Compositions and methods using a combination of oleuropein or metabolite thereof and adenosylcobalamin

Abstract

The present invention relates to methods and compositions comprising a combination of oleuropein and / or metabolite thereof and adenosylcobalamin in an effective amount for use in achieving at least one result selected from the group consisting of i) preventing and / or treating a mitochondria-related disease or condition associated with altered mitochondrial function and / or ii) at least one physical state selected from the group consisting of oxidative stress or a condition associated with oxidative stress in an individual; (iii) increasing mitochondrial energy, cellular energy, mitochondrial function and mitochondrial calcium uptake in one or more cells, (iv) increasing resistance to age-related pathologies; (v) improving a physiological state or disorder related to cell ageing or metabolic fatigue in one or more cells; (vi) improving mobility and / or (vii) improving healthspan and / or lifespan in an individual.

Description

TITLECOMPOSITIONS AND METHODS USING A COMBINATION OF OLEUROPEIN OR METABOLITE THEREOF AND ADENOSYLCOBALAMINTECHNICAL FIELD

[0001] The present disclosure generally relates to compositions and methods that manage energy at a cellular level, treat or prevent a mitochondria-related disease or condition associated with altered mitochondrial function, oxidative stress or a condition associated with oxidative stress.BACKGROUND

[0002] Population aging has been a remarkable demographic event. As the growth of the older population has outpaced the total population due to increased longevity, the proportion of older persons relative to the rest of the population has increased considerably due to decreased fertility rates. For example, one in every twelve individuals was at least 60 years of age in 1950, and one in every ten was aged 60 years or older by the end of 2000. By the end of 2050, the number of persons worldwide that is 60 years or over is projected to be one in every five.

[0003] Mitochondrial dysfunction, oxidative stress, altered intercellular communication (including chronic low-grade inflammation), genomic instability, telomere attrition, loss of proteostasis, altered nutrient sensing, epigenetic alterations, and stem cell exhaustion have been proposed as hallmarks of aging. Moreover, free radicals - reactive oxygen species (ROS)- are the main origin of aging by causing oxidative cellular injuries. Free radicals are necessary for many biochemical processes and they are produced as by-products during some biochemical reactions or as substrates for other biochemical reactions in each cell. As mitochondria are the principal source of intracellular reactive oxygen species (ROS), this hypothesis suggested a central role for the mitochondrion in normal mammalian aging. In recent years, however, much work has questioned the importance of mitochondrial ROS in driving aging. Conversely new evidence points to other facets of mitochondrial dysfunction which may nevertheless suggest the mitochondrion retains a critical role at the center of a complex web of processes leading to cellular and organismal aging.

[0004] Altered mitochondrial calcium uptake can lead to increased production of reactive oxygen species (ROS) within the mitochondria. This increase in ROS production is often associated with oxidative stress. Excessive calcium accumulation in the mitochondria can also disrupt the electron transport chain and impair oxidative phosphorylation, leading to mitochondrial dysfunction. This dysfunction further contributes to the generation of ROS and oxidative stress. Prolonged oxidative stress can lead to cellular damage and dysfunction in various tissues and organs. This can have implications for the development and progression of numerous diseases, including neurodegenerative disorders, cardiovascular diseases, and metabolic disorders.

[0005] Examples include atherosclerosis, Parkinson's disease, heart failure, myocardial infarction, Alzheimer's disease, schizophrenia, bipolar disorder, fragile X syndrome, and chronic fatigue syndrome.

[0006] Oxidative stress contributes to tissue injury following irradiation and hyperoxia. It is suspected to be important in neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease. Oxidative stress is also thought to be linked to certain cardiovascular diseases, since oxidation of low-density lipoprotein (LDL) in the vascular endothelium is a precursor to plaque formation. Oxidative stress also plays a role in the ischemic cascade due to oxygen reperfusion injury following hypoxia. This cascade includes both strokes and heart attacks. Oxidative stress has also been implicated in chronic fatigue syndrome.

[0007] Aged or aging individuals frequently suffer some degree of physical decline and / or cognitive impairment, including decline in cognitive function, that progresses with age, and age-related changes in brain morphology and cerebrovascular function are commonly observed. Cognitive decline has been consistently reported with aging across a range of cognitive domains including processing speed, attention, episodic memory, spatial ability and executive function. Brain imaging studies have revealed that these normal age-related cognitive declines are associated with decreases in both grey and white matter volume in the brain, with the fronto-striatal system most heavily compromised with aging. These decreases in cortical volume can be attributed to a number of detrimental cellular processes involved with normal aging, such as accumulation of damage by free radicals over time leading to oxidative damage, chronic low-grade inflammation, homocysteine accumulation (which when elevated are a risk factor for cognitive impairment and dementia), and decreased mitochondrial efficiency. In addition to directcellular damage, the brain is also indirectly impaired by insults to micro-vascular structures. It is evident that the pathology of aging and also dementia involves a complexity of these interacting factors which are linked together. For example, mitochondrial dysfunction leads to increased oxidative stress, and oxidative stress can trigger inflammation and vascular insults.

[0008] 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 contributes to the orchestration of cellular metabolic homeostasis. (Glancy, B. et al. (2012). "Role of mitochondrial Ca2+ in the regulation of cellular energetics." Biochemistry 51(14): 2959-2973). Alterations in mitochondrial Ca2+ homeostasis have been linked to a variety of pathological conditions and are critical in the etiology of several human diseases (Arduino et al. Journal Physiol. 2018 Jul; 596(14):2717-2733).

[0009] Nutrition, education, physical exercise and cognitive exercise have been recently demonstrated as possible intervention to prevent physical and cognitive decline with aging. An abundance of clinical, epidemiological, and individual evidence is in favor of individual nutritional factors that reduce dementia risk and age-related neurodegeneration. However, formal trial testing of nutritional interventions has yielded mixed results (Schmitt et al., Nutrition Reviews 68: S2-S5 (2010). Moreover, there are very limited solutions to increase mitochondrial calcium uptake and thereby modulate bioenergetics through natural bioactives and vitamins in conditions of restrictions of the bioavailability of some of them.

[0010] Thus, there is a strong need to provide compositions and methods that can boost mitochondrial function and increase bioenergetics through activation of the mitochondrial calcium elevation that promote cellular activation, and thereby treat or prevent conditions and diseases linked to mitochondrial dysfunction.SUMMARY

[0011] As detailed in the experimental data set forth later herein, the inventors surprisingly found that the combination of compounds disclosed herein is able to activate the mitochondrial calcium elevation that promotes cellular activation by boosting mitochondrial function and increasing bioenergetics.

[0012] It could support mitochondrial function in oxidative stress conditions, such as aging, exercise, musculo-skeletal diseases, respiratory diseases, pain syndromes, neurodegenerative diseases and metabolic diseases. In particular, by reducing oxidative stress and improving mitochondrial function, the present composition can treat, reduce incidence of, or reduce severity of metabolic and degenerative diseases at least with a combined effect, possibly potentiating each other or providing synergy.

[0013] Reduction of oxidative stress and improvement of mitochondrial function are mechanistically linked. Mitochondrial dysfunction contributes to cellular damage, partially through reactive oxygen species (ROS) and metabolic derangements, by not being able to metabolize nutrients, in turn leading to metabolic and degenerative diseases.

[0014] Moreover, 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 composition according to the present invention improves mitochondrial function, which in turn can increase muscle energy metabolism to thereby improve cellular energy and mobility.

[0015] Accordingly, in a general embodiment, the present disclosure provides a composition comprising a combination of oleuropein and / or metabolite thereof and adenosylcobalamin in an effective amount for use in achieving at least one result selected from the group consisting of i) preventing and / or treating a mitochondria-related disease or condition associated with altered mitochondrial function and / or ii) at least one physical state selected from the group consisting of oxidative stress or a condition associated with oxidative stress in an individual; (iii) increasing mitochondrial energy, mitochondrial function and mitochondrial calcium uptake in one or more cells, (iv) increasing resistance to age-related pathologies; (v) improving a physiological state or disorder related to cell ageing or metabolic fatigue in one or more cells; (vi) improving mobility and / or (vii) improving healthspan and / or lifespan in an individual.

