Urolithin for improving heart function and health

Abstract

Methods and compositions for use in improving heart function and health are disclosed. The methods and compositions are based on urolithin.

Description

Cross - reference to related applications

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 349,327, filed Jun. 6, 2022.

Background Art

[0002] Today, rapidly increasing and aging populations are facing cardiac dysfunction such as myocardial infarction, coronary artery disease (CAD), congestive heart failure (CHF), angina, stroke, arrhythmia, atrial fibrillation, peripheral artery disease (PAD), heart disorders, arterial disorders, and disorders after heart transplantation. These heart diseases and disorders require "polymedication" in order for the affected individuals, especially middle - aged and elderly people, to maintain optimal function, reduce symptoms associated with cardiac dysfunction, and improve the quality of life. However, many commonly used pharmaceuticals that are prescribed and used to improve or treat cardiac dysfunction can cause or be associated with side effects. There remains an unmet need to improve or treat individuals suffering from cardiac dysfunction and reduce the side effects of medication.

[0003] Urolithin is a metabolite derived from ellagitannins and ellagic acid, produced, for example, by the colonic microflora (microbiota) of mammals, including human colonic microflora. Urolithin has a strong effect on the improvement of many health conditions and has been shown to be highly biologically active in vitro and in vivo. Urolithin has been proposed as a therapeutic agent for various conditions, including diseases and conditions related to cardiac dysfunction.

Summary of the Invention

Means for Solving the Problems

[0004] One aspect of the present invention is a method useful for improving and / or protecting cardiac function, reducing the rate of decline of cardiac function, preventing the decline of cardiac function, managing or treating peripheral artery disease (PAD).

[0005] Accordingly, provided herein is a method for improving cardiac function, the method comprising administering to a mammal in need thereof an effective amount of urolithin; wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg; and the mammal has myocardial infarction, coronary artery disease (CAD), congestive heart failure (CHF), angina pectoris, stroke, arrhythmia, fibrillation, peripheral artery disease (PAD), or a heart or arterial disorder.

[0006] In some embodiments, urolithin is administered after ischemia-reperfusion injury (e.g., at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, or about 1 week after ischemia-reperfusion injury).

[0007] In certain embodiments, cardiac hypertrophy caused by myocardial infarction is decreased in the mammal (e.g., cardiac hypertrophy in the left ventricle is decreased).

[0008] In certain aspects, provided herein is a method for improving cardiac function, the method comprising administering to a mammal in need thereof an effective amount of urolithin; wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0009] In certain aspects, provided herein is a method for protecting cardiac function, the method comprising administering to a mammal in need thereof an effective amount of urolithin; wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0010] In certain aspects, provided herein is a method for reducing the rate of decline of cardiac function, the method comprising administering to a mammal in need thereof an effective amount of urolithin; wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0011] In certain embodiments, provided herein is a method for preventing a decline in cardiac function, the method comprising administering to a mammal in need thereof an effective amount of urolithin; wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0012] In certain embodiments, provided herein is a method for managing or treating peripheral arterial disease (PAD), the method comprising administering to a mammal in need thereof an effective amount of urolithin; wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0013] In certain embodiments, provided herein is a method for managing or improving tissue oxygenation, the method comprising administering to a mammal in need thereof an effective amount of urolithin; wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0014] In certain embodiments, provided herein is a method for improving cardiac output (e.g., heart rate, stroke volume, heart rate variability), the method comprising administering to a mammal in need thereof an effective amount of urolithin; wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0015] In some embodiments, the cardiac output is the heart rate.

[0016] In certain embodiments, provided herein is a method for increasing ventricular ejection fraction, the method comprising administering to a mammal in need thereof an effective amount of urolithin; wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0017] In some embodiments, the left ventricular ejection fraction (LVEF) of the mammal increases.

[0018] In certain embodiments, the mammalian LVEF is less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, or less than about 25%, less than about 20% prior to administration of urolithin.

[0019] In some embodiments, the mammalian LVEF is about 5 - 75% (e.g., about 15 - 50%, about 15 - 40%, about 15 - 35%, about 20 - 45%, about 40 - 49%, or about 41 - 49%) prior to administration of urolithin.

[0020] In certain embodiments, the right ventricular ejection fraction (RVEF) of the mammal increases.

[0021] In certain embodiments, the mammalian RVEF is less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, or less than about 25%, less than about 20% prior to administration of urolithin.

[0022] In some embodiments, the mammalian RVEF is about 5 - 75% (e.g., about 15 - 50%, about 15 - 40%, about 15 - 35%, about 20 - 45%, about 40 - 49%, or about 41 - 49%) prior to administration of urolithin.

[0023] In certain embodiments, cardiac function is improved. For example, heart function with preserved ejection fraction (HFpEF) is improved, or heart function with reduced ejection fraction (HFrEF) is improved.

[0024] In some embodiments, cardiac contractile function is improved.

[0025] In certain embodiments, provided herein is a method of increasing cardiac contractile function, the method comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0026] In some embodiments, the cardiac contractile function of the mammal increases by about 20-90% (e.g., about 30-80% or 50-70%).

