WO2018085440A1 - Reduction and prevention of muscle loss by conjugated linoleic acid (cla) and vitamin d - Google Patents

Reduction and prevention of muscle loss by conjugated linoleic acid (cla) and vitamin d Download PDF

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Publication number
WO2018085440A1
WO2018085440A1 PCT/US2017/059585 US2017059585W WO2018085440A1 WO 2018085440 A1 WO2018085440 A1 WO 2018085440A1 US 2017059585 W US2017059585 W US 2017059585W WO 2018085440 A1 WO2018085440 A1 WO 2018085440A1
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Prior art keywords
cla
muscle
vitamin
tonalin
composition
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PCT/US2017/059585
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French (fr)
Inventor
Joseph M. MORITZ
Thorsten Schmeller
Mareike Katharina KAMPMANN
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Basf Corporation
Basf Se
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Publication of WO2018085440A1 publication Critical patent/WO2018085440A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/592—9,10-Secoergostane derivatives, e.g. ergocalciferol, i.e. vitamin D2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/593—9,10-Secocholestane derivatives, e.g. cholecalciferol, i.e. vitamin D3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals

Definitions

  • the present application relates to the fields of pharmaceutical chemistry, biochemistry and medicine.
  • One aspect relates to the reduction and/or prevention of muscle loss by administration of conjugated linoleic acid (CLA) and vitamin D.
  • CLA conjugated linoleic acid
  • Loss of muscle is often characterized by a deterioration of muscle quantity and quality. For example, during aging, there is a gradual decrease in the ability to maintain skeletal muscle function and mass that can eventually lead to sarcopenia. Patients with sarcopenia display a deterioration of muscle quantity and quality, leading to a decrease in functional capacity adversely affecting survival and quality of life. Muscle weakness associated with sarcopenia is also known to be associated with fatigue, a reduced ability to perform the tasks required for independent living and an increased risk of bony fractures.
  • compositions comprising conjugated linoleic acid (CLA) and Vitamin D.
  • CLA conjugated linoleic acid
  • Vitamin D Vitamin D
  • the CLA comprises between about 1% and 99% of one or more isomers of CLA.
  • at least one of the one or more isomers of CLA is in triglyceride form.
  • at least one of the one or more isomers of CLA is in free fatty acid form.
  • at least one of the one or more isomers of CLA is in ester form.
  • At least one of the one or more isomers of CLA is selected from the group comprising cis-9, cis- 11 -octadecadienoic acid, cis-9, trans- 11 -octadecadienoic acid, trans-9, cis- 11 -octadecadienoic acid, trans-9, trans- 11 -octadecadienoic acid, cis- 10, cis-12-octadecadienoic acid, cis- 10, trans- 12- octadecadienoic acid, trans- 10, cis-12-octadecadienoic acid, and trans- 10, trans- 12- octadecadienoic acid.
  • the CLA is a mixture of two or more different isomers of CLA.
  • the mixture can comprise cis-9, trans- 11 -octadecadienoic acid CLA and trans- 10, cis-12-octadecadienoic acid.
  • the mixture comprises cis-9, trans- 11 -octadecadienoic acid CLA and trans-10, cis-12-octadecadienoic acid in a 50:50 ratio.
  • the CLA is derived from safflower oil.
  • the CLA further comprises one or more tocopherols.
  • At least one of the one or more tocopherols can be, for example, ⁇ -tocopherol, ⁇ -tocopherol, a-tocopherol, or a combination thereof.
  • the CLA is or comprises one or more of a Tonalin product, a Clarinol product, Tonalin 35 WDP, Tonalin 60 WDP, Tonalin FFA 80, Tonalin SG 1000 T FFA, Tonalin SG 500 T FFA, Tonalin SG 750 T FFA, Tonalin TG 80, Tonalin TG 80, MET-Rx® CLA Tonalin 1000, Solgar Tonalin CLA, NatureWise CLA 1250, Natrol Tonalin CLA, NDS Body Toner Tonalin Censor, The Vitamin Shoppe Tonalin CLA, Natrol Tonalin Cla, Nature Made Cla Tonalin, Nature's Bounty Tonalin 1000 CLA, NDS Nutrition Censor Body Toner, BulkSupplements CLA Soft
  • the composition comprises one or more Group A agents, where the Group A agent(s) can diminish the inflammatory mechanism resulting in mitochondrial oxidative stress.
  • at least one of the one or more Group A agent is an omega-3 fatty acid, an amino acid, a vitamin, or any combination thereof.
  • at least one of the one or more Group A agents is aniline or vitamin E.
  • the omega-3 fatty acid can be an alpha-linolenic acid, stearidonic acid, eicosapentanoic acid, docosahexanoic acid, or any mixture thereof.
  • the composition comprises one or more Group B agents, wherein the Group B agent(s) can positively impact the muscle stimulating mechanism.
  • At least one of the one or more Group B agents is a vitamin D other than vitamin D3, creatine, leucine, citrulline, whey protein, or any mixture thereof.
  • the composition comprises at least one Group A agent and at least one Group B agent. In some embodiments, the composition comprises at least two Group A agents and at least two Group B agents.
  • the amount of the CLA and the vitamin D in the composition can vary.
  • the composition can comprise about 1 g to about 6 g of the CLA, and about 100 IU to about 5000 IU of vitamin D.
  • the composition comprises about 2 g to about 4 g of the CLA, and about 400 IU to about 2000 IU of vitamin D.
  • the composition comprises about 3 g to about 3.4 g of the CLA, and about 800 IU to about 1000 IU of vitamin D.
  • the composition comprises about 4 g of CLA and about 2000 IU of Vitamin D.
  • the composition comprises an effective amount of the CLA and vitamin D to treat or prevent a condition of muscle loss in a subject in need thereof.
  • the composition comprises an effective amount of the CLA and vitamin D to reduce or prevent age-related muscle loss or function in a subject in need thereof. In some embodiments, the composition comprises an effective amount of the CLA and Vitamin D to increase muscle protein synthesis in a subject in need thereof. In some embodiments, where upon administration to a subject in need thereof, the composition has a synergistic effect on one or more of treating or preventing a condition of muscle loss, increasing muscle protein synthesis, and reducing or preventing age-related loss of muscle mass and function.
  • the form of the composition can vary.
  • the composition can be an oral composition, a foodstuff, a food supplement, a pharmaceutical composition, or any mixture thereof.
  • the foodstuff can be or comprise, for example, a nutritional complete formula, a dairy product, a chilled or shelf stable beverage, a mineral water, a liquid drink, a shot, a soup, a dietary supplement, a meal replacement bar, a nutritional bar, a confectionery product, a milk or a fermented milk product, a yogurt, a pectin chew, a gummy, a milk based powder, an enteral nutrition product, a cereal product, a fermented cereal based product, an ice-cream, a chocolate, coffee, a culinary product, or any combination thereof.
  • the food supplement can be, for example, in the form of capsules, gelatin capsules, soft capsules, tablets, sugar-coated tablets, powders, pills, pastes, pastilles, gums, drinkable solutions, drinkable emulsions, syrups, gels, or a combination thereof.
  • the pharmaceutical composition can be, for example, in the form of capsules, gelatin capsules, soft capsules, tablets, chewable tablets, sugar-coated tablets, pills, pastes or pastilles, powders, softgels, chewable softgels, gums, drinkable solutions or emulsions, syrups, gels, or any combination thereof.
  • the composition can comprise, in some embodiments, one or more of binding agents, gelling agents, thickeners, colorants, taste masking agents, stabilizers, antioxidants, coatings, sweeteners, taste modifiers, aroma chemicals.
  • the composition is formulated for intravenous, intramuscular, rectal, or inhalation administration.
  • the composition comprises one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the composition is in a single unit dosage form. In some embodiments, the composition is in two or more unit dosage forms.
  • compositions disclosed herein for treating or preventing a condition of muscle loss a subject in need thereof.
  • the use comprises increasing muscle mass, increasing muscle function, increasing the rate of muscle synthesis, or decreasing the rate of muscle breakdown in a subject in need thereof.
  • the use comprises reducing or preventing age-related loss of muscle mass and function in a subject in need thereof.
  • the use comprises treating or preventing at least one of sarcopenia, insufficient muscle protein synthesis, muscle degradation, muscle proteolysis, muscle atrophy, muscle dystrophy, muscle catabolism, muscle wasting, loss of muscle strength, loss of physical capacity, loss of physical performance, impaired mobility, frailty, surgery, disability, risk of falling, and risk of fall-related fractures in a subject in need thereof.
  • the use comprises treating or preventing sarcopenia in a subject in need thereof.
  • compositions disclosed herein for the manufacture of a medicament for treating or preventing a condition of muscle loss a subject in need thereof.
  • the use comprises increasing muscle mass, increasing muscle function, increasing the rate of muscle synthesis, or decreasing the rate of muscle breakdown in a subject in need thereof.
  • the use comprises reducing or preventing age-related loss of muscle mass and function in a subject in need thereof.
  • the use comprises increasing muscle protein synthesis in a subject in need thereof.
  • the use comprises treating or preventing at least one of sarcopenia, insufficient muscle protein synthesis, muscle degradation, muscle proteolysis, muscle atrophy, muscle dystrophy, muscle catabolism, muscle wasting, loss of muscle strength, loss of physical capacity, loss of physical performance, impaired mobility, frailty, surgery, disability, risk of falling, and risk of fall-related fractures in a subject in need thereof.
  • the use comprises treating or preventing sarcopenia in a subject in need thereof.
  • the methods comprise administering the compositions disclosed herein to a subject in need thereof.
  • the method comprises increasing muscle mass, increasing muscle function, increasing the rate of muscle synthesis, decreasing the rate of muscle breakdown, or a combination thereof in the subject in need thereof.
  • the method comprises reducing or preventing muscle mass and function in the subject in need thereof.
  • the method comprises increasing muscle protein synthesis in the subject in need thereof.
  • the method comprises treating or preventing at least one of sarcopenia, insufficient muscle protein synthesis, muscle degradation, muscle proteolysis, muscle atrophy, muscle dystrophy, muscle catabolism, muscle wasting, loss of muscle strength, loss of physical capacity, loss of physical performance, impaired mobility, frailty, surgery, disability, risk of falling, and risk of fall-related fractures in a subject in need thereof.
  • the method comprises treating or preventing sarcopenia in a subject in need thereof.
  • Disclosed herein include methods of treating or preventing a condition of muscle loss, comprising administering CLA and Vitamin D to a subject in need thereof.
  • the CLA and Vitamin D are administered separately.
  • the CLA is administered before the Vitamin D is administered to the subject.
  • the CLA is administered after the Vitamin D is administered to the subject.
  • the CLA and Vitamin D are administered to the subject concurrently.
  • the method comprises increasing muscle mass, increasing muscle function, increasing the rate of muscle synthesis, or decreasing the rate of muscle breakdown in need thereof. In some embodiments, the method comprises reducing or preventing loss of muscle mass and function in a subject in need thereof. In some embodiments, the method comprises increasing muscle protein synthesis in a subject in need thereof. In some embodiments, the method comprises treating or preventing at least one of sarcopenia, insufficient muscle protein synthesis, muscle degradation, muscle proteolysis, muscle atrophy, muscle dystrophy, muscle catabolism, muscle wasting, loss of muscle strength, loss of physical capacity, loss of physical performance, impaired mobility, frailty, surgery, disability, risk of falling, and risk of fall-related fractures in a subject in need thereof.
  • the method comprises treating or preventing sarcopenia in a subject in need thereof.
  • the CLA comprises between about 1% and 99% of one or more isomers of CLA.
  • at least one of the one or more isomers of CLA is in triglyceride form, free fatty acid form, or ester form.
  • At least one of the one or more isomers of CLA is cis-9, cis-11- octadecadienoic acid, cis-9, trans- 11-octadecadienoic acid, trans-9, cis-11-octadecadienoic acid, trans-9, trans- 11 -octadecadienoic acid, cis-10, cis-12-octadecadienoic acid, cis-10, trans- 12-octadecadienoic acid, trans- 10, cis-12-octadecadienoic acid, trans- 10, trans- 12- octadecadienoic acid, or any combination thereof.
  • the CLA is a mixture of two or more different isomers of CLA.
  • the mixture can, for example, comprise cis-9, trans- 11 -octadecadienoic acid CLA, trans- 10, cis- 12-octadecadienoic acid, or any mixture thereof.
  • the mixture comprises cis-9, trans-11- octadecadienoic acid CLA and trans- 10, cis- 12-octadecadienoic acid in a 50:50 ratio.
  • the CLA is derived from safflower oil.
  • the CLA further comprises one or more tocopherols.
  • the tocopherol can be, or comprise, ⁇ - tocopherol, ⁇ -tocopherol, a-tocopherol, or any combination thereof.
  • the CLA can be, or comprise, one or more of a Tonalin product, a Clarinol product, Tonalin 35 WDP, Tonalin 60 WDP, Tonalin FFA 80, Tonalin SG 1000 T FFA, Tonalin SG 500 T FFA, Tonalin SG 750 T FFA, Tonalin TG 80, Tonalin TG 80, MET-Rx® CLA Tonalin 1000, Solgar Tonalin CLA, NatureWise CLA 1250, Natrol Tonalin CLA, NDS Body Toner Tonalin Censor, The Vitamin Shoppe Tonalin CLA, Natrol Tonalin Cla, Nature Made Cla Tonalin, Nature's Bounty Tonalin 1000 CLA, NDS Nutrition Censor Body Toner, BulkSupplements CLA Softgels, TNVit
  • the method comprises administrating one or more Group A agent that diminishes the inflammatory mechanism resulting in mitochondrial oxidative stress.
  • at least one of the one or more Group A agents is an omega-3 fatty acid, amino acid, vitamin, or any mixture thereof.
  • at least one of the one or more Group A agents is aniline or Vitamin E.
  • the omega-3 fatty acid can be, or comprise, alpha-linolenic acid, stearidonic acid, eicosapentanoic acid, docosahexanoic acid, or any mixture thereof.
  • the method can, for example, comprise administrating one or more Group B agents that positively impacts the muscle stimulating mechanism.
  • At least one of the one or more Group B agents is, or comprise, a vitamin D other than Vitamin D3, Vitamin D 2 , citrulline, creatine, leucine, whey protein, or a mixture thereof.
  • the method comprises administrating at least one Group A agent and at least one Group B agent to the subject. In some embodiments, the method comprises administrating at least two Group A agents and at least two Group B agents to the subject.
  • the CLA is administered in an amount from about 1.0 g to about 6.0 g per day and the vitamin D is administered in an amount from about 100 IU to about 5000 IU per day.
  • the CLA can be administered in an amount from about 2.0 g to about 4.0 g per day and the vitamin D can be administered in an amount from about 400 IU to about 2000 IU per day.
  • the CLA is administered in an amount from about 3.0 g to about 3.4 g per day and the vitamin D is administered in an amount from about 800 IU to about 1000 IU per day.
  • the CLA is administered in an amount of about 4 g per day and the vitamin D is administered in an amount of about 2000 IU per day.
  • the dose is administered in a single unit dosage form. In some embodiments, the dose is administered in two or more unit dosage forms.
  • the administration of one or more of the CLA, vitamin D, Group A agent(s) and Group B agent(s) is repeated one time per day. In some embodiments, the administration of one or more of the CLA, vitamin D, Group A agent(s) and Group B agent(s) is repeated more than one time per day. In some embodiments, the administration of one or more of the CLA, Vitamin D, Group A agent(s) and Group B agent(s) is oral, intravenous, intraperitoneal, intragastric, or intravascular administration. In some embodiments, the administration of one or more of the CLA, Vitamin D, Group A agent(s) and Group B agent(s) is oral administration.
  • the subject that can be treated with the methods and compositions disclosed herein can vary in age, and/or gender, and/or lifestyle.
  • the subject can be an adult.
  • the subject can be a male or a female.
  • the subject is a middle-aged adult.
  • the subject is an elderly adult.
  • the subject is human is a person of the age of 40 years or more.
  • the subject is human is a person of the age of 60 years or more.
  • the subject is with an active lifestyle.
  • the subject is with a sedentary lifestyle.
  • the subject has sarcopenia or is at risk of developing sarcopenia.
  • the method comprises identifying a subject is suffering from a condition of muscle loss or is at the risk of developing a condition of muscle loss. In some embodiments, the method comprises determining muscle weight, muscle circumference, lean muscle, body weight, fat mass, lean mass, brain water content, locomotor activity, protein synthesis rate, or any combination thereof of the subject. In some embodiments, at least one symptom of the condition of muscle loss is skeletal muscle loss, or muscle mass loss. In some embodiments, the condition of muscle loss is caused by aging, disease, injury, inactivity, or any combination thereof. In some embodiments, the condition of muscle loss is sarcopenia, muscle atrophy, cachexia, muscular dystrophy, or any combination thereof.
  • the rate of muscle protein synthesis is increased.
  • the increased muscle protein synthesis can comprise, for example, increased muscle fractional synthesis rate (FSR).
  • FSR muscle fractional synthesis rate
  • the administration of a combination of the CLA and vitamin D has a synergistic effect
  • Figure 1 depicts an outline of an exemplary muscle protein metabolism study, indicating the timing of infusions, measurements, and samples taken.
  • FIG. 2 shows bar graphs illustrating the predicted muscle protein synthetic rate results from an ongoing clinical trial, wherein elderly subjects are administered a placebo, CLA, vitamin D, or a combination of CLA and vitamin D.
  • Notation “a” indicates fractional muscle protein synthesis rate (FSR) significantly different (p ⁇ 0.05) under "clamp” infusion, no statistical significance between before and after intervention;
  • notation “b” indicates FSR is significantly different (p ⁇ 0.05) under “clamp” infusion from before clamp;
  • notation “c” indicates muscle FSR is significantly different (p ⁇ 0.05) before and after intervention;
  • notation “d” indicates “clamp” FSR is significantly different (p ⁇ 0.05) after combined CLA/Vitamin D intervention as compared to the CLA alone and vitamin D alone interventions.
  • a "subject” refers to an animal that is the object of treatment, observation or experiment.
  • Animals include cold- and warm-blooded vertebrates and invertebrates such as fish, shellfish, reptiles and, in particular, mammals.
  • “Mammal” includes, without limitation, mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates, such as monkeys, chimpanzees, and apes, and, in particular, humans.
  • a "patient” refers to a subject that has been treated by, or is being treated by, or will be treated by a medical professional, such as a Medical Doctor (i.e. Doctor of Allopathic medicine or Doctor of Osteopathic medicine) or a Doctor of Veterinary Medicine, to attempt to cure, or at least ameliorate the effects of, a particular disease or disorder or to prevent the disease or disorder from occurring in the first place.
  • a Medical Doctor i.e. Doctor of Allopathic medicine or Doctor of Osteopathic medicine
  • a Doctor of Veterinary Medicine to attempt to cure, or at least ameliorate the effects of, a particular disease or disorder or to prevent the disease or disorder from occurring in the first place.
  • administering refers to a method of giving a dosage of a pharmaceutically active ingredient to a vertebrate.
  • a "dosage” refers to the combined amount of the active ingredients (e.g., CLA and Vitamin D).
  • a "unit dosage" refers to an amount of therapeutic agent administered to a patient in a single dose.
  • a "daily dosage” refers to the total amount of therapeutic agent administered to a patient in a day.
  • terapéuticaally effective amount or “pharmaceutically effective amount” is meant an amount of therapeutic agent, which has a therapeutic effect.
  • dosages of a pharmaceutically active ingredient which are useful in treatment are therapeutically effective amounts.
  • a therapeutically effective amount means those amounts of therapeutic agent which produce the desired therapeutic effect as judged by clinical trial results and/or model animal studies.
  • a "therapeutic effect” relieves, to some extent, one or more of the symptoms of a disease or disorder.
  • a therapeutic effect may be observed by a reduction of the subjective discomfort that is communicated by a subject (e.g., reduced discomfort noted in self-administered patient questionnaire).
  • Treatment refers to administering a compound or a composition (e.g., a nutritional composition or a pharmaceutical composition) to a subject for prophylactic and/or therapeutic purposes.
  • a compound or a composition e.g., a nutritional composition or a pharmaceutical composition
  • prophylactic treatment refers to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition.
  • therapeutic treatment refers to administering treatment to a subject already suffering from a disease or condition.
  • prevent refers to a method of preventing the onset of a disease and/or its attendant symptoms or barring a subject from acquiring a disease.
  • prevent also include delaying the onset of a disease and/or its attendant symptoms and reducing a subject's risk of acquiring a disease.
  • the term "without a significant change in lifestyle” is used to describe the lifestyle of a subject before co-administration of CLA and vitamin D as described herein in comparison to that subject's lifestyle after commencing co-administration of CLA and vitamin D as described herein. This term is used to distinguish effects on muscle mass and function in a subject where a change in that subject's lifestyle materially contributes to an impact on muscle mass and function.
  • Vitamin D refers to all forms of vitamin D, for example vitamin Di, D 2 , D3, D 4 , D5, or any mixture thereof.
  • the vitamin D comprises mainly vitamin D 2 and/or vitamin D3.
  • a "synergistic" or “synergizing” effect can be such that the one or more effects of the combination compositions are greater than the one or more effects of each component alone, or they can be greater than the sum of the one or more effects of each component alone.
  • the synergistic effect can be about, or greater than about 5, 10, 20, 30, 50, 75, 100, 110, 120, 150, 200, 250, 350, or 500% or even more than the effect on a subject with one of the components alone, or the additive effects of each of the components when administered individually.
  • the effect can be any of the measurable effects described herein.
  • CLA conjugated linoleic acid
  • FSR fractional muscle protein synthesis rate
  • Disclosed herein includes methods and compositions for the prevention and reduction of loss in muscle mass and/or function, for example the age-related loss of muscle mass and/or function.
  • Muscle loss is a condition of deterioration of muscle quantity and quality.
  • Non-limiting symptoms of muscle loss can be loss or reduction of muscle mass, loss or reduction of lean muscle, loss or reduction of muscle weight, loss or reduction of muscle circumference, loss or reduction of fat mass, loss or reduction of lean mass, loss or reduction of muscle function, loss or reduction of muscle strength, loss or reduction of mobility, weight loss, reduction in muscle protein fractional synthesis rate (FSR), or any combination thereof.
  • at least one symptom of the condition of muscle loss is muscle mass loss or skeletal muscle loss.
  • at least one symptom of the condition of muscle loss is weight loss.
  • At least one symptom of the condition of muscle loss is loss or reduction in lean mass, loss or reduction of muscle circumference, or reduction in FSR.
  • the muscle loss can be caused by aging, disease (for example cancer and liver diseases), inactivity, injury (for example liver transplantation), or any combination thereof.
  • causes for muscle loss include age (e.g., age-related reduction in nerve cells responsible for sending signals from the brain to the muscles to initiate movement); a decrease in the concentration of some hormones, including but not limited to, growth hormone, testosterone, and insulin-like growth factor; a decrease in the body's ability to synthesize protein; inadequate intake of calories and/or protein to sustain muscle mass; and any combination thereof.
  • the condition of muscle loss is sarcopenia, muscle atrophy, cachexia, muscular dystrophy, muscle wasting, or any combination thereof. In patients having sarcopenia, the patients display a deterioration of muscle quantity and quality, leads to a decrease in functional capacity, adversely affecting survival, and quality of life.
  • Muscle loss can be determined by various conventional methods, for example measuring muscle size (for example circumference of the rectus femoris) by techniques such as ultrasound, measuring muscle resistance to an electrical current using electric impedance myography (EIM), measuring change in body weight, measuring muscle mass, measure lean mass or fat mass, measuring locomotor activity, measuring skeletal muscle fiber number, measuring muscle cross-sectional area (CSA), measuring fractional synthesis of protein (FSR) in muscle (e.g., with D 2 0), tracking lean body mass (LBM), or any combination thereof.
  • muscle loss can be measured by tracking the lean body mass (LBM) of a subject over time.
  • Some embodiments provide methods and compositions for preventing or reducting age-related loss of muscle mass and function that can eventually proceed to sarcopenia.
  • Sarcopenia is characterized by a loss of muscle mass, strength and function occurring during aging.
  • "Sarcopenia” is currently proposed to be defined as a “reduction of muscle function” and "mass below a certain threshold” in the elderly. Muscle mass loss typically starts from the age of 30 years at a rate of 3-8% per decade and accelerates from 60 years of age. This loss reaches up to 35-40 % in elderly over 70, and hence, sarcopenia is especially prominent in elderly.
  • Muscle mass preservation can be achieved through an adequate stimulation of protein synthesis and/or inhibition of proteolysis.
  • Several factors contribute to muscle protein synthesis among which the most important are the availability of amino acids (serving as building blocks for the newly synthesized proteins) and the activation signals generated by anabolic amino acids or anabolic amino acid derivatives (e.g. citrulline, leucine, essential amino acids (EAA), and creatine).
  • anabolic amino acids or anabolic amino acid derivatives e.g. citrulline, leucine, essential amino acids (EAA), and creatine.
  • protein intake is typically decreased in elderly, which leads to inadequate postprandial availability of amino acids.
  • aging is characterized by decreased muscle sensitivity to the anabolic effect of essential amino acids, especially leucine. This reduced responsiveness to leucine is associated with a decrease in the activation of intracellular pathways controlling muscle protein synthesis.
  • current supplemental regimens are delivered by meal replacement beverages or powdered beverages which may increase adiposity and calorie consumption and therefore may be counterproductive in those overweight or trying to watch weight while simultaneously maintaining muscle mass.
  • Current supplemental regimens (such as creatine) require the combination with resistance exercise; however, a large portion of US population is sedentary. Thus, additional approaches for reducing and preventing loss of muscle mass and function in sedentary populations are needed.
  • CLA conjugated linoleic acids
  • CLA is a collective name for a class of positional and structural isomers of linoleic acid that contain conjugated double bonds.
  • CLA are a family of at least 28 isomers of linoleic acid found mostly in the meat and dairy products derived from ruminants.
  • CLAs can be either cis- or trans-fats and the double bonds of CLAs are conjugated and separated by a single bond between them.
  • GRAS United States Food and Drug Administration
  • Supplementation with CLA for 24 months has been found to be well tolerated and reduce body fat mass in healthy, overweight humans.
  • CLA supplementation studies in younger populations have found a reduction of muscle breakdown markers after resistance exercise.
  • conjugated linoleic acid refers to any conjugated linoleic acid or octadecadienoic acid and its salts and derivatives. It is intended that this term encompass and indicate all positional and geometric isomers of linoleic acid with two conjugated carbon-carbon double bonds any place in the molecule.
  • CLA differs from ordinary linoleic acid in that ordinary linoleic acid has double bonds at carbon atoms 9 and 12.
  • Non-limiting examples of CLA include cis- and trans isomers ("E/Z isomers") of the following positional isomers: 2,4-octadecadienoic acid, 4,6- octadecadienoic acid, 6,8-octadecadienoic acid, 7,9-octadecadienoic acid, 8,10- octadecadienoic acid, 9, 11 -octadecadienoic acid and 10,12 octadecadienoic acid, and 11, 13 octadecadienoic acid.
  • CLA can encompass a single isomer, a selected mixture of two or more isomers, and/or a non-selected mixture of isomers obtained from natural sources.
  • the CLA or CLA composition can comprise synthetic and/or semisynthetic CLA.
  • CLA can comprise one or more of free fatty acid(s) of CLA, physiologically acceptable salts of CLA, and esters with physiologically acceptable, preferably naturally occurring, alcohols (e.g., ethanol and glycerol), and CLA triglycerides.
  • triglycerides of CLA can comprise an isomer of CLA at any or all of three positions on the triglyceride backbone.
  • conjugated linoleic acid or CLA is intended to include “esters” of CLA which term includes any CLA isomer bound through an ester linkage to an alcohol or any other chemical group.
  • c encompasses a chemical bond in the cis orientation
  • t refers to a chemical bond in the trans orientation. If a positional isomer of CLA is designated without a “c” or a "t", then that designation includes all four possible isomers.
  • 10,12 octadecadienoic acid encompasses cl0,tl2; tl0,cl2; tl0,tl2; and cl0,cl2 octadecadienoic acid.
  • the CLA comprises major and minor isomers, for example, one or more of cl l,tl3; tl l,cl3; tl l,tl3; and cl l,cl3 octadecadienoic acids.
  • the CLA administered to patients is or comprises an octadecadienoic acid isomer selected from the group of cis-9, trans-11; cis-9, cis-11; trans-9, cis-11; trans-9, trans-11; cis-10, cis-10, trans-12; trans-10, cis-12; trans-10, trans-12 octadecadienoic acid and mixtures thereof.
  • the CLA administered to patients contains less than 5% of minor isomers of CLA.
  • the minor isomer is cl l,tl3; tl l,cl3; tl l,tl3; or cl l,cl3 octadecadienoic acid.
  • the CLA contains less than 1% of minor isomers of CLA.
  • the CLA comprises an ester or triglyceride.
  • the CLA comprises greater than about 55% tl0,cl2 octadecadienoic acid.
  • the CLA comprises one or more of the isomers of octadecadienoic acid including the cis-9, trans-11; cis-9, cis-11; trans-9, cis-11 ; trans-9, trans-11; cis-10, cis-12; cis-10, trans-12; trans-10, cis-12; and trans-10, trans-12 isomers.
  • eight possible geometric isomers of 9,11 and 10,12 octadecadienoic acid i.e., c9,cl l; c9,tl l; t9,cl l; t9,tl l ; cl0,cl2; cl0,tl2; tl0,cl2 and tl0,tl2
  • c9,cl2-octadecadienoic acid i.e., c9,cl l; c9,tl l; t9,cl l; t9,tl l ; cl0,cl2; cl0,cl2 and tl0,tl2
  • the cis-9,trans-l l and trans-10, cis-12 isomers of CLA are thought to have the most biological activity. Therefore, in some embodiments, these isomers can be used in a purified form or a substantially purified form, or in CLA compositions containing high ratios of these isomers.
  • isomers in the CLA mixtures include 9,11-octadecadienonic acid, 10,12-octadecadienoic acid, most preferably the c9,tl l and tl0,cl2 isomers.
  • the CLA mixture contains greater than about 50% tl0,cl2 isomer.
  • the CLA mixture contains greater than about 55% tl0,cl2 isomer. In some embodiments, the CLA mixture contains greater than about 60% tl0,cl2 isomer. It is contemplated that in some embodiments, supplementation of the CLA mixture derived from isomerization of linoleic acid with purified or synthesized tl0,cl2 isomer may be necessary to achieve these percentages.
  • CLA can be free or bound through ester linkages or provided in the form of an oil comprising CLA triglycerides.
  • the triglycerides may be partially or wholly comprised of CLA attached to a glycerol backbone.
  • the CLA can, in some embodiments, be provided as a methylester or ethylester.
  • the CLA is in the form of a non-toxic salt, such as a potassium or sodium salt (e.g., a salt formed by reacting chemically equivalent amounts of the free acids with an alkali hydroxide at a pH of about 8 to 9).
