleuropein or a metabolite thereof, the unit dosage form comprising a combined amount effective for at least one result selected from: (i) improving mitochondrial calcium uptake in muscle cells, (ii) improving calcium utilization in muscle cells, (iii) increasing mitochondrial energetics in muscle cells, (iv) improving at least one of muscle function, muscle performance, or muscle strength, (v) reducing muscle fatigue, (vi) enhancing mobility, and (vii) treating or preventing muscle diseases associated with calcium depletion or deficiency (e.g., reducing incidence and / or severity).

[0018] In one embodiment, the unit dosage form consists essentially of caffeine in combination with at least one of oleuropein or a metabolite thereof.

[0019] In one embodiment, the unit dosage form consists of an excipient and a combination of caffeine and at least one of oleuropein or a metabolite thereof.

[0020] In another embodiment, the present disclosure provides a method for preparing a composition for achieving at least one result selected from the following: (i) improving mitochondrial calcium uptake in muscle cells, (ii) improving calcium utilization in muscle cells, (iii) increasing mitochondrial energy in muscle cells, (iv) improving at least one of muscle function, muscle performance, or muscle strength, (v) reducing muscle fatigue or weakness, (vi) enhancing mobility, and (vii) treating or preventing muscle diseases associated with calcium depletion or deficiency (e.g., reducing incidence and / or severity). The method comprises adding an effective amount of caffeine in combination with at least one of oleuropein or its metabolites to at least one ingredient selected from protein, carbohydrates, and fat.

[0021] In one embodiment, the method further comprises adding to at least one ingredient a food additive selected from the group consisting of acidulants, thickeners, buffers or agents for pH adjustment, chelating agents, colorants, emulsifiers, excipients, flavoring agents, minerals, osmotic agents, pharmaceutically acceptable carriers, preservatives, stabilizers, sugars, sweeteners, texturizers, vitamins, minerals, and combinations thereof.

[0022] Additional features and advantages are described herein, and will be apparent from the following figures and detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Graph showing the effect of oleuropein aglycone (10 μM, grey), caffeine (5 mM, black) and the combination of 10 μM oleuropein aglycone + 5 mM caffeine on mitochondrial activation in myotubes differentiated from C2C12 cells, measured as the integrated increase in mitochondrial calcium. Results are expressed as mean + / - SEM, n = 29 experiments. * indicates statistically significant difference, P < 0.05 (one-way ANOVA test).

[0024] Figure 2 Graph showing that oleuropein (10 μM) and caffeine (5 mM) synergistically promote the increase of mitochondrial calcium in myotubes of C2C12 cells. Figure 1The graph obtained from the data represented in compares the theoretical additive effect of caffeine supplementation plus oleuropein aglycone supplementation with the measured effect of the combination of caffeine plus oleuropein aglycone supplemented simultaneously on the increase of mitochondrial calcium. The theoretical effect was compared with the measured effect to infer a synergistic effect. The data are the average of 29 experiments. The results are expressed as mean + / - SEM. * indicates a statistically significant difference in the measured value in mitochondrial calcium relative to the theoretical difference, P < 0.05 (Student's t-test). DETAILED DESCRIPTION

[0025] definition

[0026] Some definitions are provided below. However, definitions may be located in the "Implementation" section below, and the above heading "Definitions" does not indicate that such disclosure in the "Implementation" section is not a definition.

[0027] All percentages expressed herein are by weight of the total weight of the composition, unless otherwise indicated. As used herein, "about", "approximately" and "substantially" should be understood to refer to numbers within a certain numerical range, such as within the range of -10% to +10% of the mentioned number, preferably -5% to +5% of the mentioned number, more preferably -1% to +1% of the mentioned number, and most preferably -0.1% to +0.1% of the mentioned number. All numerical ranges herein should be understood to include all integers or fractions within the range. In addition, these numerical ranges should be understood to provide support for claims involving any number or subset of numbers within the range. For example, the disclosure of 1 to 10 should be understood 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] As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a metabolite" or "the metabolite" includes one metabolite, but also includes two or more metabolites.

[0029] The words "including / comprising" will all be interpreted as inclusive rather than exclusive. Likewise, the terms "including / comprising" and "or" should all be considered inclusive unless the context clearly prohibits such an interpretation. However, the compositions disclosed herein may not contain any elements not specifically disclosed herein. Thus, disclosures of embodiments using the term "including / comprising" include disclosures of embodiments "consisting essentially of the specified components" and embodiments "consisting of the specified components."

[0030] As used herein, a "composition consisting essentially of a combination of calcium and oleuropein or at least one of its metabolites" does not include any additional compounds that affect mitochondrial calcium import other than the combination of calcium and oleuropein or at least one of its metabolites. In a specific non-limiting embodiment, the composition consists of an excipient and a combination of calcium and at least one of its metabolites.

[0031] 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". For example, "at least one of oleuropein or its metabolites" means "oleuropein" or "metabolites of oleuropein" or "both oleuropein and its metabolites".

[0032] As used herein, the terms "example" and "such as" (especially when followed by a list of terms) are exemplary and illustrative only and should not be considered exclusive or comprehensive. As used herein, "associated with" and "associated with" refer to co-occurrence, preferably to being caused by the same underlying condition, and most preferably to one of the identified conditions being caused by another identified condition.

