Compositions and methods using nicotinamide to reduce biological age of an adult animal

Abstract

Compositions and methods reduce the biological age and / or reduce the rate of increase of the biological age of an adult animal. More specifically, the present disclosure relates to administering a formulation comprising an effective amount of Nicotinamide (NAM), alone or in combination with a dietary regime such as a standard diet, caloric restriction, senior diet or high protein diet. The formulation can concomitantly reduce the biological age of a tissue, organ or physiological system in the adult animal in need; or treat, prevent, or delay the onset of an organ disease, or decline of organ function, or condition relating thereto in an adult animal in need.

Description

TITLECOMPOSITIONS AND METHODS USING NICOTINAMIDE TO REDUCE BIOLOGICAL AGE OF AN ADULT ANIMALTECHNICAL FIELD

[0001] The present disclosure generally relates to compositions and methods to reduce the biological age and / or reduce the rate of increase of the biological age of an adult animal. More specifically, the present disclosure relates to administering a formulation comprising an effective amount of nicotinamide (NAM). The formulation can concomitantly reduce the biological age of a tissue, organ or physiological system in the adult animal in need; or treat, prevent, or delay the onset of an organ disease, or decline of organ function, or condition relating thereto in an adult animal in need.BACKGROUND

[0002] Chronological age is known to be a major indicator of general health status, with increasing chronological age associated with reduced health. However, depending on genetics, nutrition, and lifestyles, individuals may age slower or faster than their chronological age.

[0003] Chronological age may therefore not always reflect an individual’s rate of aging or risk of reduced health. On the other hand, the biological age of an individual (based on e.g. clinical biochemistry and cell biology measures) can vary compared to others of the same chronological age.

[0004] Highly digestible and high-quality protein diets are generally recommended based upon the chronological age of a dog. For example, it may be recommended that a dog is switched to a senior diet around 7 or 8 years old. However, the determination of an increased biological age for a dog compared to its chronological age may allow a determination to provide additional nutritional solution to the animal.

[0005] There is thus a need for methods and compositions effective in reducing biological age and conditions related thereto, of an adult animal having an increased biological age compared to its chronological age.SUMMARY

[0006] Accordingly, in a general embodiment, the present disclosure provides a method for reducing the biological age and / or for reducing the rate of increase of the biological age of an adult animal in need, the method comprising: feeding the adult animal a composition comprising an effective amount of Nicotinamide (NAM). As detailed in the experimental data set forth later herein, the inventors surprisingly found that nicotinamide induces reduction in biological age. This reduction in biological age occurred regardless of the diet the nicotinamide was administered in combination with.

[0007] 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, the method comprising feeding the adult animal a composition comprising an effective amount of Nicotinamide (NAM).

[0008] The present disclosure further provides a method for treating, preventing, or delaying the onset of an organ disease, or decline of organ function, or condition relating thereto in an adult animal in need, the method comprising feeding the adult animal a composition comprising an effective amount of nicotinamide (NAM).

[0009] Suitably, the present methods may comprise selecting and / or applying the composition comprising an effective amount of Nicotinamide (NAM) to an animal following a determination that the animal’s biological age is greater than its chronological age. The increased biological age may be in a tissue, in an organ or in a physiological system such that immune, gastrointestinal, urinary, muscular, cardiovascular, and / or neurological system.

[0010] 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, where the biological age of the animal is greater than its chronological age. Alternatively, a worsening in the biological age of an animal may refer to an increase in the difference between the biological age and chronological age of the animal, where the biological age of the animal is greater than its chronological age.

[0011] Suitably, improving the biological age of an animal may refer to a reduction in the rate of change between the biological age and chronological age of the animal, where the biological age of the animal is greater than its chronological age. For example, an animal’s biological age may have been increasing by 1.5 years per 1 year increase in chronological age.

[0012] Suitably, whilst the present composition may be effective for animals based on chronological age, it may be particularly effective when applied to an animal with anincreased biological age compared to its chronological age. As such, the present method may advantageously be beneficial for an animal that has an increased likelihood to respond, or improved magnitude of response, to the present composition.

[0013] The composition comprising Nicotinamide (NAM) may be part of or administered together with, a dietary regime. The dietary regime may be a diet in which protein is 18% to 60% of protein / dry matter (w / w) for dogs and 26% to 60% of protein / dry matter (w / w) for cats.

