Compositions and methods using thymol and / or carvacrol for induction of autophagy

Abstract

To provide compositions and methods for administration that are capable of inducing autophagy in an individual in need thereof, and which can concomitantly promote protein synthesis and removal of damaged cellular materials.SOLUTION: Compositions and methods can use a combination of thymol and / or carvacrol, optionally with high protein for induction of autophagy in an individual in need thereof. Preferably, a formulation containing a combination of thymol and / or carvacrol, optionally with protein, is administered to the individual in an amount effective to induce autophagy, for example in muscle. The formulation can concomitantly promote protein synthesis and removal of damaged cellular materials.SELECTED DRAWING: None

Description

Background Art

[0001]

[0001] The present disclosure generally relates to compositions and methods using a combination of thymol and / or carvacrol for the induction of autophagy. More specifically, the present disclosure relates to administering a formulation comprising thymol and / or carvacrol, alone or in combination with a high protein, in an amount effective to induce autophagy, for example, in muscle. This formulation can simultaneously promote protein synthesis and the removal of damaged cellular material. The recipient of the administration can be an individual or a critically ill patient, such as a patient in an intensive care unit (ICU), an elderly patient, such as an aged individual, or a patient having sarcopenia or frailty; or an individual having chronic kidney disease (e.g., loss of amino acids due to dialysis), and / or acute kidney injury, or liver disease.

[0002]

[0002] Due to great advances in intensive care, critically ill patients often survive acute diseases that were previously fatal. However, even if they survive this initial phase, the mortality rate of patients who enter a severe chronic phase remains high. Death is often due to refractory multiple organ failure, acute kidney injury and acute renal failure, severe myopathy, or low-grade muscle weakness. To improve muscle myopathy and muscle weakness, treatment methods such as nutrition therapy, growth hormone, or androgen have been introduced, but these interventions have not been successful because they unexpectedly increase the risk of organ failure and death. Furthermore, nutritional supplementation for trauma and surgical patients can actually have harmful effects.

[0003]

[0003] There is still no effective means for providing appropriate treatment and sufficient nutrition to critically ill patients.

[0004] [Summary of the Invention]

[0004] The degradation of cytoplasmic proteins is mediated by a cellular process called macroautophagy (also simply called autophagy). The autophagy process is also involved in the inflammatory response and promotes the destruction of bacteria by the immune system. Autophagy constitutes an important lysosomal degradation pathway that recycles damaged and potentially harmful cellular materials, such as damaged mitochondria. In particular, autophagy attenuates the effects of cell death and extends lifespan in various aging models. The inventors have surprisingly found that thymol and / or carvacrol induce autophagy in muscle. Furthermore, thymol and / or carvacrol synergistically induce autophagy in muscle in combination with a high-protein isocaloric diet.

[0005]

[0005] Accordingly, in general embodiments, the present disclosure provides a composition comprising thymol and / or carvacrol for use in treating, preventing, or managing a disease or condition associated with cellular dysfunction, genomic damage, changes in mitochondrial function, or a decrease in mitochondrial density in an individual in need thereof.

[0006]

[0006] The present invention also provides a composition for use in inducing autophagy in an individual in need thereof. The composition comprises an effective amount of thymol and / or carvacrol, alone or in combination with a high protein. The high amount of protein can be an amount of protein that is at least about 25 energy % of the composition, and / or the high amount of protein can be an amount of protein that provides a protein / energy ratio of more than 6 g / 100 kcal of the composition.

[0007]

[0007] In one embodiment, thymol and carvacrol are combined with an autophagy inducer selected from the group consisting of spermidine, urolithin (e.g., urolithin A, urolithin B, or urolithin D), rapamycin, Torin1, valproic acid, polyphenols (e.g., resveratrol), caffeine, metformin, 5’AMP-activated protein kinase (AMPK) activators, L-type calcium channel inhibitors, ketones (e.g., β-hydroxybutyrate, ketone salts, or ketone ester derivatives), and mixtures thereof.

[0008]

[0008] In one embodiment, autophagy is induced in skeletal muscle.

[0009]

[0009] In one embodiment, the individual is an elderly individual.

[0010]

[0010] In one embodiment, the individual has sarcopenia or frailty, or is at risk of developing sarcopenia or frailty.

[0011]

[0011] In one embodiment, the individual is severely ill.

[0012]

[0012] In one embodiment, the individual has severe myopathy or is at risk of developing severe myopathy.

[0013]

[0013] In one embodiment, the individual is in a severe condition accompanied by acute kidney injury or is at risk of developing acute kidney injury.

[0014]

[0014] In one embodiment, the individual has a neurodegenerative disease or a stroke.

[0015]

[0015] In one embodiment, the individual has a liver disease, e.g., gastrointestinal conditions such as NAFLD, NASH, or enteritis, e.g., ulcerative colitis, Crohn's disease, mucositis, and gut dysbiosis.

[0016] In one embodiment, the individual has chronic kidney disease, with or without associated reduced muscle mass or muscle function.

[0017] In one embodiment, the individual has cachexia or muscle wasting secondary to a chronic disease such as cancer, chronic obstructive pulmonary disease (COPD), chronic heart failure (CHF), acute kidney disease or chronic kidney disease (CKD).

[0018] In one embodiment, at least a portion of the protein is selected from the group consisting of (i) proteins derived from animal sources, (ii) proteins derived from plant sources, and (iii) mixtures thereof.

[0019] In one embodiment, at least a portion of the protein is selected from the group consisting of (i) milk protein, (ii) whey protein, (iii) casein salts, (iv) casein micelles, (v) pea protein, (vi) soy protein, and (vii) mixtures thereof.

[0020] In one embodiment, the protein has a formulation selected from the group consisting of (i) a formulation in which at least 50% by weight of the protein is casein, (ii) a formulation in which at least 50% by weight of the protein is whey protein, (iii) a formulation in which at least 50% by weight of the protein is pea protein, and (iv) a formulation in which at least 50% by weight of the protein is soy protein.

[0021] In one embodiment, at least a portion of the protein is selected from the group consisting of (i) free-form amino acids, (ii) non-hydrolyzed proteins, (iii) partially hydrolyzed proteins, (iv) extensively hydrolyzed proteins, and (v) mixtures thereof. In certain non-limiting examples, at least a portion of the protein is collagen, i.e., non-hydrolyzed collagen and / or hydrolyzed collagen.

[0022]

[0022] The protein may contain one or more amino acids selected from the group consisting of histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, arginine, cysteine, glutamine, glycine, proline, ornithine, serine, tyrosine, and mixtures thereof. The protein may contain peptides having a length of 2 to 10 amino acids.

[0023]

[0023] In one embodiment, the composition contains branched-chain amino acids in at least one form selected from the group consisting of (i) the free form, (ii) the form bound to at least one additional amino acid, and (iii) mixtures thereof.

[0024]

[0024] In one embodiment, at least a portion of the protein is 5 to 95% hydrolyzed.

[0025]

[0025] In one embodiment, the protein has a formulation selected from the group consisting of (i) a formulation in which at least 50% of the protein has a molecular weight of 1 to 5 kDa, (ii) a formulation in which at least 50% of the protein has a molecular weight of 5 to 10 kDa, and (iii) a formulation in which at least 50% of the protein has a molecular weight of 10 to 20 kDa.