[0016] In an embodiment, the mitochondria-related disease or condition is selected from the group consisting of stress, obesity, reduced metabolic rate, metabolic syndrome, diabetes mellitus, complications from diabetes, cardiovascular disease, hyperlipidemia, respiratory diseases, pain syndromes, neurodegenerative disease, cognitive disorder, stress-induced or stress-related cognitive dysfunction, mood disorder, anxiety disorder, age-related neuronal death or dysfunction,musculo-skeletal disorder, muscle aging, sarcopenia, frailty, pre-frailty, chronic kidney disease, macular degeneration, and combinations thereof.

[0017] In an embodiment, the at least one physical state is selected from the group consisting of deleterious effects of aging, muscle loss, pre-diabetes, gestational diabetes, type I diabetes, type II diabetes, complications from diabetes, insulin resistance, metabolic syndrome, dyslipidemia, overweight, obesity, raised cholesterol levels, raised triglyceride levels, elevated fatty acid levels, fatty liver disease, renal disease, cardiovascular disease, musculo-skeletal diseases, respiratory diseases, pain syndromes, neurodegenerative disease, impaired cognitive function, myopathy such as statin-induced myopathy, non-alcoholic steatohepatitis, tinnitus, dizziness, alcohol hangover, hearing impairment, osteoporosis, hypertension, atherosclerosis / coronary artery disease, myocardial damage after stress, traumatic brain injury, cystic fibrosis, inflammation, cancer, and HIV infection.

[0018] In an embodiment, at least a portion of the one or more cells are part of at least one body part selected from the group consisting of a liver, a kidney, a brain, and a skeletal muscle.

[0019] In an embodiment, the physiological state or disorder related to cell ageing or metabolic fatigue comprises muscle fatigue or weakness, lack of energy, physical energy, lack of vitality or weakness.

[0020] In an embodiment, the metabolite of oleuropein, including conjugated metabolites, 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.

[0021] In an embodiment, the individual is selected from the group consisting of an adult, 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; a sportsman; and a pet.

[0022] In an embodiment, the composition according to this invention is administered orally.

[0023] In an embodiment, the combination is administered in a composition selected from the group consisting of a food product, a food for special medical purposes (FSMP), a nutritional supplement, a dairy-based drink, a low-volume liquid supplement, a meal replacement beverage, and combinations thereof.

[0024] The present invention also relates to a composition comprising a combination of oleuropein and / or metabolite thereof and adenosylcobalamin in an effective amount for use in delaying off-set of metabolic decline, decreasing oxidative stress, improving or maintaining muscle mass and function, immune function and / or cognitive function in an individual.

[0025] The present invention also relates to a composition comprising a combination of oleuropein and / or metabolite thereof and adenosylcobalamin in an effective amount for use in i) mitigating deleterious effects of aging, ii) improving at least one of muscle performance or muscle recovery from exercise, exercise capacity and / or physical function, iii) reducing severity of metabolic and / or degenerative diseases in an individual.

[0026] The present invention also relates to a method of manufacturing a nutritional composition for use according to any of the preceding claims, comprising the steps of providing one or more ingredients for a nutritional composition, oleuropein and / or metabolite thereof and adenosylcobalamin, and mixing.

[0027] The present invention also relates to a method of achieving at least one result selected from the group consisting of i) preventing and / or treating a mitochondria-related disease or condition associated with altered mitochondrial function and / or ii) at least one physical state selected from the group consisting of oxidative stress or a condition associated with oxidative stress in an individual; (iii) increasing mitochondrial energy, cellular energy, mitochondrial function and mitochondrial calcium uptake in one or more cells, (iv) increasing resistance to age-related pathologies; (v) improving a physiological state or disorder related to cell ageing or metabolic fatigue in one or more cells; (vi) improving mobility and / or (vii) improving healthspan and / or lifespan in an individual, the method comprising orally administering to the individual in need thereof or at risk thereof an effective amount of the combination of oleuropein and / or metabolite thereof and adenosylcobalamin.

[0028] In an embodiment, the combination of oleuropein and / or metabolite thereof and adenosylcobalamin is administered in a composition selected from the group consisting of food compositions, dietary supplements, nutritional compositions, beverages, nutraceuticals, powdered nutritional products to be reconstituted in water or milk before consumption, food additives, medicaments, drinks, petfood, and combinations thereof.

[0029] In an embodiment, the method comprises administering the combination of adenosylcobalamin and the metabolite of oleuropein selected from the group consisting ofoleuropein aglycone, hydroxytyrosol, homovanillyl alcohol, isohomovanillyl alcohol, glucuronidated forms thereof, sulfated forms thereof, derivatives thereof including conjugated metabolites, and mixtures thereof.

[0030] The present invention also relates to a method of treating in an individual in need thereof or preventing in an individual at risk thereof 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 comprising orally administering to the individual in need thereof or at risk thereof an effective amount of the combination of oleuropein and / or metabolite thereof and adenosylcobalamin.

[0031] The present invention also relates to a unit dosage form comprising a combination of oleuropein and / or metabolite thereof and adenosylcobalamin, the unit dosage form comprises an amount of the combination effective for at least one result selected from the group consisting of i) preventing and / or treating a mitochondria-related disease or condition associated with altered mitochondrial function and / or ii) at least one physical state selected from the group consisting of oxidative stress or a condition associated with oxidative stress in an individual; (iii) increasing mitochondrial energy, mitochondrial function and mitochondrial calcium uptake in one or more cells, (iv) increasing resistance to age-related pathologies; (v) improving a physiological state or disorder related to cell ageing or metabolic fatigue in one or more cells; (vi) improving mobility and / or (vii) improving healthspan and / or lifespan in an individual.

[0032] In an embodiment, the unit dosage form of consists essentially of the combination of oleuropein and / or metabolite thereof and adenosylcobalamin.

[0033] Additional features and advantages are described herein and will be apparent from the following Detailed Description.BRIEF DESCRIPTION OF THE FIGURES

[0034] FIG. 1 is a graph showing oleuropein (aglycone form) synergies with adenosylcobalamin to activate mitochondria, via mitochondrial Ca2+ rise, in C2C12-derived myotubes. The bar chart shows that the effect of the combination of Oleuropein aglycone (10 pM) and adenosylcobalamin (0.9 pM) is greater than the effect of oleuropein aglycone (10 pM,grey) alone or adenosylcobalamin (0.9 pM, black) alone on the integrated mitochondrial calcium rise, evoked by 5mM caffeine. Results are expressed as mean + / - SEM (Standard error of the mean) from n = 27 myotubes preparations per condition, indicates statistically significant difference at P < 0.05 (one-way ANOVA test).

[0035] FIG. 2 is a graph showing that oleuropein (aglycone form) synergizes with vitamin adenosylcobalamin to activate mitochondria, via mitochondrial Ca2+ rise, in C2C12-derived myotubes. To quantify the synergistic effect of the combination of oleuropein aglycone and adenosylcobalamin on mitochondrial activation, the expected theoretical effect (sum between oleuropein aglycone effect and adenosylcobalamin effect, extracted from the data in fig.l) and the real measured effect of the combination (oleuropein aglycone and adenosylcobalamin, extracted from data in fig. 1) were compared. Results are expressed as mean + / - SEM (Standard error of the mean), from n = 27 myotubes preparations, indicates statistically significant difference of the measured vs. theoretical difference in mitochondrial calcium at P < 0.05 (Student’s test).DETAILED DESCRIPTION

[0036] Definitions

[0037] Some definitions are provided hereafter. Nevertheless, definitions may be located in the “Embodiments” section below, and the above header “Definitions” does not mean that such disclosures in the “Embodiments” section are not definitions.