[0033] The terms "food," "food product," and "food composition" mean a product or composition that is intended for ingestion by an individual (such as a human) and that provides at least one nutrient to the individual. The compositions of the present disclosure (including the various embodiments described herein) may comprise, consist of, or consist essentially of the following elements: the elements disclosed herein, as well as any additional or optional ingredients, components, or elements described herein or that can be used in a diet.

[0034] As used herein, the term "treatment" refers to applying the compositions disclosed herein to an individual with a condition to alleviate, reduce or improve at least one symptom associated with the condition and / or to slow down, reduce or block the progression of the condition. The term "treatment" includes both preventive or preventive treatment (preventing and / or delaying the development or progression of the target pathological condition or disorder), as well as curative, therapeutic or disease-modifying treatments, including therapeutic measures that cure, delay, alleviate symptoms of confirmed pathological conditions or disorders and / or interrupt their progression; and treating patients who are at risk of contracting or suspected of contracting a disease, as well as treating patients who are ill or have been diagnosed with a disease or medical condition. The term "treatment" does not necessarily mean that an individual is treated until full recovery. The term "treatment" also refers to health maintenance and / or promotion in individuals who do not suffer from a disease but may be prone to developing an unhealthy condition. The term "treatment" is also intended to include strengthening or otherwise enhancing one or more primary preventive or therapeutic measures. As a non-limiting example, treatment can be performed by a patient, a caregiver, a doctor, a nurse, or another healthcare professional.

[0035] Both human and veterinary treatments are within the scope of the present disclosure.Preferably, the combination of caffeine and at least one of oleuropein or a metabolite thereof is administered in an amount or unit dosage form that provides a therapeutically effective amount or a prophylactically effective amount of the combination.

[0036] The term "prevention" refers to administering a composition disclosed herein to an individual who does not show any symptoms of the condition to reduce or prevent the development of at least one symptom associated with the condition. In addition, "prevention" includes reducing the risk, incidence and / or severity of a condition or disorder.

[0037] As used herein, an "effective amount" is an amount that treats or prevents a defect, treats or prevents a disease or medical condition in an individual, or more generally, reduces symptoms, manages disease progression, or provides a nutritional, physiological or medical benefit to the individual.

[0038] The relative terms "improved," "increased," "enhanced," and the like refer to the effect of a composition disclosed herein (i.e., a composition comprising an effective amount of a combination of caffeine and at least one of oleuropein or a metabolite thereof) relative to administration of an otherwise identical composition without caffeine or one of oleuropein / oleuropein metabolites over the same period of time.

[0039] As used herein, "administering" includes another individual providing the referenced composition to an individual so that the individual can consume the composition, and also includes merely the act of the individual consuming the referenced composition himself.

[0040] "Animal" includes, but is not limited to, mammals, including, but not limited to, rodents; aquatic mammals; livestock, such as dogs, cats, and other pets; farm animals, such as sheep, pigs, cattle, and horses; and humans. When "animal," "mammal," or their plural forms are used, these terms also apply to any animal that is capable of having the effect indicated or intended to be indicated by the context of the paragraph, such as an animal that benefits from improved mitochondrial calcium import. Although the terms "individual" or "subject" are often used herein to refer to humans, the present disclosure is not limited thereto. Therefore, the terms "individual" or "subject" refer to any animal, mammal, or human that can benefit from the methods and compositions disclosed herein.

[0041] The term "pet" means any animal that can benefit from or enjoy the compositions provided by the present disclosure. For example, the pet can be an avian, bovine, canine, equine, feline, caprine, lupine, murine, ovine, or porcine animal, but the pet can be any suitable animal. The term "companion animal" means a dog or a cat.

[0042] In the case of humans, the term "old age" refers to an age of at least 60 years old since birth, preferably over 63 years old, more preferably over 65 years old, and most preferably over 70 years old. In the context of non-human animals, "old age" means a non-human individual that has reached 60% of its possible lifespan, at least 70%, at least 80% or at least 90% of its possible lifespan in some embodiments. The determination of lifespan can be based on mortality statistics, calculations, or estimates, and can take into account past, present and future influences or factors that are known to cause positive or negative effects on lifespan. When determining lifespan, species, sex, body size, genetic factors, environmental factors and stressors, current and past health status, past and present nutritional status, and consideration of stressors can be taken into account.

[0043] In the context of humans, the term "elderly" refers to an age of at least 45 years from birth, preferably over 50 years, more preferably over 55 years, and includes elderly individuals.

[0044] “Mobility” refers to the ability to move independently and safely from one place to another.

[0045] Sarcopenia is defined as the age-related loss of muscle mass and function, including muscle strength and gait speed.