  • a non-toxic salt such as a potassium or sodium salt (e.g., a salt formed by reacting chemically equivalent amounts of the free acids with an alkali hydroxide at a pH of about 8 to 9).
  • the active forms of CLA include, in addition to the free acids the active isomers of CLA; non-toxic salts thereof; active esters and other active chemical derivatives thereof; and mixtures thereof.
  • the non-toxic salts of the free acids may be made by reacting the free acids with a non-toxic base.
  • the CLA can be heat stable and can be used as is, or dried and powdered.
  • the CLA can be readily converted into a non-toxic salt, such as the sodium or potassium salt, by reacting chemically equivalent amounts of the free acid with an alkali hydroxide at a pH of about 8 to 9.
  • the CLA comprises between about 1% and 99% of one or more isomers of CLA.
  • the one or more isomers of CLA is selected from the group comprising cis-9, cis-11 -octadecadienoic acid, cis-9, trans-11- octadecadienoic acid, trans-9, cis-11-octadecadienoic acid, trans-9, trans- 11-octadecadienoic acid, cis-10, cis-12-octadecadienoic acid, cis-10, trans- 12-octadecadienoic acid, trans- 10, cis- 12-octadecadienoic acid, and trans- 10, trans- 12-octadecadienoic acid.
  • the CLA is a mixture of two or more different isomers of CLA.
  • the CLA comprises cis-9, trans- 11 -octadecadienoic acid CLA and trans-10, cis-12- octadecadienoic acid at about a 10:90 to 90: 10.
  • the CLA comprises one or more of a Tonalin product, a Clarinol product, Tonalin 35 WDP, Tonalin 60 WDP, Tonalin FFA 80, Tonalin SG 1000 T FFA, Tonalin SG 500 T FFA, Tonalin SG 750 T FFA, Tonalin TG 80, Tonalin TG 80, MET-Rx® CLA Tonalin 1000, Solgar Tonalin CLA, NatureWise CLA 1250, Natrol Tonalin CLA, NDS Body Toner Tonalin Censor, The Vitamin Shoppe Tonalin CLA, Natrol Tonalin Cla, Nature Made Cla Tonalin, Nature's Bounty Tonalin 1000 CLA, NDS Nutrition Censor Body Toner, BulkSupplements CLA Softgels, TNVitamins CLA, Puritan's Pride CLA Tonalin, Barlean's Organic Oils Tonalin CLA Swirl, Solgar, Tonalin
  • the CLA is derived from safflower oil. In some embodiments, the CLA further comprises one or more tocopherols. In some embodiments, the one or more tocopherols is selected from the group consisting of consisting of ⁇ -tocopherol, ⁇ -tocopherol, a-tocopherol, and combinations thereof.
  • Vitamin D is a group of fat-soluble secosteroids, the two major physiologically relevant forms of which are vitamin D 2 (ergocalciferol) and vitamin D3 (cholecalciferol). These are known collectively as calciferol. Vitamin D without a subscript refers to all forms of vitamin D (including vitamin Di, D 2 , D3, D 4 , and D5), in particular D 2 and D3, or any mixture thereof.
  • Vitamin D from the diet or skin synthesis is biologically inactive, and an enzymatic conversion (hydroxylation) in the liver and kidney is required for activation.
  • vitamin D When ingested, vitamin D is hydroxylated in the liver (endoplasmic reticulum) to 25- hydroxycholecalciferol (25(OH)D), also known as calcidiol, by the enzyme 25 -hydroxylase, produced by hepatocytes.
  • 25(OH)D) also known as calcidiol
  • 25- hydroxycholecalciferol is then transported to the proximal tubules of the kidneys where it can be hydroxylated by one of two enzymes to different forms of vitamin D, one of which is active vitamin D (l ,25(OH)D) and another which is inactive vitamin D (24,25(OH)D).
  • the enzyme la-hydroxylase which is activated by parathyroid hormone (and additionally by low calcium or phosphate) forms the main biologically active vitamin D hormone with a CI hydroxylation forming 1,25-dihydroxy cholecalciferol (l,25(OH) 2 D, also known as calcitriol).
  • Vitamin D can be provided in an active (l,25(OH) 2 D) or inactive (Vitamin D3 or D 2 ) form.
  • VDR vitamin D receptors
  • Vitamin D Deficiency levels blood 250H-VitD3 of ⁇ 15 ng/mL
  • others continued to see improvement in lower extremity function up to and beyond 40 ng 250H-VitD 3 /mL which are levels well above what previously might have been thought necessary for maximal benefit.
  • Muscle contains VDRs for l,250H 2 -VitD 3 , found in both the nucleus and at the cell membrane, and these are also involved in non-specific binding 250H-VitD 3 as well. It has been shown that feeding D 3 to vitamin D deficient rats 7 hours prior to measurement increased protein synthesis as measured by H-leucine incorporation into muscle cell proteins. However, when the muscles were removed from the vitamin D deficient rats and studied, it was found that only 25-OH VitD 3 acts directly in the muscles.
  • Vitamin D receptors were discovered in muscle tissue, thus providing direct evidence of vitamin D's effect on muscle function. Muscle biopsies in adults with vitamin D deficiency exhibit mainly type II muscle fiber atrophy. Type II fibers are important because they are the first initiated to prevent a fall.
  • a recent randomized controlled study found that daily supplementation of 1,000 IU of vitamin D 2 in elderly stroke survivors resulted in an increase in mean type II fiber diameter and in percentage of type II fibers. There was also a correlation between serum 250H-VitD 3 level and type II fiber diameter.
  • Vitamin D conveys its action by binding to VDR.
  • VDR is expressed in particular stages of differentiation from myoblast to myotubes. Two different VDRs have been described. One is located at the nucleus and acts as a nuclear receptor and the other is located at the cell membrane and acts as a cellular receptor. VDR knockout mice are characterized by a reduction in body weight and size, as well as impaired motor coordination.
  • the nuclear VDR is a ligand-dependent nuclear transcription factor that belongs to the steroid-thyroid hormone receptor gene superfamily. It has been reported that the first in situ detection of VDR in human muscle tissue with significant associated intranuclear staining for VDR.
  • l,250H 2 -VitD3 binds to its nuclear receptor, it causes changes in mRNA transcription and subsequent protein synthesis.
  • the genomic pathway has been known to influence muscle calcium uptake, phosphate transport across the cell membrane, phospholipid metabolism, and muscle cell proliferation and differentiation.
  • MPK mitogen-activated protein kinase
  • Vitamin D can regulate formation and regeneration of tight junctions and neuromuscular junctions. Studies that shown that vitamin D regulates the expression of VDR and the neural growth factor (NGF) in Schwann cells. Recent studies have also shown that vitamin D enhances glial cell line-derived neurotrophic factor (GDNF) in rats and that this may have beneficial effects in neurodegenerative disease. Therefore, vitamin D can act through several mechanisms of cellular function and neural interaction to improve overall muscle strength and function.
  • VDR VDR
  • NGF neural growth factor
  • GDNF glial cell line-derived neurotrophic factor
  • vitamin D is the biological equivalent of 0.025 ⁇ g.
  • 1,000 IU is the biological equivalent of 25 ⁇ g.
  • vitamin D is used in an amount of 800 IU or more per daily dosage in combination with CLA, such as, for instance 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000 or 10,000 IU, or any value between any of two of said values or over, but not so high that it causes toxicity in the subject to which it is administered.
  • vitamin D is used in an amount of 20 ⁇ g or more per daily dosage in combination with CLA, such as, for instance 25, 50, 75, 100, 125, 150, 175, 200, 225 or 250 ⁇ g per daily dosage, or any value in between any of two of said values or over.
  • CLA alone has been discussed in the literature with respect to muscle maintenance in the elderly, there are currently no clinical trials testing CLA in the elderly population. Further, while CLA in combination with creatine has been tested in the elderly, this combination intervention was not compared to each intervention alone, and the combination was not shown to be superior to prior results observed with creatine. CLA has been shown to have an impact on muscle in younger and post-menopausal populations but has not been tested in older populations losing muscle. CLA feeding studies also show improvement in muscle status and muscle markers in older mice. CLA supplementation in older mice shows an improvement in oxidative and mitochondrial markers, which are markers that are affected by elderly muscle loss. Due to CLA's impact on muscle metabolism it has been termed an exercise mimetic.
  • Vitamin D supplementation has yielded unclear results in terms of its influence elderly muscle mass and functional maintenance. Individual studies of vitamin D supplementation in randomized controlled trials showed improvements in muscle function in older populations (e.g., postmenopausal women 50-65 years old). Vitamin D supplementation interacts with muscle via the receptor VDR, which hypothesized to affect downstream regulation of insulin-like growth factor signaling pathway. Studies have also found that adequate vitamin D is necessary for proper performance of muscle growth ingredient HMB in restoring muscle function in elderly persons.
  • CLA CLA
  • Vitamin D can impact muscle growth stimulatory mechanisms, and has been shown to impact falls in the elderly (a measure of elderly muscle strength).
  • vitamin D deficiency serum vitamin D levels below 25-50 nmol/L
  • insufficiency serum vitamin D levels below 75 nmol/L
  • Low serum 25- (OH)D concentrations are associated with an increased risk of sarcopenia in elderly.
  • compositions comprising CLA and vitamin D, and uses and methods of co-administering CLA and vitamin D, for the prevention and/or reduction of age-related loss of muscle mass and function that can eventually proceed to sarcopenia.
  • compositions comprising CLA and vitamin D, and methods of co-administering CLA and vitamin D, to improve muscle synthetic rate, breakdown rate, mass, strength, or function.
  • stimulating muscle protein synthesis results in the treatment or prevention of a condition of muscle loss.
  • increasing muscle mass results in the treatment or prevention of a condition of muscle loss.
  • increasing muscle function results in the treatment or prevention of the condition of muscle loss.
  • increasing the rate of muscle synthesis results in the treatment or prevention of a condition of muscle loss.
  • decreasing the rate of muscle breakdown results in the treatment or prevention of the condition of muscle loss.
  • the co-administration of CLA and vitamin D increases the rate of muscle protein synthesis.
  • increasing the rate of muscle protein synthesis results in the reduction or prevention of age-related loss of muscle mass or function.
  • increasing the rate of muscle protein synthesis results in the treatment or prevention of a condition of muscle loss.
  • increasing the rate of muscle protein synthesis results in the reduction or prevention of sarcopenia.
  • Some embodiments disclosed herein include methods of treating or preventing a condition of muscle loss by co-administering CLA and vitamin D to a subject in need thereof.
  • co-administering CLA and vitamin D to a subject in need thereof treats or prevents a condition of muscle loss by reducing or preventing age- related loss of muscle mass and function.
  • Some such embodiments include therapeutic treatment, and some embodiments include prophylactic treatment.
  • co-administration it is meant that the two or more agents (for example CLA and Vitamin D) may be found in the patient's bloodstream at the same time, regardless of when or how they are actually administered.
  • the agents are administered simultaneously.
  • administration in combination is accomplished by combining the agents in a single dosage form.
  • the agents are administered sequentially.
  • the agents for example CLA and Vitamin D
  • the agents for example CLA and Vitamin D
  • the subject in need thereof can be a patient who is suffering from a condition of muscle loss or a subject that is suspected of or at the risk of developing a condition of muscle loss.
  • the subject may have, or may not have, symptoms of sarcopenia.
  • the subject does not have sarcopenia.
  • the subject has sarcopenia.
  • the age for the subject in need thereof can vary.
  • the subject can be an adult, for example a middle-aged adult, or an elderly adult.
  • the subject is of the age of 40, 45, 50, 55, 60, 65, 70, 75, or more.
  • the subject can be a person at least 40 years old, or the subject can be a person at least 60 years old.
  • the gender of the subject in need thereof can vary.
  • the subject is a female.
  • the subject is a male.
  • the use and methods disclosed herein can also comprise identifying a subject who is suffering from a condition of muscle loss or a subject that is suspect of or at the risk of developing a condition of muscle loss; and co-administering to the subject CLA and vitamin D.
  • the methods disclosed herein include acquiring knowledge of the presence of a condition of muscle loss in a subject or the risk/potential of developing a condition of muscle loss in a subject; and co-administering to the subject CLA and vitamin D.
  • Change in muscle loss for example attenuation or acceleration of muscle loss can be detected, for example, by detecting loss in muscle mass, detecting change in body weight, detecting change in muscle lean mass and/or fat mass, determining change in locomotor activity, detecting change in muscle fiber number, detecting change in muscle cross-sectional area, or any combination thereof of the subject.
  • Some embodiments disclosed herein provide methods of treating or preventing a condition of muscle loss by co-administering CLA and vitamin D to a subject in need thereof. Some embodiments can include identifying a subject as having or at risk for developing a condition of muscle loss (e.g., sarcopenia, muscle atrophy, cachexia, or muscular dystrophy) prior to co-administering CLA and vitamin D.
  • a condition of muscle loss e.g., sarcopenia, muscle atrophy, cachexia, or muscular dystrophy
  • the co-administration of CLA with Vitamin D prevents or reduces age-related loss of muscle mass or function by synergistically combining to impact the mitochondrial oxidative stress- reductive and/or muscle stimulating mechanisms of aging-related muscle loss. Additionally, while not being bound by any particular theory, in some embodiments, the co-administration of CLA with Vitamin D prevents or relieves the condition of muscle loss by synergistically combining to impact inflammatory and/or muscle stimulating mechanisms of aging-related muscle loss.
  • the co-administration of CLA and vitamin D as described herein can, in some embodiments, allow for the regulation or maintenance of the subject's rate of muscle protein synthesis.
  • the regulation or maintenance of the subject's rate of muscle protein synthesis can allow for a subject to experience a number of beneficial effects. These beneficial effects include the reduction or prevention of age-related loss of muscle mass and function, increased muscle mass, increased muscle function, increased rate of muscle synthesis, decreased rate of muscle breakdown, and/or treatment or prevention of sarcopenia. Compared to a baseline prior to treatment, these effects can result in an improvement of about or greater than about 5, 10, 15, 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 400, or 500%. These amounts and/or levels can be maintained within 0, 1, 5, or 10% of the amounts and/or levels at the initiation of administration.
  • Some embodiments provide a method of co-administering CLA and vitamin D to a subject in need thereof such that the subject's rate of muscle protein synthesis increases.
  • the subject may or may not engage in exercise.
  • the subject coadministered CLA and Vitamin D can, in some embodiments, observe and/or feel improvement without a significant change in lifestyle.
  • exercising in conjunction with the co-administration of CLA and vitamin D results in an even greater improvement in muscle mass and function, but exercise is not necessary to improve muscle mass and function.
  • the amount of CLA and vitamin D administered that are effective for increasing the animal's muscle mass can be determined in accordance with methods known in the art.
  • the effective amount of CLA administered may be from about 1 g to about 6 g and the effective amount of vitamin D administered may be from may be from about 100 IU to about 5000 IU per 24 hour period.
  • the uses, methods and composition disclosed herein can prevent or reduce age-related loss of muscle mass (including but not limited to loss of lean muscle mass).
  • the methods and composition may prevent muscle mass loss (including but not limited to loss of lean muscle mass) from occurring.
  • the rate of muscle mass loss is reduced in a patient receiving or received treatment by at least, or at least about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% as compared to the patients received no treatment.
  • the methods and composition reduce the rate of loss of muscle mass in a patient by, or by about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values as compared to the patients received no treatment.
  • the methods and composition may reduce the final muscle mass loss (including but not limited to final loss in lean muscle mass).
  • the final muscle mass loss in the patient receiving or received treatment is at most, or at most about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the final muscle mass loss in patients received no treatment.
  • the final muscle mass loss in the patient receiving or received treatment is, or is about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values, of the final muscle mass loss in patients received no treatment.
  • the methods and composition disclosed herein can prevent or reduce age-related loss of muscle function (including but not limited to loss of lean muscle function).
  • the methods and composition may prevent muscle function loss (including but not limited to loss of lean muscle function) from occurring.
  • the rate of muscle function loss is reduced in a patient receiving or received treatment by at least, or at least about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% as compared to the patients received no treatment.
  • the methods and composition reduce the rate of loss of muscle function in a patient by, or by about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values as compared to the patients received no treatment.
  • the methods and composition may reduce the final muscle function loss (including but not limited to final loss in lean muscle function).
  • the final muscle function loss in the patient receiving or received treatment is at most, or at most about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the final muscle function loss in patients received no treatment.
  • the final muscle function loss in the patient receiving or received treatment is, or is about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values, of the final muscle function loss in patients received no treatment.
  • the methods and composition disclosed herein can prevent or reduce age-related loss of muscle weight.
  • the methods and composition may prevent muscle weight loss from occurring.
  • the rate of muscle weight loss in a patient receiving or received treatment is reduced by at least, or at least about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% as compared to the patients received no treatment.
  • the methods and composition reduce the rate of loss of muscle weight in the patient receiving or received treatment by, or by about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values as compared to patients received no treatment.
  • the methods and composition may reduce the final muscle weight loss.
  • the final muscle weight loss in the patient receiving or received treatment is at most, or at most about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the final muscle weight loss in patients received no treatment. In some embodiments, the final muscle weight loss in the patient receiving or received treatment is, or is about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values, of the final muscle weight loss in patients received no treatment.
  • the methods and composition disclosed herein can prevent or reduce age-related loss of muscle circumference.
  • the methods and composition may prevent muscle circumference loss from occurring.
  • the rate of muscle circumference loss in a patient receiving or received treatment is reduced by at least, or at least about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% as compared to the patients received no treatment.
  • the methods and composition reduce the rate of muscle circumference loss in a patient receiving or received treatment by, or by about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values as compared to the patients received no treatment.
  • the methods and composition may reduce the final muscle circumference loss.
  • the final muscle circumference loss in the patient receiving or received treatment is at most, or at most about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the final muscle circumference loss in patients received no treatment.
  • the final muscle circumference loss in the patient receiving or received treatment is, or is about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values, of the final muscle circumference loss in patients received no treatment.
  • the methods and composition disclosed herein can prevent or reduce age-related loss of muscle strength.
  • the methods and composition may prevent muscle strength loss from occurring.
  • the rate of muscle strength loss in a patient receiving or received treatment is reduced by at least, or at least about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% as compared to the patients received no treatment.
  • the methods and composition reduce the rate of muscle strength loss in a patient receiving or received treatment by, or by about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values as compared to patients received no treatment.
  • the methods and composition may reduce the final muscle strength loss.
  • the final muscle strength loss in the patient receiving or received treatment is at most, or at most about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the final muscle strength loss in patients received no treatment. In some embodiments, the final muscle strength loss in the patient receiving or received treatment is, or is about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values, of the final muscle strength loss in patients received no treatment. [0085] In some embodiments, the methods and composition disclosed herein can prevent or reduce age-related mobility loss. For example, the methods and composition may prevent mobility loss from occurring.
  • the rate of mobility loss in a patient receiving or received treatment is reduced by at least, or at least about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% as compared to the patients received no treatment.
  • the methods and composition reduce the rate of mobility loss in the patient receiving or received treatment by, or by about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values as compared to the patients received no treatment.
  • the methods and composition may reduce the final mobility loss.
  • the final mobility loss in the patient receiving or received treatment is at most, or at most about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the final mobility loss in patients received no treatment. In some embodiments, the final mobility loss in the patient receiving or received treatment is, or is about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values, of the final mobility loss in patients received no treatment.
  • the methods and composition disclosed herein can prevent age-related reduction in muscle protein FSR or reduce the rate of age-related reduction in muscle protein FSR.
  • the methods and composition may prevent the reduction in muscle protein FSR from occurring.
  • the rate of reduction in muscle protein FSR in a patient receiving or received treatment is reduced by at least, or at least about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% as compared to patients received no treatment.
  • the methods and composition reduce the rate of reduction in muscle protein FSR in a patient receiving or received treatment by, or by about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values as compared to the patients received no treatment.
  • the co-administration of CLA and vitamin D results in significant enhancements in overall muscle mass, function and strength.
  • This co-administration can be used on all age groups seeking enhancement in overall muscle mass, function and strength.
  • CLA and vitamin D are co-administered in elderly subjects. Current estimates place a large portion of the older population at risk for falls with potential significant associated morbidities.
  • the coadministration of CLA and vitamin D specifically targets muscle mass, strength and function and consequently may produce significant improvement in health, quality of life, and in particular, decreased falls and injury in this group.
  • the younger population also benefits from the co-administration of CLA and vitamin D in still further embodiments, in part due to the widespread occurrence of vitamin D deficiency.
  • Women also benefit from the coadministration of CLA and vitamin D in some embodiments, as women are prone to vitamin D deficiency.
  • synergistic effects can be such that the one or more effects of the combination compositions are greater than the one or more effects of each component alone at a comparable dosing level, or they can be greater than the predicted sum of the effects of all of the components at a comparable dosing level, assuming that each component acts independently.
  • the synergistic effect can be about, or greater than about, 5, 10, 20, 30, 50, 75, 100, 1 10, 120, 150, 200, 250, 350, or 500% better than the effect of treating a subject with one of the components alone, or the additive effects of each of the components when administered individually.
  • the effect can be any of the measurable effects described herein.
  • the composition comprising a plurality of components can be such that the synergistic effect is an enhancement in FSR and that FSR is increased to a greater degree as compared to the sum of the effects of administering each component, determined as if each component exerted its effect independently, also referred to as the predicted additive effect herein. For example, if a composition comprising component (a) yields an effect of a 20% improvement in FSR and a composition comprising component (b) yields an effect of 50% improvement in FSR, then a composition comprising both component (a) and component (b) would have a synergistic effect if the combination composition's effect on cellular metabolism was greater than 70%.
  • a synergistic combination composition can have an effect that is greater than the predicted additive effect of administering each component of the combination composition alone as if each component exerted its effect independently. For example, if the predicted additive effect is 70%, an actual effect of 140% is 70% greater than the predicted additive effect or is 1 fold greater than the predicted additive effect.
  • the synergistic effect can be at least about 20, 50, 75, 90, 100, 150, 200 or 300% greater than the predicted additive effect. In some embodiments, the synergistic effect can be at least about 0.2, 0.5, 0.9, 1.1,
  • the synergistic effect of the combination compositions can also allow for reduced dosing amounts, leading to reduced side effects to the subject and reduced cost of treatment. Furthermore, the synergistic effect can allow for results that are not achievable through any other treatments. Therefore, proper identification, specification, and use of combination compositions can allow for significant improvements in the reduction and prevention of age-related loss of muscle mass and function.
  • compositions include the combination compositions described herein, such as combination compositions comprising CLA and vitamin D.
  • combination compositions comprising CLA and vitamin D.
  • an amount of CLA and vitamin D in the levels sufficient to improve muscle strength, function, and mass is administered for an effective period of time.
  • the dosing of the combination compositions can be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times daily.
  • a subject can receive dosing for a period of about, less than about, or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • a unit dose can be chosen such that the subject is administered about or greater than about 1000 mg of CLA (e.g. about or more than about 1100 mg, 1 130 mg, 2000 mg, 2100 mg, 2200 mg, 2250 mg, 2260 mg, 3300 mg, 3390 mg, 4400 mg, 4520 mg, 4750 mg, 4900 mg, 5000 mg, 5100 mg, 5300 mg, 5530 mg, 5750 mg, 5900 mg, 6000 mg, or more) and about or greater than about 100 IU of vitamin D (e.g.
  • CLA e.g. about or more than about 1100 mg, 1 130 mg, 2000 mg, 2100 mg, 2200 mg, 2250 mg, 2260 mg, 3300 mg, 3390 mg, 4400 mg, 4520 mg, 4750 mg, 4900 mg, 5000 mg, 5100 mg, 5300 mg, 5530 mg, 5750 mg, 5900 mg, 6000 mg, or more
  • vitamin D e.g.
  • the vitamin D can comprise Vitamin D3.
  • a unit dose can be a fraction of the daily dose, such as the daily dose divided by the number of unit doses to be administered per day.
  • a unit dose can be a fraction of the daily dose that is the daily dose divided by the number of unit doses to be administered per day and further divided by the number of unit doses (e.g. tablets) per administration.
  • the number of unit doses per administration may be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
  • the number of doses per day may be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
  • the number of unit doses per day may be determined by dividing the daily dose by the unit dose, and may be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, 20, or more unit doses per day.
  • a unit dose can be about 1 ⁇ 2, 1 ⁇ 2, 1 ⁇ 4, Vs, 1 ⁇ 2, 1/7, 1 ⁇ 4, 1/9, or 1/10.
  • a unit dose can be about one- third of the daily amount and administered to the subject three times daily.
  • a unit dose can be about one- half of the daily amount and administered to the subject twice daily.
  • a unit dose can be about one-fourth of the daily amount with two unit doses administered to the subject twice daily.
  • a unit dose can have about, less than about, or more than about 250 mg, 275 mg, 500 mg, 550 mg, 750 mg, 825 mg, 1100 mg, 1125 mg, 1130 mg, 1650 mg, 2200 mg, 2550 mg, 2750 mg, 2825 mg, 3100 mg, 3300 mg, 3530 mg, 3750 mg, 3900 mg, 4000 mg, 4100 mg, 4300 mg, 4530 mg, 4750 mg, 4900 mg, 5000 mg, 5100 mg, 5300 mg, 5530 mg, 5750 mg, 5900 mg, 6000 mg, or more of CLA and about, less than about, 100 IU, 150 IU, 200 IU, 300 IU, 500 IU, 700 IU, 850 IU, 1000 IU, 1200 IU, 1300 IU, 1500 IU, 1700 IU, 1850 IU, 2000IU, 2200 IU, 2300 IU, 2500 IU, 2700 IU, 2850 IU, 3000 IU, 3200 IU, 3300
  • the co-administration of CLA to vitamin D can have a specified ratio of CLA to vitamin D.
  • the specified ratio can provide for effective stimulation of muscle protein synthesis.
  • the specified ratios can cause an increase in muscle mass of a subject, an increase in muscle function of a subject, an increase in FSR of a subject, an increase in anabolic signaling markers of a subject, a decrease in proteolysis markers of a subject, an increase in mitochondrial number and size of a subject, a decrease in oxidative stress markers of a subject, and/or a decrease in inflammatory markers of a subject.
  • the ratio of CLA to vitamin D can be a mass ratio, a molar ratio, or a volume ratio. In some embodiments, the mass ratio of CLA to vitamin D is about, greater than about, or less than about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, 175, 200, 250, 500, 750, 1000, or more.
  • the molar ratio of CLA to vitamin D co-administered is about, greater than about, or less than about 90, 95, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more.
  • the co-administration of CLA to vitamin D is effective for reducing or preventing age-related loss of muscle mass or function, as measured by an increase in the anabolic signaling of a subject, an increase in expression of growth regulatory factors of a subject, an increase in FSR of a subject, an increase in insulin sensitivity of a subject, a decrease in oxidative stress markers of a subject, and/or a decrease in inflammatory markers of a subject.
  • the administration of a combination composition can have a measured effect that is an improvement of about, or greater than about, 5, 10, 15, 20, 30, 50, 75, 100, 110, 120, 150, 200, 250, 350, 500, 700, or 1000% over a control subject or control group.
  • a subject co-administered CLA and vitamin D as described herein can experience an increase in FSR.
  • the increase in FSR can be about, or greater than about, 1, 5, 10, 15, 20, 25, 30, 35, or 40%, as compared to the subject's FSR before treatment.
  • the measurement of FSR can be for a test group as compared to a control group.
  • the effects on a subject can be determined by a variety of in vitro or in vivo methods that utilize samples taken from the subject or that directly assay for parameters indicative of the effect in the subject.
  • FSR can be measured as described in Example 1
  • intracellular signaling cascades known to be involved in the control of translational activity and known markers of proteolysis can be evaluated by western blot analysis of muscle biopsy samples
  • muscle mRNA expression of regulators of energy metabolism and proteolysis can be evaluated by reverse transcription real-time quantitative PCR
  • adipose tissue mRNA expression of markers of inflammation and oxidative stress by can be evaluated by reverse transcription real-time quantitative PCR
  • mitochondrial function in muscle can be evaluated by oxygraphy
  • total body fat and fat-free masses and appendicular lean body mass can be evaluated by body composition scans (DXA, Lunar iDXATM, GE Healthcare)
  • oxidative stress can be measured using blood samples drawn from the subject
  • inflammatory markers can be measured using ELISA.
  • compositions can be administered periodically.
  • the compositions can be administered one, two, three, four times a day, or even more frequent.
  • the subject can be administered every 1 , 2, 3, 4, 5, 6, or 7 days.
  • the compositions are administered three times daily.
  • the administration can be concurrent with meal time of a subject.
  • the period of treatment or diet supplementation can be for about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 days, 2 weeks, 1-1 1 months, or 1 year, 2 years, 5 years, or even longer.
  • the dosages that are administered to a subject can change or remain constant over the period of treatment.
  • the daily dosing amounts can increase or decrease over the period of administration.
  • the length of the period of administration and/or the dosing amounts can be determined by a physician, a nutritionist, or any other type of clinician.
  • the period of time can be one, two, three, four or more weeks. In some embodiments, the period of time can be one, two, three, four, five, six or more months.
  • the dosing level can be adjusted based on the subject's characteristics, such as weight, height, ethnicity, genetics, or baseline energy metabolism level.
  • the physician, nutritionist, or clinician can observe the subject's response to the administered compositions and adjust the dosing based on the subject's performance or measured circulating levels of CLA, vitamin D, or any other component of the composition. For example, dosing levels can be increased for subjects that show reduced effects in FSR or circulating levels of CLA or vitamin D below desired target levels.
  • the compositions administered to a subject can be optimized for a given subject.
  • the ratio of CLA to vitamin D or the particular components in a combination composition can be adjusted.
  • the ratio and/or particular components can be selected after evaluation of the subject after being administered one or more compositions with varying ratios of CLA to vitamin D or varying combination composition components.
  • the CLA and vitamin D do not have to be administered in the same composition to perform the claimed methods.
  • separate capsules, pills, mixtures, etc. of vitamin D and of CLA may be administered to a subject to carry out the claimed methods.
  • the administration of CLA and vitamin D may be at the same time or at different times provided that effective concentrations of both CLA and vitamin D are both found in the subject at the same time.
  • administration of both CLA and vitamin D is at the same time, preferably in a single composition, in order to facilitate the compliance of the subject to adhere to a schedule of administration.
  • the dosing regimen of the compositions disclosed herein is administered for a period of time, which time period can be, for example, from at least about 1 week to at least about 4 weeks, from at least about 4 weeks to at least about 8 weeks, from at least about 4 weeks to at least about 12 weeks, from at least about 4 weeks to at least about 16 weeks, or longer.
  • the dosing regimen of the compositions disclosed herein can be administered three times a day, twice a day, daily, every other day, three times a week, every other week, three times per month, once monthly, substantially continuously or continuously.
  • the amount of vitamin D and CLA needed to reach appropriate blood serum levels of vitamin D and CLA may vary from person to person, and determination of the optimum amount in each instance can be readily obtained by routine procedures.
  • the formulation, route of administration and dosage for the compositions disclosed herein can be chosen by the individual physician in view of the patient's condition.