[0026]

[0026] In one embodiment, the composition contains a carbohydrate source. The composition may have a high protein:carbohydrate ratio.

[0027]

[0027] In one embodiment, the step of administration uses at least one route selected from the group consisting of oral, enteral, parenteral, and intravenous injection.

[0028]

[0028] In another embodiment, the present disclosure provides a composition comprising a combination of thymol and / or carvacrol, optionally with high protein, wherein the composition contains the above combination in a single dose effective to induce autophagy in an individual in need of autophagy induction. The present composition may be selected from the group consisting of food compositions, dietary supplements, nutritional compositions, nutraceuticals, powdered nutritional formulations reconstituted with water or milk before ingestion, food additives, pharmaceuticals, drinks, and combinations thereof.

[0029]

[0029] In another embodiment, the present disclosure provides a method for manufacturing a therapeutic composition, the method comprising adding a combination of thymol and / or carvacrol, alone or in combination with high protein, to a base composition to form a therapeutic composition, wherein the therapeutic composition contains the above combination in a single dose effective to induce autophagy in an individual in need of autophagy induction. The base composition may be formulated for administration by at least one route selected from the group consisting of oral, enteral, parenteral, and intravenous injection.

[0030]

[0030] In another embodiment, a composition comprising thymol and / or carvacrol, alone or in combination with high protein, promotes both protein synthesis and the removal of damaged cellular material simultaneously in an individual in need of protein synthesis and the removal of damaged cellular material.

[0031]

[0031] In another embodiment, the present disclosure provides a method of obtaining at least one result selected from the group consisting of: (i) an increase in the level of LC3-II protein expression or turnover, (ii) an increase in the level of the LC3-II / LC3-I protein ratio, (iii) a decrease in the level of p62 protein, (iv) a decrease in the level of autophagosome protein, (v) an increase in the level of mRNA expression of autophagy-related genes, (vi) an increase in the number and / or size and / or intensity of LC3-positive granules, and (vii) degradation of LC3 and / or another autophagosome protein. The method includes administering a composition comprising a combination of thymol and / or carvacrol, optionally with a high protein, in a therapeutically effective amount to an individual in need of such composition.

[0032]

[0032] An advantage of one or more embodiments provided by the present disclosure is to improve the condition of an individual, a severely ill animal, a severely ill human, an aged animal, or an aged human.

[0033]

[0033] Another advantage of one or more embodiments provided by the present disclosure is to prevent or treat excessive catabolism, for example, in a severely ill patient, an individual, or an aged individual.

[0034]

[0034] Yet another advantage of one or more embodiments provided by the present disclosure is to reduce or prevent the risk of morbidity or death due to excessive catabolism.

[0035]

[0035] An additional advantage of the present disclosure is to recover, treat, or cure a severely ill patient from multiple organ dysfunction syndrome.

[0036]

[0036] An additional advantage of one or more embodiments provided by the present disclosure is to protect aged individuals from neurological diseases such as mild cognitive impairment, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, dementia, and related rare neurological diseases.

[0037]

[0037] Additional advantages of one or more embodiments provided by the present disclosure include, for example, protecting elderly individuals from muscle dysfunction such as sarcopenia, frailty, inclusion body myositis, drug-induced myopathy / rhabdomyolysis such as corticosteroids or statins, and muscle wasting induced by immobility or hospitalization.

[0038]

[0038] Additional advantages of one or more embodiments provided by the present disclosure include protecting patients suffering from genetic diseases, including but not limited to muscular dystrophies such as Duchenne muscular dystrophy or type VI collagen muscular dystrophy, mitochondrial encephalomyopathy, mitochondrial myopathy, glycogenosis, lysosomal storage diseases, and Pompe disease.

[0039]

[0039] Another advantage of one or more embodiments provided by the present disclosure is a composition that can be administered parenterally or enterally to an individual or a critically ill patient, for example, as an aqueous liquid composition, to induce autophagy.

[0040]

[0040] Yet another advantage of one or more embodiments provided by the present disclosure is shortening the period during which a critically ill patient is on a ventilator or advancing the period until the ventilator is removed.

[0041]

[0041] Another advantage of one or more embodiments provided by the present disclosure is protecting critically ill patients receiving parenteral nutrition from multiple organ failure or muscle weakness caused by, for example, parenteral nutrition delivery, particularly unbalanced nutrition or relative overnutrition.

[0042]

[0042] Additional advantages of one or more embodiments provided by the present disclosure include protecting elderly individuals from muscle weakness.

[0043]

[0043] Yet another advantage of one or more embodiments provided by the present disclosure is improving the viability of critically ill patients or elderly individuals.

[0044]

[0044] An additional advantage of one or more embodiments provided by the present disclosure is to accelerate the recovery of mobility or shorten the period of immobility after discharge from the intensive care unit.

[0045]

[0045] Yet another advantage of one or more embodiments provided by the present disclosure is that the effect is beneficial even when the critically ill patient is in a state where the life is already considerably advanced to a life-threatening state.

[0046]

[0046] Additional features and advantages will be described in the following detailed description of the invention and the drawings, and will become apparent therefrom.

Brief Description of the Drawings

[0047]

[0047]

Figure 1

Figure 2

Figure 3

Figure 4

BEST MODE FOR CARRYING OUT THE INVENTION

[0048]

[0051] Definitions

[0052] Some definitions are shown below. However, there may be cases where the definitions are in the section "Embodiments" below, and the above heading "Definitions" does not mean that such disclosure in the section "Embodiments" is not a definition.

[0049]

[0053] All percentages are by weight based on the total weight of the composition unless otherwise specified. Similarly, all ratios are by weight unless otherwise specified. When a reference is made to pH, the value corresponds to the pH measured at 25°C with a standard device. As used herein, "about", "approximately", and "substantially" are understood to refer to numbers within a numerical range, for example, within -10% to +10% of the reference number, preferably within -5% to +5% of the reference number, more preferably within -1% to +1% of the reference number, and most preferably within -0.1% to +0.1% of the reference number.

[0050]

[0054] Furthermore, all numerical ranges in this specification are to be understood to include all integers, whole numbers, or fractions within that range. Furthermore, these numerical ranges are to be interpreted as supporting claims directed to any number or subset of numbers within this range. For example, the disclosure of 1 to 10 is to be interpreted as supporting ranges such as 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0051]

[0055] As used in this specification and the appended claims, unless the context clearly indicates otherwise, singular words include the plural. Thus, references to "one", "a", and "the" generally include the plural of each such term. For example, reference to "an ingredient" or "a method" includes a plurality of such "ingredients" or "methods". The term "and / or" used in the context of "X and / or Y" should be construed as "X" or "Y" or "X and Y". Similarly, "at least one of X or Y" should be construed as "X" or "Y" or "both X and Y".

