Compositions and methods using glycine to reduce biological age in adult animals

Abstract

The present disclosure generally relates to compositions and methods for reducing the biological age and / or the rate of biological age increase in adult animals. More specifically, the present disclosure relates to administering a formulation comprising an effective amount of glycine and / or a functional derivative thereof, alone or in combination with a dietary regimen, such as a calorie-restricted diet, an aging diet, or a low-protein diet. The formulation can simultaneously reduce the biological age of a tissue, organ, or physiological system in an adult animal in need thereof; or treat, prevent, or delay the onset of organ disease, or organ function decline, or conditions associated therewith, in an adult animal in need thereof.

Description

[Technical Field]

[0001] [Background technology] The present disclosure generally relates to compositions and methods for reducing the biological age and / or the rate of biological age increase in adult animals. More specifically, the present disclosure relates to administering a formulation comprising an effective amount of glycine and / or a functional derivative thereof, alone or in combination with a dietary regimen, such as a calorie-restricted diet, an aging diet, or a low-protein diet, which can simultaneously reduce the biological age of a tissue, organ, or physiological system in an adult animal in need thereof; or treat, prevent, or delay the onset of organ disease, or organ function decline, or conditions associated therewith, in an adult animal in need thereof.

[0002] Chronological age is known to be a major indicator of overall health, with increasing chronological age being associated with decreased health, however, genetics, nutrition, and lifestyle factors can cause individuals to age slower or faster than their chronological age.

[0003] Thus, chronological age may not necessarily reflect an individual's rate of aging or risk of declining health, whereas an individual's biological age (e.g., based on measures of clinical biochemistry and cell biology) may vary compared to other individuals of the same chronological age.

[0004] Highly digestible, high-quality protein diets are generally recommended based on the dog's chronological age. For example, a dog may be recommended to switch to a senior diet around age 7 or 8. However, an assessment of a dog's increasing biological age relative to its chronological age may dictate the decision to provide the animal with additional nutritional solutions.

[0005] Thus, there is a need for methods and compositions that are effective in reducing biological age and its associated conditions in adult animals that have increased biological age compared to their chronological age.

[0006] [Summary of the Invention] The present inventors have surprisingly found that glycine induces a decrease in biological age at the individual and tissue levels.

[0007] Thus, in a general embodiment, the present disclosure provides a method of reducing the biological age and / or reducing the rate of biological age accrual in an adult animal in need thereof, comprising feeding said adult animal a composition comprising an effective amount of glycine and / or a functional derivative thereof.

[0008] The present disclosure also provides a method for reducing the biological age of a tissue, organ, or physiological system in an adult animal in need thereof, comprising feeding the adult animal a composition comprising an effective amount of glycine and / or a functional derivative thereof.

[0009] The present disclosure further provides a method for treating, preventing, or delaying the onset of organ disease, or decreased organ function, or conditions associated therewith, in an adult animal in need thereof, comprising feeding the adult animal a composition comprising an effective amount of glycine and / or functional derivatives.

[0010] Suitably, the method may comprise the step of selecting and / or applying to the animal a composition comprising an effective amount of glycine and / or a functional derivative thereof in accordance with an assessment that the animal's biological age is greater than its chronological age. The increased biological age may be in a tissue, organ, or physiological system, such as the immune system, gastrointestinal system, urinary system, muscular system, cardiovascular system, and / or nervous system.

[0011] In one embodiment, the tissue is a glycine deficient tissue.

[0012] Suitably, improving the biological age of an animal may refer to a reduction in the difference between the biological age and chronological age of the animal when the biological age of the animal is higher than the chronological age. Alternatively, worsening the biological age of an animal may refer to an increase in the difference between the biological age and chronological age of the animal when the biological age of the animal is higher than the chronological age.

[0013] Suitably, improving the biological age of an animal may refer to reducing the rate of change between the biological age and chronological age of an animal when the biological age of the animal is higher than the chronological age.For example, the biological age of an animal may increase by 1.5 years for every 1 year increase in chronological age.

[0014] Suitably, the compositions of the present invention may be effective for animals based on their chronological age, but may be particularly effective when applied to animals that have an increased biological age compared to their chronological age. Thus, the method may advantageously be beneficial for animals that are more likely to respond to the composition, or that have an improved degree of response.