  • the dose range of the composition administered to the patient can be from about 0.1 to about 4000 mg/kg of the patient's body weight.
  • the dosage may be a single one or a series of two or more given in the course of one or more days, as is needed by the patient.
  • human dosages for the compositions have been established for at least some condition, the present disclosure will use those same dosages, or dosages that are between about 0.1% and about 5000%, more preferably between about 25% and about 1000% of the established human dosage.
  • a suitable human dosage can be inferred from ED 50 or ID 50 values, or other appropriate values derived from in vitro or in vivo studies, as qualified by toxicity studies and efficacy studies in animals.
  • the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity).
  • the magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.
  • the composition is administered 1 to 4 times per day.
  • the compositions of the compositions disclosed herein can be administered by continuous intravenous infusion, preferably at a dose of each active ingredient up to 100 g per day.
  • the compositions disclosed herein are administered for a period of continuous therapy, for example for a week or more, or for months or years.
  • the methods and compositions disclosed herein can comprise, in some embodiments, at least one other ingredient which positively impacts the inflammatory mechanism resulting in mitochondrial oxidative stress (collectively termed "Group A" agents herein).
  • the Group A agent is selected from the group comprising omega-3 fatty acid-containing substances (e.g., oils), amino acids (e.g. alanine), and vitamins (e.g., vitamin E).
  • the omega-3 fatty acid is selected from the group consisting of alpha-linolenic acid, stearidonic acid, eicosapentanoic acid, docosahexanoic acid, and mixtures thereof.
  • the methods and compositions disclosed herein can comprise, in some embodiments, at least one other ingredient which positively impacts the muscle stimulating mechanism (collectively termed "Group B” agents herein).
  • the Group B agent is selected from the group comprising another vitamin D other than D3, creatine, leucine, and whey protein.
  • the Group B agent is an anabolic amino acid derivative stimulus, herein defined as a chemical compound derived from or which is a precursor to an amino acid (hence, amino acid derivative) which promotes (hence, anabolic) muscle growth by increasing net protein synthesis (hence, stimulus).
  • the anabolic amino acid derivative stimulus is one or more selected from the group comprising L-leucine, citrulline, and creatine.
  • L-leucine (hereafter also called leucine, since the R-form of leucine is biologically not relevant in the context of the present disclosure) is an essential amino acid, being part of a diverse number of proteins and, together with valine and isoleucine, belongs to the group of branched-chain amino acids.
  • Leucine may be used as a free amino acid, or in a bound form, such as a dipeptide, an oligopeptide, a polypeptide or a protein.
  • Common protein sources of leucine are dairy proteins such as whey, casein, micellar casein, caseinate, and glycomacroprotein (GMP), and vegetable proteins such as wheat, rice, pea, lupine and soy proteins.
  • the Group B agent is citrulline.
  • Citrulline is an a- amino acid.
  • Citrulline in the form of citrulline malate, is sold as a performance-enhancing athletic dietary supplement was been suggested to promote aerobic energy production and to increase athletic performance and decreasing muscle soreness.
  • citrulline is produced from ornithine and carbamoyl phosphate in one of the central reactions in the urea cycle. It is also produced from arginine in the body as a by-product of the reaction catalyzed by NOS family.
  • Citrulline is also capable of promoting muscle protein synthesis and has been described in human and animal studies.
  • the Group B agent is creatine (N-(amino-imino- methyl)-N-methyl-glycine; methylglycocy amine).
  • Creatine is a nitrogenous organic acid that is produced in vertebrates, in particular the human body from L-arginine, glycine, and L- methionine and helps to supply energy to muscles.
  • the anabolic amino acid derivative stimulus is provided in a daily dosage of 0.5 to 20 g, preferably 1 to 10 g. In some embodiments, such daily dosage is administered as a single serving.
  • leucine is provided in a daily dosage of 1 to 10 g.
  • leucine When leucine is provided as proteinaceous matter, it should comprise at least about 11 weight% of leucine. It was found that about 11 weight % of total leucine based on the total amount of proteinaceous matter is a minimum amount present in the nutritional composition.
  • said proteinaceous matter comprises at least about 12 weight%, preferably at least about 12.5 weight%, more preferably at least about 13 weight% of leucine.
  • total leucine comprises at least about 20 weight%, preferably at least about 22.5 weight%, preferably at least about 25 weight% of leucine in a free form, relative to the total amount of leucine.
  • free form is meant a peptide comprising 1 to 5 amino acids, preferably 1 to 3 amino acids, more preferably 1 amino acid.
  • leucine is a free amino acid, either as a base, a salt or a chelate.
  • citrulline is provided in a daily dosage of 0.5 to 10 g.
  • creatine is provided in a daily dosage of 0.5 to 20 g.
  • any combination of leucine, citrulline, and creatine is provided in a daily dosage of 0.5 to 20 g, preferably 1 to 10 g. Any combination is a combination selected from the group of leucine and citrulline; leucine and creatine; citrulline and creatine; and leucine, citrulline and creatine.
  • the Group B agent is whey protein.
  • combination further comprises at least about 80 weight% of whey protein, preferably at least about 85 weight% of whey protein, preferably at least about 90 weight%, and most preferably about 95 weight% of whey protein.
  • Whey protein is considered a "fast" protein referring to the rate of appearance in the circulation of the amino acids following whey ingestion.
  • the whey protein may be an intact whey protein, a hydrolysed whey protein, a microparticular whey protein, a nanoparticular whey protein, a micellar whey protein, and the like.
  • the whey protein is an intact whey protein, i.e. a whey protein in its intact form, such as present in fresh milk.
  • whey protein source i.e. whey obtained by any process for the preparation of whey known in the art, as well as whey protein fractions prepared thereof, or the proteins that constitute the bulk of the whey proteins being ⁇ -lactoglobulin, ct- lactalbumin and serum albumin, such as liquid whey, or whey in powder form, such as whey protein isolate (WPI) or whey protein concentrate (WPC).
  • Whey protein concentrate is rich in whey proteins, but also contains other components such as fat, lactose and glycomacroprotein (GMP), a casein-related non-globular protein.
  • whey protein concentrate is produced by membrane filtration.
  • whey protein isolate consists primarily of whey proteins with minimal amounts of fat and lactose. Whey protein isolate usually requires a more rigorous separation process such as a combination of microfiltration and ultra-filtration or ion exchange chromatography. It is generally understood that a whey protein isolate refers to a mixture in which at least 90 weight% of the solids are whey proteins. A whey protein concentrate is understood as having a percentage of whey proteins between the initial amount in the by-product (about 12 weight%) and a whey protein isolate.
  • sweet whey obtained as a by-product in the manufacturing of cheese
  • acid whey obtained as a by-product in the manufacturing of acid casein
  • native whey obtained by milk microfiltration or rennet whey, obtained as a byproduct in the manufacturing of rennet casein
  • at least one other ingredient selected from the Group A and/or Group B is present within the oral composition.
  • the at least one other ingredient is provided in amounts sufficient to elicit noticeable effects (which can be measured as described herein).
  • the combination compositions can be formulated for oral administration in the form of a tablet, a capsule, or any other form described herein.
  • the compositions can be administered to a subject orally or by any other methods.
  • Methods of oral administration include, in some embodiments, administering the composition as a liquid, a solid, or a semisolid that can be taken in the form of a dietary supplement or a foodstuff.
  • the administration of CLA and vitamin D is oral.
  • the CLA and vitamin D may be formulated together with suitable carriers such as starch, sucrose or lactose in tablets, pills, dragees, capsules, solutions, liquids, slurries, suspensions and emulsions.
  • suitable carriers such as starch, sucrose or lactose in tablets, pills, dragees, capsules, solutions, liquids, slurries, suspensions and emulsions.
  • the vitamin D and CLA may be administered separately or together, provided that the total amount of CLA and vitamin D is an effective amount in combination per day to have a substantial impact on the rate of muscle protein synthesis.
  • some embodiments relate to an oral composition comprising CLA and Vitamin D in a foodstuff, in a food supplement, or in a pharmaceutical preparation.
  • a food composition for human consumption is supplemented by the above composition.
  • the food composition can be, or comprise, a nutritional complete formula, a dairy product, a chilled or shelf stable beverage, a powdered beverage, a mineral or purified water, a liquid drink, a soup, a dietary supplement, a meal replacement, a nutritional bar, a confectionery, a milk, a fermented milk product, a yoghurt, a milk based powder, an enteral nutrition product, an infant formula, an infant nutritional product, a cereal product or a fermented cereal-based product, an ice cream, a chocolate, coffee, a culinary product such as mayonnaise, tomato puree, salad dressings, a pet food, or any combination thereof.
  • oral compositions and in particular of food supplements are possible. They are formulated by means of the usual methods for producing sugar-coated tablets, pills, pastes, gums, gelatin capsules, gels, emulsions, tablets, capsules or drinkable solutions or emulsions, which can then be taken directly with water or by any other known means.
  • the nutritional supplement for oral administration may be in capsules, gelatin capsules, soft capsules, tablets, sugar-coated tablets, pills, pastes or pastilles, gums, or drinkable solutions or emulsions, syrups or gels, with a dose of about 0.001 to 100% of the primary composition, which can then be taken directly with water or by any other known means.
  • This supplement may also include a sweetener, a stabilizer, an additive, a flavoring or a colorant.
  • a supplement for cosmetic purpose can additionally comprises a compound active with respect to the skin. Methods for preparing them are common knowledge.
  • the formulation as described above may be incorporated into any other forms of food supplements or of enriched foods, for example food bars, or compacted or non- compacted powders. Methods for preparing them are common knowledge.
  • the food composition or food supplement may also include a sweetener, a stabilizer, an antioxidant, an additive, a flavoring or a colorant.
  • the composition may also contain synthetic or natural bioactive ingredients such as amino acids, fatty acids, vitamins, minerals, carotenoids, polyphenols, etc. that can be added either by dry or by wet mixing to said composition before pasteurization and/or drying.
  • the composition disclosed herein can be used cosmetically.
  • cosmetic use is meant a non- therapeutic use which may improve the aesthetic aspect or comfort of the skin, coat and/or hair of humans or pets.
  • a pharmaceutical composition can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient already suffering from a disease, as described herein under, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications.
  • An amount adequate to accomplish this is defined as "a therapeutically effective dose”. Amounts effective for this will depend on the severity of the disease and the weight and general state of the patient.
  • compositions disclosed herein are administered to a patient susceptible to or otherwise at risk of a particular disease. Such an amount is defined to be "a prophylactic effective dose”. In this use, the precise amounts again depend on the patient's state of health and weight.
  • compositions disclosed herein are, in some embodiments, administered with a pharmaceutically acceptable carrier, the nature of the carrier differing with the mode of administration, for example, enteral, oral and topical (including ophthalmic) routes.
  • the desired formulation can be made using a variety of excipients including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate.
  • This composition may be a tablet, a capsule, a pill, a solution, a suspension, a syrup, a dried oral supplement, a wet oral supplement.
  • the composition can be intravenously administered in any suitable manner.
  • the composition is preferably in a water-soluble non-toxic form.
  • Intravenous administration is particularly suitable for hospitalized patients that are undergoing intravenous (IV) therapy.
  • the composition can be dissolved in an IV solution (e.g., a saline or glucose solution) being administered to the patient.
  • the composition can be added to nutritional IV solutions, which may include amino acids and/or lipids.
  • the amounts of the composition to be administered intravenously can be similar to levels used in oral administration. Intravenous infusion may be more controlled and accurate than oral administration.
  • the combination of CLA and vitamin D can be formulated for administration in a pharmaceutical composition comprising a physiologically acceptable surface active agents, carriers, diluents, excipients, smoothing agents, suspension agents, film forming substances, coating assistants, or a combination thereof.
  • the combination of CLA and vitamin D are formulated for administration with a pharmaceutically acceptable carrier or diluent.
  • the combination of CLA and vitamin D can be formulated as a medicament with a standard pharmaceutically acceptable carrier(s) and/or excipient(s) as is routine in the pharmaceutical art. The exact nature of the formulation will depend upon several factors including the desired route of administration.
  • combination of CLA and vitamin D are formulated for oral, intravenous, intragastric, intravascular or intraperitoneal administration.
  • Standard pharmaceutical formulation techniques may be used, such as those disclosed in Remington's The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (2005), incorporated herein by reference in its entirety.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • substances which can serve as pharmaceutically- acceptable carriers or components thereof, are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the TWEENS; wetting agents, such sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline;
  • composition choice of a pharmaceutically-acceptable carrier to be used in conjunction with a composition can be determined by the way the composition is to be administered.
  • compositions described herein are preferably provided in unit dosage form.
  • a "unit dosage form" is a composition that is suitable for administration to an animal, preferably mammal subject, in a single dose, according to good medical practice.
  • the preparation of a single or unit dosage form does not imply that the dosage form is administered once per day or once per course of therapy.
  • Such dosage forms are contemplated to be administered once, twice, thrice or more per day and may be administered as infusion over a period of time (e.g., from about 30 minutes to about 2-6 hours), or administered as a continuous infusion, and may be given more than once during a course of therapy, though a single administration is not specifically excluded.
  • the skilled artisan will recognize that the formulation does not specifically contemplate the entire course of therapy and such decisions are left for those skilled in the art of treatment rather than formulation.
  • compositions useful as described above may be in any of a variety of suitable forms for a variety of routes for administration, for example, for oral, nasal, rectal, topical (including transdermal), ocular, intracerebral, intracranial, intrathecal, intra-arterial, intravenous, intramuscular, or other parental routes of administration.
  • routes for administration for example, for oral, nasal, rectal, topical (including transdermal), ocular, intracerebral, intracranial, intrathecal, intra-arterial, intravenous, intramuscular, or other parental routes of administration.
  • oral and nasal compositions include compositions that are administered by inhalation, and made using available methodologies.
  • a variety of pharmaceutically-acceptable carriers well-known in the art may be used.
  • Pharmaceutically-acceptable carriers include, for example, solid or liquid fillers, diluents, hydrotropies, surface-active agents, and encapsulating substances.
  • Optional pharmaceutically-active materials may be included, which do not substantially interfere with the inhibitory activity of the composition.
  • the amount of carrier employed in conjunction with the composition is sufficient to provide a practical quantity of material for administration per unit dose of the composition.
  • Various oral dosage forms can be used, including such solid forms as tablets, capsules, and granules. Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed, containing suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents.
  • Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules, containing suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, melting agents, coloring agents and flavoring agents.
  • suitable solvents preservatives, emulsifying agents, suspending agents, diluents, sweeteners, melting agents, coloring agents and flavoring agents.
  • Tablets typically comprise conventional pharmaceutically-compatible adjuvants as inert diluents, such as calcium carbonate, sodium carbonate, mannitol, lactose and cellulose; binders such as starch, gelatin and sucrose; disintegrants such as starch, alginic acid and croscarmelose; lubricants such as magnesium stearate, stearic acid and talc. Glidants such as silicon dioxide can be used to improve flow characteristics of the powder mixture. Coloring agents, such as the FD&C dyes, can be added for appearance. Sweeteners and flavoring agents, such as aspartame, saccharin, menthol, peppermint, and fruit flavors, are useful adjuvants for chewable tablets. Capsules typically comprise one or more solid diluents disclosed above. The selection of carrier components depends on secondary considerations like taste, cost, and shelf stability, which are not critical, and can be readily made by a person skilled in the art.
  • Peroral compositions also include liquid solutions, emulsions, suspensions, and the like.
  • the pharmaceutically-acceptable carriers suitable for preparation of such compositions are well known in the art.
  • Typical components of carriers for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water.
  • typical suspending agents include sodium carboxymethyl cellulose, AVICEL RC-591, tragacanth and sodium alginate; typical wetting agents include lecithin and polysorbate 80; and typical preservatives include methyl paraben and sodium benzoate.
  • Peroral liquid compositions may also contain one or more components such as sweeteners, flavoring agents and colorants disclosed above.
  • compositions useful for attaining systemic delivery can be in, for example, sublingual, buccal and/or nasal dosage forms.
  • Such compositions typically comprise one or more of soluble filler substances such as sucrose, sorbitol and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose and hydroxypropyl methyl cellulose. Glidants, lubricants, sweeteners, colorants, antioxidants and flavoring agents disclosed above may also be included.
  • Topical formulations may generally be comprised of a pharmaceutical carrier, co-solvent, emulsifier, penetration enhancer, preservative system, and emollient.
  • a pharmaceutically acceptable diluent such as a saline or dextrose solution.
  • Suitable excipients may be included to achieve the desired pH, including but not limited to NaOH, sodium carbonate, sodium acetate, HC1, and citric acid.
  • the pH of the final composition ranges from 2 to 8, or preferably from 4 to 7.
  • Antioxidant excipients may include sodium bisulfite, acetone sodium bisulfite, sodium formaldehyde, sulfoxylate, thiourea, and EDTA.
  • Other non-limiting examples of suitable excipients found in the final intravenous composition may include sodium or potassium phosphates, citric acid, tartaric acid, gelatin, and carbohydrates such as dextrose, mannitol, and dextran.
  • Antimicrobial agents may also be included to achieve a bacteriostatic or fungistatic solution, including but not limited to phenylmercuric nitrate, thimerosal, benzethonium chloride, benzalkonium chloride, phenol, cresol, and chlorobutanol.
  • compositions for intravenous administration may be provided to caregivers in the form of one more solids that are reconstituted with a suitable diluent such as sterile water, saline or dextrose in water shortly prior to administration.
  • a suitable diluent such as sterile water, saline or dextrose in water shortly prior to administration.
  • the compositions are provided in solution ready to administer parenterally.
  • the compositions are provided in a solution that is further diluted prior to administration.
  • the combination may be provided to caregivers as a mixture, or the caregivers may mix the two agents prior to administration, or the two agents may be administered separately.
  • CLA is somewhat susceptible to oxidation. Therefore, it is desirable to package CLA and vitamin D with suitable antioxidants such as lecithin, tocopherols, ascorbate, ascorbyl palmitate or spice extracts such as rosemary extract.
  • suitable antioxidants such as lecithin, tocopherols, ascorbate, ascorbyl palmitate or spice extracts such as rosemary extract.
  • a combination composition comprises CLA, Vitamin D, and one or more additional ingredients.
  • An additional ingredient may serve one or more functions.
  • an additional ingredient accounts for about, less than about, or more than about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the mass or volume of the combination composition.
  • Non-limiting examples of additional ingredients include sweeteners, bulking agents, stabilizers, acidulants, preservatives, binders, lubricants, disintegrants, fillers, solubilizers, coloring agents (such as fruit juice and vegetable juice), and other additives and excipients known in the art.
  • a combination composition comprises one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more) sweeteners.
  • sweeteners include, but are not limited to, sucrose, fructose, dextrose, maltose, lactose, high fructose corn syrup solids, invert sugar, sugar alcohols, sorbitol, saccharin, cyclamates, sweeteners derived from stevia, sweeteners derived from momordica grosvenorii, sweeteners derived from mogrosides, acesulfame K, L-aspartyl-L- phenylalanine lower alkyl ester sweeteners, L-aspartyl-D-alanine amide sweeteners, L- aspartyl-D-serine amide sweeteners, L-aspartyl-L-l-hydroxymethylalkaneamide sweeteners, L-aspartyl-1 -hydroxy ethyalkaneamide sweeteners, L-aspartyl-D-phenylglycine ester and amide sweeteners, rebaudioside A, rebaudioside B, re
  • the sweetener is a polyol additive, such as a sugar alcohol, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol (glycerine), threitol, galactitol, palatinose, reduced isomalto-oligosaccharides, reduced xylo-oligosaccharides, reduced gentio- oligosaccharides, reduced maltose syrup, or reduced glucose syrup.
  • a combination composition comprises one or more (e.g.
  • bulking agents 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more
  • bulking agents include guar gum, locust bean gum, cassia gum, pectin from botanical sources, high molecular weight carboxymethylcellulose, carrageenan, alginate, and xanthane.
  • one or more bulking agents may be added to enhance the viscosity of a liquid formulation.
  • a combination composition comprises one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more) stabilizers.
  • stabilizers include pectin, polysaccharide hydrolysates comprising dextrin, agar, can- ageenan, tamarind seed polysaccharides, angelica gum, karaya gum, xanthan gum, sodium alginate, tragacanth gum, guar gum, locust bean gum, pullulan, gellan gum, gum arabic, carboxymethylcellulose, and propylene glycol alginate ester.
  • one or more stabilizers are added to the combination composition to enhance the shelf-life of the combination composition.
  • shelf-life refers to the amount of time the container and composition therein can be held at ambient conditions (approximately room temperature, e.g. about 18-28° C.) or less, without degradation of the composition and/or container occurring to the extent that the composition cannot be used in the manner and for the purpose for which it was intended.
  • the combination composition has a shelf life of about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 14, 30, 60, 90, or more days; or about, less than about, or more than about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months or years.
  • the combination composition remains non-perishable for a period of time after opening a container containing the composition.
  • perishability refers to degradation to an extent that the composition cannot be used in the manner and purpose for which it was designed.
  • the combination composition remains non-perishable for about, less than about, or more than about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 18, 24, 30, 36, 48, 60, 72, 90, or more hours or days after opening; or about, less than about, or more than about 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, or more months or years after opening.
  • the combination composition remains nonperishable for a period of time at room temperature (e.g. about 18-28° C).
  • the combination composition remains non-perishable for a period of time upon refrigeration, such as storage below about 20° C, 15° C, 10° C, 5° C, 4° C, 3° C, 2° C, 1° C, 0° C, -1° C, -2° C, -3° C, -4° C, -5° C, -10° C, -20° C, or lower.
  • a combination composition comprises one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more) acidulants.
  • acidulants include C2-C30 carboxylic acids, substituted hydroxyl C1-C30 carboxylic acids, benzoic acid, substituted benzoic acids (e.g.
  • 2,4-dihydroxybenzoic acid substituted cinnamic acids, hydroxyacids, substituted hydroxybenzoic acids, substituted cyclohexyl carboxylic acids, tannic acid, lactic acid, tartaric acid, citric acid, gluconic acid, glucoheptonic acids, adipic acid, hydroxycitric acid, malic acid, fruitaric acid (a blend of malic, fumaric, and tartaric acids), fimaric acid, maleic acid, succinic acid, chlorogenic acid, salicylic acid, creatine, glucosamine hydrochloride, glucono delta lactone, caffeic acid, bile acids, acetic acid, ascorbic acid, alginic acid, erythorbic acid, polyglutamic acid, and their alkali or alkaline earth metal salt derivatives thereof.
  • a combination composition comprises one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more) preservatives.
  • preservatives include sorbic acid, benzoic acid, and salts thereof, including (but not limited to) calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, and mixtures thereof.
  • the compositions can be a food product, for example a snack bar, or a beverage, comprising CLA and vitamin D.
  • the snack bar can be a chocolate bar, a granola bar, or a trail mix bar.
  • the present dietary supplement or food compositions are formulated to have suitable and desirable taste, texture, and viscosity for consumption. Any suitable food carrier can be used in the present food compositions. Food carriers of the compositions described herein include practically any food product.
  • Such food carriers include, but are not limited to food bars (granola bars, protein bars, candy bars, etc.), cereal products (oatmeal, breakfast cereals, granola, etc.), bakery products (bread, donuts, crackers, bagels, pastries, cakes, etc.), beverages (milk-based beverage, sports drinks, fruit juices, alcoholic beverages, bottled waters), pastas, grains (rice, corn, oats, rye, wheat, flour, etc.), egg products, snacks (candy, chips, gum, chocolate, etc.), meats, fruits, and vegetables.
  • food carriers employed herein can mask the undesirable taste (e.g., bitterness).
  • liquid food carriers can be used to obtain the present food compositions in the form of beverages, such as supplemented juices, coffees, teas, shakes (e.g., milk shakes), smoothies, and the like.
  • solid food carriers can be used to obtain the present food compositions in the form of meal replacements, such as supplemented snack bars, pasta, breads, and the like.
  • semi-solid food carriers can be used to obtain the present food compositions in the form of gums, chewy candies or snacks, and the like.
  • CLA and Vitamin D can be administered separately or simultaneously (e.g., in a single dosage form).
  • CLA, vitamin D, and/or the combination of CLA and vitamin D are administered as pharmaceutically acceptable salts.
  • pharmaceutically acceptable salt refers to salts that retain the biological effectiveness and properties of a compound and, which are not biologically or otherwise undesirable for use in a pharmaceutical.
  • the compounds disclosed herein are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.
  • Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids.
  • Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
  • Pharmaceutically acceptable salts can also be formed using inorganic and organic bases.
  • Inorganic bases from which salts can be derived include, for example, bases that contain sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts.
  • treatment of the compounds disclosed herein with an inorganic base results in loss of a labile hydrogen from the compound to afford the salt form including an inorganic cation such as Li , Na , K , Mg and Ca and the like.
  • Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
  • substituted amines including naturally occurring substituted amines
  • cyclic amines cyclic amines
  • basic ion exchange resins and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
  • Many such salts are known in the art, as described in WO 87/05297 published September 11, 1987 (incorporated by reference herein in its entirety).
  • kits comprising one or more compositions described herein, in suitable packaging, and may further comprise written material that can include instructions for use, discussion of clinical studies, listing of side effects, and the like.
  • Such kits may also include information, such as scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the health care provider.
  • Such information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials.
  • a kit may comprise one or more unit doses described herein.
  • a kit comprises about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 31, 60, 90, 120, 150, 180, 210, or more unit doses.
  • Instructions for use can comprise dosing instructions, such as instructions to take 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more unit doses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times per day.
  • a kit may comprise a unit dose supplied as a tablet, with each tablet package separately, multiples of tablets packaged separately according to the number of unit doses per administration (e.g. pairs of tablets), or all tablets packaged together (e.g. in a bottle).
  • a kit may comprise a unit dose supplied as a bottled drink, the kit comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 24, 28, 36, 48, 72, or more bottles.
  • the kit can further contain another agent.
  • the CLA and vitamin D are provided as separate compositions in separate containers within the kit.
  • the CLA and vitamin D are provided as a single composition within a container in the kit.
  • Suitable packaging and additional articles for use e.g., measuring cup for liquid preparations, foil wrapping to minimize exposure to air, and the like) are known in the art and may be included in the kit. Kits described herein can be provided, marketed and/or promoted to health providers, including but not limited to, physicians, nurses, pharmacists, formulary officials, and the like. Kits can also, in some embodiments, be marketed directly to the consumer.
  • a kit can comprise a multi-day supply of unit dosages.
  • the unit dosages can be any unit dosage described herein.
  • the kit can comprise instructions directing the administration of the multi-day supply of unit dosages over a period of multiple days.
  • the multi-day supply can be a one-month supply, a 30-day supply, or a multi-week supply.
  • the multi-day supply can be a 90-day, 180-day, 3 -month or 6-month supply.
  • the kit can include packaged daily unit dosages, such as packages of 1, 2, 3, 4, or 5 unit dosages.
  • the kit can be packaged with, for example, other dietary supplements, vitamins, and meal replacement bars, mixes, and beverages.
  • This example describe a study based on a randomized, double-blind, controlled clinical trial to determine the independent and combined effects of dietary CLA and vitamin D supplementation on anabolic signalling, the expression of growth regulatory factors, and muscle protein turnover in older adults.
  • Forty (40) 65-85 year old men and women (“Subjects") are randomized to receive: i) placebo (4,000 mg corn oil per day), ii) CLA (4,000 mg Tonalin FFA 80 per day), iii) vitamin D 3 (2,000 IU per day), or iv) CLA (4,000 mg Tonalin FFA 80 per day) plus vitamin D 3 (2,000 IU per day) for 8 weeks.
  • Study supplements are provided in finished bottles according to labeling guidelines.
  • Subjects who fulfil the following inclusion criteria i) age: >65 and ⁇ 85 years; ii) BMI: >18.5 and ⁇ 30.0 kg/m 2 ; iii) weight stable ( ⁇ 2% change) and untrained ( ⁇ 1 h of structured exercise/wk) for at least 3 months before entering the study; and iv) serum 250H- Vit D3 ⁇ 35 ng/ml are included.
  • Subjects are admitted to the Clinical Study Unit in the late afternoon on the day before the study. At 2000 h, they consume a standardized meal and then fast (except for water) and rest in bed until the completion of the study (outlined in Figure 1) the next day.
  • a catheter is inserted into an antecubital vein for the infusion of amino acids and insulin; two additional catheters are inserted for blood sampling: one into the radial artery of the opposite arm and another in retrograde fashion into the brachial vein.
  • constant infusions of [ring- 2 H5]phenylalanine (priming dose: 7.9 ⁇ /kg FFM, infusion rate: 0.07 ⁇ -kg FFM "1 min "1 ) and [6,6- Eyglucose (priming dose: 22 ⁇ /kg body weight, infusion rate: 0.22 ⁇ 1 ⁇ 13 ⁇ 4 body weight "1 min "1 ) are started and maintained for 420 min.
  • the infusion rate of [ring- 2 H5]phenylalanine is increased to 0.14 ⁇ ⁇ FFM "1 min "1 to match the increased availability of phenylalanine during the mixed amino acid infusion and the infusion rate of [6,6- 2 H 2 ]glucose is reduced to 0.11 ⁇ ⁇ body weight "1 min "1 to account for the insulin-mediated decrease in hepatic glucose production.
  • Blood samples are collected immediately before the start of the phenylalanine tracer infusion and then at 60, 120, 180, 210, 220, 230, 240, 300, 360, 390, 400, 410 and 420 min.
  • Data average values collected at 210, 220, 230, and 240 min (basal period) and 390, 400, 410, and 420 min (clamp) are used to calculate forearm amino acid net balance, protein breakdown and protein synthesis.
  • Data obtained from the remaining samples are used to confirm isotopic and metabolic steady state.
  • Muscle biopsies from the quadricep femor is obtained by using a conchotome forceps (during local anesthesia) 60 min after the start of the isotope infusion and at the end of the basal period (at 240 min) and at the end of the hyperaminoacidemic- hyperinsulinemic clamp (at 420 min) to measure the incorporation rate of labelled phenylalanine into muscle protein (myofibrillar, sarcoplasmic, and mitochondrial) and the muscle tissue free phenylalanine labelling to measure the fractional muscle protein synthesis rate (FSR), and to evaluate gene expression and intracellular signaling events and markers of proteolysis and muscle morphology.
  • Subcutaneous adipose tissue biopsies from the periumbilical region are obtained (during local anesthesia) immediately after the first and third muscle biospies at 60 and 420 min.
  • Forearm blood flow are measured at multiple time points during basal conditions (i.e., between 195 and 210 min and between 225 and 240 min) and at multiple time points during the hyperaminoacidemic-hyperinsulinemic-euglycemic clamp (i.e., between 375 and 390 min and between 405 and 420 min) by using the ultrasound Doppler technique.