[0014] An advantage of one or more embodiments provided by the present disclosure is to improve the condition of animals, ageing animals, or ageing humans.

[0015] Still another advantage of one or more embodiments provided by the present disclosure is to increase the survivability of an ageing individual.

[0016] An additional advantage of one or more embodiments provided by the present disclosure is to increase mobility in the animal.

[0017] Additional features and advantages are described in, and will be apparent from, the following Detailed Description and the Figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 (A, B) shows the effect of NAM on biological age in C. elegans measured using the BitAge (Fig. 1A) and Raptor transcriptomics clock (Fig.1 B). Worms (n=1270 ± 331 worms per conditions, divided in 5 replicates) were cultured on plates of 5 days and treated daily with NAM. RNA sequencing allowed to determine BitAge and Raptor Age. Data were analyzed using one-way ANOVA followed by Dunnett’s post-hoc test.DETAILED DESCRIPTION

[0019] Definitions

[0020] Some definitions are provided hereafter. Nevertheless, definitions may be located in the “Embodiments” section below, and the above header “Definitions” does not mean that such disclosures in the “Embodiments” section are not definitions.

[0021] All percentages expressed herein are by weight of the total weight of the composition unless expressed otherwise. As used herein, “about,” “approximately” and “substantially” are understood to refer to numbers in a range of numerals, for example the range of -10% to +10% of the referenced number, preferably -5% to +5% of the referenced number, more preferably -1% to +1% of the referenced number, most preferably -0.1% to +0.1% of the referenced number. All numerical ranges herein should be understood to include all integers,whole or fractions, within the range. Moreover, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

[0022] As used in this disclosure and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to “an ingredient” or “a method” includes a plurality of such “ingredients” or “methods.”

[0023] The words “comprise,” “comprises” and “comprising” are to be interpreted inclusively rather than exclusively. Likewise, the terms “include,” “including” and “or” should all be construed to be inclusive, unless such a construction is clearly prohibited from the context. Nevertheless, the compositions and methods disclosed herein may lack any element that is not specifically disclosed herein. Thus, a disclosure of an embodiment using the term “comprising” includes a disclosure of embodiments “consisting essentially of’ and “consisting of’ the components or steps identified.

[0024] The terms “at least one of’ and “and / or” used respectively in the context of “at least one of X and Y” and “X and / or Y” should be interpreted as “X without Y,” or “Y without X,” or “both X and Y.” Where used herein, the terms “example” and “such as,” particularly when followed by a listing of terms, are merely exemplary and illustrative and should not be deemed to be exclusive or comprehensive.

[0025] As used herein, “related to,” “associated with” and “linked with” mean occurring concurrently, preferably mean caused by the same underlying condition, more preferably mean that one of the identified conditions is at least indirectly caused by the other identified condition, and most preferably mean that one of the identified conditions is directly caused by the other identified condition.

[0026] The terms “food,” “food supplement,” “food product” and “food composition” mean a product or composition that is intended for ingestion by an individual such as a human and provides at least one nutrient to the individual. The compositions of the present disclosure, including the many embodiments described herein, can comprise, consist of, or consist essentially of the elements disclosed herein, as well as any additional or optional ingredients, components, or elements described herein or otherwise useful in a diet supplement.

[0027] “Prevention” includes reduction of risk, incidence and / or severity of a condition or disorder. The terms “treatment” and “treat” include both prophylactic or preventive treatment (that prevent and / or slow the development of a targeted pathologiccondition or disorder) and curative, therapeutic or disease-modifying treatment, including therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder; and treatment of patients at risk of contracting a disease or suspected to have contracted a disease, as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition. The terms “treatment” and “treat” do not necessarily imply that a subject is treated until total recovery. The terms “treatment” and “treat” also refer to the maintenance and / or promotion of health in an individual not suffering from a disease but who may be susceptible to the development of an unhealthy condition. The terms “treatment” and “treat” are also intended to include the potentiation or otherwise enhancement of one or more primary prophylactic or therapeutic measures. As nonlimiting examples, a treatment can be performed by a patient, a caregiver, a doctor, a nurse, or another healthcare professional.

[0028] As used herein, a prophylactically or therapeutically “effective amount” is an amount that prevents a deficiency, treats a disease or medical condition in an individual, or, more generally, reduces symptoms, manages progression of the disease, or provides a nutritional, physiological, or medical benefit to the individual.