[0052]

[0056] Similarly, the terms "comprise", "comprises", and "comprising" should be construed as non-exclusive and capable of including other elements. Similarly, the terms "include", "including", and "or" are all construed as capable of including other elements unless such construction is clearly precluded by the context. However, the embodiments provided by the present disclosure may not include any element not specifically disclosed herein. Thus, the disclosure of an embodiment defined using the term "comprising" is also a disclosure of an embodiment "consisting essentially of" and "consisting of" the disclosed components. "Consisting essentially of" means that the embodiment contains more than 50% by weight of a particular component, preferably at least 75% by weight of a particular component, more preferably at least 85% by weight of a particular component, most preferably at least 95% by weight of a particular component, for example, at least 99% by weight of a particular component.

[0053]

[0057] As used herein, the term "example" is merely illustrative and for the purpose of explanation, and should not be considered exclusive or inclusive, particularly when followed by a list of terms. All embodiments disclosed herein can be combined with any other embodiment disclosed herein, unless expressly indicated otherwise.

[0054]

[0058] Examples of "animals" include, but are not limited to, mammals such as rodents, aquatic mammals, domestic animals such as dogs and cats, livestock such as sheep, pigs, cows and horses, and humans. When the terms "animal", "mammal", or their plurals are used, these terms also apply to any animal that has the ability for the effects indicated or shown by the context of the sentence, for example, an animal capable of autophagy. As used herein, the term "patient" is understood to include an animal, such as a mammal, preferably a human, that is undergoing or scheduled to undergo treatment as defined herein. As used herein, the terms "individual" and "patient" are often used to refer to humans, but the present disclosure is not limited to humans.

[0055]

[0059] Accordingly, the terms "individual" and "patient" refer to any animal, mammal or human to which the methods and compositions disclosed herein may be beneficial. In fact, animals other than humans experience long-term serious illnesses that closely resemble those in humans. These seriously ill animals experience the same metabolic disorders, immune disorders, and endocrine gland disorders, as well as the onset of organ failure and muscle wasting, as their human counterparts. In addition, animals also experience the effects of aging.

[0056]

[0060] The term "aged" means, in relation to humans, an age of at least 55 years, preferably over 63 years, more preferably over 65 years, and most preferably over 70 years. The terms "older adult" or "ageing individual" in the context of humans mean an age of at least 45 years, preferably 50 years or older, more preferably 55 years or older, and include aged individuals.

[0057]

[0061] For other animals, "older adult" or "ageing individual" means more than 50% of the average lifespan of that individual species and / or genus within the species. An animal is considered "aged" when it exceeds 66% of its average life expectancy, preferably when it exceeds 75% of its average life expectancy, and more preferably when it exceeds 80% of its average life expectancy. An aged cat or dog is at least about 5 years old. An aged cat or dog is at least about 7 years old.

[0058]

[0062] Sarcopenia is defined as a decrease in muscle mass and muscle function (including muscle strength and walking speed) associated with aging. Sarcopenia can be evaluated by one or more of muscle mass loss, muscle strength loss, and physical performance decline.

[0059]

[0063] Sarcopenia can be diagnosed in a subject based on the definition of the AWGSOP (Asian Working Group for Sarcopenia in Older People), as described, for example, in Chen et al. (2014). Muscle mass loss can generally be based on a decrease in appendicular lean mass (ALM index) normalized to the square of height, specifically an ALM index of less than 7.00 kg / m2 for men and less than 5.40 kg / m2 for women. A decline in physical performance can generally be based on walking speed, specifically a walking speed of less than 0.8 m / second. Muscle strength loss can generally be based on a decrease in grip strength, specifically a grip strength of less than 26 kg for men and less than 18 kg for women.

[0060]

[0064] Additionally or alternatively, sarcopenia can be diagnosed in a subject based on the definition of the European Working Group for Sarcopenia in Older People (EWGSOP), as described, for example, by Crutz-Jentoft et al. (2010). Muscle mass loss can generally be based on a decrease in appendicular lean mass (ALM index) normalized to the square of height, specifically, an ALM index of less than 7.23 kg / m2 for men and less than 5.67 kg / m2 for women can be used as a criterion. Decrease in physical performance can generally be based on walking speed, specifically, a walking speed of less than 0.8 m / second can be used as a criterion. Muscle strength loss can generally be based on a decrease in grip strength, specifically, a grip strength of less than 30 kg for men and less than 20 kg for women can be used as a criterion.

[0061]

[0065] Additionally or alternatively, sarcopenia can be diagnosed in a subject based on the definition of the Foundation for the National Institutes of Health (FNIH), as described, for example, by Studenski et al. (2014). Muscle mass loss can generally be based on a decrease in appendicular lean mass (ALM) normalized to body mass index (BMI; kg / m2), specifically, an ALM to BMI of less than 0.789 for men and less than 0.512 for women can be used as a criterion. Decrease in physical performance can generally be based on walking speed, specifically, a walking speed of less than 0.8 m / second can be used as a criterion. Muscle strength loss can generally be based on a decrease in grip strength, specifically, a grip strength of less than 26 kg for men and less than 16 kg for women can be used as a criterion. Muscle strength loss can also generally be based on a decrease in grip strength to body mass index, specifically, a grip strength to body mass index of less than 1.00 for men and less than 0.56 for women can be used as a criterion.

[0062]

[0066] As used herein, "frailty" is defined as a clinically recognizable state in which, as a result of increased vulnerability due to a decline in reserve capacity and function across multiple physiological systems with aging, the ability to cope with daily or acute stressors is impaired. In the absence of established quantitative criteria, frailty is functionally defined by Fried et al. as meeting three of the following five phenotypic criteria indicating impaired energy characteristics: (1) reduced muscle strength (grip strength at baseline within the lowest 20% of the population, corrected for sex and body mass index), (2) poor endurance and energy deficiency (self-reported fatigue related to maximum VO 2 (3) slowness of movement (baseline based on 15-foot walking time within the lowest 20% of the population, corrected for sex and height), (4) reduced physical activity (weighted score of kilocalories consumed per week at baseline, lowest quintile of physical activity identified 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 (10 pounds in the 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). The pre-frail stage, in which one or two of these criteria are present, is identified as having a high risk of progressing to frailty.

[0063]

[0067] "Cachexia" is a complex metabolic syndrome associated with underlying disease, characterized by muscle wasting, which may or may not be accompanied by a reduction in fat mass. Weight loss in adults (corrected for fluid retention) or growth failure in children (excluding endocrine disorders) are prominent clinical features of cachexia.

[0064]

[0068] Cachexia is often seen in patients with diseases such as cancer, chronic heart failure, kidney failure, chronic obstructive pulmonary disease, AIDS, autoimmune disorders, chronic inflammatory disorders, cirrhosis, anorexia nervosa, chronic pancreatitis, and / or metabolic acidosis and neurodegenerative diseases.

[0065]

[0069] In certain types of cancer, such as pancreatic cancer, esophageal cancer, gastric cancer, intestinal cancer, lung cancer, and / or liver cancer, cachexia is particularly commonly seen.

[0066]

[0070] Internationally recognized diagnostic criteria for cachexia are weight loss of more than 5% over a limited period, e.g., 6 months, or weight loss of more than 2% in an individual who already shows depletion by current weight and height (body mass index [BMI] less than 20 kg / m 2 2) or skeletal muscle mass (measured by DXA, MRI, CT, or bioimpedance). Cachexia can progress gradually through various stages, i.e., from pre-cachexia to cachexia and refractory cachexia. The severity can be classified in combination with ongoing weight loss according to the degree of depletion of energy stores and body protein (BMI).