[0046] As used herein, “frailty” is defined as a clinically recognizable state of increased vulnerability resulting from aging-related declines in reserve and function across multiple physiological systems, which impairs the ability to cope with daily or acute stressors. In the absence of definitive quantitative criteria, Fried et al operationally defined frailty as meeting three of five phenotypic criteria indicating impaired energy: (1) weakness (handgrip strength in the bottom 20% of the baseline population, adjusted for sex and body mass index), (2) poor endurance and energy (self-reported exertion versus maxima), (3) slowness (bottom 20% of the baseline population, based on time to walk 15 feet, adjusted for sex and standing height), (4) low physical activity (weighted fraction of kilocalories consumed per week at baseline, in the lowest quintile of physical activity determined for each sex; e.g., less than 383 kcal / week for men and less than 270 kcal / week for women), and / or unintentional weight loss (less than 10 pounds in the past year). Fried LP, Tangen CM, Walston J, et al., "Frailty in older adults: evidence for a phenotype." J. Gerontol. Biol. Sci. Med. Sci. 56(3):M146–M156 (2001). The presence of one or both of these criteria identifies the pre-frail stage as being at high risk for progression to frailty.

[0047] "Muscle fatigue" refers to a decrease in contractile force in one or more muscles due to insufficient substrate within the muscle fiber and / or accumulation of metabolites within the muscle fiber that interfere with the release of calcium or the ability of calcium to stimulate muscle contraction.

[0048] "Muscle weakness" is a condition in which the force exerted by a muscle is less than expected. The American Medical Research Council muscle strength scoring system is widely used to determine muscle weakness and its severity. Specifically, the examiner assesses the patient's ability to move a muscle against resistance, which is provided by the examiner who has empirically developed a feel for the expected normal range. This will vary from patient to patient, depending on the individual's basic physique and condition; a well-trained athlete can be expected to perform differently than an individual of smaller build, sedentary status, or deteriorating health. The expected force should also be adjusted for the degree of atrophy in patients with wasting disorders.

[0049] The patient's effort is scored on a scale of 0 to 5. As used herein, "muscle weakness" refers to any of the scores 0-4.

[0050] 5 points: The muscle contracts normally against all resistance.

[0051] 4 points: Muscle strength is reduced, but muscle contraction can still move the joint against resistance.

[0052] 3 points: Muscle strength is further reduced so that the joint can only be moved against gravity if the examiner's resistance is completely removed. For example, the elbow can be moved from full extension to full flexion starting with the arm hanging down at the side.

[0053] 2 points: The muscle can move only when gravity resistance is removed. For example, the elbow can be fully flexed only when the arm is maintained in a horizontal plane.

[0054] 1 point: Only a trace or flash of movement is seen or felt in the muscle, or a spontaneous contraction is observed in the muscle.

[0055] 0 points: No movement observed.

[0056] As used herein, an "athlete" is an individual who participates in at least one of 1) resistance exercise, 2) anaerobic or repetitive sprint-type exercise, or 3) endurance exercise.

[0057] Resistance exercise is when an individual undergoes explosive weight movements with long rest periods, and is driven primarily by the phospholotide and glycolytic energy systems. Resistance exercise produces energy quickly, but individuals fatigue quickly. The main adaptations include increased muscle mass (hypertrophy) by increasing muscle cross-sectional area through repeated weightlifting training. Hakkinen K. 1989. Neuromuscular and hormonal adaptations during strength and powertraining. J. Sports Med. Phys. Fitness. 29: 9-26; and Hakkinen K. et al. 1987. Relationships between training volume, physical performance capacity, and serum hormone concentrations during prolonged training in elite weightlifters. Int. J. Sports Med. 8 Suppl 1: 61-65.

[0058] Repeated full-speed running type training is anaerobic, involves high-intensity exercise with limited recovery periods, and involves almost pure carbohydrate metabolism, in which muscle glycogen is broken down in large quantities (glycolytic energy production). In these cases of anaerobic energy production, such as high-intensity speed training or exercise involving repeated sprints, the increased load on the muscle is achieved by increasing the firing of type IIa fibers. Finally, under very high workloads, type IIb glycolytic muscle fibers are activated to maintain high demands for energy supply via anaerobic energy supply. However, during these situations, the high rate of anaerobic energy production exceeds the rate that can be aerobic oxidized in the mitochondria, and this leads to the production of lactate at extreme levels present in these types of training situations. Spriet LL, Howlett RA, and Heigenhauser G J. 2000. An enzymatic approach to lactate production in human skeletal muscle during exercise. Med. Sci. Sports Exerc. 32: 756-763.

[0059] Endurance training is characterized by individuals performing low-intensity training for a long time (e.g., >15 minutes). The energy system representing endurance training includes an aerobic system that primarily uses aerobic metabolism of fat and carbohydrates to produce the required energy in the presence of sufficient oxygen in the mitochondria. The main adaptations include increasing muscle glycogen storage and glycogen saving under submaximal workloads via increased fat oxidation, increased lactic acid dynamics and morphological changes (including larger type I fibers per muscle area), and increased capillary and mitochondrial density. Holloszy JO and Coyle EF. 1984. Adaptations of skeletal muscle to endurance exercise and their metabolic consequences. J. Appl. Physiol. 56:831-838; exercise.Ann.NYAcad.Sci.301:440-450.