[0027] In certain embodiments, the cardiac contractile function of the mammal increases by about 56%.

[0028] In some embodiments, the cardiac contractile function of the mammal increases by about 64%.

[0029] In certain embodiments, the diastolic dysfunction of the mammal is reduced.

[0030] In some embodiments, the diastolic dysfunction of the mammal is reduced by about 5-10%.

[0031] In certain embodiments, provided herein is a method of increasing cardiac contractile function and skeletal muscle function, the method comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg; and the mammal is an aging mammal.

[0032] In some embodiments, the cardiac contractile function of the mammal increases by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60%.

[0033] In certain embodiments, the skeletal muscle function (e.g., skeletal muscle strength) of the mammal increases by about 1-50% (e.g., about 2-5%).

[0034] In certain embodiments, provided herein is a method of maintaining, supporting, or improving blood circulation, the method comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0035] In certain embodiments, provided herein is a method of maintaining, supporting, or improving cardiac function, the method comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0036] In certain embodiments, provided herein is a method of maintaining, supporting, or improving myocardial function, the method comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0037] In some embodiments, myocardial contractility increases.

[0038] In some embodiments, heart fibrosis decreases.

[0039] In some embodiments, cardiac mitochondrial function is improved.

[0040] In some embodiments, cardiac mitophagy is improved.

[0041] In some embodiments, plasma ceramide levels, acylcarnitine levels, brain natriuretic peptide (BNP) levels, creatine kinase (CK) levels, C-reactive protein (CRP) levels, troponin levels, or galectin-2 levels decrease.

[0042] In some embodiments, heart rate is improved.

[0043] In some embodiments, the heart rate variability increases.

[0044] In some embodiments, the circulation (e.g., blood flow) is improved.

[0045] In certain embodiments, the above method further comprises transplanting mitochondria into a mammal.

[0046] In some embodiments, the mitochondrial damage is improved.

[0047] In some embodiments, urolithin is administered orally.

[0048] In some embodiments, urolithin is administered in the form of tablets or capsules.

[0049] In some embodiments, urolithin is administered over a period of at least about 1 month (e.g., at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months).

[0050] In some embodiments, the mammal is a human.

[0051] In some embodiments, the human is an elderly person.

[0052] In some embodiments, the human is at least about 40 years old (e.g., at least about 50 years old, at least about 60 years old, at least about 70 years old, at least about 80 years old, or at least about 90 years old).

[0053] In some embodiments, the effective amount of urolithin is about 25 mg / kg.

[0054] In some embodiments, urolithin is selected from the group consisting of urolithin A, urolithin B, urolithin C, urolithin D, and any combination thereof.

[0055] In some embodiments, urolithin is selected from the group consisting of urolithin A, urolithin B, and combinations of urolithin A and urolithin B.

[0056] In certain embodiments, urolithin is urolithin A.

[0057] In certain embodiments, urolithin is urolithin B.

[0058] In certain embodiments, urolithin is urolithin C.

[0059] In certain embodiments, urolithin is urolithin D.

[0060] In some embodiments, the methods described herein further comprise conjointly administering an additional agent to a mammal.

[0061] In certain embodiments, the additional agent is a therapeutic agent (e.g., an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin receptor-neprilysin inhibitor (ARNi), a β-blocker, a mineralocorticoid receptor antagonist (MRA), a sodium-glucose cotransporter 2 (SGLT2) inhibitor (e.g., dapagliflozin, empagliflozin), an angiotensin-2 receptor antagonist (ARB), a diuretic, an If channel blocker, an aldosterone antagonist, aspirin, a P2Y12 inhibitor, a calcium channel blocker, a cholesterol-lowering agent (e.g., a statin), an antiarrhythmic agent, a hydrazine containing nitrate, digoxin, and an anticoagulant).

[0062] In some embodiments, the additional agent is a nutritional supplement (e.g., omega (ω)-3 fatty acids, nitrate-rich beet root, nitric oxide, red yeast rice, β-glucan, vitamin A, vitamin C, vitamin E, vitamin K, potassium salts, magnesium salts, calcium salts, iron salts, manganese salts, copper salts, zinc salts, and phosphates).

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0064] The development of methods for improving cardiac function and health by urolithin required substantial innovation. Quantitative analysis of cardiac contractile function, diastolic function, hypertrophy, and skeletal muscle function was performed, demonstrating that urolithin A has a protective effect against heart failure and a cardioprotective effect during natural aging.

[0065] Definitions The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element or a plurality of elements.

[0066] The phrase "and / or" as used in the present specification and claims is to be understood to mean "either or both" of the elements so conjoined, i.e., elements that may be present conjunctively in some cases and disjunctively in other cases. Multiple elements listed using "and / or" are to be construed in like manner, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present whether or not they are related to or unrelated to the specifically identified elements specifically identified by the "and / or" clause. Thus, by way of non-limiting example, reference to "A and / or B", when used in conjunction with open-ended language such as "comprising", in one embodiment refers to only A (optionally including elements other than B); in another embodiment, to only B (optionally including elements other than A); and in yet another embodiment, to both A and B (optionally including other elements).