[0029] “Animal” includes, but is not limited to, mammals, which includes but is not limited to rodents, aquatic mammals, domestic animals such as dogs and cats, farm animals such as sheep, pigs, cows and horses, and humans.

[0030] The term “elderly” in the context of a human means an age from birth of at least 55 years, preferably above 63 years, more preferably above 65 years, and most preferably above 70 years. The term “older adult” or “ageing individual” in the context of a human means an age from birth of at least 45 years, preferably above 50 years, more preferably above 55 years, and includes elderly individuals.

[0031] For other animals, an “older adult” or “ageing individual” has exceeded 50% of the average lifespan for its particular species and / or breed within a species. An animal is considered “elderly” if it has surpassed 66% of the average expected lifespan, preferably if it has surpassed the 75% of the average expected lifespan, more preferably if it has surpassed 80% of the average expected lifespan. An ageing cat or dog has an age from birth of at least about 5 years. An elderly cat or dog has an age from birth of at least about 7 years.

[0032] Chronological age may be defined as the amount of time that has passed from the subject’s birth to the given date. Chronological age may be expressed in terms of 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, at least about 3, at least about 4, at least about5, at least about 6, at least about 7, at least about 8, at least about 9 or at least about 10 years old.

[0033] A biological age for a particular tissue, organ, or physiological system may be determined using a DNA methylation profile comprising, or consisting of, methylation sites from genes that are preferentially or specifically expressed by that tissue, organ, or physiological system. Classifications of genes by a particular tissue, organ, or physiological system are publically available at, for example, 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).

[0034] The term “composition” may mean a food, beverage, dietary supplement, complete nutrition or oral nutritional supplement (ONS) or medical food composition, or mixture thereof.

[0035] As used herein, “complete nutrition” contains sufficient types and levels of macronutrients (protein, fats and carbohydrates) and micronutrients to be sufficient to be a sole source of nutrition for the animal to which the composition is administered. Individuals can receive 100% of their nutritional requirements from such complete nutritional compositions.

[0036] The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the composition disclosed herein in an amount sufficient to produce the desired effect, in association with an acceptable diluent, carrier or vehicle. The specifications for the unit dosage form depend on the particular compounds employed, the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0037] Embodiments

[0038] An aspect of the present disclosure is a method for reducing the biological age and / or reducing the rate of increase of the biological age of an adult animal in need. The method comprises feeding the adult animal a composition comprising an effective amount of Nicotinamide (NAM). The present methods may comprise determining a DNA methylation profile to determine a biological age of an animal, e.g. a human or a dog using any suitable method.

[0039] The composition comprising an effective amount of Nicotinamide (NAM) can be administered to an animal such as a human, e.g., an ageing individual or a pet animal, in a therapeutically effective dose. The therapeutically effective dose can be determined bythe person skilled in the art and will depend on a number of factors known to those of skill in the art, such as the severity of the condition and the weight and general state of the individual.

[0040] Nicotinamide, also known as niacinamide or nicotinic acid amide, is the water-soluble, active form of vitamin B3.

[0041] The method can comprise administering, in particular to a pet animal, a daily amount of nicotinamide (NAM) in the weight range of 0.05 mg - 1 g per kg body weight, preferably 1 mg -500 mg per kg body weight, more preferably 5 mg - 250 mg per kg body weight, even more preferably 4 mg - 125 mg per kg body weight, or most preferably 2 mg - 80 mg per kg body weight.

[0042] In some embodiments, the method can comprise administering to small breed dogs, an amount of about 100 mg / day to 3600 mg / day, preferably about 50 mg / day to 1800 mg / day, more preferably about 20 mg / day to 840 mg / day of nicotinamide (NAM) per serving of a food item or supplement or treat.

[0043] In some embodiments, the method can comprise administering to medium breed dogs, an amount of about 800 mg / day to 8000 mg / day, preferably about 400 mg / day to 5000 mg / day, more preferably about 100 mg / day to 2400 mg / day of nicotinamide (NAM) per serving of a food item or supplement or treat.

[0044] In some embodiments, the method can comprise administering to large breed dogs, an amount of about 1800 mg / day to 24000 mg / day, preferably about 1200 mg / day to 16000 mg / day, more preferably about 600 mg / day to 8000 mg / day of nicotinamide (NAM) per serving of a food item or supplement or treat.