[0060] As used herein, the terms "portion" or "unit dosage form" are interchangeable and refer to physically discrete units suitable as unit dosages for human subjects and animal subjects, each unit preferably containing a predetermined amount of a composition comprising a combination of calcium and at least one of oleuropein or its metabolites as disclosed herein, in association with a pharmaceutically acceptable diluent, carrier or vehicle, in an amount sufficient to produce the desired effect. The specifications for the unit dosage form depend on the specific compound used, the effect to be achieved, and the pharmacodynamics associated with each compound in the host. In one embodiment, the unit dosage form can be a predetermined amount of liquid contained in a container such as a bottle.

[0061] An "oral nutritional supplement" or "ONS" is a composition comprising at least one macronutrient and / or at least one micronutrient, e.g., in sterile liquid, semisolid, or powdered form, and intended to supplement other nutritional intake, such as that from food. Non-limiting examples of commercially available ONS products include and In some embodiments, the ONS may be a beverage in liquid form that can be consumed without further addition of liquid, such as a certain amount of liquid in a serving of the composition.

[0062] As used herein, "incomplete nutrition" preferably refers to a nutritional product that does not contain sufficient amounts of macronutrients (protein, fat and carbohydrates) or micronutrients to be sufficient as a sole source of nutrition for the animal to which the nutritional product is administered. The term "complete nutrition" refers to a product that can be the sole source of nutrition for an individual. From a complete nutritional composition, an individual can obtain 100% of his nutritional requirements.

[0063] "Kit" means that the multiple components of the kit are physically associated in or with one or more containers and are considered a unit for manufacture, distribution, sale, or use. Containers include, but are not limited to, bags, boxes, cartons, bottles, outer packaging, shrink wrap, attached components (e.g., binding components, adhesive components, etc.), packaging of any type, design, or material, or a combination thereof.

[0064] Implementation

[0065] One aspect of the present disclosure is a method of achieving at least one result selected from the group consisting of: (i) improving mitochondrial calcium uptake in muscle cells, (ii) improving calcium utilization in muscle cells, (iii) increasing mitochondrial energy in muscle cells, (iv) improving at least one of muscle function, muscle performance, or muscle strength, (v) reducing muscle fatigue or weakness, (vi) enhancing mobility, and (vii) treating or preventing muscle diseases associated with calcium depletion or deficiency (e.g., reducing incidence and / or severity). The method comprises orally administering to an individual an effective amount of a combination of caffeine and at least one of oleuropein or a metabolite thereof.

[0066] Another aspect of the present disclosure is a method of treating in an individual in need thereof or preventing (e.g., reducing incidence and / or severity) in an individual at risk thereof at least one condition selected from (i) impairment of at least one of muscle function, muscle performance, or muscle strength, (ii) muscle fatigue or weakness, (iii) pre-frailty, weakness, sarcopenia, or impaired mobility, and (iv) muscle disease associated with calcium depletion or deficiency. The method comprises orally administering to an individual in need thereof or at risk thereof an effective amount of a combination of caffeine and at least one of oleuropein or its metabolites.

[0067] This also results in improved vitality and / or energy in the individual.

[0068] In another embodiment, reducing muscle fatigue is in an individual participating in exercise, the exercise comprising at least one of 1) resistance exercise, 2) anaerobic or repetitive sprinting type exercise, or 3) endurance exercise, the method comprising orally administering to the individual an effective amount of a combination of caffeine and at least one of oleuropein or its metabolites. The combination of caffeine and at least one of oleuropein or its metabolites is administered to the individual before, and / or during, and / or after exercise, preferably less than two hours before, and / or during, and / or less than two hours after exercise. In a more preferred embodiment, the combination of caffeine and at least one of oleuropein or its metabolites is administered to the individual less than one hour before exercise.

[0069] The effective amount of the combination of caffeine and oleuropein or at least one of its metabolites varies with the specific composition, the age and condition of the recipient, and the specific disorder or disease being treated. However, in general embodiments, 0.001 mg to 1.0 g of oleuropein or at least one of its metabolites may be administered to an individual per day, preferably 0.01 mg to 0.9 g of oleuropein or at least one of its metabolites per day, more preferably 0.1 mg to 750 mg of oleuropein or at least one of its metabolites per day, more preferably 0.5 mg to 500 mg of oleuropein or at least one of its metabolites per day, and most preferably 1.0 mg to 200 mg of oleuropein or at least one of its metabolites per day.

[0070] In one embodiment, at least a portion of oleuropein is obtained by extraction, for example by extraction from plants such as plants belonging to the family Oleaceae, preferably from plants belonging to the family Oleaceae such as olive (olive tree), plants of the genus Ligustrum, plants of the genus Syringa, plants of the genus Fraxinus, plants of the genus Jasminum and plants of the genus Oleaceae One or more of the stems, leaves, fruits or kernels. In addition or alternatively, at least a portion of oleuropein can be obtained by chemical synthesis.

[0071] Non-limiting examples of suitable metabolites of oleuropein include oleuropein aglycone, hydroxytyrosol, homovanillyl alcohol, isohomovanillyl alcohol, and mixtures thereof.

[0072] In some embodiments, oleuropein or at least one of its metabolites is the only polyphenol administered to the subject and / or the only polyphenol in a composition administered to the subject.