  • Plasma glucose concentration is determined on an automated glucose analyzer (Yellow Spring Instruments Co, Yellow Springs, OH). Plasma insulin concentration is measured by using a commercially available ELISA kit (Diagnostic Systems Labs, Webster, TX). The phenylalanine tracer to tracee ratio (TTR) and concentration in blood and the muscle free and protein-bound phenylalanine TTR will be determined by GC-MS (MSD 5973 System, Hewlett-Packard) by using enrichment standards.
  • GC-MS MSD 5973 System, Hewlett-Packard
  • Reverse transcription real-time quantitative PCR (ABI 7500 real-time PCR system, Invitrogen, Life Technologies Inc.) is used to evaluate the muscle mRNA expression of peroxisome proliferator-activated receptor alpha [PPARA] and gamma [PPARG], regulators of energy metabolism; myogenic differentiation 1 [MYOD1], a myogenic growth factor; myostatin [MSTN], a muscle growth inhibitor; follistatin [FST], which binds to and thereby inhibits myostatin; forkhead box 03 [FOX03], which induces the transcription of ubiquitin ligases; MaFbx and MuRFl, two components of the ubiquitin- proteasome pathway; Calpains and Calpastatin, a family of calcium-dependent, non- lysosomal cysteine proteases; tripeptidyl peptidase II, an enzyme responsible for the terminal steps of proteolysis; and the lysosomal peptidases, cathepsin L
  • mRNA expression of markers of inflammation and oxidative stress e.g. TNF, IL6
  • adipokines e.g. AdipoQ, LEP
  • adipogenesis e.g. PPARG
  • mitochondrial function e.g. PPARA, uncoupling protein 1 [UCPl]
  • NAMPT nicotinamide phosphoribosyltransferase
  • SIRTl SIRTl
  • measure adipose tissue NAD + concentration are evaluated.
  • GAPDH is used as a house keeping gene after verifying the stability of its expression against the expression of cyclophilin A and/or ⁇ -actin.
  • Muscle fiber-type (1, 2a and 2x) composition, fiber size, capillary density, lipid content, mitochondrial density, number and size, and mitochondrial cristae density are determined according to current American College of Pathologists guidelines. Mitochondrial function in muscle is evaluated by oxygraphy (Oroboros 02k, Innsbruck, Austria). All samples are coded in ways that do not reveal subjects' treatment status to ensure unbiased analysis.
  • Subject randomization and dietary supplementation with CLA and/or vitamin D [0173] After completing the baseline metabolic study, subjects are randomized to either: i) placebo (corn oil), or ii) CLA, or iii) vitamin D, or iv) CLA plus vitamin D for 8 weeks.
  • a block randomization scheme is used to ensure our study groups are balanced with respect to sex, racial/ethnical distribution, and adiposity status (lean vs overweight).
  • Subjects randomized to receive placebo receive 4,000 mg corn oil daily (2 softgels containing 1,000 mg corn oil each in the morning and 2 in the evening), those randomized to CLA receive 4,000 mg Tonalin FFA 80 daily (2 softgels containing 750 mg CLA isomers each in the morning and 2 in the evening), those randomized to vitamin D receive 2,000 IU vitamin D3 daily (2 softgels containing 500 IU vitamin D3 each in the morning and 2 in the evening), and those randomized to CLA plus vitamin D receive both 4,000 mg Tonalin FFA 80 and 2,000 IU vitamin D3 daily (2 softgels containing 750 mg CLA isomers and 500 iU vitamin D3 each in the morning and 2 in the evening).
  • CLA Tropon 80 FFA
  • GRAS generally-recognized-as-safe
  • the dose of vitamin D given to our study subjects (2,000 IU per day) is below the Tolerable Upper Limit (4,000 IU) set by the Institute of Medicine; it is expected to increase serum 250H-Vit D3 concentration by 15-20 ng/ml and will therefore not raise serum 250H-Vit D3 concentrations above 100 ng/ml.
  • Both the active and placebo treatments are dispensed by a Study nurse in our Center for Human Nutrition, who is not part of the Study team so that both the subjects and investigators are blinded to the treatment. Compliance with the treatment is assessed by pill count at the end of the study. In previous studies using a similar protocol, compliance was >90%. To avoid “tampering" with the pill count, subjects are given an (to them) unknown amount of pills in excess of needs at the beginning of the study and will be asked to return any remaining pills at the end of the study. Subjects have weekly contact with a member of the study team to review body weight, food records, any signs of supplement intolerance, life events, and any medical complaints. Any indication of a potential adverse event is reviewed with the PI and study physician.
  • Subjects maintain their habitual diet and physical activity during the intervention.
  • the study coordinator instructs the subjects to do so and follow-up with them weekly to remind them and help them maintain their habitual life-style.
  • physical activity and dietary intake are monitored and potential changes are taken into account in the statistical analyses.
  • Subjects are asked to keep detailed food records (by using notebooks and/or mobile apps), which is reviewed by the PI and study coordinator and/or a dietician in our nutrition center.
  • Urine and stool are collected before and at the end of the intervention to assess 24-h urinary nitrogen excretion and fecal isovaleric acid content, two markers of dietary protein intake.
  • each subject undergo an orientation session to explain in detail the nature, purpose, and demands of the study; ii) subjects are encouraged to review and discuss the consent form with their husband/wife or domestic partners before agreeing to participate; iii) subjects are screened with a "barriers interview" that we have developed to help ensure we select subjects who will likely be able to complete the study; iv) subjects are required to complete a diet and physical activity history form and return for at least one pre-study visit after they passed screening before being able to enroll in the study, which helps demonstrate their commitment and ability to complete the study; v) once enrolled, subjects have regular (weekly for the first month, biweekly to monthly for the subsequent months) contact with a member of the study team which reinforces participation; the Study coordinator will also help subjects overcome barriers that can lead to non-compliance and/or study withdrawal, and vi) a philosophy of partnership and collaborative relationships is encouraged between Study personnel and study participants (e.
  • FIG 2 depicts the expected results from the ongoing clinical trial on muscle synthetic rate in elderly subjects. It is expected that those subjects receiving either placebo or Vitamin D alone will have a statistically significant difference in FSR under "clamp” infusion relative to the basal period, but will have not have a statistically significant difference in FSR before and after intervention (indicated in Figure 2 by "a").
  • Method Make a blend of the above ingredients to homogenize same and encapsulate using standard softgel methodology.
  • Tonalin FFA 80 may be substituted with any other pharmaceutically acceptable product containing CLA.
  • Vitamin D 3 may be substituted with Vitamin D 2 .
  • antioxidant E 306 may be substituted with tocopherols, lecithins, or other suitable antioxidants that will counter the slight susceptibility of CLA to oxidation.
  • a subject having sarcopenia is identified.
  • the subject is then administered four of the softgels described in Example 2 on a daily basis (with two softgels taken morning and evening daily with meals).
  • a subject having a condition of muscle loss that can be treated by the coadministration of CLA and vitamin D is identified.
  • the subject is then administered, once per day, a meal replacement bar comprising about 4 g of a CLA mixture (cis-9, trans-11 - octadecadienoic acid CLA and trans-10, cis-12-octadecadienoic acid in a 50: 50 ratio) and about 2000 IU of Vitamin D 3 .

Abstract

Disclosed herein include compositions comprising conjugated linoleic acid (CLA) and vitamin D, and the uses thereof. Methods of administering CLA and vitamin D to a subject are also provided in some embodiments. Vitamin D and CLA can be co-administered to reduce and prevent muscle loss, for example age-related loss of muscle mass and functions. Co-administration of vitamin D and CLA can have a synergistic effect, which results in a surprising and unexpected level of improvement in muscle mass, strength and functionality.

Description

BASC.001WO PATENT REDUCTION AND PREVENTION OF MUSCLE LOSS BY CONJUGATED LINOLEIC ACID (CLA) AND VITAMIN D
RELATED APPLICATIONS
[0001] The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/416586, filed on November 2, 2016. The content of this related application is herein expressly incorporated by reference in its entirety.
BACKGROUND
Field
[0002] The present application relates to the fields of pharmaceutical chemistry, biochemistry and medicine. One aspect relates to the reduction and/or prevention of muscle loss by administration of conjugated linoleic acid (CLA) and vitamin D.
Description of the Related Art
[0003] Loss of muscle is often characterized by a deterioration of muscle quantity and quality. For example, during aging, there is a gradual decrease in the ability to maintain skeletal muscle function and mass that can eventually lead to sarcopenia. Patients with sarcopenia display a deterioration of muscle quantity and quality, leading to a decrease in functional capacity adversely affecting survival and quality of life. Muscle weakness associated with sarcopenia is also known to be associated with fatigue, a reduced ability to perform the tasks required for independent living and an increased risk of bony fractures.
[0004] Various prevention, treatment and management strategies for reducing and/or preventing muscle loss, for example age-related muscle loss, are currently available depending upon the severity of the symptoms. There is still a need for effective therapies for treating and preventing muscle loss.
SUMMARY
[0005] Disclosed herein includes compositions comprising conjugated linoleic acid (CLA) and Vitamin D. In some embodiments, the CLA comprises between about 1% and 99% of one or more isomers of CLA. In some embodiments, at least one of the one or more isomers of CLA is in triglyceride form. In some embodiments, at least one of the one or more isomers of CLA is in free fatty acid form. In some embodiments, at least one of the one or more isomers of CLA is in ester form. In some embodiments, at least one of the one or more isomers of CLA is selected from the group comprising cis-9, cis- 11 -octadecadienoic acid, cis-9, trans- 11 -octadecadienoic acid, trans-9, cis- 11 -octadecadienoic acid, trans-9, trans- 11 -octadecadienoic acid, cis- 10, cis-12-octadecadienoic acid, cis- 10, trans- 12- octadecadienoic acid, trans- 10, cis-12-octadecadienoic acid, and trans- 10, trans- 12- octadecadienoic acid. In some embodiments, the CLA is a mixture of two or more different isomers of CLA. For example, the mixture can comprise cis-9, trans- 11 -octadecadienoic acid CLA and trans- 10, cis-12-octadecadienoic acid. In some embodiments, the mixture comprises cis-9, trans- 11 -octadecadienoic acid CLA and trans-10, cis-12-octadecadienoic acid in a 50:50 ratio. In some embodiments, the CLA is derived from safflower oil. In some embodiments, the CLA further comprises one or more tocopherols. At least one of the one or more tocopherols can be, for example, δ-tocopherol, γ-tocopherol, a-tocopherol, or a combination thereof. In some embodiments, the CLA is or comprises one or more of a Tonalin product, a Clarinol product, Tonalin 35 WDP, Tonalin 60 WDP, Tonalin FFA 80, Tonalin SG 1000 T FFA, Tonalin SG 500 T FFA, Tonalin SG 750 T FFA, Tonalin TG 80, Tonalin TG 80, MET-Rx® CLA Tonalin 1000, Solgar Tonalin CLA, NatureWise CLA 1250, Natrol Tonalin CLA, NDS Body Toner Tonalin Censor, The Vitamin Shoppe Tonalin CLA, Natrol Tonalin Cla, Nature Made Cla Tonalin, Nature's Bounty Tonalin 1000 CLA, NDS Nutrition Censor Body Toner, BulkSupplements CLA Softgels, TNVitamins CLA, Puritan's Pride CLA Tonalin, Barlean's Organic Oils Tonalin CLA Swirl, Solgar, Tonalin CLA, TLS Tonalin CLA, Swanson Tonalin Cla, Iron Tek Essential Cla Pure Tonalin Complex, Trunature® Tonalin® CLA, VIROCUT EXTREME CLA, BlueBonnet Tonalin CLA, Labrada Nutrition EFA Lean Gold Essential Fatty Acid Softgel Capsules, Solgar Tonalin CLA Supplement, Davinci Labs Tonalin CLA 90, Vitacost Tonalin XS-CLA, GNC Total Lean™ CLA Soft Chew, ALLMAX CLA, GNC Total Lean™ CLA PM, Xenadrine NextGen, GNC Total Lean™ Thermo CLA, Puregel's Clarinol® CLA, LiveGreat Foods ACCLAIM® drink, Progressive Labs CLA, Jamieson Slim Down CLA 95, AST CLA 100, GNC ProPerformance AMP Ripped Vita Pak, Muscle Feast CLA, Jarrow Formulas CLA Slim, BQuick Nutrition Lean Capsules, Trophic Slim Fit CLA, GNC Total Lean CLA, Bluebonnet Clarinol® CLA Softgels, Nature's Plus Mega CLA 1200, PhytoCeutical Formulations' OptiCLA, BQuick BURN Clarinol® CLA Powder, SYLAB CLARINOL CLA, GoldNutrition® Slim Ultimate Raspberry, PROZIS CLA Clarinol®, Eurosup Clarinol, MegaCLA GoldNutrition, CLA Figurel with Chromium, Performance CLA, Bluebonnet Clarinol® CLA Softgels, Pro Figur CLA, Protech CLA 1000, LIPOcap, Reflex Nutrition Reflex CLA, Body Shape CLA, and Clarinol Xs Body Reshape. In some embodiments, the vitamin D is or comprises vitamin D3, vitamin D2, their biologically active metabolites and precursors, or any mixtures thereof.
[0006] In some embodiments, the composition comprises one or more Group A agents, where the Group A agent(s) can diminish the inflammatory mechanism resulting in mitochondrial oxidative stress. In some embodiments, at least one of the one or more Group A agent is an omega-3 fatty acid, an amino acid, a vitamin, or any combination thereof. In some embodiments, at least one of the one or more Group A agents is aniline or vitamin E. For example, the omega-3 fatty acid can be an alpha-linolenic acid, stearidonic acid, eicosapentanoic acid, docosahexanoic acid, or any mixture thereof. In some embodiments, the composition comprises one or more Group B agents, wherein the Group B agent(s) can positively impact the muscle stimulating mechanism. In some embodiments, at least one of the one or more Group B agents is a vitamin D other than vitamin D3, creatine, leucine, citrulline, whey protein, or any mixture thereof. In some embodiments, the composition comprises at least one Group A agent and at least one Group B agent. In some embodiments, the composition comprises at least two Group A agents and at least two Group B agents.
[0007] The amount of the CLA and the vitamin D in the composition can vary. For example, the composition can comprise about 1 g to about 6 g of the CLA, and about 100 IU to about 5000 IU of vitamin D. In some embodiments, the composition comprises about 2 g to about 4 g of the CLA, and about 400 IU to about 2000 IU of vitamin D. In some embodiments, the composition comprises about 3 g to about 3.4 g of the CLA, and about 800 IU to about 1000 IU of vitamin D. In some embodiments, the composition comprises about 4 g of CLA and about 2000 IU of Vitamin D. In some embodiments, the composition comprises an effective amount of the CLA and vitamin D to treat or prevent a condition of muscle loss in a subject in need thereof. In some embodiments, the composition comprises an effective amount of the CLA and vitamin D to reduce or prevent age-related muscle loss or function in a subject in need thereof. In some embodiments, the composition comprises an effective amount of the CLA and Vitamin D to increase muscle protein synthesis in a subject in need thereof. In some embodiments, where upon administration to a subject in need thereof, the composition has a synergistic effect on one or more of treating or preventing a condition of muscle loss, increasing muscle protein synthesis, and reducing or preventing age-related loss of muscle mass and function.
[0008] The form of the composition can vary. For example, the composition can be an oral composition, a foodstuff, a food supplement, a pharmaceutical composition, or any mixture thereof. The foodstuff can be or comprise, for example, a nutritional complete formula, a dairy product, a chilled or shelf stable beverage, a mineral water, a liquid drink, a shot, a soup, a dietary supplement, a meal replacement bar, a nutritional bar, a confectionery product, a milk or a fermented milk product, a yogurt, a pectin chew, a gummy, a milk based powder, an enteral nutrition product, a cereal product, a fermented cereal based product, an ice-cream, a chocolate, coffee, a culinary product, or any combination thereof. The food supplement can be, for example, in the form of capsules, gelatin capsules, soft capsules, tablets, sugar-coated tablets, powders, pills, pastes, pastilles, gums, drinkable solutions, drinkable emulsions, syrups, gels, or a combination thereof. The pharmaceutical composition can be, for example, in the form of capsules, gelatin capsules, soft capsules, tablets, chewable tablets, sugar-coated tablets, pills, pastes or pastilles, powders, softgels, chewable softgels, gums, drinkable solutions or emulsions, syrups, gels, or any combination thereof. The composition can comprise, in some embodiments, one or more of binding agents, gelling agents, thickeners, colorants, taste masking agents, stabilizers, antioxidants, coatings, sweeteners, taste modifiers, aroma chemicals. In some embodiments, the composition is formulated for intravenous, intramuscular, rectal, or inhalation administration. In some embodiments, the composition comprises one or more pharmaceutically acceptable carriers, diluents or excipients. In some embodiments, the composition is in a single unit dosage form. In some embodiments, the composition is in two or more unit dosage forms.
[0009] Also disclosed herein include uses of the compositions disclosed herein for treating or preventing a condition of muscle loss a subject in need thereof. In some embodiments, the use comprises increasing muscle mass, increasing muscle function, increasing the rate of muscle synthesis, or decreasing the rate of muscle breakdown in a subject in need thereof. In some embodiments, the use comprises reducing or preventing age-related loss of muscle mass and function in a subject in need thereof. In some embodiments, the use comprises treating or preventing at least one of sarcopenia, insufficient muscle protein synthesis, muscle degradation, muscle proteolysis, muscle atrophy, muscle dystrophy, muscle catabolism, muscle wasting, loss of muscle strength, loss of physical capacity, loss of physical performance, impaired mobility, frailty, surgery, disability, risk of falling, and risk of fall-related fractures in a subject in need thereof. In some embodiments, the use comprises treating or preventing sarcopenia in a subject in need thereof.
[0010] Disclosed herein includes uses of the compositions disclosed herein for the manufacture of a medicament for treating or preventing a condition of muscle loss a subject in need thereof. In some embodiments, the use comprises increasing muscle mass, increasing muscle function, increasing the rate of muscle synthesis, or decreasing the rate of muscle breakdown in a subject in need thereof. In some embodiments, the use comprises reducing or preventing age-related loss of muscle mass and function in a subject in need thereof. In some embodiments, the use comprises increasing muscle protein synthesis in a subject in need thereof. In some embodiments, the use comprises treating or preventing at least one of sarcopenia, insufficient muscle protein synthesis, muscle degradation, muscle proteolysis, muscle atrophy, muscle dystrophy, muscle catabolism, muscle wasting, loss of muscle strength, loss of physical capacity, loss of physical performance, impaired mobility, frailty, surgery, disability, risk of falling, and risk of fall-related fractures in a subject in need thereof. In some embodiments, the use comprises treating or preventing sarcopenia in a subject in need thereof.
[0011] Further disclosed herein include methods of treating or preventing a condition of muscle loss. In some embodiments, the methods comprise administering the compositions disclosed herein to a subject in need thereof. In some embodiments, the method comprises increasing muscle mass, increasing muscle function, increasing the rate of muscle synthesis, decreasing the rate of muscle breakdown, or a combination thereof in the subject in need thereof. In some embodiments, the method comprises reducing or preventing muscle mass and function in the subject in need thereof. In some embodiments, the method comprises increasing muscle protein synthesis in the subject in need thereof. In some embodiments, the method comprises treating or preventing at least one of sarcopenia, insufficient muscle protein synthesis, muscle degradation, muscle proteolysis, muscle atrophy, muscle dystrophy, muscle catabolism, muscle wasting, loss of muscle strength, loss of physical capacity, loss of physical performance, impaired mobility, frailty, surgery, disability, risk of falling, and risk of fall-related fractures in a subject in need thereof. In some embodiments, the method comprises treating or preventing sarcopenia in a subject in need thereof.
[0012] Disclosed herein include methods of treating or preventing a condition of muscle loss, comprising administering CLA and Vitamin D to a subject in need thereof. In some embodiments, the CLA and Vitamin D are administered separately. In some embodiments, the CLA is administered before the Vitamin D is administered to the subject. In some embodiments, the CLA is administered after the Vitamin D is administered to the subject. In some embodiments, the CLA and Vitamin D are administered to the subject concurrently.
[0013] In some embodiments, the method comprises increasing muscle mass, increasing muscle function, increasing the rate of muscle synthesis, or decreasing the rate of muscle breakdown in need thereof. In some embodiments, the method comprises reducing or preventing loss of muscle mass and function in a subject in need thereof. In some embodiments, the method comprises increasing muscle protein synthesis in a subject in need thereof. In some embodiments, the method comprises treating or preventing at least one of sarcopenia, insufficient muscle protein synthesis, muscle degradation, muscle proteolysis, muscle atrophy, muscle dystrophy, muscle catabolism, muscle wasting, loss of muscle strength, loss of physical capacity, loss of physical performance, impaired mobility, frailty, surgery, disability, risk of falling, and risk of fall-related fractures in a subject in need thereof.
[0014] In some embodiments, the method comprises treating or preventing sarcopenia in a subject in need thereof. In some embodiments, the CLA comprises between about 1% and 99% of one or more isomers of CLA. In some embodiments, at least one of the one or more isomers of CLA is in triglyceride form, free fatty acid form, or ester form. In some embodiments, at least one of the one or more isomers of CLA is cis-9, cis-11- octadecadienoic acid, cis-9, trans- 11-octadecadienoic acid, trans-9, cis-11-octadecadienoic acid, trans-9, trans- 11 -octadecadienoic acid, cis-10, cis-12-octadecadienoic acid, cis-10, trans- 12-octadecadienoic acid, trans- 10, cis-12-octadecadienoic acid, trans- 10, trans- 12- octadecadienoic acid, or any combination thereof. In some embodiments, the CLA is a mixture of two or more different isomers of CLA. The mixture can, for example, comprise cis-9, trans- 11 -octadecadienoic acid CLA, trans- 10, cis- 12-octadecadienoic acid, or any mixture thereof. In some embodiments, the mixture comprises cis-9, trans-11- octadecadienoic acid CLA and trans- 10, cis- 12-octadecadienoic acid in a 50:50 ratio. In some embodiments, the CLA is derived from safflower oil. In some embodiments, the CLA further comprises one or more tocopherols. The tocopherol can be, or comprise, δ- tocopherol, γ-tocopherol, a-tocopherol, or any combination thereof. In some embodiments, the CLA can be, or comprise, one or more of a Tonalin product, a Clarinol product, Tonalin 35 WDP, Tonalin 60 WDP, Tonalin FFA 80, Tonalin SG 1000 T FFA, Tonalin SG 500 T FFA, Tonalin SG 750 T FFA, Tonalin TG 80, Tonalin TG 80, MET-Rx® CLA Tonalin 1000, Solgar Tonalin CLA, NatureWise CLA 1250, Natrol Tonalin CLA, NDS Body Toner Tonalin Censor, The Vitamin Shoppe Tonalin CLA, Natrol Tonalin Cla, Nature Made Cla Tonalin, Nature's Bounty Tonalin 1000 CLA, NDS Nutrition Censor Body Toner, BulkSupplements CLA Softgels, TNVitamins CLA, Puritan's Pride CLA Tonalin, Barlean's Organic Oils Tonalin CLA Swirl, Solgar, Tonalin CLA, TLS Tonalin CLA, Swanson Tonalin Cla, Iron Tek Essential Cla Pure Tonalin Complex, Trunature® Tonalin® CLA, VIROCUT EXTREME CLA, BlueBonnet Tonalin CLA, Labrada Nutrition EFA Lean Gold Essential Fatty Acid Softgel Capsules, Solgar Tonalin CLA Supplement, Davinci Labs Tonalin CLA 90, Vitacost Tonalin XS-CLA, GNC Total Lean™ CLA Soft Chew, ALLMAX CLA, GNC Total Lean™ CLA PM, Xenadrine NextGen, GNC Total Lean™ Thermo CLA, Puregel's Clarinol® CLA, LiveGreat Foods ACCLAIM® drink, Progressive Labs CLA, Jamieson Slim Down CLA 95, AST CLA 100, GNC ProPerformance AMP Ripped Vita Pak, Muscle Feast CLA, Jarrow Formulas CLA Slim, BQuick Nutrition Lean Capsules, Trophic Slim Fit CLA, GNC Total Lean CLA, Bluebonnet Clarinol® CLA Softgels, Nature's Plus Mega CLA 1200, PhytoCeutical Formulations' OptiCLA, BQuick BURN Clarinol® CLA Powder, SYLAB CLARINOL CLA, GoldNutntion® Slim Ultimate Raspberry, PROZIS CLA Clarinol®, Eurosup Clarinol, MegaCLA GoldNutrition, CLA Figurel with Chromium, Performance CLA, Bluebonnet Clarinol® CLA Softgels, Pro Figur CLA, Protech CLA 1000, LIPOcap, Reflex Nutrition Reflex CLA, Body Shape CLA, and Clarinol Xs Body Reshape. In some embodiments, the vitamin D comprises vitamin D3, vitamin D2, their biologically active metabolites and precursors, or any mixture thereof.
[0015] In some embodiments, the method comprises administrating one or more Group A agent that diminishes the inflammatory mechanism resulting in mitochondrial oxidative stress. In some embodiments, at least one of the one or more Group A agents is an omega-3 fatty acid, amino acid, vitamin, or any mixture thereof. In some embodiments, at least one of the one or more Group A agents is aniline or Vitamin E. In some embodiments, the omega-3 fatty acid can be, or comprise, alpha-linolenic acid, stearidonic acid, eicosapentanoic acid, docosahexanoic acid, or any mixture thereof. The method can, for example, comprise administrating one or more Group B agents that positively impacts the muscle stimulating mechanism. In some embodiments, at least one of the one or more Group B agents is, or comprise, a vitamin D other than Vitamin D3, Vitamin D2, citrulline, creatine, leucine, whey protein, or a mixture thereof. In some embodiments, the method comprises administrating at least one Group A agent and at least one Group B agent to the subject. In some embodiments, the method comprises administrating at least two Group A agents and at least two Group B agents to the subject.
[0016] In some embodiments, the CLA is administered in an amount from about 1.0 g to about 6.0 g per day and the vitamin D is administered in an amount from about 100 IU to about 5000 IU per day. For example, the CLA can be administered in an amount from about 2.0 g to about 4.0 g per day and the vitamin D can be administered in an amount from about 400 IU to about 2000 IU per day. In some embodiments, the CLA is administered in an amount from about 3.0 g to about 3.4 g per day and the vitamin D is administered in an amount from about 800 IU to about 1000 IU per day. In some embodiments, the CLA is administered in an amount of about 4 g per day and the vitamin D is administered in an amount of about 2000 IU per day. In some embodiments, the dose is administered in a single unit dosage form. In some embodiments, the dose is administered in two or more unit dosage forms.
[0017] In some embodiments, the administration of one or more of the CLA, vitamin D, Group A agent(s) and Group B agent(s) is repeated one time per day. In some embodiments, the administration of one or more of the CLA, vitamin D, Group A agent(s) and Group B agent(s) is repeated more than one time per day. In some embodiments, the administration of one or more of the CLA, Vitamin D, Group A agent(s) and Group B agent(s) is oral, intravenous, intraperitoneal, intragastric, or intravascular administration. In some embodiments, the administration of one or more of the CLA, Vitamin D, Group A agent(s) and Group B agent(s) is oral administration.
[0018] The subject that can be treated with the methods and compositions disclosed herein can vary in age, and/or gender, and/or lifestyle. For example, the subject can be an adult. The subject can be a male or a female. In some embodiments, the subject is a middle-aged adult. In some embodiments, the subject is an elderly adult. In some embodiments, the subject is human is a person of the age of 40 years or more. In some embodiments, the subject is human is a person of the age of 60 years or more. In some embodiments, the subject is with an active lifestyle. In some embodiments, the subject is with a sedentary lifestyle. In some embodiments, the subject has sarcopenia or is at risk of developing sarcopenia.
[0019] In some embodiments, the method comprises identifying a subject is suffering from a condition of muscle loss or is at the risk of developing a condition of muscle loss. In some embodiments, the method comprises determining muscle weight, muscle circumference, lean muscle, body weight, fat mass, lean mass, brain water content, locomotor activity, protein synthesis rate, or any combination thereof of the subject. In some embodiments, at least one symptom of the condition of muscle loss is skeletal muscle loss, or muscle mass loss. In some embodiments, the condition of muscle loss is caused by aging, disease, injury, inactivity, or any combination thereof. In some embodiments, the condition of muscle loss is sarcopenia, muscle atrophy, cachexia, muscular dystrophy, or any combination thereof.
[0020] In some embodiments, upon administration to a subject in need thereof, the rate of muscle protein synthesis is increased. The increased muscle protein synthesis can comprise, for example, increased muscle fractional synthesis rate (FSR). In some embodiments, the administration of a combination of the CLA and vitamin D has a synergistic effect
BRIEF DESCRIPTION OF THE DRAWINGS [0021] Figure 1 depicts an outline of an exemplary muscle protein metabolism study, indicating the timing of infusions, measurements, and samples taken.
[0022] Figure 2 shows bar graphs illustrating the predicted muscle protein synthetic rate results from an ongoing clinical trial, wherein elderly subjects are administered a placebo, CLA, vitamin D, or a combination of CLA and vitamin D. Notation "a" indicates fractional muscle protein synthesis rate (FSR) significantly different (p<0.05) under "clamp" infusion, no statistical significance between before and after intervention; notation "b" indicates FSR is significantly different (p<0.05) under "clamp" infusion from before clamp; notation "c" indicates muscle FSR is significantly different (p<0.05) before and after intervention; notation "d" indicates "clamp" FSR is significantly different (p<0.05) after combined CLA/Vitamin D intervention as compared to the CLA alone and vitamin D alone interventions.
DETAILED DESCRIPTION
[0023] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects of the present disclosure, as generally described herein, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this disclosure.
Definitions
[0024] As used herein, a "subject" refers to an animal that is the object of treatment, observation or experiment. "Animals" include cold- and warm-blooded vertebrates and invertebrates such as fish, shellfish, reptiles and, in particular, mammals. "Mammal" includes, without limitation, mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates, such as monkeys, chimpanzees, and apes, and, in particular, humans.
[0025] As used herein, a "patient" refers to a subject that has been treated by, or is being treated by, or will be treated by a medical professional, such as a Medical Doctor (i.e. Doctor of Allopathic medicine or Doctor of Osteopathic medicine) or a Doctor of Veterinary Medicine, to attempt to cure, or at least ameliorate the effects of, a particular disease or disorder or to prevent the disease or disorder from occurring in the first place.
[0026] As used herein, "administration" or "administering" refers to a method of giving a dosage of a pharmaceutically active ingredient to a vertebrate.
[0027] As used herein, a "dosage" refers to the combined amount of the active ingredients (e.g., CLA and Vitamin D).
[0028] As used herein, a "unit dosage" refers to an amount of therapeutic agent administered to a patient in a single dose.
[0029] As used herein, a "daily dosage" refers to the total amount of therapeutic agent administered to a patient in a day.
[0030] As used herein, "therapeutically effective amount" or "pharmaceutically effective amount" is meant an amount of therapeutic agent, which has a therapeutic effect. The dosages of a pharmaceutically active ingredient which are useful in treatment are therapeutically effective amounts. Thus, as used herein, a therapeutically effective amount means those amounts of therapeutic agent which produce the desired therapeutic effect as judged by clinical trial results and/or model animal studies.