[0067] As used in this specification and the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one of the number of elements or items in the list, but also more than one, and optionally also including additional unlisted items. Only terms that clearly indicate the contrary, such as "only one of" or "exactly one of", or "consisting of" as used in the claims, refer to exactly one element of the number of elements or the list. Generally, the term "or" as used in this specification should be interpreted as indicating an exclusive alternative (i.e., "either one and not both") only when preceded by terms of exclusive alternatives such as "either", "one", "only one", "exactly one", etc. When used in the claims, the term "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0068] As used in this specification and the claims, the phrase "at least one" shall be understood to mean at least one element selected from any one or more of the elements in a list of one or more elements, but not necessarily including at least one of each and every element specifically recited in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for the possibility that elements other than those specifically identified in the list of elements referred to by the phrase "at least one" may optionally be present, whether or not they are related to the specifically identified elements. Thus, by way of non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B", or, equivalently, "at least one of A and / or B") may, in one embodiment, include at least one (optionally more than one) A where B is not present (and optionally includes elements other than B); in another embodiment, include at least one (optionally more than one) B where A is not present (and optionally includes elements other than A); in yet another embodiment, include at least one (optionally more than one) A and at least one (optionally more than one) B (and optionally includes other elements); and so forth.

[0069] It should also be understood that, unless the contrary is clearly indicated, in any method claimed in this specification that includes a plurality of steps or acts, the order of the steps or acts of that method is not necessarily limited to the order in which the steps or acts of that method are recited.

[0070] In the claims and the above specification, all transitional phrases such as "comprising", "including", "carrying", "having", "containing", "involving", "holding", "composed of", etc. are open-ended, i.e., it should be understood to mean including but not limited to. As described in Section 2111.03 of the Patent Examining Procedures of the United States Patent and Trademark Office, only the transitional phrases "consisting of" and "consisting essentially of" are closed or semi-closed transitional phrases, respectively.

[0071] As used herein, the term "prodrug" encompasses compounds that are converted to a therapeutically active agent under physiological conditions. A common method for making a prodrug is to include a selected moiety that is hydrolyzed under physiological conditions to expose the desired molecule. In other embodiments, the prodrug is converted by the enzymatic activity of the host animal.

[0072] As used herein, the terms "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" mean pharmaceutically acceptable materials, compositions or vehicles such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials that are involved in transporting or delivering the subject chemical substance from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, not injurious to the patient, and substantially nonpyrogenic. Some examples of substances that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical formulations. In certain embodiments, the pharmaceutical compositions of the invention are nonpyrogenic, i.e., they do not induce a significant increase in temperature when administered to a patient.

[0073] The term "pharmaceutically acceptable salts" refers to relatively non-toxic inorganic and organic acid addition salts of a compound. These salts can be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt so formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and laurylsulfonate (see, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).

[0074] In other cases, the compounds useful in the methods of the present invention may contain one or more acidic functional groups and can thus form pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these cases refers to relatively non-toxic inorganic and organic base addition salts of the compound. These salts can similarly be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free acid form with a suitable base, such as a hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, ammonia, or a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Representative alkali or alkaline earth salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, and aluminum salts. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, etc. (see, e.g., Berge et al. supra).

[0075] The "therapeutically effective amount" (or "effective amount") of a compound for use in therapy refers to the amount of the compound in a formulation that, when administered (to a mammal, preferably a human) as part of a desired dosage regimen, alleviates symptoms, improves a condition, or delays the onset of a disease condition, according to clinically acceptable criteria for the disorder or condition being treated or for cosmetic purposes, e.g., at a reasonable benefit / risk ratio applicable to any medical treatment.

[0076] The terms "prophylactic" or "therapeutic" treatment are recognized in the art and include administration of one or more subject compositions to a host. When administered before the clinical signs of an undesirable condition (e.g., a disease or other undesirable condition in the host animal) appear, the treatment is prophylactic (i.e., it protects the host from developing the undesirable condition), whereas when administered after the signs of the undesirable condition appear, the treatment is therapeutic (i.e., it is intended to alleviate, improve, or stabilize the existing undesirable condition or its side effects).

[0077] The terms "patient" or "subject" refer to a mammal in need of a particular treatment. In certain embodiments, the patient is a primate, dog, cat, or horse. In certain embodiments, the patient is a human.

[0078] "Effective amount" means an amount sufficient to produce a beneficial or desired result. For example, a therapeutic amount is an amount that achieves the desired therapeutic effect. This amount may or may not be the same as a prophylactically effective amount, which is the amount necessary to prevent the onset of a disease or disease symptom. An effective amount can be administered in one or more administrations, applications, or dosages. The therapeutically effective amount of a composition depends on the composition selected. The composition can be administered once or multiple times a day, or once or multiple times a week, including once every other day. One of ordinary skill in the art will understand that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the overall health status and / or age of the subject, and other diseases present, can affect the dosage and timing required to effectively treat the subject. Further, treatment of a subject with a therapeutically effective amount of the compositions described herein may include a single treatment or a series of treatments.