[0073] The effective amount of caffeine also varies with a particular composition, the age and condition of the recipient, and the particular disorder or disease being treated. However, in a general embodiment, up to 400 mg of caffeine may be administered to an individual every day, preferably from about 20 mg to about 380 mg every day, more preferably from about 40 mg to about 300 mg every day. Alternatively, caffeine may also be present in the composition in an amount of from about 0.05 g / portion to 0.3 g / portion, from about 0.1 g / portion to 0.2 g / portion, from about 0.125 g / portion to 0.175 g / portion, or from about 0.14 g / portion.

[0074] In some embodiments, a combination of caffeine and at least one of oleuropein or its metabolites is administered to an individual selected from the group consisting of: an aging individual; an elderly individual; an individual with muscle fatigue or weakness; an individual with impaired mobility; a frail individual; a pre-frail individual; a sarcopenic individual; an individual recovering from pre-frailty, weakness, sarcopenia, or impaired mobility; an individual undergoing physical rehabilitation (e.g., recovering from an injury to one or more of the muscles, bones, ligaments, or nervous systems); an athlete; and a pet. In some embodiments, the individual is healthy. In some embodiments, the individual suffers from sarcopenia, weakness, muscle fatigue, or weakness, or one or more of muscle function, muscle performance, or muscle strength is impaired, but is optionally otherwise healthy.

[0075] For example, a combination of caffeine and at least one of oleuropein or a metabolite thereof can be administered to an athlete before, during, and / or after exercise, e.g., less than two hours before exercise or less than one hour before exercise, and less than two hours after exercise or less than one hour after exercise.

[0076] In one embodiment, at least a portion of the muscle cells are part of a skeletal muscle selected from the group consisting of the gastrocnemius, tibialis, soleus, extensor digitorum longus (EDL), biceps femoris, semitendinosus, semimembranosus, gluteus maximus, and combinations thereof.

[0077] The combination of caffeine and at least one of oleuropein or its metabolites can be administered in any composition suitable for human and / or animal consumption. In a preferred embodiment, the combination of caffeine and at least one of oleuropein or its metabolites is administered to the individual orally or enterally (e.g., by tube feeding). For example, the combination of caffeine and at least one of oleuropein or its metabolites can be administered to the individual in a beverage, food product, capsule, tablet, powder, or suspension.

[0078] Non-limiting examples of suitable compositions include food compositions, dietary supplements, dietary supplements (e.g., liquid ONS), complete nutritional compositions, beverages, medicaments, nutraceuticals, powdered nutritional products to be reconstituted with water or milk before consumption, food additives, pharmaceuticals, drinks, pet foods, and combinations thereof.

[0079] Food products according to the invention may include dairy products, such as fermented milk products, e.g. yoghurt, buttermilk etc.; ice cream; condensed milk; milk; dairy cream; flavoured milk drinks; whey-based drinks; toppings; coffee creamer; chocolate; cheese-based products; soups; sauces; purees; condiments; puddings; custards; baby foods; nutritional formulas, such as those for complete nutrition, e.g. for infants, children, teenagers, adults, the elderly or the critically ill; e.g. cereals and cereal bars.

[0080] The drinks may include, for example, milk or yoghurt based drinks, fermented milks, protein drinks, coffee, tea, energy drinks, soy drinks, fruit and / or vegetable drinks, fruit and / or vegetable juices.

[0081] The combination of caffeine and at least one of oleuropein or a metabolite thereof may be administered in a food product further comprising a component selected from the group consisting of protein, carbohydrate, fat, and mixtures thereof.

[0082] In some cases where oral or enteral administration is not possible or advisable, the composition may be administered parenterally.

[0083] Preferably, the muscle function that can be improved by the methods disclosed herein comprises a characteristic selected from the group consisting of muscle strength, walking speed, and combinations thereof.Muscle function is generally defined as strength per unit of vertical skeletal muscle mass or per muscle volume.

[0084] Non-limiting examples of muscle diseases associated with calcium depletion or deficiency that can be treated by the methods disclosed herein include muscular dystrophy, congenital core myopathies, and mitochondrial myopathies. Specific non-limiting examples include: Barth syndrome; chronic progressive external ophthalmoplegia (cPEO); Kearns-Sayre syndrome (KSS); Leigh syndrome; mitochondrial DNA depletion syndrome (MDDS); mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS); mitochondrial neurogastrointestinal encephalomyopathy (MNGIE); myoclonic epilepsy with ruffled red fibers (MERRF); neuropathy, ataxia, and retinitis pigmentosa (NARP); and Pearson syndrome.

[0085] The individual may be at risk for a disorder or condition (e.g., sarcopenia, weakness, muscle fatigue or weakness, or one or more of impaired muscle function, muscle performance, or muscle strength), in which case an effective amount of the composition is a preventive effective dose; or the individual may suffer from a disorder or condition, in which case an effective amount of the composition is a therapeutically effective dose. In some embodiments, the method includes identifying the individual as suffering from or at risk for the condition prior to administration.

[0086] In another embodiment, the present disclosure provides a method for treating or preventing impaired mobility in the elderly. The method comprises orally administering to the elderly an effective amount of a combination of caffeine and at least one of oleuropein or its metabolites. The elderly may be elderly individuals. In some embodiments, the elderly suffer from a condition selected from the following: frailty, pre-frailty, sarcopenia, recovery from sarcopenia, osteoporosis, osteoarthritis, malnutrition, risk of malnutrition, undergoing rehabilitation, planning to undergo rehabilitation within the next year, and a combination thereof.