[0067]

[0071] In particular, cancer cachexia is defined as weight loss of more than 5% in the past 6 months (in the absence of simple starvation); or BMI less than 20 and weight loss of more than 2%; or appendicular lean mass that is not inconsistent with muscle mass reduction (less than 7.26 kg / m 2 in men and less than 5.45 kg / m 2 in women), and any degree of weight loss of more than 2% (Fearon et al., 2011).

[0068]

[0072] "Pre-cachexia" can be defined as weight loss of 5% or less in combination with anorexia and metabolic changes. Currently, there are no strong biomarkers to identify patients with pre-cachexia who are likely to progress further or the rate at which pre-cachexia progresses. Refractory cachexia is essentially defined based on the clinical characteristics and situation of the patient.

[0069]

[0073] The terms "treatment" and "treating" include any effect that brings about an improvement in a condition or disease (disorder), such as alleviating, reducing, controlling, or eliminating a condition or disease. This term does not necessarily mean that the subject is treated until complete cure. Non-limiting examples of "treating" a condition or disease include (1) inhibiting a condition or disease, i.e., stopping the onset of a condition or disease or its clinical symptoms, and (2) alleviating a condition or disease, i.e., causing a temporary or permanent remission of a condition or disease or its clinical symptoms. Treatment may be relevant to a patient or to a physician.

[0070]

[0074] The term "prevention" or "preventing" means not causing the clinical symptoms of a stated condition or disorder in an individual who may be exposed to or is prone to developing a condition or disorder but who has not yet presented or manifested the symptoms of the condition or disorder. The terms "condition" and "disease / disorder" mean any disease, condition, symptom, or sign.

[0071]

[0075] Relative terms such as "improved", "increased", and "enhanced" refer to the effect of comparing a composition of the present invention comprising a combination of an autophagy inducer (e.g., spermidine) and a high protein (disclosed herein) with a composition that is identical except for having less protein.

[0072]

[0076] 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 such an individual. The compositions of the present disclosure, including many embodiments described herein, may include, consist of, or consist essentially of any additional or optional ingredients, components, or limitations described herein or useful in a dietary regimen, in addition to the essential elements and limitations described herein.

[0073]

[0077] As used herein, "complete nutrition" contains sufficient types and levels of macronutrients (protein, lipid, and carbohydrate) and micronutrients to be a sufficient sole source of nutrition for the animal to which the composition is administered. An individual can receive 100% of the nutrients the individual needs from such a complete nutrition composition.

[0074]

[0078] As used herein, the term "critically ill patient" is an individual who has developed acute life-threatening symptoms or who has been diagnosed as being at imminent risk of such development. Critically ill patients are medically unstable and have a high likelihood of death (e.g., greater than 50% mortality rate) if not treated.

[0075]

[0079] Non-limiting examples of critically ill patients include patients who have, or are at risk of having, persistent acute single or multiple organ system failure due to a disease or injury, patients who have undergone surgery and are experiencing complications, and patients who have had major surgery on a vital organ or major surgery during the past week.

[0076]

[0080] More specific non-limiting examples of critically ill patients include patients who have, or are at risk of having, acute single or multiple organ system failure threatening life due to a disease or injury, and patients who have undergone surgery and are suffering from complications. Further specific non-limiting examples of critically ill patients include patients who require one or more of cardiac surgery, brain surgery, thoracic surgery, abdominal surgery, vascular surgery, or transplantation, and patients suffering from one or more of neurological diseases, brain trauma, respiratory failure, peritonitis, multiple trauma, severe burns, or severe polyneuropathy.

[0077]

[0081] The term "intensive care unit (ICU)" refers to a part of a hospital that treats critically ill patients. The term "intensive care unit" also includes, if the treatment activities carried out there are the same as or similar to those of an ICU, nursing homes; clinics (e.g., private clinics), etc. An "ICU patient" is included in the term "critically ill patient".

[0078]

[0082] The term "multiple organ dysfunction" refers to a condition resulting from infections, decreased blood flow, metabolic hyperactivity, or injuries such as accidents or surgeries. "Multiple organ failure" in critically ill patients leading to death is considered a descriptive clinical syndrome defined as the dysfunction or failure of at least two major organ systems. The major organ systems that are uniformly and most specifically affected are the liver, kidneys, lungs, as well as the cardiovascular, nervous, and hematological systems. Non-limiting examples of multiple organ dysfunction include acute respiratory distress syndrome, heart failure, liver failure, kidney failure, respiratory failure, intensive care, shock, extensive burns, sepsis (e.g., systemic inflammatory response syndrome), and stroke.

[0079]

[0083] The term "enteral administration" includes oral administration (including oral forced nutrition) and rectal administration, with oral administration being preferred. The term "parenteral administration" refers to the delivery of substances administered by routes other than the gastrointestinal tract, including intravenous, intra-arterial, intramuscular, intraventricular, intraosseous, intradermal, subarachnoid, and also intraperitoneal administration, intravesical instillation, and intracorporal injection, etc.

[0080]

[0084] Preferred parenteral administration is intravenous administration. A specific form of parenteral administration is delivery by intravenous administration of nutrition. Parenteral nutrition is "total parenteral nutrition" when food is not provided by other routes. "Parenteral nutrition" preferably contains saccharides such as glucose and further contains one or more of lipids, amino acids, and vitamins, and is an isotonic aqueous solution or a hypertonic aqueous solution (or a solid composition to be dissolved, or a liquid concentrate diluted to obtain an isotonic solution or a hypertonic solution).

[0081]

[0085] Embodiment

[0086] Accordingly, in general embodiments, the present disclosure provides a composition comprising thymol and / or carvacrol for use in the treatment, prevention, or management of a disease or condition associated with cellular dysfunction, genomic damage, changes in mitochondrial function, or a decrease in mitochondrial density in an individual in need of such treatment, prevention, or management. The composition also provides a composition for inducing autophagy.

[0082]

[0087] In one aspect of the present invention, the composition increases antioxidant capacity, reduces oxidative stress, maintains immune function, and / or maintains cognitive function in healthy middle-aged and elderly individuals.

[0083]

[0088] In another aspect, the mitochondrial-related disease or condition is selected from the group consisting of stress, obesity, decreased metabolic rate, metabolic syndrome, type 2 diabetes, diabetic complications, hyperlipidemia, elevated free fatty acids, liver disease, NAFLD, NASH, neurodegenerative diseases, stroke, cognitive impairment, stress-induced or stress-related cognitive dysfunction, mood disorders, anxiety disorders, age-related neuronal death or nerve dysfunction, musculoskeletal disorders, frailty, pre-frailty, chronic kidney disease, gastrointestinal disorders, trauma, infection, cancer, macular degeneration, and combinations thereof.

[0084]

[0089] Another aspect of the present invention is a method of inducing autophagy in an individual in need of autophagy induction. The method includes administering a composition comprising thymol and / or carbachol, optionally together with a high protein (e.g., about 25% of the total energy of the composition), wherein the composition is administered in an amount effective to provide a combination effective to induce autophagy, for example, in muscle. The composition can be administered parenterally, enterally, or intravenously.