[0079] The terms "decrease", "reduce", "reduced", "reduction", and "inhibit" are all generally used herein to mean a decrease by a statistically significant amount relative to a reference. However, to avoid ambiguity, "reduce", "reduction" or "decrease" or "inhibit" typically means a decrease of at least 10% compared to a reference level, e.g., at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% decrease, e.g., up to and including complete absence of a given entity or parameter compared to a reference level, or any decrease between 10-99% compared to the absence of a given treatment.

[0080] The terms "increased", "increase", "enhance", or "activate" are all generally used herein to mean an increase by a statically significant amount; to avoid any ambiguity, the terms "increased", "increase", "enhance", or "activate" mean an increase of at least 10% compared to a reference level, for example, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to a 100% increase (including a 100% increase), or any increase between 10 - 100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2 - 10-fold or greater compared to the reference level.

[0081] As used herein, the term "modulate" includes upregulation and downregulation, for example, includes enhancing or inhibiting a response.

[0082] "Optional" or "optionally" means that the event or circumstance described below may or may not occur, and that the description includes both the case where the event or circumstance occurs and the case where it does not occur. For example, "optionally substituted aryl" means that the aryl group may or may not be substituted, and that the description includes both the substituted aryl group and the aryl group having no substitution.

[0083] As used herein, the term "treat" when used in connection with a subject disease, disorder, or condition means reducing by a detectable amount at least one clinical or objective symptom of the subject disease, disorder, or condition. In one embodiment, the term "treat" when used in connection with a subject disease, disorder, or condition means curing the subject disease, disorder, or condition.

[0084] As used herein, "food product" refers to a product prepared from natural foods. Non-limiting examples of food products include juices, wines, concentrates, jams, jellies, preserves, pastes, and extracts.

[0085] As used herein, "nutritional supplement" refers to a product suitable for ingestion or other administration primarily for its health-promoting properties rather than for its calorie content.

[0086] As used herein, the term "alkyl" refers to a straight-chain or branched-chain, acyclic or cyclic, unsaturated or saturated aliphatic hydrocarbon group containing carbon atoms. Representative saturated straight-chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, etc.; saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, etc. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.; unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, etc. Unsaturated alkyls contain at least one double bond or triple bond between adjacent carbon atoms (referred to as "alkenyl" or "alkynyl", respectively). Representative straight-chain and branched alkenyls include ethenyl, propenyl, 1-butenyl, 2-butenyl, isobutenylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, etc.; representative straight-chain and branched alkynyls include ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, etc.

[0087] As used herein, the term "aryl" refers to a hydrocarbon ring system radical containing hydrogen, 6 to 18 carbon atoms and at least one aromatic ring. For the purposes of the present invention, the aryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system and may include a fused or bridged ring system. Aryl radicals include, but are not limited to, aryl radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene and triphenylene. Unless otherwise specifically stated herein, the term "aryl" or the prefix "ar-" (such as in "aralkyl") means including an optionally substituted aryl radical.

[0088] As used herein, the term "monosaccharide" refers to a simple sugar of the formula (CH2O) n . Monosaccharides can be linear or cyclic and can contain sucrose units of the formula -CH(OH)-C(=O)-. Examples of monosaccharides include erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, erythrose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, erythro-pentulose, threo-pentulose, glycerotetrulose, glucopyranose, and fructofuranose. In certain embodiments, the monosaccharide refers to glucopyranose.

[0089] As used herein, the term "oligosaccharide" refers to a sugar consisting of at least two and up to ten glycosidically linked monosaccharide units, preferably two to eight monosaccharide units, more preferably two to seven monosaccharide units, even more preferably two to six monosaccharide units or two to five monosaccharide units.

[0090] As used herein, the term "substituted" (e.g., in the context of a substituted heterocycle or a substituted aryl) means that at least one hydrogen atom is replaced by a substituent. "Substituents" in the context of the present invention include halogen, hydroxy, oxo, cyano, nitro, imino, thioxo, amino, alkylamino, dialkylamino, alkyl, alkoxy, alkylthio, haloalkyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, heterocycle and heterocycloalkyl, and -NRaRb, -NRaC(=O)Rb, -NRaC(=O)NRaNRb, -NRaC(=O)ORb -NRaSO2Rb, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRb, -OC(=O)NRaRb, -ORa, -SRa, -SORa, -S(=O)2Ra, -OS(=O)2Ra, -S(=O)2ORa, =NSO2Ra. In the above, Ra and Rb in this context may be the same or different and may independently be hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, aralkyl, heterocyclyl. Also, the above substituents may be further substituted with one or more of the above substituents.

[0091] The method of the present invention In accordance with the present invention, it has been discovered that urolithin unexpectedly improves cardiac contractile function, diastolic function, hypertrophy, and skeletal muscle function. The methods of the present invention are useful for the improvement and / or protection of cardiac function, the reduction of the rate of decline of cardiac function, the prevention of the decline of cardiac function, the management or treatment of peripheral arterial disease (PAD).

[0092] In certain embodiments according to this and other aspects of the present invention, urolithin is urolithin A. In certain embodiments according to this and other aspects of the present invention, urolithin is urolithin B. In certain embodiments according to this and other aspects of the present invention, urolithin is urolithin C. In certain embodiments according to this and other aspects of the present invention, urolithin is urolithin D.