[0031] As used herein, a "therapeutic effect" relieves, to some extent, one or more of the symptoms of a disease or disorder. For example, a therapeutic effect may be observed by a reduction of the subjective discomfort that is communicated by a subject (e.g., reduced discomfort noted in self-administered patient questionnaire).
[0032] "Treat," "treatment," or "treating," as used herein refers to administering a compound or a composition (e.g., a nutritional composition or a pharmaceutical composition) to a subject for prophylactic and/or therapeutic purposes. The term "prophylactic treatment" refers to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition. The term "therapeutic treatment" refers to administering treatment to a subject already suffering from a disease or condition.
[0033] The terms "prevent", "preventing" and "prevention" as used herein refer to a method of preventing the onset of a disease and/or its attendant symptoms or barring a subject from acquiring a disease. As used herein, "prevent", "preventing" and "prevention" also include delaying the onset of a disease and/or its attendant symptoms and reducing a subject's risk of acquiring a disease.
[0034] The term "without a significant change in lifestyle" is used to describe the lifestyle of a subject before co-administration of CLA and vitamin D as described herein in comparison to that subject's lifestyle after commencing co-administration of CLA and vitamin D as described herein. This term is used to distinguish effects on muscle mass and function in a subject where a change in that subject's lifestyle materially contributes to an impact on muscle mass and function.
[0035] The term "Vitamin D" as used herein without a subscript, refers to all forms of vitamin D, for example vitamin Di, D2, D3, D4, D5, or any mixture thereof. In some embodiments, the vitamin D comprises mainly vitamin D2 and/or vitamin D3.
[0036] As used herein, a "synergistic" or "synergizing" effect can be such that the one or more effects of the combination compositions are greater than the one or more effects of each component alone, or they can be greater than the sum of the one or more effects of each component alone. The synergistic effect can be about, or greater than about 5, 10, 20, 30, 50, 75, 100, 110, 120, 150, 200, 250, 350, or 500% or even more than the effect on a subject with one of the components alone, or the additive effects of each of the components when administered individually. The effect can be any of the measurable effects described herein.
Abbreviations
[0037] CLA = conjugated linoleic acid
[0038] FSR = fractional muscle protein synthesis rate
Muscle Loss
[0039] Disclosed herein includes methods and compositions for the prevention and reduction of loss in muscle mass and/or function, for example the age-related loss of muscle mass and/or function.
[0040] Muscle loss is a condition of deterioration of muscle quantity and quality. Non-limiting symptoms of muscle loss can be loss or reduction of muscle mass, loss or reduction of lean muscle, loss or reduction of muscle weight, loss or reduction of muscle circumference, loss or reduction of fat mass, loss or reduction of lean mass, loss or reduction of muscle function, loss or reduction of muscle strength, loss or reduction of mobility, weight loss, reduction in muscle protein fractional synthesis rate (FSR), or any combination thereof. In some embodiments, at least one symptom of the condition of muscle loss is muscle mass loss or skeletal muscle loss. In some embodiment, at least one symptom of the condition of muscle loss is weight loss. In some embodiment, at least one symptom of the condition of muscle loss is loss or reduction in lean mass, loss or reduction of muscle circumference, or reduction in FSR. There are a variety of causes for muscle loss. For example, the muscle loss can be caused by aging, disease (for example cancer and liver diseases), inactivity, injury (for example liver transplantation), or any combination thereof.
[0041] Some non-limiting examples of causes for muscle loss include age (e.g., age-related reduction in nerve cells responsible for sending signals from the brain to the muscles to initiate movement); a decrease in the concentration of some hormones, including but not limited to, growth hormone, testosterone, and insulin-like growth factor; a decrease in the body's ability to synthesize protein; inadequate intake of calories and/or protein to sustain muscle mass; and any combination thereof. In some embodiments, the condition of muscle loss is sarcopenia, muscle atrophy, cachexia, muscular dystrophy, muscle wasting, or any combination thereof. In patients having sarcopenia, the patients display a deterioration of muscle quantity and quality, leads to a decrease in functional capacity, adversely affecting survival, and quality of life.
[0042] Muscle loss can be determined by various conventional methods, for example measuring muscle size (for example circumference of the rectus femoris) by techniques such as ultrasound, measuring muscle resistance to an electrical current using electric impedance myography (EIM), measuring change in body weight, measuring muscle mass, measure lean mass or fat mass, measuring locomotor activity, measuring skeletal muscle fiber number, measuring muscle cross-sectional area (CSA), measuring fractional synthesis of protein (FSR) in muscle (e.g., with D20), tracking lean body mass (LBM), or any combination thereof. In some embodiments, muscle loss can be measured by tracking the lean body mass (LBM) of a subject over time. [0043] Some embodiments provide methods and compositions for preventing or reducting age-related loss of muscle mass and function that can eventually proceed to sarcopenia. Sarcopenia is characterized by a loss of muscle mass, strength and function occurring during aging. "Sarcopenia" is currently proposed to be defined as a "reduction of muscle function" and "mass below a certain threshold" in the elderly. Muscle mass loss typically starts from the age of 30 years at a rate of 3-8% per decade and accelerates from 60 years of age. This loss reaches up to 35-40 % in elderly over 70, and hence, sarcopenia is especially prominent in elderly.
[0044] The better a person maintains muscle mass through middle and old age, the more able they are to withstand the insults that lead to sarcopenia (e.g., prolonged bed rest, inactivity, poor nutrition, hospitalization). Muscle loss of approximately 50% typically leads to functional impairment and progression to sarcopenia; this, in turn, increases the risk of several deleterious outcomes, such as frailty, difficulty in self-care, falls, broken bones, and mortality. Exercise, increased protein intake, and amino acid supplements have been proposed to improve outcomes of elderly muscle loss and sarcopenia.
[0045] Muscle mass preservation can be achieved through an adequate stimulation of protein synthesis and/or inhibition of proteolysis. Several factors contribute to muscle protein synthesis, among which the most important are the availability of amino acids (serving as building blocks for the newly synthesized proteins) and the activation signals generated by anabolic amino acids or anabolic amino acid derivatives (e.g. citrulline, leucine, essential amino acids (EAA), and creatine). However, protein intake is typically decreased in elderly, which leads to inadequate postprandial availability of amino acids. Furthermore, aging is characterized by decreased muscle sensitivity to the anabolic effect of essential amino acids, especially leucine. This reduced responsiveness to leucine is associated with a decrease in the activation of intracellular pathways controlling muscle protein synthesis. Hence, to counteract the deleterious effect of aging on muscle protein synthesis, it been proposed to simultaneously target the bioavailability of amino acids, especially essential amino acids, by increasing the intake of high-quality protein (rich in essential amino acids, especially leucine), and the muscle response to anabolic amino acids by improving muscle sensitivity to these amino acids. Elderly muscle fractional synthetic rate response to protein intake is deficient, however. Currently recommended supplements target stimulus of muscle growth but do not target inflammatory oxidative mechanism with respect to sarcopenia. Current recommendations typically require the administration of more than 8 g amino acids or more than 15 g protein. Such amounts can be too large to be applicable to the dietary supplement formats of pills or soft gels. Hence, current supplemental regimens are delivered by meal replacement beverages or powdered beverages which may increase adiposity and calorie consumption and therefore may be counterproductive in those overweight or trying to watch weight while simultaneously maintaining muscle mass. Current supplemental regimens (such as creatine) require the combination with resistance exercise; however, a large portion of US population is sedentary. Thus, additional approaches for reducing and preventing loss of muscle mass and function in sedentary populations are needed.
CLA
[0046] The biological activity associated with conjugated linoleic acids (termed CLA) is diverse and complex. CLA is a collective name for a class of positional and structural isomers of linoleic acid that contain conjugated double bonds. CLA are a family of at least 28 isomers of linoleic acid found mostly in the meat and dairy products derived from ruminants. CLAs can be either cis- or trans-fats and the double bonds of CLAs are conjugated and separated by a single bond between them. In 2008, the United States Food and Drug Administration categorized CLA as generally recognized as safe (GRAS). Supplementation with CLA for 24 months has been found to be well tolerated and reduce body fat mass in healthy, overweight humans. CLA supplementation studies in younger populations have found a reduction of muscle breakdown markers after resistance exercise.
[0047] As used herein, the term "conjugated linoleic acid" (abbreviated as "CLA") refers to any conjugated linoleic acid or octadecadienoic acid and its salts and derivatives. It is intended that this term encompass and indicate all positional and geometric isomers of linoleic acid with two conjugated carbon-carbon double bonds any place in the molecule. CLA differs from ordinary linoleic acid in that ordinary linoleic acid has double bonds at carbon atoms 9 and 12. Non-limiting examples of CLA include cis- and trans isomers ("E/Z isomers") of the following positional isomers: 2,4-octadecadienoic acid, 4,6- octadecadienoic acid, 6,8-octadecadienoic acid, 7,9-octadecadienoic acid, 8,10- octadecadienoic acid, 9, 11 -octadecadienoic acid and 10,12 octadecadienoic acid, and 11, 13 octadecadienoic acid. CLA can encompass a single isomer, a selected mixture of two or more isomers, and/or a non-selected mixture of isomers obtained from natural sources. The CLA or CLA composition can comprise synthetic and/or semisynthetic CLA. In some embodiments, CLA can comprise one or more of free fatty acid(s) of CLA, physiologically acceptable salts of CLA, and esters with physiologically acceptable, preferably naturally occurring, alcohols (e.g., ethanol and glycerol), and CLA triglycerides.
[0048] As used herein, "triglycerides" of CLA can comprise an isomer of CLA at any or all of three positions on the triglyceride backbone. As used herein, the term conjugated linoleic acid or CLA is intended to include "esters" of CLA which term includes any CLA isomer bound through an ester linkage to an alcohol or any other chemical group.
[0049] As used herein, "c" encompasses a chemical bond in the cis orientation, and "t" refers to a chemical bond in the trans orientation. If a positional isomer of CLA is designated without a "c" or a "t", then that designation includes all four possible isomers. For example, 10,12 octadecadienoic acid encompasses cl0,tl2; tl0,cl2; tl0,tl2; and cl0,cl2 octadecadienoic acid.
[0050] In some embodiments, the CLA comprises major and minor isomers, for example, one or more of cl l,tl3; tl l,cl3; tl l,tl3; and cl l,cl3 octadecadienoic acids. In some embodiments, the CLA administered to patients is or comprises an octadecadienoic acid isomer selected from the group of cis-9, trans-11; cis-9, cis-11; trans-9, cis-11; trans-9, trans-11; cis-10, cis-10, trans-12; trans-10, cis-12; trans-10, trans-12 octadecadienoic acid and mixtures thereof. In some embodiments, the CLA administered to patients contains less than 5% of minor isomers of CLA. In some embodiments, the minor isomer is cl l,tl3; tl l,cl3; tl l,tl3; or cl l,cl3 octadecadienoic acid. In some embodiments, the CLA contains less than 1% of minor isomers of CLA. In some embodiments, the CLA comprises an ester or triglyceride. In some embodiments, the CLA comprises greater than about 55% tl0,cl2 octadecadienoic acid.
[0051] In some embodiments, the CLA comprises one or more of the isomers of octadecadienoic acid including the cis-9, trans-11; cis-9, cis-11; trans-9, cis-11 ; trans-9, trans-11; cis-10, cis-12; cis-10, trans-12; trans-10, cis-12; and trans-10, trans-12 isomers. In some embodiments, eight possible geometric isomers of 9,11 and 10,12 octadecadienoic acid (i.e., c9,cl l; c9,tl l; t9,cl l; t9,tl l ; cl0,cl2; cl0,tl2; tl0,cl2 and tl0,tl2) would form from the isomerization of c9,cl2-octadecadienoic acid.
[0052] The cis-9,trans-l l and trans-10, cis-12 isomers of CLA are thought to have the most biological activity. Therefore, in some embodiments, these isomers can be used in a purified form or a substantially purified form, or in CLA compositions containing high ratios of these isomers. In some embodiments, isomers in the CLA mixtures include 9,11-octadecadienonic acid, 10,12-octadecadienoic acid, most preferably the c9,tl l and tl0,cl2 isomers. In some embodiments, the CLA mixture contains greater than about 50% tl0,cl2 isomer. In some embodiments, the CLA mixture contains greater than about 55% tl0,cl2 isomer. In some embodiments, the CLA mixture contains greater than about 60% tl0,cl2 isomer. It is contemplated that in some embodiments, supplementation of the CLA mixture derived from isomerization of linoleic acid with purified or synthesized tl0,cl2 isomer may be necessary to achieve these percentages.
[0053] Some embodiments also contemplate the use of derivatives of CLA. As used herein, the term "CLA" also refers to such derivatives. For example, CLA can be free or bound through ester linkages or provided in the form of an oil comprising CLA triglycerides. In these embodiments, the triglycerides may be partially or wholly comprised of CLA attached to a glycerol backbone. The CLA can, in some embodiments, be provided as a methylester or ethylester. In some embodiments, the CLA is in the form of a non-toxic salt, such as a potassium or sodium salt (e.g., a salt formed by reacting chemically equivalent amounts of the free acids with an alkali hydroxide at a pH of about 8 to 9).
[0054] The active forms of CLA include, in addition to the free acids the active isomers of CLA; non-toxic salts thereof; active esters and other active chemical derivatives thereof; and mixtures thereof. The non-toxic salts of the free acids may be made by reacting the free acids with a non-toxic base. The CLA can be heat stable and can be used as is, or dried and powdered. The CLA can be readily converted into a non-toxic salt, such as the sodium or potassium salt, by reacting chemically equivalent amounts of the free acid with an alkali hydroxide at a pH of about 8 to 9.
[0055] In some embodiments, the CLA comprises between about 1% and 99% of one or more isomers of CLA. In still further embodiments, the one or more isomers of CLA is selected from the group comprising cis-9, cis-11 -octadecadienoic acid, cis-9, trans-11- octadecadienoic acid, trans-9, cis-11-octadecadienoic acid, trans-9, trans- 11-octadecadienoic acid, cis-10, cis-12-octadecadienoic acid, cis-10, trans- 12-octadecadienoic acid, trans- 10, cis- 12-octadecadienoic acid, and trans- 10, trans- 12-octadecadienoic acid. In some embodiments, the CLA is a mixture of two or more different isomers of CLA. In some embodiments, the CLA comprises cis-9, trans- 11 -octadecadienoic acid CLA and trans-10, cis-12- octadecadienoic acid at about a 10:90 to 90: 10.
[0056] In some embodiments, the CLA comprises one or more of a Tonalin product, a Clarinol product, Tonalin 35 WDP, Tonalin 60 WDP, Tonalin FFA 80, Tonalin SG 1000 T FFA, Tonalin SG 500 T FFA, Tonalin SG 750 T FFA, Tonalin TG 80, Tonalin TG 80, MET-Rx® CLA Tonalin 1000, Solgar Tonalin CLA, NatureWise CLA 1250, Natrol Tonalin CLA, NDS Body Toner Tonalin Censor, The Vitamin Shoppe Tonalin CLA, Natrol Tonalin Cla, Nature Made Cla Tonalin, Nature's Bounty Tonalin 1000 CLA, NDS Nutrition Censor Body Toner, BulkSupplements CLA Softgels, TNVitamins CLA, Puritan's Pride CLA Tonalin, Barlean's Organic Oils Tonalin CLA Swirl, Solgar, Tonalin CLA, TLS Tonalin CLA, Swanson Tonalin Cla, Iron Tek Essential Cla Pure Tonalin Complex, Trunature® Tonalin® CLA, VIROCUT EXTREME CLA, BlueBonnet Tonalin CLA, Labrada Nutrition EFA Lean Gold Essential Fatty Acid Softgel Capsules, Solgar Tonalin CLA Supplement, Davinci Labs Tonalin CLA 90, Vitacost Tonalin XS-CLA, GNC Total Lean™ CLA Soft Chew, ALLMAX CLA, GNC Total Lean™ CLA PM, Xenadrine NextGen, GNC Total Lean™ Thermo CLA, Puregel's Clarinol® CLA, LiveGreat Foods ACCLAIM® drink, Progressive Labs CLA, Jamieson Slim Down CLA 95, AST CLA 100, GNC ProPerformance AMP Ripped Vita Pak, Muscle Feast CLA, Jarrow Formulas CLA Slim, BQuick Nutrition Lean Capsules, Trophic Slim Fit CLA, GNC Total Lean CLA, Bluebonnet Clarinol® CLA Softgels, Nature's Plus Mega CLA 1200, PhytoCeutical Formulations' OptiCLA, BQuick BURN Clarinol® CLA Powder, SYLAB CLARINOL CLA, GoldNutrition® Slim Ultimate Raspberry, PROZIS CLA Clarinol®, Eurosup Clarinol, MegaCLA GoldNutrition, CLA Figurel with Chromium, Performance CLA, Bluebonnet Clarinol® CLA Softgels, Pro Figur CLA, Protech CLA 1000, LIPOcap, Reflex Nutrition Reflex CLA, Body Shape CLA, and Clarinol Xs Body Reshape. In some embodiments, the CLA is derived from safflower oil. In some embodiments, the CLA further comprises one or more tocopherols. In some embodiments, the one or more tocopherols is selected from the group consisting of consisting of δ-tocopherol, γ-tocopherol, a-tocopherol, and combinations thereof.
Vitamin D
[0057] Vitamin D is a group of fat-soluble secosteroids, the two major physiologically relevant forms of which are vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). These are known collectively as calciferol. Vitamin D without a subscript refers to all forms of vitamin D (including vitamin Di, D2, D3, D4, and D5), in particular D2 and D3, or any mixture thereof.
[0058] Vitamin D from the diet or skin synthesis is biologically inactive, and an enzymatic conversion (hydroxylation) in the liver and kidney is required for activation. When ingested, vitamin D is hydroxylated in the liver (endoplasmic reticulum) to 25- hydroxycholecalciferol (25(OH)D), also known as calcidiol, by the enzyme 25 -hydroxylase, produced by hepatocytes. Once made, the product is stored in the hepatocytes until it is needed and can be released into the plasma where it will be bound to an a-globulin. 25- hydroxycholecalciferol is then transported to the proximal tubules of the kidneys where it can be hydroxylated by one of two enzymes to different forms of vitamin D, one of which is active vitamin D (l ,25(OH)D) and another which is inactive vitamin D (24,25(OH)D). The enzyme la-hydroxylase which is activated by parathyroid hormone (and additionally by low calcium or phosphate) forms the main biologically active vitamin D hormone with a CI hydroxylation forming 1,25-dihydroxy cholecalciferol (l,25(OH)2D, also known as calcitriol). A separate enzyme hydroxylates the C24 atom forming 24R,25(OH)2D3 when la-hydroxylase is not active, this inactivates the molecule from any biological activity. In some embodiments, the Vitamin D can be provided in an active (l,25(OH)2D) or inactive (Vitamin D3 or D2) form.
[0059] Muscle strength declines with age and a recently characterized deficiency symptom of vitamin D is skeletal muscle weakness. Deficiency of vitamin D, which has hormonal effects on muscle mass and strength, has been described as a risk factor in falls and bone fractures in the elderly. Loss of muscle strength has been correlated with a loss of vitamin D receptors (VDR) in muscle cells. Supplemental vitamin D of at least 800 IU per day may result in a clinically significant increase in VDR in muscle cells, and this may be in part be the mechanism whereby other studies have shown improvement in body-sway, muscle strength and falling risk were seen with vitamin D supplementation at this level. While this muscular weakness associated with vitamin D may not be surprising at classical Vitamin D Deficiency levels (blood 250H-VitD3 of <15 ng/mL), others continued to see improvement in lower extremity function up to and beyond 40 ng 250H-VitD3/mL which are levels well above what previously might have been thought necessary for maximal benefit.
[0060] While the exact mechanism is still unclear, it is clear that both the active metabolite, l,250H2-VitD3 and its precursor, 250H-VitD3, play a significant role in normal functioning of muscle. Muscle contains VDRs for l,250H2-VitD3, found in both the nucleus and at the cell membrane, and these are also involved in non-specific binding 250H-VitD3 as well. It has been shown that feeding D3 to vitamin D deficient rats 7 hours prior to measurement increased protein synthesis as measured by H-leucine incorporation into muscle cell proteins. However, when the muscles were removed from the vitamin D deficient rats and studied, it was found that only 25-OH VitD3 acts directly in the muscles.
[0061] Early clinical evidence pointed to a reversible myopathy associated with Vitamin D deficiency. Vitamin D receptors were discovered in muscle tissue, thus providing direct evidence of vitamin D's effect on muscle function. Muscle biopsies in adults with vitamin D deficiency exhibit mainly type II muscle fiber atrophy. Type II fibers are important because they are the first initiated to prevent a fall. A recent randomized controlled study found that daily supplementation of 1,000 IU of vitamin D2 in elderly stroke survivors resulted in an increase in mean type II fiber diameter and in percentage of type II fibers. There was also a correlation between serum 250H-VitD3 level and type II fiber diameter.
[0062] Vitamin D conveys its action by binding to VDR. VDR is expressed in particular stages of differentiation from myoblast to myotubes. Two different VDRs have been described. One is located at the nucleus and acts as a nuclear receptor and the other is located at the cell membrane and acts as a cellular receptor. VDR knockout mice are characterized by a reduction in body weight and size, as well as impaired motor coordination. The nuclear VDR is a ligand-dependent nuclear transcription factor that belongs to the steroid-thyroid hormone receptor gene superfamily. It has been reported that the first in situ detection of VDR in human muscle tissue with significant associated intranuclear staining for VDR. Once l,250H2-VitD3 binds to its nuclear receptor, it causes changes in mRNA transcription and subsequent protein synthesis. The genomic pathway has been known to influence muscle calcium uptake, phosphate transport across the cell membrane, phospholipid metabolism, and muscle cell proliferation and differentiation. Recent data indicate that l,250H-VitD3 has a fast activation of mitogen-activated protein kinase (MAPK) signaling pathways, which in turn forward signals to their intracellular targets that effect the initiation of myogenesis, cell proliferation, differentiation, or apoptosis.
[0063] Vitamin D can regulate formation and regeneration of tight junctions and neuromuscular junctions. Studies that shown that vitamin D regulates the expression of VDR and the neural growth factor (NGF) in Schwann cells. Recent studies have also shown that vitamin D enhances glial cell line-derived neurotrophic factor (GDNF) in rats and that this may have beneficial effects in neurodegenerative disease. Therefore, vitamin D can act through several mechanisms of cellular function and neural interaction to improve overall muscle strength and function.
[0064] As used herein, 1 IU of vitamin D is the biological equivalent of 0.025 μg. Hence, 1,000 IU is the biological equivalent of 25 μg. In some embodiments, vitamin D is used in an amount of 800 IU or more per daily dosage in combination with CLA, such as, for instance 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000 or 10,000 IU, or any value between any of two of said values or over, but not so high that it causes toxicity in the subject to which it is administered. In some embodiments, vitamin D is used in an amount of 20 μg or more per daily dosage in combination with CLA, such as, for instance 25, 50, 75, 100, 125, 150, 175, 200, 225 or 250 μg per daily dosage, or any value in between any of two of said values or over.
CLA and Vitamin D Supplementation for Muscle Maintenance
[0065] While the literature provides various nutritional recommendations for sarcopenia, including amino acids, HMB, vitamin D, CLA, and omega-3 amino acids, specific combinations of these compounds are not discussed. Resveratrol, carotenoids and EGCG have also been proposed for combating muscle wasting/sarcopenia. Studies suggest that vitamin D metabolite calcefidiol slows muscle loss in elderly populations (optionally with vitamin D3 cholecalciferol). Another study demonstrated the short term use of high concentrated omega-3 fatty acids (4g for 2 months) stimulates muscle synthesis in an elderly population. This was followed by another study that demonstrated impact of long term intake of omega-3 fatty acids (6 months) on skeletal muscle mass and functions in elderly populations. These dosages, however, are too high in view of FDA recommendations in the United States market.
[0066] While CLA alone has been discussed in the literature with respect to muscle maintenance in the elderly, there are currently no clinical trials testing CLA in the elderly population. Further, while CLA in combination with creatine has been tested in the elderly, this combination intervention was not compared to each intervention alone, and the combination was not shown to be superior to prior results observed with creatine. CLA has been shown to have an impact on muscle in younger and post-menopausal populations but has not been tested in older populations losing muscle. CLA feeding studies also show improvement in muscle status and muscle markers in older mice. CLA supplementation in older mice shows an improvement in oxidative and mitochondrial markers, which are markers that are affected by elderly muscle loss. Due to CLA's impact on muscle metabolism it has been termed an exercise mimetic.
[0067] Studies of vitamin D supplementation have yielded unclear results in terms of its influence elderly muscle mass and functional maintenance. Individual studies of vitamin D supplementation in randomized controlled trials showed improvements in muscle function in older populations (e.g., postmenopausal women 50-65 years old). Vitamin D supplementation interacts with muscle via the receptor VDR, which hypothesized to affect downstream regulation of insulin-like growth factor signaling pathway. Studies have also found that adequate vitamin D is necessary for proper performance of muscle growth ingredient HMB in restoring muscle function in elderly persons.
[0068] Both CLA and Vitamin D separately have been discussed with respect to elderly muscle maintenance but the combination has not been disclosed in a patent or other prior art document, nor has it been the object of any clinical studies. CLA is hypothesized to activate mechanisms similar to resistance exercise and impact mitochondrial energy production in muscle, deficiency in which due to systemic inflammation and resultant oxidative stress is a key mechanism in muscle loss in the elderly. Without being bound by any particular theory, it is believed that CLA can impact the utilization of fat as an energy source in muscle. Also without being bound by any particular theory, it is believed that Vitamin D can impact muscle growth stimulatory mechanisms, and has been shown to impact falls in the elderly (a measure of elderly muscle strength). Of interest, aging is also characterized by a high prevalence of vitamin D deficiency (serum vitamin D levels below 25-50 nmol/L) and insufficiency (serum vitamin D levels below 75 nmol/L). Low serum 25- (OH)D concentrations are associated with an increased risk of sarcopenia in elderly.
Administration of CLA and Vitamin D
[0069] Some embodiments relate to compositions comprising CLA and vitamin D, and uses and methods of co-administering CLA and vitamin D, for the prevention and/or reduction of age-related loss of muscle mass and function that can eventually proceed to sarcopenia. Also provided, in some embodiments, are compositions comprising CLA and vitamin D, and methods of co-administering CLA and vitamin D, to improve muscle synthetic rate, breakdown rate, mass, strength, or function. In some embodiments, stimulating muscle protein synthesis results in the treatment or prevention of a condition of muscle loss. In some embodiments, increasing muscle mass results in the treatment or prevention of a condition of muscle loss. In some embodiments, increasing muscle function results in the treatment or prevention of the condition of muscle loss. In some embodiments, increasing the rate of muscle synthesis results in the treatment or prevention of a condition of muscle loss. In some embodiments, decreasing the rate of muscle breakdown results in the treatment or prevention of the condition of muscle loss.
[0070] In some embodiments, the co-administration of CLA and vitamin D increases the rate of muscle protein synthesis. In some embodiments, increasing the rate of muscle protein synthesis results in the reduction or prevention of age-related loss of muscle mass or function. In some embodiments, increasing the rate of muscle protein synthesis results in the treatment or prevention of a condition of muscle loss. In some embodiments, increasing the rate of muscle protein synthesis results in the reduction or prevention of sarcopenia.
[0071] Some embodiments disclosed herein include methods of treating or preventing a condition of muscle loss by co-administering CLA and vitamin D to a subject in need thereof. In some embodiments, co-administering CLA and vitamin D to a subject in need thereof treats or prevents a condition of muscle loss by reducing or preventing age- related loss of muscle mass and function. Some such embodiments include therapeutic treatment, and some embodiments include prophylactic treatment.
[0072] By "co-administration," it is meant that the two or more agents (for example CLA and Vitamin D) may be found in the patient's bloodstream at the same time, regardless of when or how they are actually administered. In some embodiments, the agents are administered simultaneously. In some embodiments, administration in combination is accomplished by combining the agents in a single dosage form. In some embodiments, the agents are administered sequentially. In some embodiments, the agents (for example CLA and Vitamin D) are administered through the same route, such as orally. In some embodiments, the agents (for example CLA and Vitamin D) are administered through different routes, such as one being administered orally and another being administered i.v.
[0073] The subject in need thereof can be a patient who is suffering from a condition of muscle loss or a subject that is suspected of or at the risk of developing a condition of muscle loss. The subject may have, or may not have, symptoms of sarcopenia. In some embodiments, the subject does not have sarcopenia. In some embodiments, the subject has sarcopenia. The age for the subject in need thereof can vary. For example, the subject can be an adult, for example a middle-aged adult, or an elderly adult. In some embodiments, the subject is of the age of 40, 45, 50, 55, 60, 65, 70, 75, or more. For example, the subject can be a person at least 40 years old, or the subject can be a person at least 60 years old. The gender of the subject in need thereof can vary. In some embodiments, the subject is a female. In some embodiments, the subject is a male.
[0074] The use and methods disclosed herein can also comprise identifying a subject who is suffering from a condition of muscle loss or a subject that is suspect of or at the risk of developing a condition of muscle loss; and co-administering to the subject CLA and vitamin D. In some embodiments, the methods disclosed herein include acquiring knowledge of the presence of a condition of muscle loss in a subject or the risk/potential of developing a condition of muscle loss in a subject; and co-administering to the subject CLA and vitamin D.
[0075] Change in muscle loss, for example attenuation or acceleration of muscle loss can be detected, for example, by detecting loss in muscle mass, detecting change in body weight, detecting change in muscle lean mass and/or fat mass, determining change in locomotor activity, detecting change in muscle fiber number, detecting change in muscle cross-sectional area, or any combination thereof of the subject.
[0076] Some embodiments disclosed herein provide methods of treating or preventing a condition of muscle loss by co-administering CLA and vitamin D to a subject in need thereof. Some embodiments can include identifying a subject as having or at risk for developing a condition of muscle loss (e.g., sarcopenia, muscle atrophy, cachexia, or muscular dystrophy) prior to co-administering CLA and vitamin D.
[0077] While not being bound by any particular theory, in some embodiments, the co-administration of CLA with Vitamin D prevents or reduces age-related loss of muscle mass or function by synergistically combining to impact the mitochondrial oxidative stress- reductive and/or muscle stimulating mechanisms of aging-related muscle loss. Additionally, while not being bound by any particular theory, in some embodiments, the co-administration of CLA with Vitamin D prevents or relieves the condition of muscle loss by synergistically combining to impact inflammatory and/or muscle stimulating mechanisms of aging-related muscle loss.