[0085]

[0090] In a preferred embodiment, the composition further contains an autophagy inducer selected from the group consisting of spermidine, urolithin (e.g., urolithin A, urolithin B, or urolithin D), rapamycin, torin 1, valproic acid, polyphenols (e.g., resveratrol), caffeine, metformin, 5'-AMP-activated protein kinase (AMPK) activators, L-type calcium channel inhibitors, and mixtures thereof. Non-limiting examples of suitable autophagy inducers are spermidine, palmitic acid, 5-aminoimidazole-4-carboxamide riboside (AICAR), verapamil, nifedipine, diltiazem, piperazine phenothiazine derivatives (e.g., trifluoperazine), ketones (e.g., β-hydroxybutyrate, ketone salts, or ketone ester derivatives), and mixtures thereof. Non-limiting examples of suitable forms of spermidine include spermidine trihydrochloride, spermidine phosphate hexahydrate, spermidine phosphate hexahydrate, and L-arginyl-3,4-spermidine.

[0086]

[0091] In one embodiment, the composition has a protein / energy ratio greater than 6 g protein / 100 kcal, preferably greater than 9 g protein / 100 kcal. In one embodiment, the protein is at least 24 energy % of the composition, more preferably at least 36 energy % of the composition.

[0087]

[0092] As a non-limiting example, the composition can be administered in a daily dose that provides an amount of protein that exceeds 1.0 g of protein per body weight (kg) per day, preferably exceeds 1.2 g of protein per body weight (kg) per day; for example, up to 2.5 g of protein per body weight (kg) per day (e.g., 1.0 - 2.5 g of protein per body weight (kg) per day; 1.2 - 2.5 g of protein per body weight (kg) per day; or 1.5 - 2.5 g of protein per body weight (kg) per day), preferably up to 2.0 g of protein per body weight (kg) per day (e.g., 1.0 - 2.0 g of protein per body weight (kg) per day; 1.2 - 2.0 g of protein per body weight (kg) per day; or 1.5 - 2.0 g of protein per body weight (kg) per day), and more preferably up to 1.5 g of protein per body weight (kg) per day (e.g., 1.0 - 1.5 g of protein per body weight (kg) per day or 1.2 - 1.5 g of protein per body weight (kg) per day). The daily dose of protein can be provided by ingesting the composition one or more times per day.

[0088]

[0093] When the composition is in liquid form, non-limiting examples of suitable high protein concentrations include 6 - 20 g of protein / 100 mL, for example, 6 - 11 g of protein / 100 mL; 7 - 14 g of protein / 100 mL; 7 - 12 g of protein / 100 mL; 8 - 11 g of protein / 100 mL, 8 - 20 g of protein / 100 mL; 9 - 20 g of protein / 100 mL; and 11 - 20 g of protein / 100 mL.

[0089]

[0094] The composition can contain a pharmacologically effective amount of thymol and / or carvacrol in a pharmaceutically suitable carrier. In an aqueous liquid composition, the concentration is preferably in the range of about 0.05 wt% to about 4 wt%, or about 0.5 wt% to about 2 wt%, or about 1.0 wt% to about 1.5 wt% of the aqueous liquid composition.

[0090]

[0095] In certain embodiments, the method is a process of increasing the plasma thymol and / or carvacrol values in an individual to values of, for example, 50 to 6000 nmol, preferably 100 to 6000 nmol, per liter of plasma. The method may include a step of administering thymol and / or carvacrol daily in a weight range of 0.05 mg to 1 g, preferably 1 mg to 200 mg, more preferably 5 mg to 150 mg, even more preferably 10 mg to 120 mg, or most preferably 40 mg to 80 mg per kilogram of body weight.

[0091]

[0096] Typically, 50 μg to 10 g of thymol and / or carvacrol per day is administered to the individual in divided doses more than once.

[0092] Thymol (10 - 64%) is one of the major components of the essential oil of Thymus vulgaris L, Lamiaceae. Carvacrol is present in the essential oil of Origanum vulgare (oregano), thyme oil, oil obtained from Capsicum annuum, and Pogostemon cablin. The essential oil of thyme subspecies contains 5% - 75% carvacrol, while Satureja subspecies has a content of 1% - 45%. Origanum majorana and Dittany of Crete are rich in carvacrol, 50% and 60 - 80% respectively. Thus, some embodiments of the present composition include plants and / or concentrated plant extracts, essential oils or fractions that provide at least a portion of the thymol and / or carvacrol in the composition, particularly from thyme and oregano.

[0093]

[0097] The present composition can induce autophagy in muscles, such as skeletal muscles. Non-limiting examples of such muscles include one or more of the following: vastus lateralis, gastrocnemius, tibialis anterior, soleus, extensor digitorum longus (EDL), biceps femoris, semitendinosus, semimembranosus, gluteus maximus, extraocular muscles, facial muscles, or diaphragm.

[0094]

[0098] Individuals in need of autophagy induction can be individuals with mitochondrial-related diseases or conditions selected from the group consisting of stress, obesity, decreased metabolic rate, metabolic syndrome, type 2 diabetes, diabetic complications, hyperlipidemia, elevated free fatty acids, liver diseases, NAFLD, NASH, neurodegenerative diseases, stroke, cognitive impairment, stress-induced or stress-related cognitive dysfunction, mood disorders, anxiety disorders, age-related neuronal death or nerve dysfunction, musculoskeletal disorders, frailty, pre-frailty, chronic kidney disease, gastrointestinal disorders (such as ulcerative colitis, Crohn's disease, mucositis, and intestinal microbiota dysbiosis), trauma, infection, cancer, macular degeneration, and combinations thereof.

[0095]

[0099] Individuals in need of autophagy induction can be elderly individuals such as elderly animals or elderly humans. In some embodiments, the individuals in need of autophagy induction are elderly animals or elderly humans.

[0096]

[0100] Individuals in need of autophagy induction can be critically ill patients. In various embodiments, the method can treat or prevent multiple organ dysfunction in critically ill patients, for example, when homeostasis fails or is impaired, such as when the patient receives parenteral nutrition; protect critically ill patients from multiple organ dysfunction; treat or prevent the onset of lactic acidosis, such as lactic acidosis induced by parenteral nutrition; treat or prevent muscle weakness in critically ill patients; reduce or prevent morbidity or death due to deterioration of nutritional status caused by parenteral nutrition; and / or prevent the collapse of the body system.

[0097]

[0101] In some embodiments, a critically ill patient has at least one life-threatening condition selected from the group consisting of lactic acidosis, muscle weakness, hyperglycemia, multiple organ failure, failure of homeostasis, and disruption of homeostasis. In one embodiment, the critically ill patient has a non-infectious disease. In one embodiment, the critically ill patient has multiple organ dysfunction that is not caused by or related to sepsis. Multiple organ dysfunction and muscle weakness are common in the emergency rescue scene and can be caused or exacerbated by uncoordinated parenteral nutrition delivery or relative or absolute overnutrition delivered parenterally.