[0087] The composition can be administered to the elderly in an amount sufficient to prevent, at least partially reduce the risk of developing frailty or sarcopenia, and / or at least partially reduce the severity of pre-frailty, frailty, sarcopenia or impaired mobility if the condition has not yet developed in the individual. Such an amount is defined as a "prophylactically effective dose". Again, the exact amount depends on a variety of factors related to the individual, such as their weight, health status, and degree of loss of muscle function (e.g., muscle strength, walking speed, etc.).

[0088] In one embodiment, the combination of caffeine and at least one of oleuropein or its metabolites is administered to an individual for a period of at least one month; preferably at least two months, more preferably at least three, four, five, or six months; and most preferably at least one year. During this period, the combination of caffeine and at least one of oleuropein or its metabolites may be administered to the individual at least one day per week; preferably at least two days per week, more preferably at least three, four, five, or six days per week; and most preferably seven days per week. The combination of caffeine and at least one of oleuropein or its metabolites may be administered in a single dose per day or in multiple separate doses per day.

[0089] The above application examples do not require continuous daily application without interruption. Instead, there may be some brief interruptions in the application, such as two to four days of interruption during the application. The ideal duration of application of the composition can be determined by those skilled in the art.

[0090] In one embodiment, caffeine and at least one of oleuropein or a metabolite thereof may be administered in the same composition, such as a unit dosage form containing both caffeine and at least one of oleuropein or a metabolite thereof.

[0091] In an alternative embodiment, caffeine and at least one of oleuropein or its metabolites may be administered sequentially in separate compositions. The term "sequentially" means that caffeine and at least one of oleuropein or its metabolites are administered in a sequential manner, such that at least one of oleuropein or its metabolites is administered without caffeine at a first time, and caffeine is administered without at least one of oleuropein or its metabolites at a second time (before or after the first time). The time between sequential administrations can be, for example, one second or seconds, one minute or minutes, or one hour or hours in the same day; one day or days or one week or weeks in the same month; or one or more months in the same year.

[0092] Another aspect of the present disclosure is a method of preparing a composition for achieving an effect selected from the group consisting of: (i) improving mitochondrial calcium uptake in muscle cells, (ii) improving calcium utilization in muscle cells, (iii) increasing mitochondrial energetics in muscle cells, (iv) improving at least one of muscle function, muscle performance, or muscle strength, (v) reducing muscle fatigue, (vi) enhancing mobility, and (vii) treating muscle diseases associated with calcium depletion or deficiency.

[0093] The method comprises adding a combination of caffeine and at least one of oleuropein or its metabolites to an ingredient selected from the group consisting of proteins, carbohydrates, lipids, and combinations thereof. The composition (e.g., food product) can be prepared prior to administration (e.g., the composition is prepared, packaged, and then purchased by a consumer who administers the composition to themselves or another individual), or can be prepared substantially simultaneously with administration (the composition is prepared by an individual who administers the composition to themselves or another person less than 30 minutes prior to administration, preferably less than 15 minutes prior to administration, more preferably less than 10 minutes prior to administration, and most preferably less than 5 minutes prior to administration).

[0094] The composition may include an effective amount of a combination of caffeine and at least one of oleuropein or a metabolite thereof. For example, a single serving or single dose of the composition may include an effective amount of the combination, and the package may include one or more servings, or one or more doses.

[0095] The composition may comprise a food additive selected from the group consisting of acidulants, thickeners, buffers or agents for pH adjustment, chelating agents, colorants, emulsifiers, excipients, flavoring agents, minerals, osmotic agents, pharmaceutically acceptable carriers, preservatives, stabilizers, sugars, sweeteners, texturizers, vitamins, minerals, and combinations thereof.

[0096] In addition to the combination of caffeine and at least one of oleuropein or its metabolites, the composition may further comprise a protein source from animal or plant origin, such as milk protein, soy protein and / or pea protein. In a preferred embodiment, the protein source is selected from the group consisting of whey protein; casein protein; pea protein; soy protein; wheat protein; corn protein; rice protein; protein from legumes, cereals and grains; and combinations thereof. Additionally or alternatively, the protein source may comprise protein from nuts and / or seeds.

[0097] The protein source preferably comprises whey protein. The whey protein may be unhydrolyzed or hydrolyzed whey protein. The whey protein may be any whey protein, for example, the whey protein may be selected from whey protein concentrate, whey protein isolate, whey protein micelles, whey protein hydrolysates, acid whey, sweet whey, modified sweet whey (sweet whey from which casein-glycomacropeptide has been removed), fractions of whey protein and any combination thereof. In a preferred embodiment, the whey protein comprises whey protein isolate and / or modified sweet whey.

[0098] As mentioned above, the protein source may be from an animal or plant source, such as milk protein, soy protein and / or pea protein. In one embodiment, the protein source comprises casein. Casein may be obtained from any mammal, but is preferably obtained from cow's milk, and is preferably micellar casein.