[0015] The compositions comprising glycine and / or functional derivatives may be part of or administered in conjunction with a dietary regimen, which may be a calorie-restricted diet, a geriatric diet, or a low-protein diet.

[0016] An advantage of one or more embodiments provided by the present disclosure is that it improves the condition of an animal, an elderly animal, or an elderly human.

[0017] Yet another advantage of one or more embodiments provided by the present disclosure is that they improve survival in elderly individuals.

[0018] An additional advantage of one or more embodiments provided by the present disclosure is that they improve mobility in animals.

[0019] Additional features and advantages are described in, and will be apparent from, the following detailed description and drawings. [Brief explanation of the drawings]

[0020] [Figure 1]The effects of glycine supplementation on glycine levels in muscle of old mice compared to young mice are shown. Results are expressed as mean ± SEM, with each data point representing a biological replicate. p values ​​are graphically interpreted using a conventional one-way analysis of variance. [Figure 2] Figure 1 shows the effect of glycine supplementation on biological age in muscle. Biological age was estimated by DNA methylation analysis using a muscle-specific clock. Results are expressed as mean ± SEM, with each data point representing a biological replicate. p-values ​​are graphically interpreted using a one-sided parametric t-statistic.

[0021] [Mode for Carrying Out the Invention] Various preferred features and embodiments of the present invention will now be described by way of non-limiting example, it being understood that those skilled in the art can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed.

[0022] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0023] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," or "containing," "contains," are inclusive, or open-ended, and do not exclude additional, unspecified members, elements, or method steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0024] Numeric ranges are inclusive of the numbers defining the range.

[0025] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Any publication cited herein should not be construed as an admission that such publication constitutes prior art to the claims appended hereto.

[0026] subject The method is directed to adult animals. Thus, the animal according to the present invention is a human, a pet animal, preferably a dog or a cat.

[0027] Chronological age Chronological age may be defined as the amount of time that has elapsed since a subject's birth to a given date. Chronological age may be expressed in years, months, days, etc. In certain embodiments, the animal is a dog and may be at least about 2 years old. Suitably, the dog may be at least about 2 years old, at least about 3 years old, at least about 4 years old, at least about 5 years old, at least about 6 years old, at least about 7 years old, at least about 8 years old, at least about 9 years old, or at least about 10 years old.

[0028] Biological age assessment The method may involve determining a DNA methylation profile to assess the biological age of an animal, such as a human or dog, using any suitable method.

[0029] The biological age of a particular tissue, organ, or physiological system may be assessed using a DNA methylation profile that includes or consists of methylation sites in genes that are preferentially or specifically expressed by that tissue, organ, or physiological system. Gene classifications by tissue, organ, or physiological system are publicly available, for example, from Gene Ontology (http: / / geneontology.org / ), the KEGG pathway database (https: / / www.genome.jp / kegg / ), or MSIgDB (https: / / www.gsea-msigdb.org / gsea / msigdb / index.jsp).

[0030] The biological age of a dog may be expressed in years, months, days, etc.

[0031] composition A composition comprising an effective amount of glycine and / or a functional derivative can be administered to an animal, e.g., a human (e.g., an elderly individual), or a companion animal, in a therapeutically effective dose. A therapeutically effective dose can be determined by one skilled in the art and can vary depending on a number of factors known to those skilled in the art, such as the severity of the condition and the weight and overall condition of the individual.

[0032] Glycine is preferably L-glycine and / or L-glycine ethyl ester. Non-limiting examples of suitable functional derivatives of glycine include D-allylglycine, N-[bis(methylthio)methylene]glycine methyl ester, Boc-allyl-Gly-OH (dicyclohexylammonium) salt, Boc-D-Chg-OH, Boc-Chg-OH, (R)-N-Boc-(2'-chlorophenyl)glycine, Boc-L-cyclopropylglycine, Boc-L-cyclopropylglycine, (R)-N-Boc-4-fluorophenylglycine, Boc-D-propargylglycine, Boc-(S)-3- Thienylglycine, Boc-(R)-3-thienylglycine, Da-cyclohexylglycine, La-cyclopropylglycine, N-(2-fluorophenyl)-N-(methylsulfonyl)glycine, N-(4-fluorophenyl)-N-(methylsulfonyl)glycine, Fmoc-N-(2,4-dimethoxybenzyl)-Gly-OH, N-(2-furoyl)glycine, La-neopentylglycine, D-propargylglycine, sarcosine, Za-phosphonoglycine trimethyl ester, and mixtures thereof.