[0038] All percentages expressed herein are by weight of the total weight of the composition unless expressed otherwise. 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. 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.

[0039] 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 two or more metabolites.

[0040] 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. Nevertheless, the compositions disclosed herein may lack any element that is not specifically disclosed herein. Thus, a disclosure of an embodiment using the term “comprising” includes a disclosure of embodiments “consisting essentially of’ and “consisting of’ the components identified.

[0041] As used herein, a “composition consisting essentially of a combination” does not include any additional compound that affects mitochondrial calcium import other than the combination of oleuropein or metabolite thereof and adenosylcobalamin. In a particular non-limiting embodiment, the composition consists of an excipient and the combination of adenosylcobalamin and oleuropein or metabolite thereof.

[0042] 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, “oleuropein or metabolite thereof’ means “oleuropein,” or “a metabolite of oleuropein,” or “both oleuropein and a metabolite thereof.”

[0043] Where used herein, the terms “example” and “such as,” particularly when followed by a listing of terms, are merely exemplary and illustrative and should not be deemed to be exclusive or comprehensive. As used herein, “associated with” and “linked with” mean occurring concurrently, preferably means caused by the same underlying condition, and most preferably means that one of the identified conditions is caused by the other identified condition.

[0044] 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 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 elements disclosed herein, as well as any additional or optional ingredients, components, or elements described herein or otherwise useful in a diet.

[0045] As used herein, the terms “treat” and "treatment" mean to administer a composition as disclosed herein to a subject having a condition in order to lessen, 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 terms “treatment” and “treat” include both prophylactic or preventive treatment (that prevent and / or slow the development or progression of a targeted pathologic condition or disorder) and curative, therapeutic or disease-modifying treatment, including therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder; and treatment of patients at risk of contracting a disease or suspected to have contracted a disease, as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition. The terms “treatment” and “treat” do not necessarily imply that a subject is treated until total recovery. The terms “treatment” and “treat” also refer to the maintenance and / or promotion of health in an individual not suffering from a disease but who may be susceptible to the development of an unhealthy condition. The terms “treatment” and “treat” are also intended to include the potentiation or otherwise enhancement of one or more primary prophylactic or therapeutic measures. As non-limiting examples, a treatment can be performed by a patient, a caregiver, a doctor, a nurse, or another healthcare professional.

[0046] Both human and veterinary treatments are within the scope of the present disclosure. Preferably the combination of adenosylcobalamin and oleuropein or metabolite thereof is administered in a serving or unit dosage form that provides a therapeutically effective or prophylactically effective amount of the combination.

[0047] The terms "prevent" and “prevention” mean to administer a composition as disclosed herein to a subject is not showing any symptoms of the condition to reduce or prevent development of at least one symptom associated with the condition. Furthermore, “prevention” includes reduction of risk, incidence and / or severity of a condition or disorder.

[0048] As used herein, an “effective amount” is an amount that treats or prevents a deficiency, treats or prevents a disease or medical condition in an individual, or, more generally, reduces symptoms, manages progression of the disease, or provides a nutritional, physiological, or medical benefit to the individual.

[0049] The relative terms “improved,” “increased,” “enhanced” and the like refer to the effects of the composition disclosed herein, namely a composition comprising an effective amount of acombination of adenosylcobalamin and at least one of oleuropein or metabolite thereof, relative to administration over the same time period of a composition lacking one of the adenosylcobalamin or the oleuropein / oleuropein metabolite but otherwise identical.

[0050] As used herein, “administering” includes another individual providing a referenced composition to an individual so that the individual can consume the composition and also includes merely the act of the individual themselves consuming a referenced composition.

[0051] ‘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; and humans. Where “animal,” “mammal” or a plural thereof is used, these terms also apply to any animal that is capable of the effect exhibited or intended to be exhibited by the context of the passage, e.g., an animal benefitting from improved mitochondrial calcium import. While the term “individual” or “subject” is often used herein to refer to a human, the present disclosure is not so limited. Accordingly, the term “individual” or “subject” refers to any animal, mammal or human that can benefit from the methods and compositions disclosed herein.

[0052] The term “pet” means any animal which could benefit from or enjoy the compositions provided by the present disclosure. For example, the pet can be an avian, bovine, canine, equine, feline, hircine, lupine, murine, ovine, or porcine animal, but the pet can be any suitable animal. The term “companion animal” means a dog or a cat.

[0053] The term “elderly” in the context of a human means an age from birth of at least 60 years, preferably above 63 years, more preferably above 65 years, and most preferably above 70 years. In the context of non-human animals, “elderly” means a non-human 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.

[0054] The term “older adult” in the context of a human means an age from birth of at least 45 years, preferably above 50 years, more preferably above 55 years, and includes elderly individuals.

[0055] "Mobility" refers to an individual's ability to move freely and easily, encompassing the coordinated function as well as the strength of joints, bones, and muscles. “Mobility” refers also to the ability to carry out everyday tasks and maintain a good quality of life. Good mobility is linked to independence and autonomy.

[0056] “Sarcopenia” is defined as the age-associated loss of muscle mass and functionality (including muscle strength and gait speed).

[0057] 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. In the absence of an established quantitative standard, frailty has been operationally defined by Fried et al. as meeting three out of five phenotypic criteria indicating compromised energetics: (1) weakness (grip strength in the lowest 20% of population at baseline, adjusted for gender and body mass index), (2) poor endurance and energy (self-reported exhaustion associated with VO2 max), (3) slowness (lowest 20% of population at baseline, based on time to walk 15 feet, adjusting for gender and standing height), (4) low physical activity (weighted score of kilocalories expended per week at baseline, lowest quintile of physical activity identified for each gender; e.g., less than 383 kcal / week for males and less than 270 kcal / week for females), and / or unintentional weight loss (10 lbs. in past year). Fried LP, Tangen CM, Walston J, et al., “Frailty in older adults: evidence for a phenotype.” J. Gerontol. A. Biol. Sci. Med. Sci. 56(3):M146-M156 (2001). A pre-frail stage, in which one or two of these criteria are present, identifies a high risk of progressing to frailty.

[0058] ‘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.

[0059] ‘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 deconditionedindividual. The expected strength should also be adjusted for degree of atrophy in patients with wasting illnesses.

[0060] The patient’s effort is graded on a scale of 0 to 5. As used herein, “muscle weakness” refers to any of grades 0-4.Grade 5: Muscle contracts normally against full resistance.Grade 4: Muscle strength is reduced, but muscle contraction can still move joint against resistance. Grade 3: Muscle strength is further reduced, such that the joint can be moved only against gravity with the examiner’s resistance completely removed. As an example, the elbow can be moved from full extension to full flexion starting with the arm hanging down at the side.Grade 2: Muscle can move only if the resistance of gravity is removed. As an example, the elbow can be fully flexed only if the arm is maintained in a horizontal plane.Grade 1 : Only a trace or flicker of movement is seen or felt in the muscle, or fasciculations are observed in the muscle.Grade 0: No movement is observed.

[0061] As used herein, a “sportsman” is an individual who participates in at least one of 1) resistance exercise, 2) anaerobic or repeated sprint-type exercise, or 3) endurance exercise.

[0062] Resistance exercise is when a subject undertakes explosive movements of weight, with long periods of rest. Resistance exercise can produce energy quickly, but the subject fatigues quickly. The primary adaptations include increases in muscle mass (hypertrophy) by increased muscle cross-section area through repeated weightlifting training. Hakkinen K. 1989. Neuromuscular and hormonal adaptations during strength and power training. 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.