[0099] The composition may include one or more branched-chain amino acids. For example, the composition may include leucine, isoleucine and / or valine. The protein source in the composition may include leucine in free form and / or leucine (such as dairy, animal or plant protein) bound to peptides and / or proteins. In one embodiment, the composition includes leucine in an amount up to 10% by weight of the dry matter of the composition. Leucine can be present in the form of D-leucine or L-leucine, preferably in the L-form. If the composition includes leucine, the composition can be administered in a daily dose of 0.01 to 0.04 g leucine per kg body weight, preferably 0.02 to 0.035 g leucine per kg body weight. Such dosages are particularly suitable for complete nutritional compositions, but ordinary technicians will easily recognize how to adjust these dosages for oral nutritional supplements (ONS).

[0100] One or more other minerals may also be used in the composition. Non-limiting examples of suitable minerals include calcium, boron, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, selenium, silicon, tin, vanadium, zinc, and combinations thereof.

[0101] One or more other vitamins other than any may be used in the composition. Non-limiting examples of suitable vitamins include vitamin A, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin or niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxal, pyridoxamine or pyridoxine hydrochloride), vitamin B7 (biotin), vitamin B9 (folic acid) and vitamin B12 (various cobalamines; typically cyanocobalamin in vitamin supplements), vitamin C, vitamin D, vitamin E, vitamin K, folic acid, biotin) and combinations thereof. "Vitamins" include such compounds, provitamins, derivatives thereof, and analogs thereof, obtained naturally from plant and animal foods or prepared synthetically.

[0102] The composition may also include carbohydrate and / or fat sources. Non-limiting examples of suitable fats include canola oil, corn oil and high oleic sunflower oil. Non-limiting examples of suitable carbohydrates include sucrose, lactose, glucose, fructose, corn syrup solids, maltodextrin and mixtures thereof. In addition or alternatively, dietary fiber may be added. Dietary fiber passes through the small intestine without being digested by enzymes and functions as a natural filler and laxative. Dietary fiber may be soluble or insoluble, and generally speaking a mixture of two types is preferred. Non-limiting examples of suitable dietary fiber include soybean, pea, oat, pectin, guar gum, partially hydrolyzed guar gum, gum arabic, oligofructose, acid oligosaccharides, oligogalactose, sialyllactose and oligosaccharides from animal milk. A preferred fiber mixture is a mixture of inulin and shorter chain oligofructose. In one embodiment, the fiber content is between 2 and 40 g / L composition, for example, between 4 and 10 g / L.

[0103] One or more food grade emulsifiers such as diacetyl tartaric acid esters of mono- and di-glycerides, lecithin and / or mono- and di-glycerides may be incorporated into the composition. Suitable salts and stabilizers may also be included.

[0104] Example

[0105] The following non-limiting examples provide experimental data supporting the compositions and methods disclosed herein.

[0106] Example 1

[0107] To test the synergistic effect of the combined effect of oleuropein + caffeine on mitochondrial calcium elevation, the inventors measured mitochondrial calcium elevation in myotubes differentiated from C2C12 cells. C2C12 cells were purchased from ATCC. C2C12 cells were seeded in DMEM high glucose (Gibco) + 10% fetal bovine serum in 96-well plates at a density of 8000 cells / well. Myotubes were differentiated from C2C12 cells by growing the cells in DMEM containing 2% horse serum for 4 days. Mitochondrial calcium measurements were performed using myotubes infected with an adenovirus (from Sirion biotech) expressing a mitochondrial-targeted calcium sensor mitochondrial mutant aequorin (Montero et al., 2004). For aequorin reconstruction, 24 hours after infection, myotubes were incubated for 2 hours at room temperature (22 ± ℃) in standard culture medium (145mM NaCl, 5mM KCl, 1mM MgCl2, 1mM CaCl2, 10mM glucose and 10mM Hepes, pH7.4) with 1μM wild-type coelenterazine. For treatment, the compound was directly added to cell culture or myotube culture 2 hours before measurement. Luminescence was measured on FLIPR Tetra Aequorin (Molecular Devices). Luminescence data were calibrated to calcium concentration (Alvarez & Montero, 2002) using the algorithm as described above. Customized module analysis based on Excel (Microsoft) and GhaphPad Prism 7.02 (GraphPad) software was used for quantitative analysis.

[0108] like Figure 1 As shown in , simultaneous supplementation with oleuropein aglycone and caffeine induced mitochondrial activation (measured as elevation in mitochondrial calcium) greater than the effect of each compound alone.

[0109] As the Lord Figure 2 To calculate the synergistic effect of oleuropein aglycone + caffeine, the theoretical effect of the sum of two different compounds (oleuropein aglycone and caffeine) was compared with the measured effect of the combination. Figure 2 As shown, oleuropein acts synergistically with caffeine to promote mitochondrial calcium elevation during stimulation.

[0110] References

[0111] Glancy, B. and RS Balaban (2012). "Role of mitochondrial Ca2+ in theregulation of cellular energetics." Biochemistry, 51(14), 2959-2973.

[0112] Alvarez,J.,&Montero,M.(2002).Measuring[Ca2+] in the endoplasmicreticulum with aequorin.Cell Calcium,32(5-6),251-260.