[0098]

[0102] In some embodiments, an individual or critically ill patient has at least one disorder selected from the group consisting of severe trauma, multiple trauma, stroke, neurodegenerative disease, high-risk surgery, extensive surgery, traumatic brain injury, intracerebral hemorrhage, respiratory failure, peritonitis, acute kidney injury, acute liver injury, NAFLD, NASH, gastrointestinal disorders (such as ulcerative colitis, Crohn's disease, mucositis, and dysbiosis of intestinal flora), severe burns, severe polyneuropathy, severe myopathy, and ICU-acquired muscle weakness.

[0099]

[0103] In some embodiments, an individual or critically ill patient is receiving enteral or parenteral nutrition. In some embodiments, the composition treats or prevents mitochondrial dysfunction, such as mitochondrial dysfunction induced by inadequate or uncoordinated parenteral nutrition in critically ill patients.

[0100]

[0104] In another aspect of the disclosure, the method comprises obtaining at least one result selected from the group consisting of: an increase in the level of LC3-II protein expression or turnover (e.g., measurable by Western blot, mass spectrometry, ELISA, aptamer- or nanobody-based proteomics); an increase in the level of the LC3-II / LC3-I protein ratio (e.g., measurable by any of the methods described above); a decrease in the level of p62 protein (e.g., measurable by the methods described above); a decrease in the level of autophagosome proteins (e.g., but not limited to, Atg5, Beclin-1, Atg7, Atg12); an increase in the level of mRNA expression of autophagy-related genes (e.g., but not limited to, MAP1LC3, GABARAP, Atg5, Beclin-1, Atg7, Atg12); and an increase in the number and / or size and / or intensity of LC3-positive granules (evaluable by immunofluorescence, or by labeling LC3 with a fluorescent reporter protein such as GFP, or by flow cytometry); degradation of LC3 and / or another autophagosome protein (evaluable by assessing its lysosomal degradation as evaluated by microscopy or flow cytometry, e.g., by fusing the protein to a pH-sensitive fluorescent reporter that changes color when it reaches the lysosome; or by comparing the fluorescence intensity of the WT protein to that of a mutant protein that cannot be inserted into the autophagosome (e.g., the LC3ΔG mutant that cannot be lipidated and inserted into the autophagosome)). The method comprises administering to an individual in need of induction of autophagy a composition comprising a combination of an autophagy inducer and a high protein in a therapeutically effective amount.

[0101]

[0105] As used herein, the term "protein" includes free amino acids, molecules of 2 to 20 amino acids (referred to herein as "peptides"), and, similarly, longer amino acid chains. Small peptides, i.e., chains of 2 to 10 amino acids, are suitable for the present composition either alone or in combination with other proteins. The "free form" of an amino acid is the monomeric form of the amino acid. Suitable amino acids include both natural and non-natural amino acids. The present composition may contain one or more proteins, for example, a mixture of one or more (i) peptides, (ii) longer amino acid chains, or (iii) free amino acids; this mixture is preferably formulated to obtain the desired amino acid profile / content.

[0102]

[0106] At least a portion of the protein can be of animal origin or plant origin, for example, dairy proteins such as milk protein concentrate or milk protein isolate; casein salts or casein, such as casein micelle concentrate or casein micelle isolate; or whey protein such as whey protein concentrate or whey protein isolate, etc., and can be derived from milk proteins, among others. Additionally or alternatively, at least a portion of the protein can be a vegetable protein such as one or more of soy protein or pea protein.

[0103]

[0107] Mixtures of these proteins, for example, mixtures in which casein is a majority but not all of the protein, mixtures in which whey protein is a majority but not all of the protein, mixtures in which pea protein is a majority but not all of the protein, and mixtures in which soy protein is a majority but not all of the protein are also suitable. In one embodiment, at least 10% by weight, preferably at least 20% by weight, more preferably at least 30% by weight of the protein is whey protein. In one embodiment, at least 10% by weight, preferably at least 20% by weight, more preferably at least 30% by weight of the protein is casein. In one embodiment, at least 10% by weight, preferably at least 20% by weight, more preferably at least 30% by weight of the protein is plant protein.

[0104]

[0108] Whey protein can be any whey protein selected from the group consisting of, for example, whey protein concentrate, whey protein isolate, whey protein micelle, whey protein hydrolyzate, acid whey, sweet whey, denatured sweet whey (sweet whey from which caseinoglycomacropeptide has been removed), fractions of whey protein, and any combination thereof.

[0105]

[0109] Casein can be obtained from any mammal, preferably from milk, and preferably obtained as casein micelles.

[0106]

[0110] The protein may not be hydrolyzed, may be partially hydrolyzed (i.e., peptides having a molecular weight of 3 kDa to 10 kDa and an average molecular weight of less than 5 kDa), or may be significantly hydrolyzed (i.e., 90% are peptides having a molecular weight of less than 3 kDa), for example, 5% to 95% of the range may be hydrolyzed. In some embodiments, the peptide profile of the hydrolyzed protein can be within different molecular weight ranges. For example, most of the peptides (more than 50 mol% or more than 50% by weight) can have a molecular weight within 1 to 5 kDa or 5 to 10 kDa or 10 to 20 kDa.

[0107]

[0111] The protein can contain essential amino acids and / or conditionally essential amino acids, for example, amino acids that may be insufficiently delivered in a calorie-restricted regimen. For example, the protein can contain one or more essential amino acids selected from the group consisting of histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine; each of these amino acids (if present) can be administered in the composition at a daily dose of about 0.0476 to about 47.6 mg per kg of body weight. In particular, when the intake of methionine is low, the translation level of the protein decreases, and ultimately the level of muscle synthesis decreases. The protein can contain one or more conditionally essential amino acids (for example, amino acids that are essential conditional on illness or stress) selected from the group consisting of arginine, cysteine, glutamine, glycine, proline, ornithine, serine, and tyrosine; each of these amino acids (if present) can be administered in the composition at a daily dose of about 0.0476 to about 47.6 mg per kg of body weight.

[0108]

[0112] The composition can contain one or more branched-chain amino acids (BCAAs). For example, the composition can contain leucine, isoleucine, and / or valine in free form and / or bound as peptides and / or proteins (such as milk protein, animal protein, or plant protein). The daily dose of branched-chain amino acids can include one or more of 0.35 to 142.85 mg of leucine per kg of body weight, preferably 0.175 to 71.425 mg of leucine per kg of body weight; 0.175 to 71.425 mg of isoleucine per kg of body weight; and 0.175 to 71.425 mg of valine per kg of body weight. The daily dose of one or more branched-chain amino acids can be provided by ingesting the composition more than once a day.

[0109]

[0113] Whey protein is rich in BCAA. Accordingly, some embodiments of the composition contain whey protein that provides at least a portion of the BCAA in the composition.

[0110]

[0114] In one embodiment, the composition comprises a carbohydrate source. Any suitable carbohydrate may be used in the composition, including, but not limited to, starch (e.g., modified starch, amylose starch, tapioca starch, corn starch), sucrose, lactose, glucose, fructose, solid corn syrup, maltodextrin, xylitol, sorbitol, or combinations thereof.