[0045] In some embodiments, the method can comprise administering to small breed cats, an amount of about 150 mg / day to 1200 mg / day, preferably about 100 mg / day to 750 mg / day, more preferably about 50 mg / day to 360 mg / day of nicotinamide (NAM) per serving of a food item or supplement or treat.

[0046] In some embodiments, the method can comprise administering to medium breed cats, an amount of about 250 mg / day to 1500 mg / day, preferably about 150 mg / day to 900 mg / day, more preferably about 75 mg / day to 450 mg / day of nicotinamide (NAM) per serving of a food item or supplement or treat.

[0047] In some embodiments, the method can comprise administering to large breed cats, an amount of about 300 mg / day to 3000 mg / day, preferably about 200 mg / day to 2000 mg / day, more preferably about 90 mg / day to 1000 mg / day of nicotinamide (NAM) per serving of a food item or supplement or treat.

[0048] The nicotinamide can be administered in particular to a human, in an amount of about 0.001 mg / day to about 3000 mg / day, for example 1 mg / day to about 3000 mg / day, preferably about 10 mg / day to about 2000 mg / day, more preferably from 500 mg / day to about 1000 mg / day. Of course, the daily dose can be administered in portions at various hours of the day. However, in any given case, the amount of compound administered will depend on such factors as the solubility of the active component, the formulation used, subject condition (such as weight), and / or the route of administration. For example, the daily doses of nicotinamide disclosed above are non-limiting and, in some embodiments, may be different; in particular, the compositions disclosed herein can be utilized as an acute care food for special medical purposes (FSMP) and contain up to about 3.0 g nicotinamide / day.

[0049] The composition is preferably administered to the individual at least two days per week, more preferably at least three days per week, most preferably all seven days of the week; for at least one week, at least one month, at least two months, at least three months, at least six months, or even longer. In some embodiments, the composition is administered to the individual consecutively for a number of days, for example at least until a therapeutic effect is achieved. In an embodiment, the composition can be administered to the individual daily for at least 30, 60 or 90 consecutive days.

[0050] The above examples of administration do not require continuous daily administration with no interruptions. Instead, there may be some short breaks in the administration, such as a break of two to four days during the period of administration. The ideal duration of the administration of the composition can be determined by those of skill in the art.

[0051] In a preferred embodiment, the composition is administered to the individual orally or enterally (e.g. tube feeding). For example, the composition can be administered to the individual as a beverage, a capsule, a tablet, a powder or a suspension.

[0052] The composition can be any kind of composition that is suitable for human and / or animal consumption. For example, the composition may be selected from the group consisting of food compositions, dietary supplements, nutritional compositions, nutraceuticals, powdered nutritional products to be reconstituted in water or milk before consumption, food additives, medicaments, beverages and drinks. In an embodiment, the composition is an oral nutritional supplement (ONS), a complete nutritional formula, a pharmaceutical, a medical or a food product. In a preferred embodiment, the composition is administered to the individual as a beverage. The composition may be stored in a sachet as a powder and then suspended in a liquid such as water for use.

[0053] In another aspect of the present disclosure, a nutritional supplement comprises a therapeutically effective amount of any of the compositions disclosed herein. In an embodiment, the nutritional supplement is an oral nutritional supplement (ONS). The nutritional supplement can be in a form of a solid powder, a powdered stick, a capsule, or a solution. In an embodiment, the nutritional supplement comprises Nicotinamide in a total daily dosage about 1 mg / day to about 3000 mg / day, preferably about 10 mg / day to about 2000 mg / day, more preferably from 500 mg / day to about 1000 mg / day.

[0054] In another aspect of the present disclosure, a food product comprises any of the compositions disclosed herein. In an embodiment, the food product is a food for special medical purpose (FSMP). The food product can comprise Nicotinamide in a total daily dosage about 0.1 mg / day to about 3000 mg / day, preferably about 10 mg / day to about 2000 mg / day, more preferably from 500 mg / day to about 1000 mg / day.

[0055] In an embodiment, the composition includes a source of protein. The composition can comprise a mixture of one or more types of protein, for example one or more (i) peptides, (ii) longer chains of amino acids, or (iii) free form amino acids; and the mixture is preferably formulated to achieve a desired amino acid profile / content.