[0113] Montero, M., Lobaton, CD, Hernandez-Sanmiguel, E., Santodomingo, J., Vay, L., Moreno, A., & Alvarez, J. (2004). Direct activation of the mitochondrial calcium uniporter by natural plant flavonoids. Biochem J, 384 (Pt 1),19-24.doi:10.1042 / BJ20040990.

[0114] It should be understood that various changes and modifications made to the presently preferred embodiments described herein will be apparent to those skilled in the art. These changes and modifications may be made without departing from the spirit and scope of the subject matter of the present invention and without diminishing its intended advantages. Therefore, such changes and modifications are intended to be covered by the appended claims.

Claims

1. A method for achieving at least one result selected from the group consisting of: (i) improving mitochondrial calcium uptake in muscle cells, (ii) improving calcium utilization in muscle cells, (iii) increasing mitochondrial energetics in muscle cells, (iv) improving at least one of muscle function, muscle performance, or muscle strength, (v) reducing muscle fatigue, (vi) enhancing mobility, and (vii) treating a muscle disease associated with calcium depletion or deficiency, the method comprising orally administering to a subject an effective amount of a combination of caffeine and at least one of oleuropein or a metabolite thereof.

2. The method of claim 1, wherein the individual is selected from the group consisting of: aging individuals; elderly individuals; individuals with muscle fatigue or weakness; individuals with impaired mobility; frail individuals; pre-frail individuals; sarcopenic individuals; individuals recovering from pre-frailty, frailty, sarcopenia or impaired mobility; individuals undergoing physical rehabilitation; athletes; and pets.

3. The method of claim 1, wherein reducing muscle fatigue is in an individual participating in 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 individual an effective amount of a combination of caffeine and at least one of oleuropein or a metabolite thereof.

4. The method of claim 3, wherein the combination of caffeine and at least one of oleuropein or a metabolite thereof is administered to the individual before the exercise, and / or during the exercise, and / or after the exercise.

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

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

7. The method according to any one of the 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.

8. The method according to any one of the preceding claims, wherein the combination of caffeine and at least one of oleuropein or its metabolites is administered in the form of a composition selected from the group consisting of a food composition, a dietary supplement, a nutritional composition, a beverage, a nutraceutical, a powdered nutritional product to be reconstituted with water or milk before consumption, a food additive, a medicament, a drink, a pet food, and combinations thereof.

9. The method according to any one of the preceding claims, wherein the caffeine is administered together with oleuropein or at least one of its metabolites in the same composition.

10. The method according to any one of the preceding claims, wherein the caffeine and at least one of oleuropein or a metabolite thereof are administered separately in different compositions.

11. The method according to any one of the preceding claims, wherein the caffeine is administered together with oleuropein or at least one of its metabolites in the form of a food product further comprising a component selected from the group consisting of protein, carbohydrates, fat and mixtures thereof.

12. A method for treating in an individual in need thereof or preventing in an individual at risk thereof at least one condition selected from: (i) impaired muscle function, muscle performance or muscle strength, (ii) muscle fatigue or weakness, (iii) pre-frailty, weakness, sarcopenia or impaired mobility, and (iv) muscle diseases associated with calcium depletion or deficiency, the method comprising orally administering to the individual in need thereof or at risk thereof an effective amount of a combination of caffeine and at least one of oleuropein or its metabolites.

13. A unit dosage form comprising a combination of caffeine and at least one of oleuropein or a metabolite thereof, the unit dosage form comprising a combined amount effective for at least one outcome selected from: (i) improving mitochondrial calcium uptake in muscle cells, (ii) improving calcium utilization in muscle cells, (iii) increasing mitochondrial energetics in muscle cells, (iv) improving at least one of muscle function, muscle performance or muscle strength, (v) reducing muscle fatigue, (vi) enhancing mobility, and (vii) treating muscle diseases associated with calcium depletion or deficiency.

14. The unit dosage form of claim 13, consisting essentially of said combination of caffeine and at least one of oleuropein or a metabolite thereof.

15. The unit dosage form according to claim 13 or 14, consisting of an excipient and said combination of caffeine and at least one of oleuropein or a metabolite thereof.

16. A method of preparing a composition for achieving at least one result selected from the group consisting of: (i) improving mitochondrial calcium uptake in muscle cells, (ii) improving calcium utilization in muscle cells, (iii) increasing mitochondrial energetics in muscle cells, (iv) improving at least one of muscle function, muscle performance, or muscle strength, (v) reducing muscle fatigue or weakness, (vi) enhancing mobility, and (vii) treating muscle diseases associated with calcium depletion or deficiency, the method comprising adding an effective amount of caffeine in combination with at least one of oleuropein or a metabolite thereof to at least one ingredient selected from the group consisting of protein, carbohydrates, and fat.

17. The method of claim 16, further comprising adding to the at least one ingredient a food additive selected from the group consisting of acidulants, thickeners, buffers or agents for pH adjustment, chelating agents, colorants, emulsifiers, excipients, flavoring agents, minerals, osmotic agents, pharmaceutically acceptable carriers, preservatives, stabilizers, sugars, sweeteners, texturizers, vitamins, minerals, and combinations thereof.

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