[0111]

[0115] The carbohydrate source is preferably 50 energy % or less of the composition, more preferably 36 energy % or less of the composition, and most preferably 30 energy % or less of the composition. The composition can have a high protein:carbohydrate energy ratio, for example, greater than 0.66, preferably greater than 0.9, more preferably greater than 1.2.

[0112]

[0116] In one embodiment, the composition comprises a fat source. The fat source can include any suitable fat or fat mixture. Non-limiting examples of suitable fat sources include vegetable fats (e.g., olive oil, corn oil, sunflower oil, high oleic sunflower oil, rapeseed oil, canola oil, hazelnut oil, soybean oil, palm oil, coconut oil, croton tiglium seed oil, perilla oil, lecithin, etc.), animal fats (e.g., milk fat); or combinations thereof.

[0113]

[0117] A composition comprising a combination of thymol and / or carvacrol, optionally in combination with a high protein, can be administered in a therapeutically effective amount to a human, e.g., an elderly or severely ill individual. A therapeutically effective dose can be determined by one of ordinary skill in the art and can vary depending on several factors known to those of ordinary skill in the art, such as the severity of the condition and the weight and general condition of the individual.

[0114]

[0118] The composition is preferably administered to an individual at least 2 days per week, more preferably at least 3 days per week, and most preferably 7 days per week; for at least 1 week, at least 1 month, at least 2 months, at least 3 months, at least 6 months, or even longer periods. In some embodiments, the composition is administered to the individual continuously for several days, for example, until at least a therapeutic effect is achieved. In some embodiments, the composition can be administered to the individual daily continuously for at least 30 days, 60 days, or 90 days.

[0115]

[0119] The above dosing examples do not require continuous daily dosing without interruption. Rather, there may be several short interruptions in dosing, such as a 2 - 4 day interruption during the dosing period. The ideal dosing duration of the composition can be determined by those skilled in the art.

[0116]

[0120] In a preferred embodiment, the composition is administered to the individual orally or enterally (e.g., by tube feeding). For example, the composition can be administered to the individual as a beverage, capsule, tablet, powder, or suspension.

[0117]

[0121] The composition can be any type of composition suitable for ingestion by humans and / or animals. For example, the composition can be selected from the group consisting of food compositions, dietary supplements, nutritional compositions, nutraceuticals, powder nutritional preparations reconstituted with water or milk before ingestion, food additives, pharmaceuticals, beverages, and drinks. In one embodiment, the composition is an oral nutritional supplement (ONS), a complete nutritional formulation, a pharmaceutical, a drug, or a food product. In a preferred embodiment, the composition is administered to the individual as a beverage. The composition can be stored in sachets as a powder and then suspended in a liquid such as water when used.

[0118]

[0122] In some cases where oral or enteral administration is not possible or not recommended, the composition may also be administered parenterally.

[0119]

[0123] In some embodiments, the composition is administered to an individual in a single-dose formulation, i.e., all the compounds are present in one formulation that is given to the individual with a meal. In other embodiments, the composition is co-administered in separate dosage forms, e.g., at least one of the components and one or more of the other components of the composition are co-administered separately.

[0124]

Examples

[0120]

[0125] Example 1: In Vitro Experiment

[0126] Materials and Methods

[0127] Autophagic flux in vitro was measured using the human lymphocyte T cell line Jurkat (clone E6.1, ATCC TIB-152). Cells were grown in RPMI medium using standard conditions (5% CO2, 37°C) in a humidified atmosphere. For the experiment, cells were washed, counted, and incubated in duplicate at 1×105 cells / well in a 96-well flat-bottom plate. Experimental conditions included a negative control (0.5% DMSO), rapamycin treatment (1 μM) as a positive control, and Earle's balanced salt solution (EBSS) as a starvation medium. Cells were treated with different concentrations of thymol (in RPMI) in the range of 1.95 μM to 250 μM. In parallel, the same experimental conditions were prepared for incubation using Solution A of the Merck Flow Cellect Autophagy Kit (now the Guava Autophagy LC3 Antibody-based Kit). Treatment with Compound A blocks the lysosomal degradation of LC3 vesicles and is used for the measurement of autophagic flux. Cells were incubated with thymol at 37°C for 1.30 hours. Solution A was added for an additional 30 minutes. Cells were then transferred to a 96-well V-bottom plate for LC3 antibody detection using the Guava Autophagy LC3 Antibody-based Kit according to the manufacturer's instructions. Briefly, cells were permeabilized to remove cytosolic LC3 (LC3-I) and incubated with a monoclonal anti-LC3 antibody labeled with the fluorophore fluorescein isothiocyanate (FITC). Samples were then collected using a Becton Dickinson LSORP Fortessa flow cytometry analyzer. Offline analysis was performed using FCS Express software and the results were expressed as the median fluorescence intensity in the FITC channel (related to the amount of LC3-II present in the cells).

[0121]

[0128] The results are shown in Figure 1. This figure shows that thymol induced autophagy in a dose-dependent manner starting from a concentration of 125 μM in human Jurkat cells.

[0122]

[0129] Example 2: Experiment on Autophagy Reporter Zebrafish

[0130] Materials and Methods

[0131] The autophagy reporter zebrafish line was generated by stable expression of LC3 protein fused to ZsGreen under the control of a skeletal muscle-specific promoter. Larvae of outbred transgenic zebrafish were raised at 28°C under standard laboratory conditions and treated with various concentrations of thymol shown in Figure 3 at 48 hours post-fertilization in 96-well plates. After 16 hours of treatment, the larvae were anesthetized with 0.016% tricaine and imaged at 20x magnification using an ImageXpress confocal system (Molecular Devices). Z-stack images of each larva were captured and maximum projection images were generated. Ammonium chloride was added for an additional 4 hours to block lysosomal degradation. Images were acquired again as before. To quantify autophagic flux, the number of LC3 puncta was calculated using MetaXpress software (Molecular Devices) in the presence and absence of ammonium chloride and normalized by the area of the zebrafish.

[0123]

[0132] The results are reported in Figure 2. This graph shows that thymol induced autophagy in a dose-dependent manner starting from a concentration of 50 μM in zebrafish larvae.

[0124]

[0133] Example 3: In Vivo Experiments

[0134] Materials and Methods

[0135] Short-term Treatment

[0136] Two types of treatments using thymol at concentrations of 20 mg / kg / body weight and 100 mg / kg / body weight were continuously performed for 2 days on 10 - 15-week-old C57bl6 / J before tissue extraction. After sacrificing the mice by isoflurane inhalation, bloodletting was performed. The liver was harvested and frozen in liquid nitrogen.

[0125]

[0137] Long-term Treatment in an Obesity Model

[0138] Mice were fed a high-fat diet (Research Diets D12492: 60% fat, 20% protein, 20% carbohydrate) for 8 weeks. After sacrificing the mice by isoflurane inhalation, bloodletting was performed. The liver was collected, embedded in OCT, and frozen in isopentane. To visualize lipid droplets, 10-μm liver sections were cut out and stained with Oil Red O. The size of the lipids was calculated using ImageJ software.