[0093] In certain embodiments, provided herein is a method of improving cardiac function, the method comprising administering to a mammal in need thereof an effective amount of urolithin; wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg; and wherein the mammal has myocardial infarction, coronary artery disease (CAD), congestive heart failure (CHF), angina pectoris, stroke, arrhythmia, fibrillation, peripheral artery disease (PAD), or a heart or arterial disorder.

[0094] Ischemia / reperfusion (I / R) injury is a causative factor contributing to morbidity and mortality. The vulnerability of the liver to I / R injury has been a major obstacle to liver resection and transplantation surgeries where reperfusion after prolonged ischemia is inevitable during hepatectomy and vascular reconstruction. Mitochondrial dysfunction is known to be one of the important downstream events leading to I / R-mediated cell death.

[0095] In some embodiments, urolithin is administered after ischemia-reperfusion injury (e.g., at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days or about 1 week after ischemia-reperfusion injury).

[0096] In certain embodiments, cardiac hypertrophy caused by myocardial infarction is decreased in the mammal (e.g., cardiac hypertrophy in the left ventricle is decreased).

[0097] In certain embodiments, provided herein is a method of improving cardiac function, the method comprising administering to a mammal in need thereof an effective amount of urolithin; wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0098] In certain embodiments, provided herein is a method of protecting cardiac function, the method comprising administering to a mammal in need thereof an effective amount of urolithin; wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0099] In certain embodiments, provided herein is a method of reducing the rate of decline of cardiac function, the method comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0100] In certain embodiments, provided herein is a method of preventing decline of cardiac function, the method comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0101] In certain embodiments, provided herein is a method of managing or treating peripheral arterial disease (PAD), the method comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0102] In certain embodiments, provided herein is a method of managing or improving tissue oxygenation, the method comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0103] In certain embodiments, provided herein is a method of improving cardiac output (e.g., heart rate, stroke volume, heart rate variability), the method comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0104] In some embodiments, the cardiac output is the heart rate.

[0105] In certain embodiments, provided herein is a method of increasing ventricular ejection fraction, the method comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0106] In some embodiments, the left ventricular ejection fraction (LVEF) of the mammal increases.

[0107] In certain embodiments, the LVEF of the mammal is less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, or less than about 25%, less than about 20% prior to administration of urolithin.

[0108] In some embodiments, the LVEF of the mammal is from about 5 - 75% (e.g., about 15 - 50%, about 15 - 40%, about 15 - 35%, about 20 - 45%, about 40 - 49%, or about 41 - 49%) prior to administration of urolithin.

[0109] In certain embodiments, the right ventricular ejection fraction (RVEF) of the mammal increases.

[0110] In certain embodiments, the RVEF of the mammal is less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, or less than about 25%, less than about 20% prior to administration of urolithin.

[0111] In some embodiments, the RVEF of the mammal is from about 5 - 75% (e.g., about 15 - 50%, about 15 - 40%, about 15 - 35%, about 20 - 45%, about 40 - 49%, or about 41 - 49%) prior to administration of urolithin.

[0112] In certain embodiments, cardiac function is improved. For example, cardiac function with preserved ejection fraction (HFpEF) is improved or cardiac function with reduced ejection fraction (HFrEF) is improved.

[0113] In some embodiments, cardiac contractile function is improved.

[0114] In certain aspects, provided herein is a method of increasing cardiac contractile function, the method comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0115] In some embodiments, the cardiac contractile function of the mammal increases by about 20 - 90% (e.g., about 30 - 80%, or 50 - 70%).

[0116] In certain embodiments, the cardiac contractile function of the mammal increases by about 56%.

[0117] In some embodiments, the cardiac contractile function of the mammal increases by about 64%.

[0118] In certain embodiments, diastolic dysfunction of the mammal is reduced.

[0119] In some embodiments, diastolic dysfunction of the mammal decreases by about 5 - 10%.

[0120] The myocardium undergoes a progressive decline in mitochondrial function, an increase in reactive oxygen species, and an increase in the accumulation of defective organelles, similar to that observed in skeletal muscle. The clearance of these damaged organelles by autophagy is important for maintaining myocardial function. Since autophagy decreases with aging, promotion of autophagy can help protect myocardial function.

[0121] The myocardium is also strongly exposed to ischemic episodes during myocardial infarction. The level of myocardial damage caused by these ischemic episodes strongly depends on the ability of the cells to initiate an effective autophagy response to clear damaged organelles. In aged animals, defects in the autophagy response result in an increase in myocardial damage after ischemic events. Therefore, promoting autophagy during these acute events can help protect the myocardium from damage.

[0122] In certain embodiments, provided herein is a method of increasing cardiac contractile function and skeletal muscle function, the method comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg, and the mammal is an aged mammal.

[0123] In some embodiments, the cardiac contractile function of the mammal increases by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60%.

[0124] In certain embodiments, the skeletal muscle function of the mammal increases by about 1 - 50% (e.g., about 2 - 5%).