[0063] Repeated sprint-type training is anaerobic, involves high-intensity exercise with limited recovery periods, and involves nearly purely carbohydrate metabolism with a large breakdown in muscle glycogen (glycolytic energy production). During these situations of anaerobic energy production, such as high intensity speed training or sports involving repeated sprints, the increased load on the muscles is accomplished by an increased firing of Type Ila fibers. Finally, at very high workloads, type lib glycolytic muscle fibers become activated to maintain the high demand of energy provision via anaerobic energy provision. However, during thesesituations, the high rate of anaerobic energy production exceeds the rate at which it can be oxidized aerobically within the mitochondria, and this leads to the extreme levels of lactate production found in these types of training situations. Spriet L L, Howlett R A, and Heigenhauser G J. 2000. An enzymatic approach to lactate production in human skeletal muscle during exercise. Med. Sci. Sports Exerc. 32: 756-763.

[0064] Endurance training is characterized by individuals performing low-intensity training over prolonged periods (e.g., >15 minutes). The energy system represented for endurance training includes the aerobic system, which primarily uses aerobic metabolism of fats and carbohydrates to produce the required energy within the mitochondria when ample oxygen is present. The primary adaptations include increased muscle glycogen stores and glycogen sparing at sub-maximal workloads via increased fat oxidation, enhanced lactate kinetics and morphological alterations, including greater type I fiber per muscle area, and increased capillary and mitochondrial density. Holloszy J O, and Coyle E F. 1984. Adaptations of skeletal muscle to endurance exercise and their metabolic consequences. J. Appl. Physiol. 56: 831-838; and Holloszy J O, Rennie M J, Hickson R C, Conlee R K, and Hagberg J M. 1977. Physiological consequences of the biochemical adaptations to endurance exercise. Ann. N.Y. Acad. Sci. 301: 440-450.

[0065] “Overweight” is defined for a human as a body mass index (BMI) between 25 and 30 kg / m2. “Obese” is defined for a human as a BMI of at least 30 kg / m2, for example 30-39.9 kg / m2“Weight loss” is a reduction of the total body weight. Weight loss may, for example, refer to the loss of total body mass in an effort to improve one or more of health, fitness or appearance.

[0066] Diabetes" encompasses both the type I and type II forms of the disease. Non-limiting examples of risk factors for diabetes include: waistline of more than 40 inches for men or 35 inches for women, blood pressure of 130 / 85 mmHg or higher, triglycerides above 150 mg / dl, fasting blood glucose greater than 100 mg / dl or high-density lipoprotein of less than 40 mg / dl in men or 50 mg / dl in women.

[0067] As used herein, the term "metabolic syndrome" refers to a combination of medical disorders that, when occurring together, increase the risk of developing cardiovascular disease and diabetes. It affects one in five people in the United States and prevalence increases with age. Some studies have shown the prevalence in the United States to be an estimated 25% of the population. In accordance with the International Diabetes Foundation consensus worldwide definition (2006), metabolic syndrome is central obesity plus any two of the following:

[0068] Raised triglycerides: > 150 mg / dL (1.7 mmol / L), or specific treatment for this lipid abnormality;

[0069] Reduced HDL cholesterol: < 40 mg / dL (1.03 mmol / L) in males, < 50 mg / dL (1.29 mmol / L) in females, or specific treatment for this lipid abnormality;

[0070] Raised blood pressure: systolic BP > 130 or diastolic BP >85 mm Hg, or treatment of previously diagnosed hypertension; and

[0071] Raised fasting plasma glucose: (FPG) > 100 mg / dL (5.6 mmol / L), or previously diagnosed type 2 diabetes.

[0072] As used herein, "neurodegenerative disease" or " neurodeg enerative disorder" refers to any condition involving progressive loss of functional neurons in the central nervous system. In an embodiment, the neurodegenerative disease is associated with age-related cell death. Non-limiting examples of neurodegenerative diseases include mild cognitive impairment, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (also known as ALS and as Lou Gehrig's disease), peripheral neuropathy, AIDS dementia complex, adrenoleukodystrophy, Alexander disease, Alper's disease, ataxia telangiectasia, Batten disease, bovine spongiform encephalopathy (BSE), Canavan disease, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia with Lewy bodies, fatal familial insomnia, frontotemporal lobar degeneration, Kennedy's disease, Krabbe disease, Lyme disease, Machado-Joseph disease, multiple sclerosis, multiple system atrophy, neuroacanthocytosis, Niemann-Pick disease, Pick's disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum disease, Sandhoff disease, diffuse myelinoclastic sclerosis, spinocerebellar ataxia, subacute combined degeneration of spinal cord, tabes dorsalis, Tay-Sachs disease, toxic encephalopathy, transmissible spongiform encephalopathy, and wobbly hedgehog syndrome. The present disclosure is not limited to a specific embodiment of the neurodegenerative disease, and the neurodegenerative disease can be any neurologically-related condition known to one skilled in this art.

[0073] As used herein, "cognitive function" refers to any mental process that involves symbolic operations, e.g., perception, memory, attention, speech comprehension, speech generation, reading comprehension, creation of imagery, learning, and reasoning, preferably at least memory.

[0074] Methods for measuring cognitive function are well-known and can include, for example, individual or battery tests for any aspect of cognitive function. One such test is thePrudhoe Cognitive Function Test by Margallo-Lana et al. (2003) J. Intellect. Disability Res. 47:488-492. Another such test is the Mini Mental State Exam (MMSE), which is designed to assess orientation to time and place, registration, attention and calculation, recall, language use and comprehension, repetition, and complex commands. Folstein et al. (1975) J. Psych. Res. 12:189-198. Such tests can be used to assess cognitive function in an objective manner, so that changes in cognitive function, for example in response to treatment in accordance with methods disclosed herein, can be measured and compared.

[0075] As used herein, a “cognitive disorder” refers to any condition that impairs cognitive function. Non-limiting examples of a cognitive disorder include delirium, dementia, learning disorder, attention deficit disorder (ADD), and attention deficit hyperactivity disorder (ADHD).

[0076] The terms “serving” or "unit dosage form," as used herein, are interchangeable and refer to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the composition comprising a combination of adenosylcobalamin and at least one of oleuropein or metabolite thereof, as disclosed herein, in an amount sufficient to produce the desired effect, preferably in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage form depend on the particular compounds employed, the effect to be achieved, and the pharmacodynamics associated with each compound in the host. In an embodiment, the unit dosage form can be a predetermined amount of liquid housed within a container such as a bottle.

[0077] An “oral nutrition supplement” or “ONS” is a composition comprising at least one macronutrient and / or at least one micronutrient, for example in a form of sterile liquids, semi-solids or powders, and intended to supplement other nutritional intake such as that from food. Non-limiting examples of commercially available ONS products include MERITENE®, BOOST®, NUTREN® and SUSTAGEN®. In some embodiments, an ONS can be a beverage in liquid form that can be consumed without further addition of liquid, for example an amount of the liquid that is one serving of the composition.

[0078] As used herein, “incomplete nutrition” refers to preferably nutritional products that do not contain sufficient levels of macronutrients (protein, fats and carbohydrates) or micronutrients to be sufficient to be a sole source of nutrition for the animal to which the nutritional product is being administered. The term "complete nutrition" refers to a product which is capable of beingthe sole source of nutrition for the subject. An individual can receive 100% of their nutritional requirements from a complete nutrition composition.

[0079] A “kit” means that the components of the kit are physically associated in or with one or more containers and considered a unit for manufacture, distribution, sale, or use. Containers include, but are not limited to, bags, boxes, cartons, bottles, packages of any type or design or material, over- wrap, shrink-wrap, affixed components (e.g., stapled, adhered, or the like), or combinations thereof.

[0080] Embodiments

[0081] An aspect of the present disclosure is a composition comprising a combination of oleuropein and / or metabolite thereof and adenosylcobalamin in an effective amount for use in achieving at least one result selected from the group consisting of i) preventing and / or treating a mitochondria-related disease or condition associated with altered mitochondrial function and / or ii) at least one physical state selected from the group consisting of oxidative stress or a condition associated with oxidative stress in an individual; (iii) increasing mitochondrial energy, cellular energy, mitochondrial function and mitochondrial calcium uptake in one or more cells, (iv) increasing resistance to age-related pathologies; (v) improving a physiological state or disorder related to cell ageing or metabolic fatigue in one or more cells; (vi) improving mobility and / or (vii) improving healthspan and / or lifespan in an individual.