[0056] At least a portion of the protein can be from animal or plant origin, for example dairy protein such as one or more of milk protein, e.g., milk protein concentrate or milk protein isolate; caseinates or casein, e.g., micellar casein concentrate or micellar casein isolate; or whey protein, e.g., whey protein concentrate or whey protein isolate. Additionally or alternatively, at least a portion of the protein can be plant protein such as one or more of soy protein or pea protein.

[0057] In an embodiment, the composition includes a source of carbohydrates. Any suitable carbohydrate may be used in the composition including, but not limited to, starch (e.g., modified starch, amylose starch, tapioca starch, com starch), sucrose, lactose, glucose, fructose, corn syrup solids, maltodextrin, xylitol, sorbitol or combinations thereof.

[0058] The source of carbohydrates is preferably not greater than 50 energy % of the composition, more preferably not greater than 36 energy % of the composition, and most preferably not greater than 30 energy % of the composition. The composition can have a high proteimcarbohydrate energy ratio, for example greater than 0.66, preferably greater than 0.9 and more preferably greater than 1.2.

[0059] In an embodiment, the composition includes a source of fat. The source of fat may include any suitable fat or fat mixture. Non-limiting examples of suitable fat sources include vegetable fat, such as olive oil, corn oil, sunflower oil, high-oleic sunflower, rapeseedoil, canola oil, hazelnut oil, soy oil, palm oil, coconut oil, blackcurrant seed oil, borage oil, lecithins, and the like, animal fats such as milk fat; or combinations thereof. In some embodiments, the source of fat is less than 20% of the composition.

[0060] In a preferred embodiment, the composition further contains an autophagy inducer selected from the group consisting of urolithin (e.g., Urolithin A, B or D), rapamycin, Torinl, valproic acid, polyphenols (e.g., resveratrol), caffeine, metformin, 5' AMP-activated protein kinase (AMPK) activators, L-type calcium channel inhibitors, and mixtures thereof. Non-limiting examples of suitable autophagy inducers are palmitic acid, 5-aminoimidazole-4- carboxamide riboside (AICAR), verapamil, nifedipine, diltiazem, piperazine phenothiazine derivatives (e.g., trifluoperazine), ketones (e.g., beta-hydroxybutyrate, ketone salts, or ketone ester derivatives) and mixtures thereof.

[0061] In a preferred embodiment, the composition further contains glycine and / or derivatives, thymol, carvacrol, spermidine and / or combinations thereof.

[0062] The glycine (GLY) or functional derivative thereof can be administered in an amount of about 0.1 - 100 milligram (mg) of glycine or functional derivative thereof per kilogram (kg) of body weight of the subject. In some embodiments, these amounts are provided at least partially by a dipeptide comprising both the N-acetylcysteine and the glycine or functional derivative thereof. In another embodiment, collagen and collagen peptides may be used as source of glycine. Additionally, whey protein is rich in glycine and cysteine, so that in another embodiment, the glycine and N-acetylcysteine are provided by whey protein. Some plant-based protein source may also provide source of glycine. In a particular non-limiting example, the daily doses of glycine for a 60 kg subject can be from 6 to 6,000 mg / day

[0063] Thymol (10-64%) is one of the major constituent of essential oils of thyme (Thymus vulgaris L., Lamiaceae). Carvacrol is present in the essential oil of Origanum vulgare (oregano), oil of thyme, oil obtained from pepperwort, and wild bergamot. The essential oil of thyme subspecies contains between 5% and 75% of carvacrol, while Satureja (savory) subspecies have a content between 1% and 45%. Origanum majorana (marjoram) and Dittany of Crete are rich in carvacrol, 50% and 60-80% respectively. Therefore, some embodiments of the composition comprise such plant and / or enriched plant extracts, essential oils or fractions that provide at least a portion of thymol and / carvacrol in the composition, in particular from thyme and oregano. The composition can comprise a pharmacologically effective amount of thymol and / or carvacrol in a pharmaceutically suitable carrier. In aqueous liquid compositions, concentration preferably ranges from about 0.05wt.% to about 4 wt.%, or from about 0.5 wt.% to about 2 wt.% or from about 1.0 wt.% to about 1.5 wt.% of the aqueous liquid composition.

[0064] In particular embodiments, the amount of administration daily of thymol and / or carvacrol is in the weight range of 0.05 mg - 1 g per kg body weight, preferably 1 mg - 200 mg per kg body weight, more preferably 5 mg - 150 mg per kg body weight, even more preferably 10 mg - 120 mg per kg body weight, or most preferably 40 mg - 80 mg per kg body weight. Typically between 50 pg to 10 g of thymol and / or carvacrol, per daily serving in one or more portions is administered to an individual.