[0078] The co-administration of CLA and vitamin D as described herein, can, in some embodiments, allow for the regulation or maintenance of the subject's rate of muscle protein synthesis. The regulation or maintenance of the subject's rate of muscle protein synthesis can allow for a subject to experience a number of beneficial effects. These beneficial effects include the reduction or prevention of age-related loss of muscle mass and function, increased muscle mass, increased muscle function, increased rate of muscle synthesis, decreased rate of muscle breakdown, and/or treatment or prevention of sarcopenia. Compared to a baseline prior to treatment, these effects can result in an improvement of about or greater than about 5, 10, 15, 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 400, or 500%. These amounts and/or levels can be maintained within 0, 1, 5, or 10% of the amounts and/or levels at the initiation of administration.
[0079] Some embodiments provide a method of co-administering CLA and vitamin D to a subject in need thereof such that the subject's rate of muscle protein synthesis increases. The subject may or may not engage in exercise. The subject coadministered CLA and Vitamin D can, in some embodiments, observe and/or feel improvement without a significant change in lifestyle. In some embodiments, exercising in conjunction with the co-administration of CLA and vitamin D results in an even greater improvement in muscle mass and function, but exercise is not necessary to improve muscle mass and function. The amount of CLA and vitamin D administered that are effective for increasing the animal's muscle mass can be determined in accordance with methods known in the art. In some embodiments, the effective amount of CLA administered may be from about 1 g to about 6 g and the effective amount of vitamin D administered may be from may be from about 100 IU to about 5000 IU per 24 hour period.
[0080] In some embodiments, the uses, methods and composition disclosed herein can prevent or reduce age-related loss of muscle mass (including but not limited to loss of lean muscle mass). For example, the methods and composition may prevent muscle mass loss (including but not limited to loss of lean muscle mass) from occurring. In some embodiments, the rate of muscle mass loss is reduced in a patient receiving or received treatment by at least, or at least about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% as compared to the patients received no treatment. In some embodiments, the methods and composition reduce the rate of loss of muscle mass in a patient by, or by about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values as compared to the patients received no treatment. As another example, the methods and composition may reduce the final muscle mass loss (including but not limited to final loss in lean muscle mass). In some embodiments, the final muscle mass loss in the patient receiving or received treatment is at most, or at most about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the final muscle mass loss in patients received no treatment. In some embodiments, the final muscle mass loss in the patient receiving or received treatment is, or is about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values, of the final muscle mass loss in patients received no treatment.
[0081] In some embodiments, the methods and composition disclosed herein can prevent or reduce age-related loss of muscle function (including but not limited to loss of lean muscle function). For example, the methods and composition may prevent muscle function loss (including but not limited to loss of lean muscle function) from occurring. In some embodiments, the rate of muscle function loss is reduced in a patient receiving or received treatment by at least, or at least about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% as compared to the patients received no treatment. In some embodiments, the methods and composition reduce the rate of loss of muscle function in a patient by, or by about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values as compared to the patients received no treatment. As another example, the methods and composition may reduce the final muscle function loss (including but not limited to final loss in lean muscle function). In some embodiments, the final muscle function loss in the patient receiving or received treatment is at most, or at most about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the final muscle function loss in patients received no treatment. In some embodiments, the final muscle function loss in the patient receiving or received treatment is, or is about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values, of the final muscle function loss in patients received no treatment.
[0082] In some embodiments, the methods and composition disclosed herein can prevent or reduce age-related loss of muscle weight. For example, the methods and composition may prevent muscle weight loss from occurring. In some embodiments, the rate of muscle weight loss in a patient receiving or received treatment is reduced by at least, or at least about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% as compared to the patients received no treatment. In some embodiments, the methods and composition reduce the rate of loss of muscle weight in the patient receiving or received treatment by, or by about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values as compared to patients received no treatment. As another example, the methods and composition may reduce the final muscle weight loss. In some embodiments, the final muscle weight loss in the patient receiving or received treatment is at most, or at most about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the final muscle weight loss in patients received no treatment. In some embodiments, the final muscle weight loss in the patient receiving or received treatment is, or is about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values, of the final muscle weight loss in patients received no treatment.
[0083] In some embodiments, the methods and composition disclosed herein can prevent or reduce age-related loss of muscle circumference. For example, the methods and composition may prevent muscle circumference loss from occurring. In some embodiments, the rate of muscle circumference loss in a patient receiving or received treatment is reduced by at least, or at least about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% as compared to the patients received no treatment. In some embodiments, the methods and composition reduce the rate of muscle circumference loss in a patient receiving or received treatment by, or by about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values as compared to the patients received no treatment. As another example, the methods and composition may reduce the final muscle circumference loss. In some embodiments, the final muscle circumference loss in the patient receiving or received treatment is at most, or at most about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the final muscle circumference loss in patients received no treatment. In some embodiments, the final muscle circumference loss in the patient receiving or received treatment is, or is about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values, of the final muscle circumference loss in patients received no treatment.
[0084] In some embodiments, the methods and composition disclosed herein can prevent or reduce age-related loss of muscle strength. For example, the methods and composition may prevent muscle strength loss from occurring. In some embodiments, the rate of muscle strength loss in a patient receiving or received treatment is reduced by at least, or at least about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% as compared to the patients received no treatment. In some embodiments, the methods and composition reduce the rate of muscle strength loss in a patient receiving or received treatment by, or by about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values as compared to patients received no treatment. As another example, the methods and composition may reduce the final muscle strength loss. In some embodiments, the final muscle strength loss in the patient receiving or received treatment is at most, or at most about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the final muscle strength loss in patients received no treatment. In some embodiments, the final muscle strength loss in the patient receiving or received treatment is, or is about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values, of the final muscle strength loss in patients received no treatment. [0085] In some embodiments, the methods and composition disclosed herein can prevent or reduce age-related mobility loss. For example, the methods and composition may prevent mobility loss from occurring. In some embodiments, the rate of mobility loss in a patient receiving or received treatment is reduced by at least, or at least about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% as compared to the patients received no treatment. In some embodiments, the methods and composition reduce the rate of mobility loss in the patient receiving or received treatment by, or by about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values as compared to the patients received no treatment. As another example, the methods and composition may reduce the final mobility loss. In some embodiments, the final mobility loss in the patient receiving or received treatment is at most, or at most about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the final mobility loss in patients received no treatment. In some embodiments, the final mobility loss in the patient receiving or received treatment is, or is about, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values, of the final mobility loss in patients received no treatment.
[0086] In some embodiments, the methods and composition disclosed herein can prevent age-related reduction in muscle protein FSR or reduce the rate of age-related reduction in muscle protein FSR. For example, the methods and composition may prevent the reduction in muscle protein FSR from occurring. In some embodiments, the rate of reduction in muscle protein FSR in a patient receiving or received treatment is reduced by at least, or at least about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% as compared to patients received no treatment. In some embodiments, the methods and composition reduce the rate of reduction in muscle protein FSR in a patient receiving or received treatment by, or by about, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a range between any two of these values as compared to the patients received no treatment.
[0087] In some embodiments, the co-administration of CLA and vitamin D results in significant enhancements in overall muscle mass, function and strength. This co-administration can be used on all age groups seeking enhancement in overall muscle mass, function and strength. The methods described herein, in some embodiments, yield increased overall muscle mass, function and strength even in non- exercising subjects. In some embodiments, CLA and vitamin D are co-administered in elderly subjects. Current estimates place a large portion of the older population at risk for falls with potential significant associated morbidities. In some embodiments, the coadministration of CLA and vitamin D specifically targets muscle mass, strength and function and consequently may produce significant improvement in health, quality of life, and in particular, decreased falls and injury in this group. The younger population also benefits from the co-administration of CLA and vitamin D in still further embodiments, in part due to the widespread occurrence of vitamin D deficiency. Women also benefit from the coadministration of CLA and vitamin D in some embodiments, as women are prone to vitamin D deficiency.
Synergistic Effects of Co-administration of CLA and Vitamin D
[0088] As disclosed herein, co-administration of particular ratios and/or amounts of CLA and vitamin D can result in synergistic effects in reducing or preventing muscle loss. These synergistic effects can be such that the one or more effects of the combination compositions are greater than the one or more effects of each component alone at a comparable dosing level, or they can be greater than the predicted sum of the effects of all of the components at a comparable dosing level, assuming that each component acts independently. The synergistic effect can be about, or greater than about, 5, 10, 20, 30, 50, 75, 100, 1 10, 120, 150, 200, 250, 350, or 500% better than the effect of treating a subject with one of the components alone, or the additive effects of each of the components when administered individually. The effect can be any of the measurable effects described herein. The composition comprising a plurality of components can be such that the synergistic effect is an enhancement in FSR and that FSR is increased to a greater degree as compared to the sum of the effects of administering each component, determined as if each component exerted its effect independently, also referred to as the predicted additive effect herein. For example, if a composition comprising component (a) yields an effect of a 20% improvement in FSR and a composition comprising component (b) yields an effect of 50% improvement in FSR, then a composition comprising both component (a) and component (b) would have a synergistic effect if the combination composition's effect on cellular metabolism was greater than 70%. [0089] A synergistic combination composition can have an effect that is greater than the predicted additive effect of administering each component of the combination composition alone as if each component exerted its effect independently. For example, if the predicted additive effect is 70%, an actual effect of 140% is 70% greater than the predicted additive effect or is 1 fold greater than the predicted additive effect. The synergistic effect can be at least about 20, 50, 75, 90, 100, 150, 200 or 300% greater than the predicted additive effect. In some embodiments, the synergistic effect can be at least about 0.2, 0.5, 0.9, 1.1,
I .5, 1.7, 2, or 3 fold greater than the predicted additive effect.
[0090] In some embodiments, the synergistic effect of the combination compositions can also allow for reduced dosing amounts, leading to reduced side effects to the subject and reduced cost of treatment. Furthermore, the synergistic effect can allow for results that are not achievable through any other treatments. Therefore, proper identification, specification, and use of combination compositions can allow for significant improvements in the reduction and prevention of age-related loss of muscle mass and function.
Methods of Administration
[0091] Methods of regulating the rate of muscle protein synthesis by administering one or more compositions are disclosed herein. The compositions include the combination compositions described herein, such as combination compositions comprising CLA and vitamin D. In general, an amount of CLA and vitamin D in the levels sufficient to improve muscle strength, function, and mass is administered for an effective period of time.
[0092] The dosing of the combination compositions can be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times daily. A subject can receive dosing for a period of about, less than about, or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
I I, 12, 13, 14, or more days, weeks or months. A unit dose can be chosen such that the subject is administered about or greater than about 1000 mg of CLA (e.g. about or more than about 1100 mg, 1 130 mg, 2000 mg, 2100 mg, 2200 mg, 2250 mg, 2260 mg, 3300 mg, 3390 mg, 4400 mg, 4520 mg, 4750 mg, 4900 mg, 5000 mg, 5100 mg, 5300 mg, 5530 mg, 5750 mg, 5900 mg, 6000 mg, or more) and about or greater than about 100 IU of vitamin D (e.g. 150 IU, 200 IU, 300 IU, 500 IU, 700 IU, 850 IU, 1000 IU, 1200 IU, 1300 IU, 1500 IU, 1700 IU, 1850 IU, 2000IU, 2200 IU, 2300 IU, 2500 IU, 2700 IU, 2850 IU, 3000 IU, 3200 IU, 3300 IU, 3500 IU, 3700 IU, 3850 IU, 4000 IU, 4200 IU, 4300 IU, 4500 IU, 4700 IU, 4850 IU, 5000 IU, or more) daily. The vitamin D can comprise Vitamin D3. A unit dose can be a fraction of the daily dose, such as the daily dose divided by the number of unit doses to be administered per day. A unit dose can be a fraction of the daily dose that is the daily dose divided by the number of unit doses to be administered per day and further divided by the number of unit doses (e.g. tablets) per administration. The number of unit doses per administration may be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The number of doses per day may be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The number of unit doses per day may be determined by dividing the daily dose by the unit dose, and may be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, 20, or more unit doses per day. For example, a unit dose can be about ½, ½, ¼, Vs, ½, 1/7, ¼, 1/9, or 1/10. A unit dose can be about one- third of the daily amount and administered to the subject three times daily. A unit dose can be about one- half of the daily amount and administered to the subject twice daily. A unit dose can be about one-fourth of the daily amount with two unit doses administered to the subject twice daily. For example, a unit dose can have about, less than about, or more than about 250 mg, 275 mg, 500 mg, 550 mg, 750 mg, 825 mg, 1100 mg, 1125 mg, 1130 mg, 1650 mg, 2200 mg, 2550 mg, 2750 mg, 2825 mg, 3100 mg, 3300 mg, 3530 mg, 3750 mg, 3900 mg, 4000 mg, 4100 mg, 4300 mg, 4530 mg, 4750 mg, 4900 mg, 5000 mg, 5100 mg, 5300 mg, 5530 mg, 5750 mg, 5900 mg, 6000 mg, or more of CLA and about, less than about, 100 IU, 150 IU, 200 IU, 300 IU, 500 IU, 700 IU, 850 IU, 1000 IU, 1200 IU, 1300 IU, 1500 IU, 1700 IU, 1850 IU, 2000IU, 2200 IU, 2300 IU, 2500 IU, 2700 IU, 2850 IU, 3000 IU, 3200 IU, 3300 IU, 3500 IU, 3700 IU, 3850 IU, 4000 IU, 4200 IU, 4300 IU, 4500 IU, 4700 IU, 4850 IU, 5000 IU, or more of vitamin D.
[0093] In some embodiments, the co-administration of CLA to vitamin D, either as separate compositions or a combined composition, can have a specified ratio of CLA to vitamin D. The specified ratio can provide for effective stimulation of muscle protein synthesis. For example, the specified ratios can cause an increase in muscle mass of a subject, an increase in muscle function of a subject, an increase in FSR of a subject, an increase in anabolic signaling markers of a subject, a decrease in proteolysis markers of a subject, an increase in mitochondrial number and size of a subject, a decrease in oxidative stress markers of a subject, and/or a decrease in inflammatory markers of a subject. Such beneficial effects can result from, in part, an increase in mitochondrial biogenesis, changes in mitochondrial oxidative stress-reductive pathways, changes in inflammation and inflammatory pathways, or a variety of other changes in cellular metabolism or the energy metabolism pathway. The ratio of CLA to vitamin D can be a mass ratio, a molar ratio, or a volume ratio. In some embodiments, the mass ratio of CLA to vitamin D is about, greater than about, or less than about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, 175, 200, 250, 500, 750, 1000, or more. In some embodiments, the molar ratio of CLA to vitamin D co-administered is about, greater than about, or less than about 90, 95, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more.
[0094] In some embodiments, the co-administration of CLA to vitamin D, either as separate compositions or a combined composition, is effective for reducing or preventing age-related loss of muscle mass or function, as measured by an increase in the anabolic signaling of a subject, an increase in expression of growth regulatory factors of a subject, an increase in FSR of a subject, an increase in insulin sensitivity of a subject, a decrease in oxidative stress markers of a subject, and/or a decrease in inflammatory markers of a subject. The administration of a combination composition can have a measured effect that is an improvement of about, or greater than about, 5, 10, 15, 20, 30, 50, 75, 100, 110, 120, 150, 200, 250, 350, 500, 700, or 1000% over a control subject or control group.
[0095] In some embodiments, a subject co-administered CLA and vitamin D as described herein can experience an increase in FSR. The increase in FSR can be about, or greater than about, 1, 5, 10, 15, 20, 25, 30, 35, or 40%, as compared to the subject's FSR before treatment. In some embodiments, the measurement of FSR can be for a test group as compared to a control group.
[0096] The effects on a subject, as described herein, can be determined by a variety of in vitro or in vivo methods that utilize samples taken from the subject or that directly assay for parameters indicative of the effect in the subject. For example, FSR can be measured as described in Example 1, intracellular signaling cascades known to be involved in the control of translational activity and known markers of proteolysis can be evaluated by western blot analysis of muscle biopsy samples, muscle mRNA expression of regulators of energy metabolism and proteolysis can be evaluated by reverse transcription real-time quantitative PCR, adipose tissue mRNA expression of markers of inflammation and oxidative stress by can be evaluated by reverse transcription real-time quantitative PCR, mitochondrial function in muscle can be evaluated by oxygraphy, total body fat and fat-free masses and appendicular lean body mass can be evaluated by body composition scans (DXA, Lunar iDXA™, GE Healthcare), oxidative stress can be measured using blood samples drawn from the subject, and inflammatory markers can be measured using ELISA.
[0097] The compositions can be administered periodically. For example, the compositions can be administered one, two, three, four times a day, or even more frequent. The subject can be administered every 1 , 2, 3, 4, 5, 6, or 7 days. In some embodiments, the compositions are administered three times daily. The administration can be concurrent with meal time of a subject. The period of treatment or diet supplementation can be for about 1 , 2, 3, 4, 5, 6, 7, 8, or 9 days, 2 weeks, 1-1 1 months, or 1 year, 2 years, 5 years, or even longer. In some embodiments disclosed herein, the dosages that are administered to a subject can change or remain constant over the period of treatment. For example, the daily dosing amounts can increase or decrease over the period of administration.
[0098] The length of the period of administration and/or the dosing amounts can be determined by a physician, a nutritionist, or any other type of clinician. The period of time can be one, two, three, four or more weeks. In some embodiments, the period of time can be one, two, three, four, five, six or more months.
[0099] In some embodiments, the dosing level can be adjusted based on the subject's characteristics, such as weight, height, ethnicity, genetics, or baseline energy metabolism level.
[0100] The physician, nutritionist, or clinician can observe the subject's response to the administered compositions and adjust the dosing based on the subject's performance or measured circulating levels of CLA, vitamin D, or any other component of the composition. For example, dosing levels can be increased for subjects that show reduced effects in FSR or circulating levels of CLA or vitamin D below desired target levels.
[0101] In some embodiments, the compositions administered to a subject can be optimized for a given subject. For example, the ratio of CLA to vitamin D or the particular components in a combination composition can be adjusted. The ratio and/or particular components can be selected after evaluation of the subject after being administered one or more compositions with varying ratios of CLA to vitamin D or varying combination composition components.
[0102] As disclosed herein, the CLA and vitamin D do not have to be administered in the same composition to perform the claimed methods. For example, separate capsules, pills, mixtures, etc. of vitamin D and of CLA may be administered to a subject to carry out the claimed methods. The administration of CLA and vitamin D may be at the same time or at different times provided that effective concentrations of both CLA and vitamin D are both found in the subject at the same time. In some embodiments, administration of both CLA and vitamin D is at the same time, preferably in a single composition, in order to facilitate the compliance of the subject to adhere to a schedule of administration.
[0103] It is contemplated that there will be some variation in effectiveness due to differences among individuals in physiological and biochemical parameters (e.g., body weight and basal metabolism), exercise, and other aspects (e.g., diet). In some embodiments, it is contemplated that individuals beginning treatment will be given a 2.0 gram dose of CLA in combination with a 500 IU dose of vitamin D daily for an initial two month period, and then, if no improvement in the rate of muscle protein synthesis, gradually increase the CLA dose to above 4 gram per day and the vitamin D dose up to about 2000 IU per day or higher.
[0104] In some embodiments, the dosing regimen of the compositions disclosed herein is administered for a period of time, which time period can be, for example, from at least about 1 week to at least about 4 weeks, from at least about 4 weeks to at least about 8 weeks, from at least about 4 weeks to at least about 12 weeks, from at least about 4 weeks to at least about 16 weeks, or longer. The dosing regimen of the compositions disclosed herein can be administered three times a day, twice a day, daily, every other day, three times a week, every other week, three times per month, once monthly, substantially continuously or continuously.
[0105] The amount of vitamin D and CLA needed to reach appropriate blood serum levels of vitamin D and CLA may vary from person to person, and determination of the optimum amount in each instance can be readily obtained by routine procedures.
[0106] The formulation, route of administration and dosage for the compositions disclosed herein can be chosen by the individual physician in view of the patient's condition. Typically, the dose range of the composition administered to the patient can be from about 0.1 to about 4000 mg/kg of the patient's body weight. The dosage may be a single one or a series of two or more given in the course of one or more days, as is needed by the patient. In instances where human dosages for the compositions have been established for at least some condition, the present disclosure will use those same dosages, or dosages that are between about 0.1% and about 5000%, more preferably between about 25% and about 1000% of the established human dosage. Where no human dosage is established, as will be the case for newly-discovered pharmaceutical compounds, a suitable human dosage can be inferred from ED50 or ID50 values, or other appropriate values derived from in vitro or in vivo studies, as qualified by toxicity studies and efficacy studies in animals.
[0107] It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.
[0108] Although the exact dosage will be determined on a drug-by-drug basis, in most cases, some generalizations regarding the dosage can be made. In cases of administration of a pharmaceutically acceptable salt, dosages may be calculated as the free base. In some embodiments, the composition is administered 1 to 4 times per day. In some embodiments, the compositions of the compositions disclosed herein can be administered by continuous intravenous infusion, preferably at a dose of each active ingredient up to 100 g per day. As will be understood by those of skill in the art, in certain situations it may be necessary to administer the compositions disclosed herein in amounts that exceed, or even far exceed, the above-stated, preferred dosage range in order to effectively and aggressively treat particularly aggressive diseases or infections. In some embodiments, the compositions disclosed herein are administered for a period of continuous therapy, for example for a week or more, or for months or years. Group A Agents and Group B Agents
[0109] The methods and compositions disclosed herein can comprise, in some embodiments, at least one other ingredient which positively impacts the inflammatory mechanism resulting in mitochondrial oxidative stress (collectively termed "Group A" agents herein). In some embodiments, the Group A agent is selected from the group comprising omega-3 fatty acid-containing substances (e.g., oils), amino acids (e.g. alanine), and vitamins (e.g., vitamin E). In some embodiments, the omega-3 fatty acid is selected from the group consisting of alpha-linolenic acid, stearidonic acid, eicosapentanoic acid, docosahexanoic acid, and mixtures thereof.
[0110] The methods and compositions disclosed herein can comprise, in some embodiments, at least one other ingredient which positively impacts the muscle stimulating mechanism (collectively termed "Group B" agents herein). In some embodiments, the Group B agent is selected from the group comprising another vitamin D other than D3, creatine, leucine, and whey protein.
[0111] In some embodiments, the Group B agent is an anabolic amino acid derivative stimulus, herein defined as a chemical compound derived from or which is a precursor to an amino acid (hence, amino acid derivative) which promotes (hence, anabolic) muscle growth by increasing net protein synthesis (hence, stimulus).
[0112] In some embodiments, the anabolic amino acid derivative stimulus is one or more selected from the group comprising L-leucine, citrulline, and creatine.
[0113] L-leucine (hereafter also called leucine, since the R-form of leucine is biologically not relevant in the context of the present disclosure) is an essential amino acid, being part of a diverse number of proteins and, together with valine and isoleucine, belongs to the group of branched-chain amino acids. Leucine may be used as a free amino acid, or in a bound form, such as a dipeptide, an oligopeptide, a polypeptide or a protein. Common protein sources of leucine are dairy proteins such as whey, casein, micellar casein, caseinate, and glycomacroprotein (GMP), and vegetable proteins such as wheat, rice, pea, lupine and soy proteins. Said sources of protein may provide intact proteins, hydrolysates or mixtures thereof, hereafter further called proteinaceous matter. Leucine is known as a potent activator of muscle protein synthesis. [0114] In some embodiments, the Group B agent is citrulline. Citrulline is an a- amino acid. Citrulline, in the form of citrulline malate, is sold as a performance-enhancing athletic dietary supplement was been suggested to promote aerobic energy production and to increase athletic performance and decreasing muscle soreness. In the human body, citrulline is produced from ornithine and carbamoyl phosphate in one of the central reactions in the urea cycle. It is also produced from arginine in the body as a by-product of the reaction catalyzed by NOS family. Citrulline is also capable of promoting muscle protein synthesis and has been described in human and animal studies.
[0115] In some embodiments, the Group B agent is creatine (N-(amino-imino- methyl)-N-methyl-glycine; methylglycocy amine). Creatine is a nitrogenous organic acid that is produced in vertebrates, in particular the human body from L-arginine, glycine, and L- methionine and helps to supply energy to muscles.
[0116] In some embodiments, the anabolic amino acid derivative stimulus is provided in a daily dosage of 0.5 to 20 g, preferably 1 to 10 g. In some embodiments, such daily dosage is administered as a single serving.
[0117] In some embodiments, leucine is provided in a daily dosage of 1 to 10 g. When leucine is provided as proteinaceous matter, it should comprise at least about 11 weight% of leucine. It was found that about 11 weight % of total leucine based on the total amount of proteinaceous matter is a minimum amount present in the nutritional composition. Preferably, said proteinaceous matter comprises at least about 12 weight%, preferably at least about 12.5 weight%, more preferably at least about 13 weight% of leucine.
[0118] In some embodiments, total leucine comprises at least about 20 weight%, preferably at least about 22.5 weight%, preferably at least about 25 weight% of leucine in a free form, relative to the total amount of leucine. In the context of this application, with "free form" is meant a peptide comprising 1 to 5 amino acids, preferably 1 to 3 amino acids, more preferably 1 amino acid. Preferably, leucine is a free amino acid, either as a base, a salt or a chelate. In some embodiments, citrulline is provided in a daily dosage of 0.5 to 10 g. In some embodiments, creatine is provided in a daily dosage of 0.5 to 20 g. In some embodiments, any combination of leucine, citrulline, and creatine is provided in a daily dosage of 0.5 to 20 g, preferably 1 to 10 g. Any combination is a combination selected from the group of leucine and citrulline; leucine and creatine; citrulline and creatine; and leucine, citrulline and creatine.
[0119] In some embodiments, the Group B agent is whey protein. In some embodiments, combination further comprises at least about 80 weight% of whey protein, preferably at least about 85 weight% of whey protein, preferably at least about 90 weight%, and most preferably about 95 weight% of whey protein.
[0120] Whey protein is considered a "fast" protein referring to the rate of appearance in the circulation of the amino acids following whey ingestion. The whey protein may be an intact whey protein, a hydrolysed whey protein, a microparticular whey protein, a nanoparticular whey protein, a micellar whey protein, and the like. Preferably, the whey protein is an intact whey protein, i.e. a whey protein in its intact form, such as present in fresh milk.
[0121] Any commercially available whey protein source may be used, i.e. whey obtained by any process for the preparation of whey known in the art, as well as whey protein fractions prepared thereof, or the proteins that constitute the bulk of the whey proteins being β-lactoglobulin, ct- lactalbumin and serum albumin, such as liquid whey, or whey in powder form, such as whey protein isolate (WPI) or whey protein concentrate (WPC). Whey protein concentrate is rich in whey proteins, but also contains other components such as fat, lactose and glycomacroprotein (GMP), a casein-related non-globular protein. Typically, whey protein concentrate is produced by membrane filtration. On the other hand, whey protein isolate consists primarily of whey proteins with minimal amounts of fat and lactose. Whey protein isolate usually requires a more rigorous separation process such as a combination of microfiltration and ultra-filtration or ion exchange chromatography. It is generally understood that a whey protein isolate refers to a mixture in which at least 90 weight% of the solids are whey proteins. A whey protein concentrate is understood as having a percentage of whey proteins between the initial amount in the by-product (about 12 weight%) and a whey protein isolate. In particular, sweet whey, obtained as a by-product in the manufacturing of cheese, acid whey, obtained as a by-product in the manufacturing of acid casein, native whey, obtained by milk microfiltration or rennet whey, obtained as a byproduct in the manufacturing of rennet casein, may be used as a source of whey proteins. [0122] In some embodiments, at least one other ingredient selected from the Group A and/or Group B is present within the oral composition. In some embodiments, the at least one other ingredient is provided in amounts sufficient to elicit noticeable effects (which can be measured as described herein).
Compositions for Administration
[0123] The combination compositions can be formulated for oral administration in the form of a tablet, a capsule, or any other form described herein. The compositions can be administered to a subject orally or by any other methods. Methods of oral administration include, in some embodiments, administering the composition as a liquid, a solid, or a semisolid that can be taken in the form of a dietary supplement or a foodstuff.
[0124] In some embodiments, the administration of CLA and vitamin D is oral. The CLA and vitamin D may be formulated together with suitable carriers such as starch, sucrose or lactose in tablets, pills, dragees, capsules, solutions, liquids, slurries, suspensions and emulsions. The vitamin D and CLA may be administered separately or together, provided that the total amount of CLA and vitamin D is an effective amount in combination per day to have a substantial impact on the rate of muscle protein synthesis.
[0125] According to a further aspect, some embodiments relate to an oral composition comprising CLA and Vitamin D in a foodstuff, in a food supplement, or in a pharmaceutical preparation.
[0126] In some embodiments, a food composition for human consumption is supplemented by the above composition. For example, the food composition can be, or comprise, a nutritional complete formula, a dairy product, a chilled or shelf stable beverage, a powdered beverage, a mineral or purified water, a liquid drink, a soup, a dietary supplement, a meal replacement, a nutritional bar, a confectionery, a milk, a fermented milk product, a yoghurt, a milk based powder, an enteral nutrition product, an infant formula, an infant nutritional product, a cereal product or a fermented cereal-based product, an ice cream, a chocolate, coffee, a culinary product such as mayonnaise, tomato puree, salad dressings, a pet food, or any combination thereof.
[0127] For ingestion, many embodiments of oral compositions and in particular of food supplements are possible. They are formulated by means of the usual methods for producing sugar-coated tablets, pills, pastes, gums, gelatin capsules, gels, emulsions, tablets, capsules or drinkable solutions or emulsions, which can then be taken directly with water or by any other known means.
[0128] The nutritional supplement for oral administration may be in capsules, gelatin capsules, soft capsules, tablets, sugar-coated tablets, pills, pastes or pastilles, gums, or drinkable solutions or emulsions, syrups or gels, with a dose of about 0.001 to 100% of the primary composition, which can then be taken directly with water or by any other known means. This supplement may also include a sweetener, a stabilizer, an additive, a flavoring or a colorant. A supplement for cosmetic purpose can additionally comprises a compound active with respect to the skin. Methods for preparing them are common knowledge.
[0129] Also, the formulation as described above may be incorporated into any other forms of food supplements or of enriched foods, for example food bars, or compacted or non- compacted powders. Methods for preparing them are common knowledge.
[0130] The food composition or food supplement may also include a sweetener, a stabilizer, an antioxidant, an additive, a flavoring or a colorant. The composition may also contain synthetic or natural bioactive ingredients such as amino acids, fatty acids, vitamins, minerals, carotenoids, polyphenols, etc. that can be added either by dry or by wet mixing to said composition before pasteurization and/or drying. According to some embodiments, the composition disclosed herein can be used cosmetically. By "cosmetic use" is meant a non- therapeutic use which may improve the aesthetic aspect or comfort of the skin, coat and/or hair of humans or pets.