[0033] In one embodiment, glycine can be provided as a dipeptide, such as N-acetylcysteinylglycine (GlyNAC) or cysteinylglycine.

[0034] Glycine (GLY) or a functional derivative thereof can be administered in an amount of about 0.1 to 100 milligrams (mg) of glycine or a functional derivative thereof per kilogram (kg) of the subject's body weight, in some embodiments, these amounts are provided, at least in part, by a dipeptide comprising both N-acetylcysteine ​​and glycine or a functional derivative thereof.

[0035] In another embodiment, collagen and collagen peptides may be used as a glycine source. Furthermore, whey proteins are rich in glycine and cysteine, so in another embodiment, glycine and N-acetylcysteine ​​are provided by whey proteins. Some plant-based protein sources may also provide a glycine source.

[0036] In a specific, non-limiting example, the daily dose of glycine for a 60 kg subject may be 6-6,000 mg / day.

[0037] The compositions of the present invention are preferably administered to an individual at least two days a week, more preferably at least three days a week, and most preferably seven days a week; for at least one week, at least one month, at least two months, at least three months, at least six months, or even longer. In some embodiments, the compositions of the present invention are administered to an individual continuously over several days, for example, until at least a therapeutic effect is achieved. In some embodiments, the compositions of the present invention can be administered to an individual daily for at least 30, 60, or 90 consecutive days.

[0038] The above dosing examples do not require uninterrupted daily administration. Instead, administration may be interrupted for several short periods, e.g., 2-4 days between administration periods. The ideal duration of administration of the compositions of the present invention can be determined by one of skill in the art.

[0039] In a preferred embodiment, the compositions of the present invention are administered to an individual orally or enterally (e.g., by tube feeding). For example, the compositions of the present invention can be administered to an individual as a drink, capsule, tablet, powder, or suspension.

[0040] The composition of the present invention may be any type of composition suitable for human and / or animal consumption. For example, the composition of the present invention may be selected from the group consisting of food compositions, dietary supplements, nutritional compositions, nutraceuticals, powdered nutritional products to be reconstituted with water or milk before ingestion, food additives, pharmaceuticals, beverages, and drinks. In one embodiment, the composition of the present invention is an oral nutritional supplement (ONS), a complete nutritional formula, a pharmaceutical, a drug, or a food product. In a preferred embodiment, the composition of the present invention is administered to an individual as a beverage. The composition of the present invention may be stored as a powder in a sachet and then suspended in a liquid such as water for use.

[0041] In one embodiment, the composition comprises a protein source. The composition may comprise a mixture of one or more proteins, such as a mixture of one or more of (i) peptides, (ii) longer amino acid chains, or (iii) free form amino acids; the mixture is preferably formulated to obtain a desired amino acid profile / content.

[0042] At least a portion of the protein can be of animal or plant origin, such as a dairy protein, including one or more milk proteins, such as a milk protein concentrate or a milk protein isolate; a caseinate or casein, such as a casein micelle concentrate or a casein micelle isolate; or a whey protein, such as a whey protein concentrate or a whey protein isolate. Additionally or alternatively, at least a portion of the protein can be a vegetable protein, such as one or more soy proteins or pea proteins.

[0043] In one embodiment, the composition comprises a carbohydrate source. Any suitable carbohydrate may be used in the compositions of the present invention, including, but not limited to, starch (e.g., modified starch, amylose starch, tapioca starch, corn starch), sucrose, lactose, glucose, fructose, corn syrup solids, maltodextrin, xylitol, sorbitol, or combinations thereof.