[0065] Non-limiting examples of suitable forms of spermidine include spermidine trihydrochloride, spermidine phosphate hexahydrate, spermidine phosphate hexahydrate, and L-arginyl-3,4-spermidine. Wheat germ is rich in spermidine. Therefore, some embodiments of the composition further comprise wheat germ and / or enriched wheat germ extracts that provide at least a portion of the spermidine in the composition. In particular embodiments, the composition further comprise administering daily the spermidine in the weight range of 0.05 mg - 1 g per kg body weight, preferably 1 mg -200 mg per kg body weight, more preferably 5 mg - 150 mg per kg body weight, even more preferably 10 mg - 120 mg per kg body weight, or most preferably 40 mg - 80 mg per kg body weight. In some embodiments, the method can comprise administering 10 mg - 100 mg of spermidine per serving, for example about 16 mg per serving of a food item or about 80 mg per serving of a supplement or treat.

[0066] In some embodiments, the composition is administered to the individual in a single dosage form, i.e. all compounds are present in one product to be given to an individual in combination with a dietary regime.

[0067] The dietary regime may be a meal, a regime of meals, a supplement or a regime of supplements, or combinations of a meal and a supplement, or combinations of a meal and multiple supplements.

[0068] The dietary intervention or dietary product described herein may be any suitable dietary regime, for example, a standard diet, a calorie-restricted diet, a senior diet, a high protein diet, a phosphorous diet, potassium supplement diet, polyunsaturated fatty acids (PUFA) supplement diet, anti-oxidant supplement diet, a vitamin B supplement diet, liquid diet, selenium supplement diet, or diets supplemented with carnitine, branched chain amino acids or derivatives, or any combination of the above.

[0069] A dietary intervention may be determined based on the baseline maintenance energy requirement (MER) of the dog. Suitably, the MER may be the amount of food that stabilizes the dog’s body weight (less than 5% change over three weeks).

[0070] Suitably, a calorie-restricted diet may comprise about 50%, about 55%, about 60%, about 65%, about 75%, about 80%, about 85%, or about 90% of the dog’s MER. Suitably, a calorie-restricted diet may comprise about 60% or about 75% of the dog’s MER.

[0071] In an embodiment, the composition has a protein / energy ratio greater than 6 g protein / 100 kcal, preferably greater than 9 g protein / 100 kcal. In an embodiment, the protein is between 18% and 60% (w / w) of the composition on a dry matter basis.

[0072] The dietary regime may comprise a food, supplement and / or drink that comprises a nutrient and / or bioactive that mimics the benefits of caloric restriction (CR) without limiting daily caloric intake. For example, the food, supplement and / or drink may comprise a functional ingredient(s) having CR-like benefits. Suitably, the food, supplement and / or drink may comprise an autophagy inducer. Suitably, the food, supplement and / or drink may comprise fruit and / or nuts (or extracts thereof). Suitable examples include, but are not limited to, pomegranate, strawberries, blackberries, camu-camu, walnuts, chestnuts, pistachios, pecans. Suitably, the food, supplement and / or drink may comprise probiotics with or without fruit extracts or nut extracts.

[0073] The methods described herein can be useful for i) reducing the biological age and / or reducing the rate of increase of the biological age of an animal or ii) reducing the biological age of a tissue, organ or physiological system as well as iii) treating, preventing, or delaying onset of an organ disease or organ functional decline, or conditions relating thereto for any animal.

[0074] The individual in need thereof may be an elderly animal or an elderly human. The individual in need thereof may be an ageing human or an ageing animal. The individual in need thereof may have a biological age that is greater than a chronological age. Additionally, the individual in need thereof may have a rate of change between the biological age that is greater than a rate of change to the chronological age.

[0075] In an embodiment, the increased biological age may be in a tissue, in an organ or in a physiological system such that immune, gastrointestinal, urinary, muscular, cardiovascular, and / or neurological system. In a preferred embodiment, the tissue is liver, kidney or skeletal muscle. Non-limiting examples of such muscle include one or more of the following: vastus lateralis, gastrocnemius, tibialis, soleus, extensor, digitorum longus (EDL), biceps femoris, semitendinosus, semimembranosus, gluteus maximus, extra-ocular muscles, face muscles or diaphragm.