[0125] In certain embodiments, provided herein is a method of maintaining, supporting, or improving blood circulation, the method comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0126] In certain embodiments, provided herein is a method of maintaining, supporting, or improving cardiac function, the method comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0127] In certain embodiments, provided herein is a method of maintaining, supporting, or improving myocardial function, the method comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

[0128] In some embodiments, myocardial contractility is increased.

[0129] In some embodiments, cardiac fibrosis is decreased.

[0130] In some embodiments, cardiac mitochondrial function is improved.

[0131] In some embodiments, the quality of cardiac mitochondria is improved.

[0132] In some embodiments, the area of cardiac mitochondria is increased.

[0133] In some embodiments, the expression of genes related to mitochondrial function in cardiac tissue is upregulated.

[0134] In some embodiments, the gene is a gene of a mitochondrial protein-containing complex or a gene of a respirasome.

[0135] In some embodiments, cardiac mitophagy is improved.

[0136] In some embodiments, plasma ceramide levels, acylcarnitine levels, brain natriuretic peptide (BNP) levels, creatine kinase (CK) levels, C-reactive protein (CRP) levels, troponin levels, or galectin-2 levels are decreased.

[0137] In some embodiments, the heart rate is improved.

[0138] In some embodiments, the heart rate variability increases.

[0139] In some embodiments, the circulation is improved.

[0140] In certain embodiments, the method further comprises transplanting mitochondria into a mammal.

[0141] In some embodiments, the mitochondrial damage is improved.

[0142] In some embodiments, urolithin is administered orally.

[0143] In some embodiments, urolithin is administered in the form of tablets or capsules.

[0144] In some embodiments, urolithin is administered over a period of at least about 1 month (e.g., at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months).

[0145] In some embodiments, the mammal is a human.

[0146] In some embodiments, the human is an elderly person.

[0147] In some embodiments, the human is at least about 40 years old (e.g., at least about 50 years old, at least about 60 years old, at least about 70 years old, at least about 80 years old, at least about 90 years old).

[0148] In some embodiments, the effective amount of urolithin is about 25 mg / kg.

[0149] In some embodiments, urolithin is selected from the group consisting of urolithin A, urolithin B, urolithin C, urolithin D, and any combination thereof.

[0150] In some embodiments, urolithin is selected from the group consisting of urolithin A, urolithin B, and a combination of urolithin A and urolithin B.

[0151] In certain embodiments, urolithin is urolithin A.

[0152] In certain embodiments, urolithin is urolithin B.

[0153] In certain embodiments, urolithin is urolithin C.

[0154] In certain embodiments, urolithin is urolithin D.

[0155] In some embodiments, the methods described herein further comprise co-administering an additional agent to a mammal.

[0156] In certain embodiments, the additional agent is a therapeutic agent (e.g., an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin receptor-neprilysin inhibitor (ARNi), a β-blocker, a mineralocorticoid receptor antagonist (MRA), a sodium-glucose cotransporter 2 (SGLT2) inhibitor (e.g., dapagliflozin, empagliflozin), an angiotensin-2 receptor antagonist (ARB), a diuretic, an If channel blocker, an aldosterone antagonist, aspirin, a P2Y12 inhibitor, a calcium channel blocker, a cholesterol-lowering agent (e.g., a statin), an antiarrhythmic agent, hydralazine containing nitrate, digoxin, and an anticoagulant).

[0157] In some embodiments, the additional agent is a nutritional supplement (e.g., fish oil (e.g., omega (ω)-3 fatty acids)), nitrate-rich beetroot, nitric oxide, red yeast rice, β-glucan, vitamin A, vitamin C, vitamin E, vitamin K, potassium salts, magnesium salts, calcium salts, iron salts, manganese salts, copper salts, zinc salts, phosphates, coenzyme q10 (Coq10), carnitine, grape seed extract, or cocoa).

[0158] Exemplary compounds of the present invention Urolithin can be used to carry out any of the methods herein, including, but not limited to, improving and / or protecting heart function, reducing the rate of decline of heart function, preventing decline of heart function, managing or treating peripheral arterial disease (PAD).

[0159] As used herein, "urolithin" refers to a compound of formula I:

Chemical formula

[0160] In one embodiment, "urolithin" refers to any one or any combination of urolithin A, urolithin B, urolithin C, and urolithin D.

[0161] In some embodiments, "urolithin" is glucuronidated, methylated, or sulfated urolithin, or in the case of a combination, may include them.

[0162] In some embodiments, urolithin is urolithin A.

[0163] In some embodiments, urolithin is urolithin B.

[0164] In some embodiments, urolithin is urolithin C.

[0165] In some embodiments, urolithin is urolithin D.

[0166] Formulations and clinical use Urolithin can be administered to a subject (e.g., a mammal) in various ways, either alone or together with another agent. For example, urolithin can be administered orally or parenterally. Parenteral administration includes, but is not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, intra-articular, intrasynovial, intraocular, intrathecal, topical, or by inhalation. Thus, the dosage forms of urolithin can be in various forms, including natural foods, processed foods, natural juices, concentrates and extracts, injection solutions, microcapsules, nanocapsules, liposomes, plasters, inhalation forms, nasal sprays, nasal drops, eye drops, sublingual tablets, and sustained release (or extended release) formulations.