[0131] In some embodiments, a pharmaceutical composition can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, compositions are administered to a patient already suffering from a disease, as described herein under, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as "a therapeutically effective dose". Amounts effective for this will depend on the severity of the disease and the weight and general state of the patient. In prophylactic applications, compositions disclosed herein are administered to a patient susceptible to or otherwise at risk of a particular disease. Such an amount is defined to be "a prophylactic effective dose". In this use, the precise amounts again depend on the patient's state of health and weight. [0132] The compositions disclosed herein are, in some embodiments, administered with a pharmaceutically acceptable carrier, the nature of the carrier differing with the mode of administration, for example, enteral, oral and topical (including ophthalmic) routes. The desired formulation can be made using a variety of excipients including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate. This composition may be a tablet, a capsule, a pill, a solution, a suspension, a syrup, a dried oral supplement, a wet oral supplement.
[0133] Furthermore, in some embodiments the composition can be intravenously administered in any suitable manner. For administration via intravenous infusion, the composition is preferably in a water-soluble non-toxic form. Intravenous administration is particularly suitable for hospitalized patients that are undergoing intravenous (IV) therapy. For example, the composition can be dissolved in an IV solution (e.g., a saline or glucose solution) being administered to the patient. Also, the composition can be added to nutritional IV solutions, which may include amino acids and/or lipids. The amounts of the composition to be administered intravenously can be similar to levels used in oral administration. Intravenous infusion may be more controlled and accurate than oral administration.
[0134] As disclosed herein, the combination of CLA and vitamin D can be formulated for administration in a pharmaceutical composition comprising a physiologically acceptable surface active agents, carriers, diluents, excipients, smoothing agents, suspension agents, film forming substances, coating assistants, or a combination thereof. In some embodiments, the combination of CLA and vitamin D are formulated for administration with a pharmaceutically acceptable carrier or diluent. The combination of CLA and vitamin D can be formulated as a medicament with a standard pharmaceutically acceptable carrier(s) and/or excipient(s) as is routine in the pharmaceutical art. The exact nature of the formulation will depend upon several factors including the desired route of administration. Typically, combination of CLA and vitamin D are formulated for oral, intravenous, intragastric, intravascular or intraperitoneal administration. Standard pharmaceutical formulation techniques may be used, such as those disclosed in Remington's The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (2005), incorporated herein by reference in its entirety. [0135] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. In addition, various adjuvants such as are commonly used in the art may be included. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Gilman et al. (Eds.) (1990); Goodman and Gilman' s: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press, which is incorporated herein by reference in its entirety.
[0136] Some examples of substances, which can serve as pharmaceutically- acceptable carriers or components thereof, are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the TWEENS; wetting agents, such sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffer solutions.
[0137] The choice of a pharmaceutically-acceptable carrier to be used in conjunction with a composition can be determined by the way the composition is to be administered.
[0138] The compositions described herein are preferably provided in unit dosage form. As used herein, a "unit dosage form" is a composition that is suitable for administration to an animal, preferably mammal subject, in a single dose, according to good medical practice. The preparation of a single or unit dosage form however, does not imply that the dosage form is administered once per day or once per course of therapy. Such dosage forms are contemplated to be administered once, twice, thrice or more per day and may be administered as infusion over a period of time (e.g., from about 30 minutes to about 2-6 hours), or administered as a continuous infusion, and may be given more than once during a course of therapy, though a single administration is not specifically excluded. The skilled artisan will recognize that the formulation does not specifically contemplate the entire course of therapy and such decisions are left for those skilled in the art of treatment rather than formulation.
[0139] The compositions useful as described above may be in any of a variety of suitable forms for a variety of routes for administration, for example, for oral, nasal, rectal, topical (including transdermal), ocular, intracerebral, intracranial, intrathecal, intra-arterial, intravenous, intramuscular, or other parental routes of administration. The skilled artisan will appreciate that oral and nasal compositions include compositions that are administered by inhalation, and made using available methodologies. Depending upon the particular route of administration desired, a variety of pharmaceutically-acceptable carriers well-known in the art may be used. Pharmaceutically-acceptable carriers include, for example, solid or liquid fillers, diluents, hydrotropies, surface-active agents, and encapsulating substances. Optional pharmaceutically-active materials may be included, which do not substantially interfere with the inhibitory activity of the composition. The amount of carrier employed in conjunction with the composition is sufficient to provide a practical quantity of material for administration per unit dose of the composition. Techniques and compositions for making dosage forms useful in the methods described herein are described in the following references, all incorporated by reference herein: Modern Pharmaceutics, 4th Ed., Chapters 9 and 10 (Banker & Rhodes, editors, 2002); Lieberman et ah, Pharmaceutical Dosage Forms: Tablets (1989); and Ansel, Introduction to Pharmaceutical Dosage Forms 8th Edition (2004).
[0140] Various oral dosage forms can be used, including such solid forms as tablets, capsules, and granules. Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed, containing suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules, containing suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, melting agents, coloring agents and flavoring agents. [0141] The pharmaceutically-acceptable carriers suitable for the preparation of unit dosage forms for peroral administration is well-known in the art. Tablets typically comprise conventional pharmaceutically-compatible adjuvants as inert diluents, such as calcium carbonate, sodium carbonate, mannitol, lactose and cellulose; binders such as starch, gelatin and sucrose; disintegrants such as starch, alginic acid and croscarmelose; lubricants such as magnesium stearate, stearic acid and talc. Glidants such as silicon dioxide can be used to improve flow characteristics of the powder mixture. Coloring agents, such as the FD&C dyes, can be added for appearance. Sweeteners and flavoring agents, such as aspartame, saccharin, menthol, peppermint, and fruit flavors, are useful adjuvants for chewable tablets. Capsules typically comprise one or more solid diluents disclosed above. The selection of carrier components depends on secondary considerations like taste, cost, and shelf stability, which are not critical, and can be readily made by a person skilled in the art.
[0142] Peroral compositions also include liquid solutions, emulsions, suspensions, and the like. The pharmaceutically-acceptable carriers suitable for preparation of such compositions are well known in the art. Typical components of carriers for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water. For a suspension, typical suspending agents include sodium carboxymethyl cellulose, AVICEL RC-591, tragacanth and sodium alginate; typical wetting agents include lecithin and polysorbate 80; and typical preservatives include methyl paraben and sodium benzoate. Peroral liquid compositions may also contain one or more components such as sweeteners, flavoring agents and colorants disclosed above.
[0143] Other compositions useful for attaining systemic delivery can be in, for example, sublingual, buccal and/or nasal dosage forms. Such compositions typically comprise one or more of soluble filler substances such as sucrose, sorbitol and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose and hydroxypropyl methyl cellulose. Glidants, lubricants, sweeteners, colorants, antioxidants and flavoring agents disclosed above may also be included.
[0144] For topical use, creams, ointments, gels, solutions or suspensions, etc., containing the compositions disclosed herein are employed. Topical formulations may generally be comprised of a pharmaceutical carrier, co-solvent, emulsifier, penetration enhancer, preservative system, and emollient. [0145] For intravenous administration, the compositions described herein may be dissolved or dispersed in a pharmaceutically acceptable diluent, such as a saline or dextrose solution. Suitable excipients may be included to achieve the desired pH, including but not limited to NaOH, sodium carbonate, sodium acetate, HC1, and citric acid. In various embodiments, the pH of the final composition ranges from 2 to 8, or preferably from 4 to 7. Antioxidant excipients may include sodium bisulfite, acetone sodium bisulfite, sodium formaldehyde, sulfoxylate, thiourea, and EDTA. Other non-limiting examples of suitable excipients found in the final intravenous composition may include sodium or potassium phosphates, citric acid, tartaric acid, gelatin, and carbohydrates such as dextrose, mannitol, and dextran. Further acceptable excipients are described in Powell, et al, Compendium of Excipients for Parenteral Formulations, PDA J Pharm Sci and Tech 1998, 52 238-311 and Nema et al., Excipients and Their Role in Approved Injectable Products: Current Usage and Future Directions, PDA J Pharm Sci and Tech 2011, 65 287-332, both of which are incorporated herein by reference in their entirety. Antimicrobial agents may also be included to achieve a bacteriostatic or fungistatic solution, including but not limited to phenylmercuric nitrate, thimerosal, benzethonium chloride, benzalkonium chloride, phenol, cresol, and chlorobutanol.
[0146] The compositions for intravenous administration may be provided to caregivers in the form of one more solids that are reconstituted with a suitable diluent such as sterile water, saline or dextrose in water shortly prior to administration. In some embodiments, the compositions are provided in solution ready to administer parenterally. In some embodiments, the compositions are provided in a solution that is further diluted prior to administration. In embodiments that include administering the compositions described herein and another agent(s) (for example, agent A, agent B, or a combination thereof), the combination may be provided to caregivers as a mixture, or the caregivers may mix the two agents prior to administration, or the two agents may be administered separately.
[0147] CLA is somewhat susceptible to oxidation. Therefore, it is desirable to package CLA and vitamin D with suitable antioxidants such as lecithin, tocopherols, ascorbate, ascorbyl palmitate or spice extracts such as rosemary extract.
[0148] In some embodiments, a combination composition comprises CLA, Vitamin D, and one or more additional ingredients. An additional ingredient may serve one or more functions. In some embodiments, an additional ingredient accounts for about, less than about, or more than about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the mass or volume of the combination composition. Non-limiting examples of additional ingredients include sweeteners, bulking agents, stabilizers, acidulants, preservatives, binders, lubricants, disintegrants, fillers, solubilizers, coloring agents (such as fruit juice and vegetable juice), and other additives and excipients known in the art. In some embodiments, a combination composition comprises one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more) sweeteners. Examples of sweeteners include, but are not limited to, sucrose, fructose, dextrose, maltose, lactose, high fructose corn syrup solids, invert sugar, sugar alcohols, sorbitol, saccharin, cyclamates, sweeteners derived from stevia, sweeteners derived from momordica grosvenorii, sweeteners derived from mogrosides, acesulfame K, L-aspartyl-L- phenylalanine lower alkyl ester sweeteners, L-aspartyl-D-alanine amide sweeteners, L- aspartyl-D-serine amide sweeteners, L-aspartyl-L-l-hydroxymethylalkaneamide sweeteners, L-aspartyl-1 -hydroxy ethyalkaneamide sweeteners, L-aspartyl-D-phenylglycine ester and amide sweeteners, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, dulcoside A, dulcoside B, rubusoside, stevia, stevia extract, stevioside, mogroside IV, mogroside V, siamenoside, monatin and their salts (monatin SS, RR, RS, SR), curculin, glycyrrhizic acid and its salts, thaumatin, monellin, mabinlin, brazzein, hemandulcin, phyllodulcin, glycyphyllin, phloridzin, trilobatin, baiyunoside, osladin, polypodoside A, pterocaryoside A, pterocaryoside B, mukurozioside, phlomisoside I, periandrin I, abrusoside A, cyclocarioside I, sucralose, potassium acesulfame, aspartame, alitame, saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, N— [N-[3-(3- hydroxy-4-methoxyphenyl)propyl]-L-a-aspartyl]L-phenylalanine-l -methyl ester, N— [N-[3- (3-hydroxy-4-methoxyphenyl)-3-methylbutyl]-L-a-aspartyl]-L-phenylalanine-l -methyl ester, N— [N-[3-(3-methoxy-4-hydroxyphenyl)propyl]-L-a-aspartyl]-L-phenylalanine-l -methyl ester, salts thereof, or combinations thereof. In some embodiments, the sweetener is a polyol additive, such as a sugar alcohol, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol (glycerine), threitol, galactitol, palatinose, reduced isomalto-oligosaccharides, reduced xylo-oligosaccharides, reduced gentio- oligosaccharides, reduced maltose syrup, or reduced glucose syrup. [0149] In some embodiments, a combination composition comprises one or more (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more) bulking agents. Non-limiting examples of bulking agents include guar gum, locust bean gum, cassia gum, pectin from botanical sources, high molecular weight carboxymethylcellulose, carrageenan, alginate, and xanthane. In some embodiments, one or more bulking agents may be added to enhance the viscosity of a liquid formulation.
[0150] In some embodiments, a combination composition comprises one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more) stabilizers. Non-limiting examples of stabilizers include pectin, polysaccharide hydrolysates comprising dextrin, agar, can- ageenan, tamarind seed polysaccharides, angelica gum, karaya gum, xanthan gum, sodium alginate, tragacanth gum, guar gum, locust bean gum, pullulan, gellan gum, gum arabic, carboxymethylcellulose, and propylene glycol alginate ester. In some embodiments, one or more stabilizers are added to the combination composition to enhance the shelf-life of the combination composition. In general, shelf-life refers to the amount of time the container and composition therein can be held at ambient conditions (approximately room temperature, e.g. about 18-28° C.) or less, without degradation of the composition and/or container occurring to the extent that the composition cannot be used in the manner and for the purpose for which it was intended. In some embodiments, the combination composition has a shelf life of about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 14, 30, 60, 90, or more days; or about, less than about, or more than about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months or years. In some embodiments, the combination composition remains non-perishable for a period of time after opening a container containing the composition. In general, perishability refers to degradation to an extent that the composition cannot be used in the manner and purpose for which it was designed. In some embodiments, the combination composition remains non-perishable for about, less than about, or more than about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 18, 24, 30, 36, 48, 60, 72, 90, or more hours or days after opening; or about, less than about, or more than about 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, or more months or years after opening. In some embodiments, the combination composition remains nonperishable for a period of time at room temperature (e.g. about 18-28° C). In some embodiments, the combination composition remains non-perishable for a period of time upon refrigeration, such as storage below about 20° C, 15° C, 10° C, 5° C, 4° C, 3° C, 2° C, 1° C, 0° C, -1° C, -2° C, -3° C, -4° C, -5° C, -10° C, -20° C, or lower.
[0151] In some embodiments, a combination composition comprises one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more) acidulants. Non-limiting examples of acidulants include C2-C30 carboxylic acids, substituted hydroxyl C1-C30 carboxylic acids, benzoic acid, substituted benzoic acids (e.g. 2,4-dihydroxybenzoic acid), substituted cinnamic acids, hydroxyacids, substituted hydroxybenzoic acids, substituted cyclohexyl carboxylic acids, tannic acid, lactic acid, tartaric acid, citric acid, gluconic acid, glucoheptonic acids, adipic acid, hydroxycitric acid, malic acid, fruitaric acid (a blend of malic, fumaric, and tartaric acids), fimaric acid, maleic acid, succinic acid, chlorogenic acid, salicylic acid, creatine, glucosamine hydrochloride, glucono delta lactone, caffeic acid, bile acids, acetic acid, ascorbic acid, alginic acid, erythorbic acid, polyglutamic acid, and their alkali or alkaline earth metal salt derivatives thereof.
[0152] In some embodiments, a combination composition comprises one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more) preservatives. Non-limiting examples of preservatives include sorbic acid, benzoic acid, and salts thereof, including (but not limited to) calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, and mixtures thereof.
[0153] In some embodiments, the compositions can be a food product, for example a snack bar, or a beverage, comprising CLA and vitamin D. For example, the snack bar can be a chocolate bar, a granola bar, or a trail mix bar. In some embodiments, the present dietary supplement or food compositions are formulated to have suitable and desirable taste, texture, and viscosity for consumption. Any suitable food carrier can be used in the present food compositions. Food carriers of the compositions described herein include practically any food product. Examples of such food carriers include, but are not limited to food bars (granola bars, protein bars, candy bars, etc.), cereal products (oatmeal, breakfast cereals, granola, etc.), bakery products (bread, donuts, crackers, bagels, pastries, cakes, etc.), beverages (milk-based beverage, sports drinks, fruit juices, alcoholic beverages, bottled waters), pastas, grains (rice, corn, oats, rye, wheat, flour, etc.), egg products, snacks (candy, chips, gum, chocolate, etc.), meats, fruits, and vegetables. In some embodiments, food carriers employed herein can mask the undesirable taste (e.g., bitterness). Where desired, the food composition presented herein exhibit more desirable textures and aromas than that of any of the components described herein. For example, liquid food carriers can be used to obtain the present food compositions in the form of beverages, such as supplemented juices, coffees, teas, shakes (e.g., milk shakes), smoothies, and the like. In some embodiments, solid food carriers can be used to obtain the present food compositions in the form of meal replacements, such as supplemented snack bars, pasta, breads, and the like. In some embodiments, semi-solid food carriers can be used to obtain the present food compositions in the form of gums, chewy candies or snacks, and the like.
Salts
[0154] As disclosed herein, CLA and Vitamin D can be administered separately or simultaneously (e.g., in a single dosage form). In some embodiments, CLA, vitamin D, and/or the combination of CLA and vitamin D are administered as pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of a compound and, which are not biologically or otherwise undesirable for use in a pharmaceutical. In many cases, the compounds disclosed herein are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable salts can also be formed using inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, bases that contain sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts. In some embodiments, treatment of the compounds disclosed herein with an inorganic base results in loss of a labile hydrogen from the compound to afford the salt form including an inorganic cation such as Li , Na , K , Mg and Ca and the like. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. Many such salts are known in the art, as described in WO 87/05297 published September 11, 1987 (incorporated by reference herein in its entirety).
Kits
[0155] Also provides herein are kits comprising one or more compositions described herein, in suitable packaging, and may further comprise written material that can include instructions for use, discussion of clinical studies, listing of side effects, and the like. Such kits may also include information, such as scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the health care provider. Such information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials. A kit may comprise one or more unit doses described herein. In some embodiments, a kit comprises about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 31, 60, 90, 120, 150, 180, 210, or more unit doses. Instructions for use can comprise dosing instructions, such as instructions to take 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more unit doses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times per day. For example, a kit may comprise a unit dose supplied as a tablet, with each tablet package separately, multiples of tablets packaged separately according to the number of unit doses per administration (e.g. pairs of tablets), or all tablets packaged together (e.g. in a bottle). As a further example, a kit may comprise a unit dose supplied as a bottled drink, the kit comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 24, 28, 36, 48, 72, or more bottles.
[0156] The kit can further contain another agent. In some embodiments, the CLA and vitamin D are provided as separate compositions in separate containers within the kit. In some embodiments, the CLA and vitamin D are provided as a single composition within a container in the kit. Suitable packaging and additional articles for use (e.g., measuring cup for liquid preparations, foil wrapping to minimize exposure to air, and the like) are known in the art and may be included in the kit. Kits described herein can be provided, marketed and/or promoted to health providers, including but not limited to, physicians, nurses, pharmacists, formulary officials, and the like. Kits can also, in some embodiments, be marketed directly to the consumer.
[0157] In some embodiments, a kit can comprise a multi-day supply of unit dosages. The unit dosages can be any unit dosage described herein. The kit can comprise instructions directing the administration of the multi-day supply of unit dosages over a period of multiple days. The multi-day supply can be a one-month supply, a 30-day supply, or a multi-week supply. The multi-day supply can be a 90-day, 180-day, 3 -month or 6-month supply. The kit can include packaged daily unit dosages, such as packages of 1, 2, 3, 4, or 5 unit dosages. The kit can be packaged with, for example, other dietary supplements, vitamins, and meal replacement bars, mixes, and beverages.
Examples
[0158] Embodiments of the present application are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the present disclosure.
Example 1
Randomized Controlled Trial to Evaluate the Independent and Combined Effects of CLA and
Vitamin D on the Rate of Muscle Protein Synthesis in Older Adults
[0159] This example describe a study based on a randomized, double-blind, controlled clinical trial to determine the independent and combined effects of dietary CLA and vitamin D supplementation on anabolic signalling, the expression of growth regulatory factors, and muscle protein turnover in older adults. Forty (40) 65-85 year old men and women ("Subjects") are randomized to receive: i) placebo (4,000 mg corn oil per day), ii) CLA (4,000 mg Tonalin FFA 80 per day), iii) vitamin D3 (2,000 IU per day), or iv) CLA (4,000 mg Tonalin FFA 80 per day) plus vitamin D3 (2,000 IU per day) for 8 weeks. Study supplements are provided in finished bottles according to labeling guidelines. The study is conducted by a protocol described in Smith et al, Am. J. Clin. Nutr., 2011 Feb, 93(2):402- 412. [0160] Anabolic signaling, the expression of growth regulatory factors, and muscle protein turnover are evaluated before and at the end of the Study by using intravenous stable isotope labeled tracer infusions in conjunction with a hyperinsulinemic- hyperaminoacidemic-euglycemic clamp procedure and muscle biopsies. It is expected that both CLA and vitamin D will stimulate muscle protein anabolism and the effect of CLA plus vitamin D will be greater than the effect of either intervention alone.
APPROACH
Subjects and preliminary testing
[0161] 40-50 (40 completers) 65-85 year old men and women of all races and ethnicities are included in the study. Written informed consent is obtained from all subjects before they are enrolled in the study. From many years of experience with this type of study, it is expected about a 20% drop-out rate; therefore, a total of 50 subjects is recruited. All subjects completes a body composition scan to determine total body fat and fat- free masses and appendicular lean body mass (DXA, Lunar iDXA™, GE Healthcare), a grip strength test, and a detailed medical examination (including a history and physical examination, a 12- lead electrocardiogram, an oral glucose tolerance and standard blood tests) to determine eligibility. Subjects who fulfil the following inclusion criteria: i) age: >65 and <85 years; ii) BMI: >18.5 and <30.0 kg/m2; iii) weight stable (<2% change) and untrained (<1 h of structured exercise/wk) for at least 3 months before entering the study; and iv) serum 250H- Vit D3 <35 ng/ml are included. Within this cohort, subjects who: i) use medications that are incompatible with the study procedures (e.g., those that affect blood coagulation) or are known to affect muscle protein metabolism (e.g., steroids); ii) use controlled substances; iii) use dietary supplements that contain >200 IU vitamin D and/or are known or suspected to affect muscle protein metabolism; iv) consume >2 alcoholic beverages per day; v) consume >10 cigarettes (or other tobacco products) per week; vi) have evidence of significant organ system dysfunction or disease (e.g., diabetes, congestive heart failure; chronic obstructive pulmonary disease; major neurological or neuromuscular disorders; cancer; uncontrolled hypertension; etc.); vii) have contraindications to any of the procedures performed during the study; viii) are intolerant to any ingredient in the dietary supplements or the intravenous infusions used in the study; ix) are unwilling or unable to provide informed consent or comply with the study protocol (e.g., those with dementia); x) are prisoners; and/or xi) the study physician considers unable to safely complete the study protocol are excluded.
Muscle protein metabolism study
[0162] Subjects are admitted to the Clinical Study Unit in the late afternoon on the day before the study. At 2000 h, they consume a standardized meal and then fast (except for water) and rest in bed until the completion of the study (outlined in Figure 1) the next day.
[0163] At 0600 h on the following day, a catheter is inserted into an antecubital vein for the infusion of amino acids and insulin; two additional catheters are inserted for blood sampling: one into the radial artery of the opposite arm and another in retrograde fashion into the brachial vein. At 0700 h, constant infusions of [ring-2H5]phenylalanine (priming dose: 7.9 μηιοΐ/kg FFM, infusion rate: 0.07 μΓηοΙ-kg FFM"1 min"1) and [6,6- Eyglucose (priming dose: 22 μηιοι/kg body weight, infusion rate: 0.22 μηιο1·1¾ body weight"1 min"1) are started and maintained for 420 min.
[0164] 240 min after the start of the tracer infusion, an infusion of insulin at a rate of 20 mU/m body-surface area (BSA) is started (after initiation with a two-step priming
2 2
dose: 80 mU/in BSA for 5 min followed by 40 mU/m BSA for 5 min) and maintained until the end of the study. This is expected to result in plasma insulin concentrations of -30 mU \ Dextrose is infused at a variable rate to maintain plasma glucose concentration (monitored every 10 min) at ~5.5 mM during insulin infusion. Also at 240 min, a primed constant infusion of a mixed balanced amino acid solution (Travasol 10%, Baxter, Deerfield, IL) is started (priming dose: 35 mg amino acid/kg FFM, infusion rate 105 mg amino acid kg FFM" ^h"1) and maintained until the end of the study (at 420 min) to increase plasma amino acid concentration. The infusion rate of [ring-2H5]phenylalanine is increased to 0.14 μιηοΐ ^ FFM"1 min"1 to match the increased availability of phenylalanine during the mixed amino acid infusion and the infusion rate of [6,6-2H2]glucose is reduced to 0.11 μιηοΐ ^ body weight"1 min"1 to account for the insulin-mediated decrease in hepatic glucose production.
[0165] Blood samples are collected immediately before the start of the phenylalanine tracer infusion and then at 60, 120, 180, 210, 220, 230, 240, 300, 360, 390, 400, 410 and 420 min. Data (average values) collected at 210, 220, 230, and 240 min (basal period) and 390, 400, 410, and 420 min (clamp) are used to calculate forearm amino acid net balance, protein breakdown and protein synthesis. Data obtained from the remaining samples are used to confirm isotopic and metabolic steady state.
[0166] Muscle biopsies from the quadricep femor is obtained by using a conchotome forceps (during local anesthesia) 60 min after the start of the isotope infusion and at the end of the basal period (at 240 min) and at the end of the hyperaminoacidemic- hyperinsulinemic clamp (at 420 min) to measure the incorporation rate of labelled phenylalanine into muscle protein (myofibrillar, sarcoplasmic, and mitochondrial) and the muscle tissue free phenylalanine labelling to measure the fractional muscle protein synthesis rate (FSR), and to evaluate gene expression and intracellular signaling events and markers of proteolysis and muscle morphology. Subcutaneous adipose tissue biopsies from the periumbilical region are obtained (during local anesthesia) immediately after the first and third muscle biospies at 60 and 420 min.
[0167] Forearm blood flow are measured at multiple time points during basal conditions (i.e., between 195 and 210 min and between 225 and 240 min) and at multiple time points during the hyperaminoacidemic-hyperinsulinemic-euglycemic clamp (i.e., between 375 and 390 min and between 405 and 420 min) by using the ultrasound Doppler technique.
[0168] All sample analyses are routinely performed in our laboratory. Plasma glucose concentration is determined on an automated glucose analyzer (Yellow Spring Instruments Co, Yellow Springs, OH). Plasma insulin concentration is measured by using a commercially available ELISA kit (Diagnostic Systems Labs, Webster, TX). The phenylalanine tracer to tracee ratio (TTR) and concentration in blood and the muscle free and protein-bound phenylalanine TTR will be determined by GC-MS (MSD 5973 System, Hewlett-Packard) by using enrichment standards.
[0169] Western blot analysis is used to measure the amount and activity (as phosphorylation) of the following components of the intracellular signaling cascade known to be involved in the control of translational activity and known markers of proteolysis in muscle biopsy samples: i) Akt (total and phospho Thr308); ii) mTOR (total and phosphor mTORSer2448. ^ p7()S6k phospho ^β^. ^ eIF4E_Bpi (total and phospho
Thr37/46); v) eIF4E (total and phospho Ser209); vi) eEF2 (total and phospho Thr56); vi) p38 MAPK (total and phospho- Thr180/Tyr182), vn) ERK1/2 (total and phosphor-Tyr202/204), vin) the C2 proteasome subunit (total protein); ix) MuRF-1 (total protein); x) MaFbx (total protein); and xi) the 14-kD actin fragment (total protein). Protein bands are visualized and quantified by densitometry ensuring no pixel saturation. All data are expressed relative to GAPDH after confirming a stable GAPDH/a-actin ratio.
[0170] Reverse transcription real-time quantitative PCR (ABI 7500 real-time PCR system, Invitrogen, Life Technologies Inc.) is used to evaluate the muscle mRNA expression of peroxisome proliferator-activated receptor alpha [PPARA] and gamma [PPARG], regulators of energy metabolism; myogenic differentiation 1 [MYOD1], a myogenic growth factor; myostatin [MSTN], a muscle growth inhibitor; follistatin [FST], which binds to and thereby inhibits myostatin; forkhead box 03 [FOX03], which induces the transcription of ubiquitin ligases; MaFbx and MuRFl, two components of the ubiquitin- proteasome pathway; Calpains and Calpastatin, a family of calcium-dependent, non- lysosomal cysteine proteases; tripeptidyl peptidase II, an enzyme responsible for the terminal steps of proteolysis; and the lysosomal peptidases, cathepsin L and cathepsin B.
[0171] In adipose tissue, mRNA expression of markers of inflammation and oxidative stress (e.g. TNF, IL6), adipokines (e.g. AdipoQ, LEP), adipogenesis (e.g. PPARG), and mitochondrial function (e.g. PPARA, uncoupling protein 1 [UCPl]) are evaluated, In addition, the mRNA/protein expression of nicotinamide phosphoribosyltransferase (NAMPT) and its downstream targets, such as SIRTl, and measure adipose tissue NAD+ concentration are evaluated. GAPDH is used as a house keeping gene after verifying the stability of its expression against the expression of cyclophilin A and/or β-actin.
[0172] Muscle fiber-type (1, 2a and 2x) composition, fiber size, capillary density, lipid content, mitochondrial density, number and size, and mitochondrial cristae density are determined according to current American College of Pathologists guidelines. Mitochondrial function in muscle is evaluated by oxygraphy (Oroboros 02k, Innsbruck, Austria). All samples are coded in ways that do not reveal subjects' treatment status to ensure unbiased analysis.
Interventions
Subject randomization and dietary supplementation with CLA and/or vitamin D [0173] After completing the baseline metabolic study, subjects are randomized to either: i) placebo (corn oil), or ii) CLA, or iii) vitamin D, or iv) CLA plus vitamin D for 8 weeks. A block randomization scheme is used to ensure our study groups are balanced with respect to sex, racial/ethnical distribution, and adiposity status (lean vs overweight). Subjects randomized to receive placebo receive 4,000 mg corn oil daily (2 softgels containing 1,000 mg corn oil each in the morning and 2 in the evening), those randomized to CLA receive 4,000 mg Tonalin FFA 80 daily (2 softgels containing 750 mg CLA isomers each in the morning and 2 in the evening), those randomized to vitamin D receive 2,000 IU vitamin D3 daily (2 softgels containing 500 IU vitamin D3 each in the morning and 2 in the evening), and those randomized to CLA plus vitamin D receive both 4,000 mg Tonalin FFA 80 and 2,000 IU vitamin D3 daily (2 softgels containing 750 mg CLA isomers and 500 iU vitamin D3 each in the morning and 2 in the evening). CLA (Tonalin 80 FFA) is a dietary supplement and generally-recognized-as-safe (GRAS) by the Food and Drug Administration. The dose of vitamin D given to our study subjects (2,000 IU per day) is below the Tolerable Upper Limit (4,000 IU) set by the Institute of Medicine; it is expected to increase serum 250H-Vit D3 concentration by 15-20 ng/ml and will therefore not raise serum 250H-Vit D3 concentrations above 100 ng/ml.
[0174] Both the active and placebo treatments are dispensed by a Study nurse in our Center for Human Nutrition, who is not part of the Study team so that both the subjects and investigators are blinded to the treatment. Compliance with the treatment is assessed by pill count at the end of the study. In previous studies using a similar protocol, compliance was >90%. To avoid "tampering" with the pill count, subjects are given an (to them) unknown amount of pills in excess of needs at the beginning of the study and will be asked to return any remaining pills at the end of the study. Subjects have weekly contact with a member of the study team to review body weight, food records, any signs of supplement intolerance, life events, and any medical complaints. Any indication of a potential adverse event is reviewed with the PI and study physician.