[0076] In an embodiment, the animal in need has a deficiency in Nicotinamide (NAM) in one or more of his tissue.

[0077] Those skilled in the art will understand that they can freely combine all features of the present invention described herein, without departing from the scope of the invention as disclosed.EXAMPLES

[0078] The following non-limiting example generally illustrates the concepts underlying the embodiments disclosed herein.

[0079] The invention will now be further described by way of examples, which are meant to serve to assist the skilled person in carrying out the invention and are not intended in any way to limit the scope of the invention.

[0080] Example 1

[0081] Material and methods

[0082] Transcriptomics for biological age clocks in C. elegans

[0083] Worm culture

[0084] For the transcriptomics clock experiment, N2 worms were synchronized by bleaching. They were then cultured on NGM plates (60 mm Petri dishes; Fisher Scientific) containing live OP50 seeded with 20 mg / mL heat-killed Escherichia coli OP50 and containing 5FU (500 pM) for 24 hours then reduced to 200 pM thereafter to stop progeny. The worms were treated for 5 days with NAM (10, 25, and 250 pM). Treatments were changed every day.

[0085] RNA Extraction

[0086] Worms were placed in a Lysing Matrix D tube (MP Biomedicals, Santa Ana, California, USA) placed on ice. Then 400 pL of lysis buffer (Agencourt RNAdvance Tissue Kit, Beckman Coulter, Indianapolis, Indiana, USA) were added. The tubes were firmly closed and agitated for 2 minutes at 8000 rpm with Precellys (Bertin Technologies SAS, Monti gny- le-Bretonneux, France). Total RNA was then extracted using the Agencourt RNAdvance Tissue Kit (Beckman Coulter, Indianapolis, Indiana, USA) following the providers recommendations. Total RNA was quantified using the Quant-iT™ RiboGreen™ RNA Assay Kit (Invitrogen, Carlsbad, California, USA) on aFilterMax F3 (Molecular Devices, Sunnyvale, California, USA). Total RNA quality assessment was done using Fragment Analyzer-96 with DNF-471-0500 Standard Sensitivity RNA Analysis Kit (Agilent Technologies, Santa Clara, California, USA).

[0087] Sequencing

[0088] RNA-seq libraries were prepared from 500 ng of total RNA with the Illumina Stranded mRNA Prep reagents (Illumina) using a unique dual indexing strategy, and following the official protocol automated on a Sciclone liquid handling robot (PerkinElmer). Libraries were quantified by a fluorometric method (QubIT, Life Technologies) and their quality assessed on a Fragment Analyzer (Agilent Technologies).

[0089] Sequencing was performed on an Element Biosciences Aviti with a 2x75 sequencing kit Cloudbreak FS High Output (run as paired end 75nt). Base calling and demultiplexing was done with bases2fastq version: 1.7 with filter mask REN15Y15N*- R2:N15Y15N*.

[0090] Analysis of transcriptomics clocks

[0091] Analysis was performed using the algorithms BitAge (Meyer et al. BiT age: A transcriptome-based aging clock near the theoretical limit of accuracy. Aging Cell, 2021. 20(3): p. el3320) and RAPToR (Bulteau et al. Real age prediction from the transcriptome with RAPToR. Nat Methods, 2022. 19(8): p. 969-975).

[0092] Data preparation:

[0093] Genes with very low counts are unlikely to be differentially expressed between groups. We filtered lowly expressed genes by selecting only genes with at least 0.256 CPM or 5 count in at least 5 samples. The threshold on the number of samples ensures that the genes will be expressed in the smallest group (or at least two samples if some groups do not have replicates). The filtering step is performed on the CPM values which takes the library size into account.

[0094] Normalization:

[0095] To account for different library size composition between samples, normalization is carried out by applying Trimmed Means of M-values (TMM). Under the assumption that most genes are not differentially expressed, normalization factors are obtained by a weighted average of the trimmed M values.

[0096] Statistical analysis for differential expression:

[0097] To assess the expression values per group of samples we first estimate genewise negative binomial dispersions by calculating the adjusted profile log-likelihood for each gene and maximize it by weighted likelihood empirical Bayes. The method gives special attention to handling of zero counts, and in particular to situations when fitted values of zero provide no useful residual degrees of freedom for estimating the QL dispersion. Then we fit a quasi -likelihood negative binomial generalized log-linear model to count data (cf equation 1) and conduct gene-wise empirical Bayes quasi -likelihood F-tests for each comparison.