[0082] In an embodiment, the mitochondria-related disease or condition is selected from the group consisting of stress, obesity, reduced metabolic rate, metabolic syndrome, diabetes mellitus, complications from diabetes, cardiovascular disease, hyperlipidemia, respiratory diseases, pain syndromes, neurodegenerative disease, cognitive disorder, stress-induced or stress-related cognitive dysfunction, mood disorder, anxiety disorder, age-related neuronal death or dysfunction, musculo-skeletal disorder, muscle aging, sarcopenia, frailty, pre-frailty, chronic kidney disease, macular degeneration, and combinations thereof.

[0083] In an embodiment, the at least one physical state is selected from the group consisting of deleterious effects of aging, muscle loss, pre-diabetes, gestational diabetes, type I diabetes, type II diabetes, complications from diabetes, insulin resistance, metabolic syndrome, dyslipidemia, overweight, obesity, raised cholesterol levels, raised triglyceride levels, elevated fatty acid levels, fatty liver disease, renal disease, cardiovascular disease, musculo-skeletal diseases, respiratory diseases, pain syndromes, neurodegenerative disease, impaired cognitive function, myopathy suchas statin-induced myopathy, non-alcoholic steatohepatitis, tinnitus, dizziness, alcohol hangover, hearing impairment, osteoporosis, hypertension, atherosclerosis / coronary artery disease, myocardial damage after stress, traumatic brain injury, cystic fibrosis, inflammation, cancer, and HIV infection.

[0084] In an embodiment, at least a portion of the one or more cells are part of at least one body part selected from the group consisting of a liver, a kidney, a brain, and a skeletal muscle.

[0085] In an embodiment, the physiological state or disorder related to cell ageing or metabolic fatigue comprises muscle fatigue or weakness, lack of energy, physical energy, lack of vitality or weakness.

[0086] 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.

[0087] The effective amount of the combination of oleuropein and / or metabolite thereof and adenosylcobalamin 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 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. The at least one of oleuropein or metabolite thereof and adenosylcobalamin may be formulated in a particular ratio. In some embodiments, the formulation may comprise these components in the following exemplary ratios: 1: 1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1 :8, 1 :9, 1: 10; 1:50; 1: 100; 1: 1000; 1:10000; 1:25000; 1:50000; 1: 100000; 1:250000 and each of these ratios can be adenosylcobalamin: oleuropein in some embodiments and adenosylcobalamin: oleuropein in other embodiments. Preferably, the oleuropein: adenosylcobalamin ratio is between 1: 10 to 1: 100.

[0088] In an embodiment, at least a portion of the oleuropein or its metabolite 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, a plant of genus Osmanthus and aplant of genus Sideroxylon (Argania spinosa). Additionally, or alternatively, at least a portion of the oleuropein or its metabolite can be obtained by chemical synthesis.

[0089] In some embodiments oleuropein or metabolite thereof is the only polyphenol in the composition and / or the only polyphenol administered to the individual.

[0090] The effective amount of adenosylcobalamin also varies with the particular composition, the age and condition of the recipient, and the particular disorder or disease being treated. Nevertheless, oral supplementation typically involves giving 1 to 10 pg up to 100 pg to 2000 pg of adenosylcobalamin daily depending on the format. When administered in the form of oral nutritional supplement the daily amount provides 1 to 10 pg, preferably 1 to 2 pg of adenosylcobalamin. When administered in the form of supplement, the daily amount provides 100 pg to 2000 pg, preferably 250 pg to 1 mg of adenosylcobalamin.

[0091] The present invention may comprise administering a probiotic supplement comprising adenosylcobalamin producing bacteria to a subject.

[0092] The probiotic supplement can include any probiotic microorganism(s) which beneficially affect the host subject by improving its intestinal microbial balance to enhance vitamin B12 uptake. The probiotic microorganism can be selected from the group comprising of Bifidobacterium, Lactobacillus, Streptococcus, Enterococcus and Saccharomyces or mixtures thereof.

[0093] The food or beverage product may comprise a probiotic supplement comprising vitamin B12 producing bacteria or other probiotics which can enhance existing microorganisms in the gut that produce vitamin B12 in situ.

[0094] Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient. The dosage is such that it is sufficient to provide required levels of active adenosyl vitamin B12.

[0095] In an embodiment, the individual is selected from the group consisting of an adult, 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; a sportsman; and a pet.

[0096] In some embodiments, the individual is healthy. In some embodiments, the individual 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.

[0097] For example, the combination can be administered to a sportsman 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.

[0098] In an embodiment, the composition according to this invention is administered orally.

[0099] In an embodiment, the combination is administered in a composition selected from the group consisting of a food product, a food for special medical purposes (FSMP), a nutritional supplement, a dairy-based drink, a low-volume liquid supplement, a meal replacement beverage, and combinations thereof.

[0100] The present invention also relates to a composition comprising a combination of oleuropein and / or metabolite thereof and adenosylcobalamin in an effective amount for use in delaying off-set of metabolic decline, decreasing oxidative stress, improving or maintaining muscle function, immune function and / or cognitive function in an individual.

[0101] The present invention also relates to a composition comprising a combination of oleuropein and / or metabolite thereof and adenosylcobalamin in an effective amount for use in i) mitigating deleterious effects of aging, ii) improving at least one of muscle performance or muscle recovery from exercise, exercise capacity and / or physical function, iii) reducing severity of metabolic and / or degenerative diseases in an individual.

[0102] The present invention also relates to a method of manufacturing a nutritional composition for use according to any of the preceding claims, comprising the steps of providing one or more ingredients for a nutritional composition, oleuropein and / or metabolite thereof and adenosylcobalamin, and mixing.

[0103] Each of the compounds can be administered at the same time as the other compounds (i.e., as a single unit) or separated by a time interval (i.e., in separate units).

[0104] Ingredients- further bioactive compound

[0105] The compositions for use according to the invention may also comprise at least one further bioactive compound selected from the group consisting of antioxidants, anti-inflammatory compounds, glycosaminoglycans, prebiotics, fibers, probiotics, fatty acids, enzymes, minerals, trace elements and / or vitamins.

[0106] The term “bioactive” in the context of the present application means that the compound contributes to the health of an individual, or has an effect on the human body, beyond that of meeting basic nutritional need. The at least one further bioactive compound may be from a naturalsource. Thus, the compounds may be from extracts of plants, animals, fish, fungi, algae, microbial fermentation. Minerals are considered as from natural source also within this definition.

[0107] Nutritional compositions

[0108] The compositions for use according to the invention may be nutritional compositions or pharmaceutical compositions, and may be for human or veterinary use. In an embodiment, the combination is administered orally.

[0109] Thus, in preferred embodiments, the composition for use according to the invention is a nutritional composition.

[0110] By "nutritional composition" is meant in the context of the present application a composition which is a source of nutrition to an individual.

[0111] The nutritional products or compositions of the invention may be a source of complete nutrition or may be a source of incomplete nutrition. As used herein, “complete nutrition” includes nutritional products and compositions that contain sufficient types and levels of macronutrients (protein, fats and carbohydrates) and micronutrients to be sufficient to be a sole source of nutrition for the animal to which it is being administered to. Patients can receive 100% of their nutritional requirements from such complete nutritional compositions. As used herein, “incomplete nutrition” includes nutritional products or compositions that do not contain sufficient levels of macronutrients (protein, fats and carbohydrates) or micronutrients to be sufficient to be a sole source of nutrition for the animal to which it is being administered to. Partial or incomplete nutritional compositions can be used as a nutritional supplement.