[0126]

[0139] Western blot

[0140] Total protein lysates were extracted from 30 - 50 mg of tissue homogenized in 20 mL / g of RIPA buffer (150 mM sodium chloride, 50 mM Tris (pH 8), 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, protease inhibitor cocktail) using a tissue dissociator (gentleMACS Miltenyi Biotec). Protein concentration was measured by BCA assay, and samples were prepared by adding 4X LDS sample buffer (Invitrogen). 20 μg of protein was separated by SDS-PAGE on a 4 - 12% gradient gel and transferred to a PVDF membrane using a dry-type iBLOT system (Invitrogen). The membrane was incubated with LC3 (Novus Biologicals 2220) and GAPDH (Cell Signaling 2118) antibodies and detected with an ECL substrate (Pierce). Protein quantification was performed by densitometry analysis of the images using ImageJ software.

[0127]

[0141] The results are shown in Figures 3 and 4 respectively. Figure 3 shows the quantification by densitometry of the LC3-II / LC3-I protein amount in the Western blot performed on the liver of mice acutely treated with thymol, and Figure 4 shows the reduction of fatty liver in obese mice treated with thymol at 20 mg / kg / day for 8 weeks.

[0128]

[0142] It should be understood that various changes and modifications to the presently preferred embodiments described in this specification 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 subject matter of the present invention and without sacrificing the intended advantages. Therefore, such changes and modifications are intended to be included within the scope of the appended claims.

Claims

1. A composition comprising thymol and / or carvacrol for use in the treatment, prevention or management of a disease or condition associated with cellular dysfunction, genomic damage, altered mitochondrial function or reduced mitochondrial density in an individual in need thereof.

2. 10. The composition of claim 1 for use in increasing antioxidant capacity, reducing oxidative stress, maintaining immune function, and / or maintaining cognitive function in healthy middle-aged and elderly individuals.

3. 2. The composition of claim 1, wherein the mitochondrial-related disease or condition is selected from the group consisting of stress, obesity, reduced metabolic rate, metabolic syndrome, diabetes mellitus, diabetic complications, hyperlipidemia, elevated free fatty acids, liver disease, NAFLD, NASH, neurodegenerative disease, stroke, cognitive impairment, stress-induced or stress-related cognitive dysfunction, mood disorder, anxiety disorder, age-related neuronal cell death or dysfunction, musculoskeletal disorder, frailty, pre-frailty, chronic kidney disease, gastrointestinal disorders, trauma, infection, cancer, macular degeneration, and combinations thereof.

4. A composition comprising an effective amount of thymol and / or carvacrol for use in inducing autophagy in an individual in need thereof.

5. A composition according to any one of claims 1 to 4, wherein thymol and / or carvacrol are combined with high protein.

6. 6. The composition of claim 5, wherein the high amount of protein is an amount of protein that is at least about 25% of the energy of the composition.

7. 7. The composition of claim 5 or 6, wherein the high amount of protein is an amount of the protein that provides a protein / energy ratio of the composition of greater than 6 g / 100 kcal.

8. 8. The composition of claim 5, wherein at least a portion of the protein is selected from the group consisting of: (i) protein from animal sources, (ii) protein from plant sources, and (iii) mixtures thereof.

9. 9. The composition of any one of claims 5 to 8, wherein at least a portion of the proteins are selected from the group consisting of: (i) milk proteins, (ii) whey proteins, (iii) caseinates, (iv) casein micelles, (v) pea proteins, (vi) soy proteins, and (vii) mixtures thereof.

10. 10. The composition of any one of claims 5 to 9, wherein the protein has a formulation selected from the group consisting of: (i) a formulation in which at least 50% by weight of the protein is casein, (ii) a formulation in which at least 50% by weight of the protein is whey protein, (iii) a formulation in which at least 50% by weight of the protein is pea protein, and (iv) a formulation in which at least 50% by weight of the protein is soy protein.

11. 11. The composition of any one of claims 5 to 10, wherein at least a portion of the proteins is selected from the group consisting of: (i) amino acids in free form, (ii) non-hydrolyzed proteins, (iii) partially hydrolyzed proteins, (iv) extensively hydrolyzed proteins, and (v) mixtures thereof.

12. The composition of claim 11, wherein the protein comprises a peptide having a length of 2 to 10 amino acids.

13. 13. The composition of any one of claims 5 to 12, wherein the protein comprises branched chain amino acids in at least one form selected from the group consisting of: (i) in a free form, (ii) in a form bound to at least one additional amino acid, and (iii) a mixture thereof.

14. The composition according to any one of claims 1 to 13, wherein the composition comprises a carbohydrate source.

15. The composition of claim 14 , wherein the composition has a high protein:carbohydrate ratio.

16. 16. The composition of any one of claims 1 to 15, comprising an autophagy inducer selected from the group consisting of spermidine, urolithin, rapamycin, torin 1, valproic acid, polyphenols, caffeine, metformin, 5'AMP-activated protein kinase (AMPK) activators, L-type calcium channel inhibitors, ketones, and mixtures thereof.

17. The composition of any one of claims 1 to 16, wherein the autophagy is induced in skeletal muscle.

18. The composition of any one of claims 1 to 17, wherein the individual is an elderly individual.

19. The composition of any one of claims 1 to 18, wherein the individual has sarcopenia or frailty or is at risk of developing sarcopenia or frailty.

20. The composition of any one of claims 1 to 19, wherein the individual is critically ill.

21. The composition according to any one of claims 1 to 20, wherein the administering step uses at least one route selected from the group consisting of oral, enteral, parenteral, and intravenous injection.

22. A composition comprising a combination of thymol and / or carvacrol and a protein in a serving effective to induce autophagy in an individual in need thereof.

23. 23. The composition of claim 22, wherein the composition is selected from the group consisting of a food composition, a dietary supplement, a nutritional composition, a nutraceutical, a powdered nutritional formulation that is reconstituted with water or milk prior to ingestion, a food additive, a pharmaceutical product, a drink, and combinations thereof.

24. The composition according to claim 22, wherein at least a portion of the protein is a protein according to any one of claims 5 to 16.

25. 1. A method of making a therapeutic composition comprising: adding a combination of thymol and / or carvacrol, optionally with high protein, to a base composition to form said therapeutic composition; The method, wherein the therapeutic composition comprises the combination in a single serving effective to induce autophagy in an individual in need thereof.

26. 1. A method comprising administering a composition comprising a combination of an autophagy inducer and a high protein, The method, wherein the composition is administered to an individual in need thereof to provide an amount of the combination that simultaneously promotes protein synthesis and the removal of damaged cellular material.

27. 1. A method for obtaining at least one result selected from the group consisting of: (i) an increase in the level of LC3-II protein expression or turnover, (ii) an increase in the level of said LC3-II / LC3-I protein ratio, (iii) a decrease in the level of p62 protein, (iv) a decrease in the level of autophagosome proteins, (v) an increase in the level of mRNA expression of autophagy-related genes, (vi) an increase in the number and / or size and / or intensity of LC3-positive granules, and (vii) degradation of LC3 and / or another autophagosomal protein, The method comprises administering to an individual in need thereof a therapeutically effective amount of a composition comprising a combination of an autophagy inducer and a high protein.

Images