[0098] equation! : Countsij = ?0 + fT!xj + e ~ NB(pi , af)

[0099] where i the gene, j the sample, / >0 the intercept, / > l the Group and u and a respectively the mean and dispersion parameters of the negative-binomial distribution.

[0100] Time Course clustering:

[0101] To visualize how the genes are changing with time and identify the different patterns of change, we performed clustering of gene expression. We selected all the genes that were found differentially expressed in the corresponding time course analysis (FDR<0.05) and selected only the samples involved in the contrast. We performed soft clustering using cmeans with a fixed number of clusters of 6. This number is arbitrary and was selected in order to get enough granularity to grasps the different patterns but is also enough to see a true difference between the different genes. As the method is a soft clustering, a gene can belong to several clusters. The membership values are used to indicate to what degree a gene belongs to each cluster.

[0102] To avoid the influence of expression level to the clustering analysis, z-score transformation is applied to convert the expression values (normalized log2 CPM) to z-scores:

[0103] z = x - u

[0104] x is the average normalized log2 CPM in one condition (eg D19-26), u is the mean (e.g., average expression value of a gene in different conditions), o is the standard deviation (e.g., standard deviation of expression of a gene in different conditions). The clustering was performed on the Euclidean distance of z-scores.

[0105] For each cluster or pattern of expression, we plot the standardized gene expression along time for each DE gene. Each trajectory is colored by cluster membership, where we filter to represent only the genes with a membership to the cluster higher than 0.5.

[0106] Statistical Analysis

[0107] Data were plotted and analyzed using GraphPad Prism 10. Normality was tested to assess distribution using and non-parametric tests were conducted when data did not follow a normal distribution. Details of the statistical tests are included in the figure legends with associated p-values depicted. P<0.05 were considered significant.

[0108] Results

[0109] We tested the effect of NAM on biological age using C. elegans cultured on plates for 5 days. The transcriptomics clocks BitAge and Raptor were used as previously developed in C. elegans (Meyer et al. BiT age: A transcriptome-based aging clock near the theoretical limit of accuracy. Aging Cell, 2021. 20(3): p. el3320) (Bulteau et al. Real ageprediction from the transcriptome with RAPToR. Nat Methods, 2022. 19(8): p. 969-975) . We observed a significant reduction in biological age with all doses of NAM treatments on both BitAge (Fig.lA) and Raptor Age clocks (Fig IB).

Claims

CLAIMS1. A method for reducing the biological age and / or for reducing the rate of increase of the biological age of an adult animal in need, the method comprising: feeding the adult animal a composition comprising an effective amount of Nicotinamide (NAM).

2. A method for reducing the biological age of a tissue, organ or physiological system in an adult animal in need, the method comprising feeding the adult animal a composition comprising an effective amount of Nicotinamide (NAM).

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

4. A method according to any of claims 2-3, wherein the tissue is skeletal muscle.

5. A method for treating, preventing, or delaying the onset of an organ disease, or decline of organ function, or condition relating thereto in an adult animal in need, the method comprising feeding the adult animal a composition comprising an effective amount of Nicotinamide (NAM).

6. A method according to any preceding claim, wherein the animal in need has an increased biological age compared to its chronological age.

7. A method according to any preceding claim, wherein the increased biological age is determined from the DNA methylation profile of the adult animal.

8. A method according to any preceding claim, wherein the animal is an ageing animal.

9. A method according to any preceding claim, wherein the animal is a pet animal.

10. A method according to any preceding claim, wherein the animal is a human.

11. A method according to any preceding claim, wherein the composition is selected from the group consisting of food compositions, dietary supplements, nutritionalcompositions, nutraceuticals, powdered nutritional products to be reconstituted in water or milk before consumption, food additives, medicaments, drinks, dry or wet pet food and combinations thereof.

12. A method according to any preceding claim, wherein the composition further contains at least one of glycine, spermidine, thymol or carvacrol.

13. A method according to any preceding claim, wherein the composition is part of, or administered together with, a dietary regime.

14. A method according to claim 13, where the dietary regime comprises a diet in which protein is 18% to 60% of protein / dry matter (w / w) for dogs.

15. A method according to claim 13, where the dietary regime comprises a diet in which protein is 26% to 60% of protein / dry matter (w / w) for cats.

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