[0112] Non-limiting examples of suitable compositions for the include food compositions, dietary supplements, dietary supplements (e.g., liquid ONS), complete nutritional compositions, beverages, pharmaceuticals, oral nutritional supplement, medical food, nutraceuticals, food for special medical purpose (FSMP), powdered nutritional products to be reconstituted in water or milk before consumption, food additives, medicaments, drinks, petfood, and combinations thereof.

[0113] In an embodiment, the compositions for use according to the invention include a source of protein. The protein source may be dietary protein including, but not limited to animal protein (such as milk protein, meat protein or egg protein), vegetable protein (such as soy protein, wheat protein, rice protein, and pea protein), or combinations thereof. In an embodiment, the protein is selected from the group consisting of whey, chicken, corn, caseinate, wheat, flax, soy, carob, pea or combinations thereof.

[0114] In an embodiment, the compositions include a source of carbohydrates. Any suitable carbohydrate may be used in the present compositions including, but not limited to, starch, sucrose, lactose, glucose, fructose, corn syrup solids, maltodextrin, modified starch, amylose starch, tapioca starch, corn starch, xylitol, sorbitol or combinations thereof.

[0115] In an embodiment, the compositions include a source of fat. The source of fat may include any suitable fat or fat mixture. For example, the fat source may include, but is not limited to, 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. The source of fat may also be less refined versions of the fats listed above (e.g., olive oil for polyphenol content).

[0116] In addition, compositions for use according to the invention may also comprise natural or artificial flavours, for example fruit flavours like banana, orange, peach, pineapple or raspberry or other plant flavours like vanilla, cocoa, coffee, etc.

[0117] Nutritional composition formats

[0118] The nutritional compositions may include, besides the main bioactive components and any further bioactive components, and optionally one or more of a protein, carbohydrate and fat source, any number of optional additional food ingredients, including conventional food additives (synthetic or natural), for example one or more acidulants, additional thickeners, buffers or agents for pH adjustment, chelating agents, colorants, emulsifiers, excipient, flavor agent, mineral, osmotic agents, a pharmaceutically acceptable carrier, preservatives, stabilizers, sugar, sweeteners, texturizers, and / or vitamins. The optional ingredients can be added in any suitable amount.

[0119] The nutritional composition may be provided in any suitable format. Examples of nutritional composition formats in which the composition for use according to the invention may be provided include solutions, ready -for-consumption compositions (e.g. ready -to-drink compositions or instant drinks), liquid comestibles, soft drinks, juice, sports drinks, milk drinks, milk-shakes, yogurt drinks, soup, etc.

[0120] In another embodiment, the nutritional compositions may be provided in the form of a concentrate, a powder, or granules (e.g. effervescent granules), which are diluted with water or other liquid, such as milk or fruit juice, to yield the ready -for-consumption composition. 1

[0121] Further nutritional composition formats include, baked products, dairy products, desserts, confectionery products, cereal bars, and breakfast cereals. Examples of dairy products include milk and milk drinks, yoghurts and other cultured milk products, ice creams and cheeses. Examples of baked products include bread, biscuits and cakes.

[0122] In one embodiment, the composition for use according to the invention may also be available in a great variety of formats designed as animal foods, in particular for the dog or the cat, whether in a wet form, semi-wet form or dry form, in particular in the form of biscuits.

[0123] The compositions disclosed herein can use any of a variety of formulations for therapeutic administration. More particularly, pharmaceutical compositions can comprise appropriate pharmaceutically acceptable carriers or diluents and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. As such, administration of the composition can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, and intratracheal administration. The active agent may be systemic after administration or may be localized by the use of regional administration, intramural administration, or use of an implant that acts to retain the active dose at the site of implantation.

[0124] 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.

[0125] For oral preparations, the compounds can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose functional derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[0126] Routes of administration

[0127] The nutritional compositions of the present disclosure may be administered by any means suitable for human administration, and in particular for administration in any part of thegastrointestinal tract. Enteral administration, oral administration, and administration through a tube or catheter are all covered by the present disclosure. The nutritional compositions may also be administered by means selected from oral, rectal, sublingual, sublabial, buccal, topical, etc.

[0128] The nutritional compositions may be administered in any known form including, for example, tablets, capsules, liquids, chewables, soft gels, sachets, powders, syrups, liquid suspensions, emulsions and solutions in convenient dosage forms. In soft capsules, the active ingredients are preferably dissolved or suspended in suitable liquids, such as fatty oils, paraffin oil or liquid polyethylene glycols. Optionally, stabilizers may be added.

[0129] If the nutritional compositions are administered by tube feeding, the nutritional compositions may be used for short term or long term tube feeding.

[0130] The composition can be administered to an individual such as a human, e.g., an ageing individual or a critically ill individual, 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 individual.

[0131] The composition is preferably administered to the individual at least one day per week, preferably 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 individual 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 individual daily for at least 30, 60 or 90 consecutive days.

[0132] In some embodiments, the administration continues for the remaining life of the individual. In other embodiments, the administration occurs until no detectable symptoms of the medical condition remain. In specific embodiments, the administration occurs until a detectable improvement of at least one symptom occurs and, in further cases, continues to remain ameliorated.

[0133] 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.

[0134] Method of treatment

[0135] Mitochondrial diseases are the result of either inherited or spontaneous mutations in mitochondrial DNA or nuclear DNA which lead to altered functions of the proteins or RNA molecules that normally reside in mitochondria. Problems with mitochondrial function, however, may only affect certain tissues as a result of factors occurring during development and growth that are not yet fully understood. Even when tissue-specific isoforms of mitochondrial proteins are considered, it is difficult to explain the variable patterns of affected organ systems in the mitochondrial disease syndromes seen clinically.

[0136] Mitochondrial diseases result from failures of the mitochondria, specialized compartments present in every cell of the body except red blood cells. Mitochondria are responsible for creating more than 90% of the energy needed by the body to sustain life and support growth. When they fail, less and less energy is generated within the cell. Cell injury and even cell death follow. If this process is repeated throughout the body, whole systems begin to fail, and the life of the person in whom this is happening is severely compromised. Mitochondrial diseases primarily affect children, but adult onset is becoming more recognized. Diseases of the mitochondria appear to cause the most damage to cells of the brain, heart, liver, skeletal muscles, kidney, and the endocrine and respiratory systems.

[0137] Many symptoms in mitochondrial disorders are non-specific. The symptoms may also show an episodic course, with periodic exacerbations. The episodic condition of migraine, as well as myalgia, gastrointestinal symptoms, tinnitus, depression, chronic fatigue, and diabetes, have been mentioned among the various manifestations of mitochondrial disorders in review papers on mitochondrial medicine (Chinnery and Turnbull (1997) QJM 90:657-67; Finsterer (2004) Eur. J. Neurol. 11 :163-86). In patients with mitochondrial disorders, clinical symptomatology typically occurs at times of higher energy demand associated with physiological stressors, such as illness, fasting, over-exercise, and environmental temperature extremes. Furthermore, psychological stressors also frequently trigger symptomatology, presumably due to higher brain energy demands for which the patient is unable to match with sufficient ATP production.

[0138] Depending on which cells are affected, symptoms may include loss of motor control, muscle weakness and pain, gastro-intestinal disorders and swallowing difficulties, poor growth,cardiac disease, liver disease, diabetes, respiratory complications, seizures, visual / hearing problems, lactic acidosis, developmental delays and susceptibility to infection.