[0044] The carbohydrate source preferably accounts for no more than 50% of the energy of the compositions of the present invention, more preferably no more than 36% of the energy of the compositions of the present invention, and most preferably no more than 30% of the energy of the compositions of the present invention. The compositions may have a high protein:carbohydrate energy ratio, for example, greater than 0.66, preferably greater than 0.9, more preferably greater than 1.2.

[0045] In one embodiment, the composition of the present invention comprises a fat source.The fat source can comprise any suitable fat or fat mixture.Non-limiting examples of suitable fat sources include vegetable fats such as olive oil, corn oil, sunflower oil, high oleic sunflower oil, rapeseed oil, canola oil, hazelnut oil, soybean oil, palm oil, coconut oil, blackcurrant seed oil, borage oil, and lecithin, animal fats such as milk fat; or combinations thereof.

[0046] In a preferred embodiment, the composition further contains an autophagy inducer selected from the group consisting of a urolithin (e.g., urolithin A, urolithin B, or urolithin D), rapamycin, torin 1, valproic acid, a polyphenol (e.g., resveratrol), caffeine, metformin, a 5'AMP-activated protein kinase (AMPK) activator, an L-type calcium channel inhibitor, and mixtures thereof. Non-limiting examples of suitable autophagy inducers are palmitic acid, 5-aminoimidazole-4-carboxamide riboside (AICAR), verapamil, nifedipine, diltiazem, a piperazine phenothiazine derivative (e.g., trifluoperazine), a ketone (e.g., β-hydroxybutyrate, a ketone salt, or a ketone ester derivative), and mixtures thereof.

[0047] In some embodiments, the compositions of the present invention are administered to an individual in a single dosage form, i.e., all compounds are present in one product that is given to an individual in conjunction with a dietary regimen.

[0048] The dietary regimen can be a single meal, a regimen of multiple meals, a dietary supplement or a regimen of dietary supplements, or a combination of a diet and a dietary supplement, or a combination of a single meal and multiple dietary supplements.

[0049] The dietary intervention or dietary product described herein may be any suitable dietary regimen, such as a calorie restricted diet, an elderly diet, a low protein diet, a phosphorus diet, a low protein diet, a potassium supplemented diet, a polyunsaturated fatty acid (PUFA) supplemented diet, an antioxidant supplemented diet, a vitamin B supplemented diet, a liquid diet, a selenium supplemented diet, or a diet supplemented with carnitine, branched chain amino acids or derivatives, or any combination of the above.

[0050] Preferably, the dietary regimen or dietary product may be a calorie-restricted diet, an elderly diet, or a low-protein diet. Preferably, the dietary intervention or dietary product may be a calorie-restricted diet. Preferably, the dietary intervention or dietary product may be a low-protein diet.

[0051] Dietary intervention can be determined based on the dog's baseline maintenance energy requirement (MER), which may suitably be the amount of food that stabilizes the dog's weight (less than a 5% change over three weeks).

[0052] For example, it is generally understood that young, growing dogs benefit from a high-energy / high-protein diet. However, older dogs may have lower energy requirements, so the diet can be appropriately modified. In particular, many manufacturers produce "senior" ranges of dog foods that are low in calories and high in fiber, but contain amounts of protein and fat appropriate for older dogs.

[0053] Preferably, the calorie-restricted diet comprises about 50%, about 55%, about 60%, about 65%, about 75%, about 80%, about 85%, or about 90% of the dog's MER. Preferably, the calorie-restricted diet comprises about 60% or about 75% of the dog's MER.

[0054] Suitably, the low protein diet may contain less than 20% protein (% dry matter), for example the low protein diet may contain less than 19% (% dry matter).

[0055] The dietary regimen may include foods, dietary supplements, and / or beverages containing nutrients and / or bioactive substances that mimic the benefits of calorie restriction (CR) without restricting daily calorie intake. For example, the foods, dietary supplements, and / or beverages may contain functional ingredients with CR-like benefits. Preferably, the foods, dietary supplements, and / or beverages may contain an autophagy inducer. Preferably, the foods, dietary supplements, and / or beverages may contain fruits and / or nuts (or extracts thereof). Suitable examples include, but are not limited to, pomegranate, strawberry, blackberry, camu camu, walnut, chestnut, pistachio, and pecan. Preferably, the foods, dietary supplements, and / or beverages may contain probiotics with or without fruit or nut extracts.