Physical activity and dietary intake during the intervention.
[0175] Subjects maintain their habitual diet and physical activity during the intervention. The study coordinator instructs the subjects to do so and follow-up with them weekly to remind them and help them maintain their habitual life-style. In addition, physical activity and dietary intake are monitored and potential changes are taken into account in the statistical analyses. Subjects are asked to keep detailed food records (by using notebooks and/or mobile apps), which is reviewed by the PI and study coordinator and/or a dietician in our nutrition center. Urine and stool are collected before and at the end of the intervention to assess 24-h urinary nitrogen excretion and fecal isovaleric acid content, two markers of dietary protein intake. To monitor physical activity, subjects wear an Acti Graph GT9X Link monitor for 7 days during the first and last week of the intervention; they also keep records of any exercise sessions in which they engage. The user is blinded to the data collected which minimizes tampering with the monitor to achieve target readings.
Non-compliance and Attrition.
[0176] To minimize drop-outs and non-compliance the following procedures are adhere: i) each subject undergo an orientation session to explain in detail the nature, purpose, and demands of the study; ii) subjects are encouraged to review and discuss the consent form with their husband/wife or domestic partners before agreeing to participate; iii) subjects are screened with a "barriers interview" that we have developed to help ensure we select subjects who will likely be able to complete the study; iv) subjects are required to complete a diet and physical activity history form and return for at least one pre-study visit after they passed screening before being able to enroll in the study, which helps demonstrate their commitment and ability to complete the study; v) once enrolled, subjects have regular (weekly for the first month, biweekly to monthly for the subsequent months) contact with a member of the study team which reinforces participation; the Study coordinator will also help subjects overcome barriers that can lead to non-compliance and/or study withdrawal, and vi) a philosophy of partnership and collaborative relationships is encouraged between Study personnel and study participants (e.g., sending birthday and anniversary cards etc.).
[0177] From many years of experience with this type of study we expect a <20% drop-out rate, which was taken into account in our recruitment targets (see Subjects section above). To account for the reasons for attrition, we plan to interview (by phone) subjects who decide to drop out. Withdrawal because of treatment-related adverse events (e.g. taste aversion, etc.) is considered treatment failure; subjects are encouraged (if there is no contraindication) to complete all primary outcome testing procedures to avoid missing data for intention-to-treat analysis.
Statistical Analyses
Data analyses
[0178] Repeated measures analyses of variance with group and time as factors are used to evaluate the effect of treatment on our outcome measures. In all analyses, careful attention is given to the appropriateness of the statistical procedure by determining whether necessary conditions are satisfied; e.g., normality and equal variance. When conditions are violated, the use of data transformations intended to produce data that satisfy normality and equal variance assumptions is explored. If an appropriate transformation cannot be found, non-parametric methods may be used as an alternative to the more standard analyses. Primary analysis is "intention-to-treat". A secondary analysis focuses on "compliant" subjects only (i.e., those who consumed >90% of the assigned treatment). A value of p<0.05 is considered significant.
Primary study endpoint
[0179] The rate of muscle protein synthesis is the primary study endpoint. The following computations demonstrate that the study is adequately powered to detect changes in these outcomes using two-sided tests, a 0.05 level of significance and n=10/group.
[0180] It is expected that the CLA and Vitamin D interventions affects the muscle synthetic rate to a small extent individually, but when administered together will synergistically impact the muscle synthetic rate. Figure 2 depicts the expected results from the ongoing clinical trial on muscle synthetic rate in elderly subjects. It is expected that those subjects receiving either placebo or Vitamin D alone will have a statistically significant difference in FSR under "clamp" infusion relative to the basal period, but will have not have a statistically significant difference in FSR before and after intervention (indicated in Figure 2 by "a"). It is expected that those subjects receiving CLA alone will have a statistically significant difference in FSR under "clamp" infusion relative to the basal period (indicated in Figure 2 by "b") and will have a statistically significant difference in FSR before and after intervention (indicated in Figure 2 by "c"). It is expected that those subjects receiving CLA plus Vitamin D will have a statistically significant difference in FSR under "clamp" infusion relative to the basal period (indicated in Figure 2 by "b"), will have a statistically significant difference in FSR before and after intervention (indicated in Figure 2 by "c"), and will have a statistically significant difference in FSR as compared to the subjects receiving either CLA or Vitamin D alone (indicated in Figure 2 by "d").
Additional study endpoints
[0181] It is expected that western blot analysis of muscle biopsy samples for above-described intracellular signaling cascades involved in the control of translational activity and the above-described markers of proteolysis will yield beneficial results that parallel the FSR results for the four study groups as exemplified in Figure 2 (i.e., the CLA and Vitamin D combined intervention will yield beneficial effects greater than the effect of either intervention alone).
[0182] It is expected that reverse transcription real-time quantitative PCR analysis will show beneficial increases in the muscle mRNA expression of PPARA, PPARG, MYODl, FST, and beneficial decreases in the in the muscle mRNA expression of MSTN, FOX03, Calpains, Calpastatin, tripeptidyl peptidase II, cathepsin L, cathepsin B that will parallel the FSR results for the four study groups as exemplified in Figure 2 (i.e., the CLA and Vitamin D combined intervention will yield beneficial effects greater than the effect of either intervention alone).
[0183] It is expected that analysis of adipose tissue will show beneficial changes in the mRNA expression of the above-described markers of inflammation and oxidative stress, adipokines, adipogenesis, and mitochondrial function that will parallel the FSR results for the four study groups as exemplified in Figure 2 (i.e., the CLA and Vitamin D combined intervention will yield beneficial effects greater than the effect of either intervention alone).
[0184] It is expected that analysis of adipose tissue will show beneficial changes in the mRNA/protein expression of NAMPT and its downstream targets, as well as show beneficial changes in adipose tissue NAD+ concentration, that will parallel the FSR results for the four study groups as exemplified in Figure 2 (i.e., the CLA and Vitamin D combined intervention will yield beneficial effects greater than the effect of either intervention alone).
[0185] It is expected analysis of the muscle fiber-type (1, 2a and 2x) composition, fiber size, capillary density, lipid content, mitochondrial density, mitochondrial number and size, and mitochondrial cristae density, and mitochondrial function (as evaluated by oxygraphy) will yield results that parallel the FSR results for the four study groups as exemplified in Figure 2 (i.e., the CLA and Vitamin D combined intervention will yield beneficial effects greater than the effect of either intervention alone).
Sample size considerations
[0186] Using the same study design, it has been found that dietary n-3 polyunsaturated fatty acid supplementation, which improves muscle mass, increased the muscle anabolic response to combined insulin and amino acid infusion (increase in fractional protein synthesis rate from basal values) by -0.022 %/h (from 0.009 ± 0.014 to 0.031 ± 0.009 %/h; mean ± SD) in older (>65 y) adults. Assuming the larger SD (0.014 %/h) and no change in the control group, a power >0.8, and a significance cut-off value of a <0.05, it is estimated that a 0.022%/h increase with 8 subjects per group can be detected.
Example 2
CLA-vitamin D Softgel Compositions
Table 1. Non-limiting exemplary composition of CLA and Vitamin D
Figure imgf000062_0001
[0187] Method: Make a blend of the above ingredients to homogenize same and encapsulate using standard softgel methodology.
[0188] Recommended Daily Dose: Four softgels, taken morning and evening daily with meals.
[0189] * Tonalin FFA 80 may be substituted with any other pharmaceutically acceptable product containing CLA.
[0190] * *Note that Vitamin D3 may be substituted with Vitamin D2. [0191] ***Note that antioxidant E 306 may be substituted with tocopherols, lecithins, or other suitable antioxidants that will counter the slight susceptibility of CLA to oxidation.
Example 3
Method of Treating Sarcopenia
[0192] A subject having sarcopenia is identified. The subject is then administered four of the softgels described in Example 2 on a daily basis (with two softgels taken morning and evening daily with meals).
Example 4
Method of Treating Using CLA and Vitamin D
[0193] A subject having a condition of muscle loss that can be treated by the coadministration of CLA and vitamin D is identified. The subject is then administered, once per day, a meal replacement bar comprising about 4 g of a CLA mixture (cis-9, trans-11 - octadecadienoic acid CLA and trans-10, cis-12-octadecadienoic acid in a 50: 50 ratio) and about 2000 IU of Vitamin D3.
[0194] In at least some of the previously described embodiments, one or more elements used in one embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.
[0195] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.
[0196] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and/or "an" should be interpreted to mean "at least one" or "one or more"); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., " a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to "at least one of A, B, or C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., " a system having at least one of A, B, or C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."
[0197] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
[0198] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non- limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as "up to," "at least," "greater than," "less than," and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.
[0199] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.
[0200] All references cited herein, including patents, patent applications, papers, text books, and the like, and the references cited herein, to the extent that they are not already, are hereby incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differ from or contradict this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.

Claims

WHAT IS CLAIMED IS:
1. A composition, comprising conjugated linoleic acid (CLA) and Vitamin D.
2. The composition of claim 1, wherein the CLA comprises between about 1% and 99% of one or more isomers of CLA.
3. The composition of claim 2, wherein at least one of the one or more isomers of CLA is in triglyceride form.
4. The composition of claim 2, wherein at least one of the one or more isomers of CLA is in free fatty acid form.
5. The composition of claim 2, wherein at least one of the one or more isomers of CLA is in ester form.
6. The composition of claim 2, wherein at least one of the one or more isomers of CLA is selected from the group comprising cis-9, cis-11 -octadecadienoic acid, cis-9, trans- 11 -octadecadienoic acid, trans-9, cis-11 -octadecadienoic acid, trans-9, trans-11- octadecadienoic acid, cis-10, cis- 12-octadecadienoic acid, cis-10, trans- 12-octadecadienoic acid, trans- 10, cis- 12-octadecadienoic acid, and trans- 10, trans- 12-octadecadienoic acid.
7. The composition of any one of claims 1-6, wherein the CLA is a mixture of two or more different isomers of CLA.
8. The composition of claim 7, wherein the mixture comprises cis-9, trans-11- octadecadienoic acid CLA and trans- 10, cis- 12-octadecadienoic acid.
9. The composition of claim 7, wherein the mixture comprises cis-9, trans-11- octadecadienoic acid CLA and trans-10, cis- 12-octadecadienoic acid in a 50:50 ratio.
10. The composition of any one of claims 1-9, wherein the CLA is derived from safflower oil.
11. The composition of any one of claims 1-10, wherein the CLA further comprises one or more tocopherols.
12. The composition of claim 11, wherein at least one of the one or more tocopherols is selected from the group consisting of consisting of δ-tocopherol, γ-tocopherol, a-tocopherol, and combinations thereof.
13. The composition of any one of claims 1-12, wherein the CLA comprises one or more of a Tonalin product, a Clarinol product, Tonalin 35 WDP, Tonalin 60 WDP, Tonalin FFA 80, Tonalin SG 1000 T FFA, Tonalin SG 500 T FFA, Tonalin SG 750 T FFA, Tonalin TG 80, Tonalin TG 80, MET-Rx® CLA Tonalin 1000, Solgar Tonalin CLA, NatureWise CLA 1250, Natrol Tonalin CLA, NDS Body Toner Tonalin Censor, The Vitamin Shoppe Tonalin CLA, Natrol Tonalin Cla, Nature Made Cla Tonalin, Nature's Bounty Tonalin 1000 CLA, NDS Nutrition Censor Body Toner, BulkSupplements CLA Softgels, TNVitamins CLA, Puritan's Pride CLA Tonalin, Barlean's Organic Oils Tonalin CLA Swirl, Solgar, Tonalin CLA, TLS Tonalin CLA, Swanson Tonalin Cla, Iron Tek Essential Cla Pure Tonalin Complex, Trunature® Tonalin® CLA, VIROCUT EXTREME CLA, BlueBonnet Tonalin CLA, Labrada Nutrition EFA Lean Gold Essential Fatty Acid Softgel Capsules, Solgar Tonalin CLA Supplement, Davinci Labs Tonalin CLA 90, Vitacost Tonalin XS-CLA, GNC Total Lean™ CLA Soft Chew, ALLMAX CLA, GNC Total Lean™ CLA PM, Xenadrine NextGen, GNC Total Lean™ Thermo CLA, Puregel's Clarinol® CLA, LiveGreat Foods ACCLAIM® drink, Progressive Labs CLA, Jamieson Slim Down CLA 95, AST CLA 100, GNC ProPerformance AMP Ripped Vita Pak, Muscle Feast CLA, Jarrow Formulas CLA Slim, BQuick Nutrition Lean Capsules, Trophic Slim Fit CLA, GNC Total Lean CLA, Bluebonnet Clarinol® CLA Softgels, Nature's Plus Mega CLA 1200, PhytoCeutical Formulations' OptiCLA, BQuick BURN Clarinol® CLA Powder, SYLAB CLARINOL CLA, GoldNutntion® Slim Ultimate Raspberry, PROZIS CLA Clarinol®, Eurosup Clarinol, MegaCLA GoldNutrition, CLA Figurel with Chromium, Performance CLA, Bluebonnet Clarinol® CLA Softgels, Pro Figur CLA, Protech CLA 1000, LIPOcap, Reflex Nutrition Reflex CLA, Body Shape CLA, Clarinol Xs Body Reshape, and any mixture thereof.
14. The composition of any one of claims 1-13, wherein the vitamin D comprises vitamin D3, vitamin D2, their biologically active metabolites and precursors, or any mixtures thereof.
15. The composition of any one of claims 1-14, comprising one or more Group A agents that diminish the inflammatory mechanism resulting in mitochondrial oxidative stress, wherein at least one of the one or more Group A agent is selected from the group comprising omega-3 fatty acids, amino acids, and vitamins.
16. The composition of claim 15, wherein at least one of the one or more Group A agents is aniline or Vitamin E.
17. The composition of claim 15, wherein the omega-3 fatty acid is selected from the group consisting of alpha-linolenic acid, stearidonic acid, eicosapentanoic acid, docosahexanoic acid, and mixtures thereof.
18. The composition of any one of claims 1-17, comprising one or more Group B agents that positively impact the muscle stimulating mechanism.
19. The composition of claim 18, wherein at least one of the one or more Group B agents is selected from the group consisting of a vitamin D other than vitamin D3, creatine, Vitamin D2, leucine, citrulline, and whey protein.
20. The composition of any one of claims 1-19, comprising at least one Group A agent and at least one Group B agent.
21. The composition of any one of claims 1-20, comprising at least two Group A agents and at least two Group B agents.
22. The composition of any one of claims 1-21, comprising about 1 g to about 6 g of the CLA and about 100 IU to about 5000 IU of vitamin D.
23. The composition of any one of claims 1-21, comprising about 2 g to about 4 g of the CLA and about 400 IU to about 2000 IU of vitamin D.
24. The composition of any one of claims 1-21, comprising about 3 g to about 3.4 g of the CLA and about 800 IU to about 1000 IU of vitamin D.
25. The composition of any one of claims 1-21, comprising about 4 g of the CLA and about 2000 IU of vitamin D.
26. The composition of any one of claims 1-25, comprising an effective amount of the CLA and vitamin D to treat or prevent a condition of muscle loss in a subject in need thereof.
27. The composition of any one of claims 1-25, comprising an effective amount of the CLA and Vitamin D to reduce or prevent age-related muscle loss or function in a subject in need thereof.
28. The composition of any one of claims 1-25, comprising an effective amount of the CLA and Vitamin D to increase muscle protein synthesis in a subject in need thereof.
29. The composition of any one of claims 1-28, wherein, upon administration to a subject in need thereof, the composition has a synergistic effect on one or more of treating or preventing a condition of muscle loss, increasing muscle protein synthesis, and reducing or preventing age-related loss of muscle mass and function.
30. The composition of any one of claims 1-29, wherein the composition is an oral composition.
31. The composition of claim 30, wherein the composition is a foodstuff, a food supplement, or a pharmaceutical composition.
32. The composition of claim 31, wherein the foodstuff comprises a nutritional complete formula, a dairy product, a chilled or shelf stable beverage, a mineral water, a liquid drink, a shot, a soup, a dietary supplement, a meal replacement bar, a nutritional bar, a confectionery product, a milk, a fermented milk product, a yogurt, a pectin chew, a gummy, a milk based powder, an enteral nutrition product, a cereal product, a fermented cereal based product, an ice cream, a chocolate, coffee, a culinary product, or an7y combination thereof.
33. The composition of claim 31, wherein the food supplement is in the form of capsules, gelatin capsules, soft capsules, tablets, sugar-coated tablets, powders, pills, pastes, pastilles, gums, drinkable solutions, drinkable emulsions, syrups, gels, or a combination thereof.
34. The composition of claim 31, wherein the pharmaceutical composition is in the form of capsules, gelatin capsules, soft capsules, tablets, chewable tablets, sugar-coated tablets, pills, pastes or pastilles, powders, softgels, chewable softgels, gums, drinkable solutions or emulsions, syrups, gels, or any combination thereof.
35. The composition of any one of claims 30-34, comprising one or more of binding agents, gelling agents, thickeners, colorants, taste masking agents, stabilizers, antioxidants, coatings, sweeteners, taste modifiers, and aroma chemicals.
36. The composition of any one of claims 1-29, wherein said composition is formulated for intravenous, intramuscular, rectal, or inhalation administration.
37. The composition of any one of claims 30-36, comprising one or more pharmaceutically acceptable carriers, diluents or excipients.
38. The composition of any one of claims 30-37, wherein the composition is in a single unit dosage form.
39. The composition of any one of claims 30-37, wherein the composition is in two or more unit dosage forms.
40. Use of the composition of any one of claims 1-39 for treating or preventing a condition of muscle loss a subject in need thereof.
41. The use of claim 40, wherein the use comprises increasing muscle mass, increasing muscle function, increasing the rate of muscle synthesis, or decreasing the rate of muscle breakdown in a subject in need thereof.
42. The use of claim 40, wherein the use comprises reducing or preventing age- related loss of muscle mass and function in a subject in need thereof.
43. The use of claim 40, wherein the use comprises treating or preventing at least one of sarcopenia, insufficient muscle protein synthesis, muscle degradation, muscle proteolysis, muscle atrophy, muscle dystrophy, muscle catabolism, muscle wasting, loss of muscle strength, loss of physical capacity, loss of physical performance, impaired mobility, frailty, surgery, disability, risk of falling, and risk of fall-related fractures in a subject in need thereof.
44. The use of claim 40, wherein the use comprises treating or preventing sarcopenia in a subject in need thereof.
45. Use of the composition of any one of claims 1-39 for the manufacture of a medicament for treating or preventing a condition of muscle loss a subject in need thereof.
46. The use of claim 45, wherein the use comprises increasing muscle mass, increasing muscle function, increasing the rate of muscle synthesis, or decreasing the rate of muscle breakdown in a subject in need thereof.
47. The use of claim 45, wherein the use comprises reducing or preventing age- related loss of muscle mass and function in a subject in need thereof.
48. The use of claim 45, wherein the use comprises increasing muscle protein synthesis in a subject in need thereof.
49. The use of claim 45, wherein the use comprises treating or preventing at least one of sarcopenia, insufficient muscle protein synthesis, muscle degradation, muscle proteolysis, muscle atrophy, muscle dystrophy, muscle catabolism, muscle wasting, loss of muscle strength, loss of physical capacity, loss of physical performance, impaired mobility, frailty, surgery, disability, risk of falling, and risk of fall-related fractures in a subject in need thereof.
50. The use of claim 40 or 45, wherein the use comprises treating or preventing sarcopenia in a subject in need thereof.
51. A method of treating or preventing a condition of muscle loss, the method comprising administering the composition of any one of claims 1-39 to a subject in need thereof.
52. The method of claim 51, wherein the method comprises increasing muscle mass, increasing muscle function, increasing the rate of muscle synthesis, decreasing the rate of muscle breakdown, or a combination thereof in the subject in need thereof.
53. The method of claim 51, wherein the method comprises reducing or preventing muscle mass and function in the subject in need thereof.
54. The method of claim 51, wherein the method comprises increasing muscle protein synthesis in the subject in need thereof.
55. The method of claim 51, wherein the method comprises treating or preventing at least one of sarcopenia, insufficient muscle protein synthesis, muscle degradation, muscle proteolysis, muscle atrophy, muscle dystrophy, muscle catabolism, muscle wasting, loss of muscle strength, loss of physical capacity, loss of physical performance, impaired mobility, frailty, surgery, disability, risk of falling, and risk of fall-related fractures in a subject in need thereof.
56. The method of claim 51, wherein the method comprises treating or preventing sarcopenia in a subject in need thereof.
57. A method of treating or preventing a condition of muscle loss, comprising administering CLA and Vitamin D to a subject in need thereof.
58. The method of claim 57, wherein the CLA and Vitamin D are administered separately.
59. The method of claim 58, wherein the CLA is administered before the Vitamin D is administered to the subject.
60. The method of claim 58, wherein the CLA is administered after the Vitamin D is administered to the subject.
61. The method of claim 57, wherein the CLA and Vitamin D are administered concurrently.
62. The method of any one of claims 57-61, comprising increasing muscle mass, increasing muscle function, increasing the rate of muscle synthesis, or decreasing the rate of muscle breakdown in need thereof.
63. The method of any one of claims 57-61, wherein the method comprises reducing or preventing loss of muscle mass and function in a subject in need thereof.
64. The method of any one of claims 57-61, wherein the method comprises increasing muscle protein synthesis in a subject in need thereof.
65. The method of any one of claims 57-61, comprising treating or preventing at least one of sarcopenia, insufficient muscle protein synthesis, muscle degradation, muscle proteolysis, muscle atrophy, muscle dystrophy, muscle catabolism, muscle wasting, loss of muscle strength, loss of physical capacity, loss of physical performance, impaired mobility, frailty, surgery, disability, risk of falling and risk of fall-related fractures in a subject in need thereof.
66. The method of any one of claims 57-61, comprising treating or preventing sarcopenia in a subject in need thereof.
67. The method of any one of claims 57-66, wherein the CLA comprises between about 1% and 99% of one or more isomers of CLA.
68. The method of claim 67, wherein at least one of the one or more isomers of CLA is in triglyceride form, free fatty acid form, or ester form.
69. The method of claim 67, wherein at least one of the one or more isomers of CLA is selected from the group comprising cis-9, cis-11 -octadecadienoic acid, cis-9, trans- 11 -octadecadienoic acid, trans-9, cis-11 -octadecadienoic acid, trans-9, trans-11- octadecadienoic acid, cis-10, cis- 12-octadecadienoic acid, cis-10, trans- 12-octadecadienoic acid, trans- 10, cis- 12-octadecadienoic acid, and trans- 10, trans- 12-octadecadienoic acid.
70. The method of any one of claims 57-69, wherein the CLA is a mixture of two or more different isomers of CLA.
71. The method of claim 70, wherein the mixture comprises cis-9, trans-11- octadecadienoic acid CLA and trans- 10, cis- 12-octadecadienoic acid.
72. The method of claim 70, wherein the mixture comprises cis-9, trans-11- octadecadienoic acid CLA and trans-10, cis- 12-octadecadienoic acid in a 50:50 ratio.
73. The method of any one of claims 57-72, wherein the CLA is derived from safflower oil.
74. The method of any one of claims 57-73, wherein the CLA further comprises one or more tocopherols.
75. The method of claim 74, wherein the tocopherol is selected from the group consisting of consisting of δ-tocopherol, γ-tocopherol, a-tocopherol, and combinations thereof.
76. The method of any one of claims 57-75, wherein the CLA comprises one or more of a Tonalin product, a Clarinol product, Tonalin 35 WDP, Tonalin 60 WDP, Tonalin FFA 80, Tonalin SG 1000 T FFA, Tonalin SG 500 T FFA, Tonalin SG 750 T FFA, Tonalin TG 80, Tonalin TG 80, MET-Rx® CLA Tonalin 1000, Solgar Tonalin CLA, NatureWise CLA 1250, Natrol Tonalin CLA, NDS Body Toner Tonalin Censor, The Vitamin Shoppe Tonalin CLA, Natrol Tonalin Cla, Nature Made Cla Tonalin, Nature's Bounty Tonalin 1000 CLA, NDS Nutrition Censor Body Toner, BulkSupplements CLA Softgels, TNVitamins CLA, Puritan's Pride CLA Tonalin, Barlean's Organic Oils Tonalin CLA Swirl, Solgar, Tonalin CLA, TLS Tonalin CLA, Swanson Tonalin Cla, Iron Tek Essential Cla Pure Tonalin Complex, Trunature® Tonalin® CLA, VIROCUT EXTREME CLA, BlueBonnet Tonalin CLA, Labrada Nutrition EFA Lean Gold Essential Fatty Acid Softgel Capsules, Solgar Tonalin CLA Supplement, Davinci Labs Tonalin CLA 90, Vitacost Tonalin XS-CLA, GNC Total Lean™ CLA Soft Chew, ALLMAX CLA, GNC Total Lean™ CLA PM, Xenadrine NextGen, GNC Total Lean™ Thermo CLA, Puregel's Clarinol® CLA, LiveGreat Foods ACCLAIM® drink, Progressive Labs CLA, Jamieson Slim Down CLA 95, AST CLA 100, GNC ProPerformance AMP Ripped Vita Pak, Muscle Feast CLA, Jarrow Formulas CLA Slim, BQuick Nutrition Lean Capsules, Trophic Slim Fit CLA, GNC Total Lean CLA, Bluebonnet Clarinol® CLA Softgels, Nature's Plus Mega CLA 1200, PhytoCeutical Formulations' OptiCLA, BQuick BURN Clarinol® CLA Powder, SYLAB CLARINOL CLA, GoldNutrition® Slim Ultimate Raspberry, PROZIS CLA Clarinol®, Eurosup Clarinol, MegaCLA GoldNutrition, CLA Figurel with Chromium, Performance CLA, Bluebonnet Clarinol® CLA Softgels, Pro Figur CLA, Protech CLA 1000, LIPOcap, Reflex Nutrition Reflex CLA, Body Shape CLA, and Clarinol Xs Body Reshape.
77. The method of any one of claims 57-76, wherein the vitamin D comprises vitamin D3, vitamin D2, their biologically active metabolites and precursors, or any mixture thereof.
78. The method of any one of claims 57-77, comprising administrating one or more Group A agent that diminishes the inflammatory mechanism resulting in mitochondrial oxidative stress, wherein at least one of the one or more Group A agents is selected from the group comprising omega-3 fatty acids, amino acids, and vitamins.
79. The method of claim 78, wherein at least one of the one or more Group A agents is aniline or vitamin E.
80. The method of claim 78, wherein the omega-3 fatty acid is selected from the group consisting of alpha-linolenic acid, stearidonic acid, eicosapentanoic acid, docosahexanoic acid, and mixtures thereof.
81. The method of any one of claims 57-80, further comprising administrating one or more Group B agents that positively impacts the muscle stimulating mechanism, wherein at least one of the one or more Group B agents is selected from the group comprising another vitamin D other than Vitamin D3, citrulline, creatine, leucine, and whey protein.
82. The method of claim 81, wherein at least one of the one or more Group B agents is creatine, leucine, citrulline, whey protein, or vitamin D2.
83. The method of any one of claims 57-82, comprising administrating at least one Group A agent and at least one Group B agent to the subject.
84. The method of any one of claims 57-83, comprising administrating at least two Group A agents and at least two Group B agents to the subject.
85. The method of any one of claims 51-84, wherein CLA is administered in an amount from about 1.0 g to about 6.0 g per day and the Vitamin D is administered in an amount from about 100 IU to about 5000 IU per day.
86. The method of any one of claims 51-84, wherein the CLA is administered in an amount from about 2.0 g to about 4.0 g per day and the Vitamin D is administered in an amount from about 400 IU to about 2000 IU per day.
87. The method of any one of claims 51-84, wherein the CLA is administered in an amount from about 3.0 g to about 3.4 g per day and the Vitamin D is administered in an amount from about 800 IU to about 1000 IU per day.
88. The method of any one of claims 51-84, wherein the CLA is administered in an amount of about 4 g per day and the Vitamin D is administered in an amount of about 2000 IU per day.
89. The method of any one of claims 51-88, wherein the dose is administered in a single unit dosage form.
90. The method of any one of claims 51-88, wherein the dose is administered in two or more unit dosage forms.
91. The method of any one of claims 51-90, wherein said administration of one or more of CLA, Vitamin D, Group A agent(s) and Group B agent(s) is repeated one time per day.
92. The method of any one of claims 51-90, wherein said administration of one or more of the CLA, vitamin D, Group A agent(s) and Group B agent(s) is repeated more than one time per day.
93. The method of any one of claims 51-92, wherein the administration of one or more of the CLA, Vitamin D, Group A agent(s) and Group B agent(s) is oral, intravenous, intraperitoneal, intragastric, or intravascular administration.
94. The method of any one of claims 51-92, wherein the administration of one or more of CLA, Vitamin D, Group A agent(s) and Group B agent(s) is oral administration.
95. The method of any one of claims 51-94, wherein the subject is an adult.
96. The method of any one of claims 51-95, wherein the subject is a male.
97. The method of any one of claims 51-95, wherein the subject is a female.
98. The method of any one of claims 51-97, wherein the subject is a middle-aged adult.
99. The method of any one of claims 51-97, wherein the subject is an elderly adult.
100. The method of any one of claims 51-97, wherein the subject is human is a person of the age of 40 years or more.
101. The method of any one of claims 51-97, wherein the subject is human is a person of the age of 60 years or more.
102. The method of any one of claims 51-101, wherein the subject is with an active lifestyle.
103. The method of any one of claims 51-101, wherein the subject is with a sedentary lifestyle.
104. The method of any one of claims 51-103, wherein the subject has sarcopenia or is at risk of developing sarcopenia.
105. The method of any one of claims 51-104, further comprising identifying a subject is suffering from a condition of muscle loss or is at the risk of developing a condition of muscle loss.
106. The method of any one of claims 51-105, further comprising determining muscle weight, muscle circumference, lean muscle, body weight, fat mass, lean mass, brain water content, locomotor activity, protein synthesis rate, or any combination thereof of the subject.
107. The method of any one of claims 51-106, at least one symptom of the condition of muscle loss is skeletal muscle loss, or muscle mass loss.
108. The method of any one of claims 51-107, wherein the condition of muscle loss is caused by aging, disease, injury, inactivity, or any combination thereof.
109. The method of any one of claims 51-108, wherein the condition of muscle loss is selected from the group comprising sarcopenia, muscle atrophy, cachexia, muscular dystrophy, or any combination thereof.
110. The method of any one of claims 51-109, wherein the condition of muscle loss is sarcopenia.
111. The method of any one of claims 51-110, wherein upon administration to a subject in need thereof, the rate of muscle protein synthesis is increased.
112. The method of any one of claims 51-111, wherein the increased muscle protein synthesis comprises increased muscle fractional synthesis rate (FSR).
113. The method of any one of claims 51-112, wherein the administration of a combination of CLA and vitamin D has a synergistic effect.
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