[0139] Mitochondrial diseases include, without limitation, Alper's disease; Barth syndrome; beta-oxidation defects; carnitine deficiency; carnitine-acyl- carnitine deficiency; chronic progressive external ophthalmoplegia syndrome; co-enzyme Q10 deficiency; Complex I deficiency; Complex II deficiency; Complex III deficiency; Complex IV deficiency; Complex V deficiency; CPT I deficiency; CPT II deficiency; adenosylcobalamin deficiency syndrome; cytochrome c oxidase deficiency; glutaric aciduria type II; Kearns-Sayre syndrome; lactic acidosis; LCHAD (long-chain acyl-CoA dehydrogenase deficiency); Leber's hereditary optic neuropathy; Leigh disease; lethal infantile cardiomyopathy; Luft disease; MAD (medium-chain acyl-CoA dehydrogenase deficiency); mitochondrial cytopathy; mitochondrial DNA depletion; mitochondrial encephalomyopathy, lactic acidosis, and stroke-like symptoms; mitochondrial encephalopathy; mitochondrial myopathy; mitochondrial recessive ataxia syndrome; muscular dystrophies, myoclonic epilepsy and ragged-red fiber disease; myoneurogenic gastrointestinal encephalopathy; neuropathy, ataxia, retinitis pigmentosa, and ptosis; Pearson syndrome; POLG mutations; pyruvate carboxylase deficiency; pyruvate dehydrogenase deficiency; SCHAD (short-chain acyl-CoA dehydrogenase deficiency); and very long-chain acyl-CoA dehydrogenase deficiency.

[0140] Accordingly, an aspect of the present disclosure is a method of achieving at least one result selected from the group consisting of i) preventing and / or treating a mitochondria-related disease or condition associated with altered mitochondrial function and / or ii) at least one physical state selected from the group consisting of oxidative stress or a condition associated with oxidative stress in an individual; (iii) increasing mitochondrial energy, cellular energy, mitochondrial function and mitochondrial calcium uptake in one or more cells, (iv) increasing resistance to age-related pathologies; (v) improving a physiological state or disorder related to cell ageing or metabolic fatigue in one or more cells; (vi) improving mobility and / or (vii) improving healthspan and / or lifespan in an individual, the method comprising orally administering to the individual in need thereof or at risk thereof an effective amount of the combination of oleuropein and / or metabolite thereof and adenosylcobalamin.

[0141] In an embodiment, the combination of oleuropein and / or metabolite thereof and adenosylcobalamin is administered in a composition selected from the group consisting of foodcompositions, dietary supplements, nutritional compositions, beverages, nutraceuticals, powdered nutritional products to be reconstituted in water or milk before consumption, food additives, medicaments, drinks, petfood, and combinations thereof.

[0142] In an embodiment, the method comprises administering the combination of adenosylcobalamin and the metabolite of oleuropein selected from the group consisting of oleuropein aglycone, hydroxytyrosol, homovanillyl alcohol, isohomovanillyl alcohol, glucuronidated forms thereof, sulfated forms thereof, derivatives including conjugated forms thereof, and mixtures thereof.

[0143] The present invention also relates to a method of treating in an individual in need thereof or preventing in an individual at risk thereof 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 comprising orally administering to the individual in need thereof or at risk thereof an effective amount of the combination of oleuropein and / or metabolite thereof and adenosylcobalamin.

[0144] Preferably, the 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.

[0145] 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 neurogastro intestinal encephalomyopathy (MNGIE); myoclonus epilepsy with ragged red fibers (MERRF); neuropathy, ataxia, and retinitis pigmentosa (NARP); and Pearson syndrome.

[0146] The individual 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 thecomposition is a prophylactically effective dose; or the individual 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 individual as having the condition or being at risk of the condition before the administration.

[0147] Further regarding muscle performance, the increased muscle performance may be one or more of improved muscle function, reduced decline in muscle function, improved muscle strength, improved muscle endurance and improved muscle recovery. The composition can improve physical endurance (e.g., ability to perform a physical task such as exercise, physical labor, sports activities), inhibit or retard physical fatigue, enhance blood oxygen levels, enhance energy in healthy individuals, enhance working capacity and endurance, reduce muscle fatigue, reduce stress, enhance cardiac and cardiovascular function, improve sexual ability, increase muscle ATP levels, and / or reduce lactic acid in blood. “Endurance capacity” refers to the time to fatigue when exercising at a constant workload, generally at an intensity <80% V02max. In some embodiments, the composition is administered in an amount that increases mitochondrial activity, increases mitochondrial biogenesis, and / or increases mitochondrial mass.

[0148] The compositions and the methods disclosed herein can also be effective in the treatment of muscle-related pathological conditions, including myopathies; neuromuscular diseases, such as Duchenne muscular dystrophy; acute sarcopenia, for example, muscle atrophy; and / or cachexia associated with burns, bed rest, limb immobilization, or major thoracic, abdominal, and / or orthopedic surgery.

[0149] In some embodiments, the composition is administered to an individual having impaired physical performance, impaired endurance capacity, and / or impaired muscle function. Improved muscle function can be particularly beneficial in elderly subjects with reduced muscle function as a result of an age-related condition. For example, a subject who may benefit from improved muscle function may experience a decline in muscle function which then leads to pre-frailty and frailty. Such subjects may not necessarily experience muscle wastage in addition to their decline in muscle function. Some subjects do experience both muscle wasting and a decline in muscle function, for example subjects with sarcopenia. The composition may enhance muscle performance in a subject who is frail or pre- frail.

[0150] 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 theolder adult an effective amount of a combination of oleuropein or metabolite thereof and adenosylcobalamin. The older adult can be an elderly individual. 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.

[0151] The composition may be administered to the older adult 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 in instances where the condition has yet not been developed in the individual. Such an amount is defined to be “a prophylactically effective dose.” Again, the precise amounts depend on a number of factors relating to the individual, such as their weight, health and how much muscle functionality (e.g., muscle strength, gait speed, etc.) is being lost.

[0152] The present invention also relates to a unit dosage form comprising a combination of oleuropein and / or metabolite thereof and adenosylcobalamin, the unit dosage form comprises an amount of the combination effective for at least one result selected from the group consisting of i) preventing and / or treating a mitochondria-related disease or condition associated with altered mitochondrial function and / or ii) at least one physical state selected from the group consisting of oxidative stress or a condition associated with oxidative stress in an individual; (iii) increasing mitochondrial energy, mitochondrial function and mitochondrial calcium uptake in one or more cells, (iv) increasing resistance to age-related pathologies; (v) improving a physiological state or disorder related to cell ageing or metabolic fatigue in one or more cells; (vi) improving mobility and / or (vii) improving healthspan and / or lifespan in an individual.

[0153] In an embodiment, the unit dosage form of consists essentially of the combination of oleuropein and / or metabolite thereof and adenosylcobalamin.

[0154] The compositions disclosed herein can also be used in the treatment of any of a variety of additional diseases and conditions in which defective or diminished mitochondrial activity participates in the pathophysiology of the disease or condition, or in which increased mitochondrial function will yield a desired beneficial effect.

[0155] It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

[0156] The compositions for use according to the invention are herein described in different parameters, such as the ingredients, nutritional composition formats, uses, target groups etc. It should be noted that embodiments and features described in the context of one of the parameters of the composition for use according to the invention, may also be combined with other embodiments and features described in the context of another parameter, unless expressly stated otherwise.

[0157] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0158] The invention will now be described in further details in the following non-limiting examples.EXAMPLE

[0159] The following non-limiting examples present experimental data supporting the compositions and methods disclosed herein.

[0160] Materials and Methods

[0161] To test the effect of oleuropein (oleuropein aglycone) 10 pM, adenosylcobalamin 0.9 pM and their combination, on mitochondrial activation in living cells, the inventors measured mitochondrial calcium elevation in myotubes differentiated from C2C12 cells.

[0162] C2C12 cells were purchased from ATCC (American Type Culture Collection). C2C12 cells were seeded in 384-well plates at a density of 4500 cells per well in DMEM (Dulbecco's Modified Eagle Medium), containing 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.