[0056] The methods described herein may be useful for i) reducing the biological age and / or reducing the rate of biological age increase in an animal, or ii) reducing the biological age of a tissue, organ, or physiological system, and iii) treating, preventing, or delaying the onset of organ disease or organ decline, or conditions related thereto, in any animal.

[0057] In one embodiment, the increased biological age may be in a tissue, organ, or physiological system, such as the immune system, gastrointestinal system, urinary system, muscular system, cardiovascular system, and / or nervous system. In a preferred embodiment, the tissue is the liver, kidney, or skeletal muscle. Non-limiting examples of such muscles include one or more of the following: vastus lateralis, gastrocnemius, tibialis, soleus, extensor digitorum longus (EDL), biceps femoris, semitendinosus, semimembranosus, gluteus maximus, extraocular muscles, facial muscles, or diaphragm.

[0058] In one embodiment, an animal in need thereof is deficient in glycine in one or more of its tissues.

[0059] Those skilled in the art will understand that they can freely combine all features of the invention described herein without departing from the scope of the invention disclosed.

[0060] [Example] The present invention will now be further described by way of examples, which are meant to be provided to aid those skilled in the art in practicing the invention, and are not intended to limit the scope of the invention in any way.

[0061] Example 1 Materials and Methods Animal care All animal experiments were performed in accordance with Swiss and EU ethical guidelines and approved by the local animal experimentation committee under license VD3519. Twenty-eight 21-month-old wild-type C57BL / 6J mice and 12 4-month-old wild-type C57BL / 6J mice were housed in a temperature- and humidity-controlled environment with a 12:12-h light:dark cycle. Mice had access to nesting and enrichment materials, and water and food were available ad libitum.

[0062] Dietary supplementation and sample collection Mice received dietary supplementation for 6 weeks. The young control group (n = 12) and old control group (n = 14) were fed a standard commercial powdered diet (Safe 150, Safe Diets) at 20% humidity. The glycine-treated group (n = 14) received the same standard commercial powdered diet homogenously mixed with 1.6 g / kg of glycine at 20% humidity. At the end of treatment, mice were fasted for 2 hours and then euthanized with isoflurane. The gastrocnemius muscles were dissected, and samples were flash-frozen in liquid nitrogen and stored at -80°C.

[0063] Metabolomic analysis of glycine levels Tissue pieces were minced on dry ice using a liquid nitrogen-cooled scalpel and stored at -80°C until extraction. For extraction, samples were homogenized in 300 μL of -20°C methanol / water (5:3) containing fully labeled 13C yeast biomass as an internal standard using 5 mm stainless steel beads in a tissue grinder (Qiagen TissueLyser II) at 23 Hz for up to 3 minutes. 500 μL of -20°C methanol / water (5:3) and 500 μL of chloroform (-20°C) were added. The samples were agitated for 10 minutes at 4°C in a thermoshaker (Thermomixer C, Eppendorf) and then centrifuged for 10 minutes. After extraction, the upper polar phase and protein pellet were collected. The protein pellet was quantified by bicinchoninic acid assay for later normalization, and the polar phase containing the metabolites of interest was dried overnight in a vacuum centrifuge at 4°C and 5 mbar and stored at -80°C until analysis. Whole blood was thawed on ice, and 20 μL was transferred to a new tube. Subsequently, 800 μL of -20°C methanol / water (5:3) containing fully labeled 13C yeast biomass as an internal standard was added. After adding 500 μL of -20°C chloroform, the extraction procedure continued as described for tissue samples. For glycine analysis, dried samples were reconstituted with 35 μL of 60% (v / v) acetonitrile / water, and the supernatant was transferred to a glass vial for LC-MS analysis. 5 μL was injected into a Vanquish UHPLC+focusing liquid chromatography system coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific). Metabolites were assigned according to exact mass, and signal intensities were normalized to the 13C internal standard and protein content. Glycine levels in muscle were analyzed using two-way ANOVA with Dunnett's multiple comparisons.