[0167] The compounds of the present invention can be provided in isolated form. As used herein, the term "isolated" means a state in which the compound of interest is substantially removed from other compounds or components that might otherwise be found together, such as when found in nature. In one embodiment, the compound is isolated when it is essentially completely removed from other compounds or components that might otherwise be found together with the compound of interest. In one embodiment, the compound is isolated when it is pure.

[0168] The compounds of the present invention can be incorporated into various formulations for therapeutic administration. More specifically, the compounds of the present invention can be formulated into pharmaceutical compositions by combining them with suitable pharmaceutically acceptable carriers or diluents, and can be formulated into preparations in solid, semi-solid, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. Therefore, administration of the compounds can be achieved by various methods such as oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, and intratracheal administration. The active agent may be systemic after administration, or may be localized by regional administration, use of intramural administration, or use of an implant that acts to maintain an active dose at the site of implantation.

[0169] The compounds of the present invention can also be formulated as food additives, food ingredients, functional foods, dietary supplements, medical foods, nutraceuticals, or food supplements.

[0170] In pharmaceutical dosage forms, the compounds can be administered in the form of their pharmaceutically acceptable salts. They can also be used in suitable association with other pharmaceutically active compounds. The following methods and excipients are merely illustrative and in no way limiting.

[0171] In the case of oral preparations, the compound can be used alone or in combination with suitable additives to prepare tablets, powders, granules or capsules. For example, it can be used in combination with conventional additives such as lactose, mannitol, corn starch or potato starch; in combination with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatin; in combination with disintegrants such as corn starch, potato starch or sodium carboxymethyl cellulose; in combination with lubricants such as talc or magnesium stearate; and, if desired, in combination with diluents, buffers, wetting agents, preservatives and flavoring agents.

[0172] The compounds can be formulated into injectable preparations by dissolving, suspending or emulsifying them in aqueous or non-aqueous solvents such as vegetable oils or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol, together with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives as required.

[0173] The compounds can also be used in aerosol preparations administered by inhalation. The compounds of the present invention can be incorporated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen.

[0174] Furthermore, the compounds can be made into suppositories by mixing with various bases such as emulsifying bases or water-soluble bases. The compounds of the present invention may be administered rectally as suppositories. Suppositories can contain vehicles such as cocoa butter, carbowax and polyethylene glycol, which melt at body temperature but solidify at room temperature.

[0175] Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, may be provided, and each dosage unit, e.g., a tablespoonful, a teaspoonful, a tablet, or a suppository, contains a predetermined amount of a composition comprising one or more compounds of the present invention. Similarly, unit dosage forms for injection or intravenous administration can contain the compounds of the present invention in the composition as a solution in sterile water, physiological saline, or another pharmaceutically acceptable carrier, and each dosage unit, e.g., mL or L, contains a predetermined amount of a composition containing one or more compounds of the present invention.

[0176] Implants for sustained release formulations are well known in the art. Implants are formulated as microspheres, slabs, etc. using biodegradable or non-biodegradable polymers. For example, polymers of lactic acid and / or glycolic acid form erodible polymers that are well tolerated by the host. Implants containing inhibitory compounds can be placed in proximity to the target site such that the local concentration of the active agent is high compared to the rest of the body.

[0177] The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, and each unit contains a predetermined amount of a compound of the present invention calculated to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound used, the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0178] Pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers, or diluents are generally readily available. Additionally, pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers, isotonic agents, stabilizers, wetting agents, etc. are also generally readily available.

[0179] For clinical use, urolithin is administered in a therapeutically effective amount.

[0180] The compounds of the present invention can also be formulated as food additives, food ingredients, functional foods, dietary supplements, medical foods, nutraceuticals, or food supplements. In certain embodiments, the compounds of the present invention can be administered in a convenient form in a daily dose by including them in nutritional beverages of various volumes. As a non-limiting example, the beverage can deliver an effective dose in a final volume ranging from 5 mL to 1,000 mL, delivered as a single dose or multiple doses. In certain embodiments, the compositions and methods of the present invention are utilized for and in non-human animals. Thus, the compounds and compositions of the present invention can be formulated as veterinary products. The compounds and compositions can also be formulated as functional foods for administration to animals such as, for example, dogs, cats, horses, etc.

[0181] Administration is generally daily to weekly. In one embodiment, administration is at least weekly. For example, a subject can receive administration once a week, twice a week, three times a week, or once every other day. In one embodiment, administration is at least daily. For example, a subject may receive administration once or multiple times a day.

[0182] Administration for obtaining maximum effectiveness in humans is considered to include daily administration over a long period. Long-term use is contemplated to include use for one month, two months, three months, four months, five months, six months, or even longer periods.

[0183] For clinical use, urolithin can be administered to treat either chronic or acute conditions. Since autophagy has been observed to increase in cells after less than one day, for example, after 8 hours, urolithin can be administered acutely to treat conditions that acutely require induction of autophagy. Such cases can include reperfusion injury (e.g., organ transplantation), heart attack, stroke, conditions due to ischemic injury, conditions during surgery (e.g., during angioplasty or heart valve replacement), or conditions after traumatic injury.