[0163] 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., 2004). For aequorin reconstitution, 48 hours after infection, myotubes were incubated for 2h at room temperature (22 ±°C) in standard Aequorin buffer (145 mM NaCl, 5 mM KC1, 1 mM MgCh, 1 mM CaCE, 10 mM glucose and 10 mM Hepes, pH 7.4) with 1 pM wild type coelenterazine. For treatment, control myotubes, adenosylcobalamin (0.9 pM) and Oleuropein aglycone (10 pM) 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. Luminescencewas 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 (Alvarez & Montero, 2002). Custom module analysis based on Excel (Microsoft) and GraphPad Prism 7.02 (GraphPad) software was used for quantification.

[0164] References

[0165] Montero et al., Direct activation of the mitochondrial calcium uniporter by natural plant flavonoids, Biochemical Journal , 2004; 384: 19-24.

[0166] Alvarez J., Montero M. Measuring [Ca2+] in the endoplasmic reticulum with aequorin. Cell Calcium. 2002; 32:251-260.

[0167] Results

[0168] As shown in Fig. 1, the simultaneous supplementation of oleuropein and adenosylcobalamin causes a mitochondrial activation (measured as mitochondrial calcium elevation) bigger than the effect of each compound alone.

[0169] As shown in the main Fig. 2, to calculate the synergistic effect of oleuropein aglycone and adenosylcobalamin, the theoretical effect of the sum of the 2 distinct compounds (oleuropein aglycone and caffeine) was compared with the measured effect of the combination. As shown in FIG. 2 oleuropein synergizes with adenosylcobalamin to promote mitochondrial calcium rise during stimulation.

[0170] It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.

Claims

CLAIMSThe invention is claimed as follows:

1. A composition comprising a combination of oleuropein and / or metabolite thereof and adenosylcobalamin in an effective amount for use in achieving at least one result selected from the group consisting of i) preventing and / or treating a mitochondria-related disease or condition associated with altered mitochondrial function and / or ii) at least one physical state selected from the group consisting of oxidative stress or a condition associated with oxidative stress in an individual; (iii) increasing mitochondrial energy, cellular energy, mitochondrial function and mitochondrial calcium uptake in one or more cells, (iv) increasing resistance to age-related pathologies; (v) improving a physiological state or disorder related to cell ageing or metabolic fatigue in one or more cells; (vi) improving mobility and / or (vii) improving healthspan and / or lifespan in an individual.

2. The composition for use according to claim 1, wherein the mitochondria-related disease or condition is selected from the group consisting of stress, obesity, reduced metabolic rate, metabolic syndrome, diabetes mellitus, complications from diabetes, cardiovascular disease, hyperlipidemia, respiratory diseases, pain syndromes, neurodegenerative disease, cognitive disorder, stress-induced or stress-related cognitive dysfunction, mood disorder, anxiety disorder, age-related neuronal death or dysfunction, musculo-skeletal disorder, muscle aging, sarcopenia, frailty, pre-frailty, chronic kidney disease, macular degeneration, and combinations thereof.

3. The composition for use according to any one of the preceding claims, wherein the at least one physical state is selected from the group consisting of deleterious effects of aging, muscle loss, pre-diabetes, gestational diabetes, type I diabetes, type II diabetes, complications from diabetes, insulin resistance, metabolic syndrome, dyslipidemia, overweight, obesity, raised cholesterol levels, raised triglyceride levels, elevated fatty acid levels, fatty liver disease, renal disease, cardiovascular disease, musculo-skeletal diseases, respiratory diseases, pain syndromes, neurodegenerative disease, impaired cognitive function, myopathy such as statin-induced myopathy, non-alcoholic steatohepatitis, tinnitus, dizziness, alcohol hangover, hearing impairment, osteoporosis, hypertension, atherosclerosis / coronary artery disease, myocardial damage after stress, traumatic brain injury, cystic fibrosis, inflammation, cancer, and HIV infection.

4. The composition for use according to any one of the preceding claims, wherein at least a portion of the one or more cells are part of at least one body part selected from the group consisting of a liver, a kidney, a brain, and a skeletal muscle.

5. The composition for use according to any one of the preceding claims, wherein the physiological state or disorder related to cell ageing or metabolic fatigue comprises muscle fatigue or weakness, lack of energy, physical energy, lack of vitality or weakness.

6. The composition for use 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.

7. The composition for use according to any one of the preceding claims, wherein the individual is selected from the group consisting of an adult, 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; a sportsman; and a pet.

8. The composition for use according to any one of the preceding claims, wherein it is administered orally.

9. The composition for use according to any one of the preceding claims, wherein the combination is administered in a composition selected from the group consisting of a food product, a food for special medical purposes (FSMP), a nutritional supplement, a dairy-based drink, a low-volume liquid supplement, a meal replacement beverage, and combinations thereof.

10. A composition comprising a combination of oleuropein and / or metabolite thereof and adenosylcobalamin in an effective amount for use in delaying off-set of metabolic decline, decreasing oxidative stress, improving or maintaining muscle mass, immune function and / or cognitive function in an individual.

11. A composition comprising a combination of oleuropein and / or metabolite thereof and adenosylcobalamin in an effective amount for use in i) mitigating deleterious effects of aging, ii) improving at least one of muscle performance or muscle recovery from exercise, exercise capacity and / or physical function, iii) reducing severity of metabolic and / or degenerative diseases in an individual.

12. A method of manufacturing a nutritional composition for use according to any of the preceding claims, comprising the steps of providing one or more ingredients for a nutritional composition, oleuropein and / or metabolite thereof and adenosylcobalamin, and mixing.

13. A method of achieving at least one result selected from the group consisting of i) preventing and / or treating a mitochondria-related disease or condition associated with altered mitochondrial function and / or ii) at least one physical state selected from the group consisting of oxidative stress or a condition associated with oxidative stress in an individual; (iii) increasing mitochondrial energy, cellular energy, mitochondrial function and mitochondrial calcium uptake in one or more cells, (iv) increasing resistance to age-related pathologies; (v) improving a physiological state or disorder related to cell ageing or metabolic fatigue in one or more cells; (vi) improving mobility and / or (vii) improving healthspan and / or lifespan in an individual, the method comprising orally administering to the individual in need thereof or at risk thereof an effective amount of the combination of oleuropein and / or metabolite thereof and adenosylcobalamin.

14. The method according to claim 13, wherein the combination of oleuropein and / or metabolite thereof and adenosylcobalamin is administered in a composition selected from the group consisting of food compositions, dietary supplements, nutritional compositions, beverages, nutraceuticals, powdered nutritional products to be reconstituted in water or milk before consumption, food additives, medicaments, drinks, petfood, and combinations thereof.

15. The method of any one of claims 13 or 14, wherein it comprises administering the combination of adenosylcobalamin and the metabolite of oleuropein selected from the group consisting of oleuropein aglycone, hydroxytyrosol, homovanillyl alcohol, isohomovanillyl alcohol, glucuronidated forms thereof, sulfated forms thereof, derivatives thereof, and mixtures thereof.

16. A method of treating in an individual in need thereof or preventing in an individual at risk thereof 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 comprising orally administering to the individual in need thereof or at risk thereof aneffective amount of the combination of oleuropein and / or metabolite thereof and adenosylcobalamin.

17. A unit dosage form comprising a combination of oleuropein and / or metabolite thereof and adenosylcobalamin, the unit dosage form comprises an amount of the combination effective for at least one result selected from the group consisting of i) preventing and / or treating a mitochondria-related disease or condition associated with altered mitochondrial function and / or ii) at least one physical state selected from the group consisting of oxidative stress or a condition associated with oxidative stress in an individual; (iii) increasing mitochondrial energy, mitochondrial function and mitochondrial calcium uptake in one or more cells, (iv) increasing resistance to age-related pathologies; (v) improving a physiological state or disorder related to cell ageing or metabolic fatigue in one or more cells; (vi) improving mobility and / or (vii) improving healthspan and / or lifespan in an individual.

18. The unit dosage form of Claim 17, consisting essentially of the combination of oleuropein and / or metabolite thereof and adenosylcobalamin.

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