[0064] DNA extraction DNA extraction was performed on 100 mg of muscle using the QIAGEN DNeasy Blood and Tissue Kit (Cat. No. 69506) according to the manufacturer's recommendations. DNA was quantified using DropSense technology (Unchained Labs).

[0065] Methylation detection Bisulfite conversion was performed on 500 ng of DNA using the Zymo EZ DNA Methylation Kit (Cat. No.: D5001) according to the manufacturer's recommendations. 200 ng of converted DNA was used to proceed with the Infinium HD Methylation Assay protocol according to the manufacturer's recommendations. DNA methylation data were generated using a custom Illumina array, HorvathMammalMethyl40 (Cat. No.: 20027399), with the consent of the Clock Foundation (Torrance, California), which developed the array. Arrays were scanned using an Illumina iScan.

[0066] Methylation analysis The raw data was sent to the Clock Foundation, which performed the analysis and calculated the biological age of the samples. The Clock Foundation applied different algorithms (clocks) to mouse species with different methylation sites used to develop the clocks. To analyze muscle, we used tissue-specific clocks trained on DNA methylation sites specific to a particular tissue. All clocks are based on different CpG methylation analyses and, although subject to variation, all demonstrate robust chronological age prediction performance. The results obtained for each sample correspond to biological age (years). Data were plotted and analyzed using R. One-tailed parametric t-tests were performed to test the effect of glycine supplementation compared to a control diet.

[0067] result The results show that tissue glycine deficiency can be restored in old mice (21 months old) by 6 weeks of dietary supplementation containing 1.6 g of glycine per kg of food, and that this supplementation also significantly reduces the biological age of the mice. Figure 1 shows that glycine levels are reduced in the muscles of old mice compared to young mice (4 months old), and that dietary supplementation with glycine significantly restores glycine levels in the muscles of old mice toward young levels. Figure 2 shows that biological age is significantly reduced after glycine supplementation compared to old mice fed a control diet.

[0068] It should be understood that various changes and modifications to the preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. Accordingly, such changes and modifications are intended to be covered by the appended claims.

Claims

1. A method for reducing the biological age of an adult animal (excluding humans) and / or reducing the rate of increase in biological age, comprising feeding the adult animal a composition comprising an effective amount of glycine and / or a functional derivative.

2. A method for reducing the biological age of tissues, organs, or physiological systems in an adult animal (excluding humans) as required, comprising the step of feeding the adult animal a composition containing an effective amount of glycine and / or a functional derivative.

3. The method according to claim 2, wherein the tissue, organ or physiological system is the immune system, gastrointestinal system, urinary system, muscular system, cardiovascular system, and / or nervous system.

4. The method according to claim 2, wherein the tissue is a glycine-deficient tissue.

5. The method according to claim 2, wherein the tissue is skeletal muscle.

6. The method according to any one of claims 1 to 5, wherein the glycine is provided as N-acetylcysteinylglycine (GlyNAC).

7. A method for treating, preventing, or delaying the onset of organ disease, impaired organ function, or related conditions in adult animals (excluding humans) that require such treatment, comprising the step of feeding the adult animals a composition containing an effective amount of glycine and / or a functional derivative.

8. The method according to any one of claims 1 to 5, 7, wherein the adult animal required has a glycine deficiency in one or more tissues.

9. The method according to any one of claims 1 to 5, 7, wherein the adult animal required has a biological age that is increased compared to its chronological age.

10. The method according to claim 9, wherein the increased biological age is evaluated from the DNA methylation profile of the adult animal.

11. The method according to any one of claims 1 to 5, 7, wherein the adult animal is an elderly animal.

12. The method according to any one of claims 1 to 5, 7, wherein the adult animal is a dog or a cat.

13. The method according to any one of claims 1 to 5, 7, wherein the composition is selected from the group consisting of food compositions, dietary supplements, nutritional compositions, nutraceuticals, powdered nutritional products that are reconstituted with water or milk before ingestion, food additives, pharmaceuticals, drinks, dry pet food or wet pet food, and combinations thereof.

14. The method according to any one of claims 1 to 5, 7, wherein the composition is part of a dietary regimen selected from a calorie-restricted diet, a diet for the elderly, or a low-protein diet, or is administered in combination with the dietary regimen.

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