[0184] For clinical use, the urolithin is usually administered in an amount equal to or equivalent to 0.2 to 2000 milligrams (mg) of urolithin per kilogram of the subject's body weight per day. In one embodiment, the urolithin is administered in a dose equal to or equivalent to 2 to 2000 mg of urolithin per kilogram of the subject's body weight per day. In one embodiment, the urolithin is administered in a dose equal to or equivalent to 20 to 2000 mg of urolithin per kilogram of the subject's body weight per day. In one embodiment, the urolithin is administered in a dose equal to or equivalent to 50 to 2000 mg of urolithin per kilogram of the subject's body weight per day. In one embodiment, the urolithin is administered in a dose equal to or equivalent to 100 to 2000 mg of urolithin per kilogram of the subject's body weight per day. In one embodiment, the urolithin is administered in a dose equal to or equivalent to 200 to 2000 mg of urolithin per kilogram of the subject's body weight per day.

[0185] Formulations of urolithin can be administered to human subjects in a therapeutically effective amount. Typical dosage ranges are from about 0.01 μg / kg body weight / day to about 2 mg / kg body weight / day. The dosage of the drug administered will likely depend on variables such as the type and degree of the disorder, the overall health of the particular subject, the particular compound being administered, the excipients used to formulate the compound, and the route of administration. Routine experimentation can be used to optimize the dosage and frequency of administration for any particular compound.

[0186] In one embodiment, urolithin is administered at a concentration in the range of from about 0.001 μg / kg to greater than about 500 mg / kg. For example, the concentration can be 0.001 μg / kg, 0.01 μg / kg, 0.05 μg / kg, 0.1 μg / kg, 0.5 μg / kg, 1.0 μg / kg, 10.0 μg / kg, 50.0 μg / kg, 100.0 μg / kg, 500 μg / kg, 1.0 mg / kg, 5.0 mg / kg, 10.0 mg / kg, 15.0 mg / kg, 20.0 mg / kg, 25.0 mg / kg, 30.0 mg / kg, 35.0 mg / kg, 40.0 mg / kg, 45.0 mg / kg, 50.0 mg / kg, 60.0 mg / kg, 70.0 mg / kg, 80.0 mg / kg, 90.0 mg / kg, 100.0 mg / kg, 150.0 mg / kg, 200.0 mg / kg, 250.0 mg / kg, 300.0 mg / kg, 350.0 mg / kg, 400.0 mg / kg, 450.0 mg / kg, greater than about 500.0 mg / kg up to and including those values, or any incremental value therebetween. It is to be understood that all values and ranges between these values and ranges are intended to be encompassed by the present invention.

[0187] In one embodiment, urolithin is administered at a dosage in the range of about 0.2 milligrams (mg) / kg / day to more than about 100 mg / kg / day. For example, the dosage can be 0.2 mg / kg / day to 100 mg / kg / day, 0.2 mg / kg / day to 50 mg / kg / day, 0.2 mg / kg / day to 25 mg / kg / day, 0.2 mg / kg / day to 10 mg / kg / day, 0.2 mg / kg / day to 7.5 mg / kg / day, 0.2 mg / kg / day to 5 mg / kg / day, 0.25 mg / kg / day to 100 mg / kg / day, 0.25 mg / kg / day to 50 mg / kg / day, 0.25 mg / kg / day to 25 mg / kg / day, 0.25 mg / kg / day to 10 mg / kg / day, 0.25 mg / kg / day to 7.5 mg / kg / day, 0.25 mg / kg / day to 5 mg / kg / day, 0.5 mg / kg / day to 50 mg / kg / day, 0.5 mg / kg / day to 25 mg / kg / day, 0.5 mg / kg / day to 20 mg / kg / day, 0.5 mg / kg / day to 15 mg / kg / day, 0.5 mg / kg / day to 10 mg / kg / day, 0.5 mg / kg / day to 7.5 mg / kg / day, 0.5 mg / kg / day to 5 mg / kg / day, 0.75 mg / kg / day to 50 mg / kg / day, 0.75 mg / kg / day to 25 mg / kg / day, 0.75 mg / kg / day to 20 mg / kg / day, 0.75 mg / kg / day to 15 mg / kg / day, 0.75 mg / kg / day to 10 mg / kg / day, 0.75 mg / kg / day to 7.5 mg / kg / day, 0.75 mg / kg / day to 5 mg / kg / day, 1.0 mg / kg / day to 50 mg / kg / day, 1.0 mg / kg / day to 25 mg / kg / day, 1.0 mg / kg / day to 20 mg / kg / day, 1.0 mg / kg / day to 15 mg / kg / day, 1.0 mg / kg / day to 10 mg / kg / day, 1.0 mg / kg / day to 7.5 mg / kg / day, 1.0 mg / kg / day to 5 mg / kg / day, 2 mg / kg / day to 50 mg / kg / day, 2 mg / kg / day to 25 mg / kg / day, 2 mg / kg / day to 20 mg / kg / day, 2 mg / kg / day to 15 mg / kg / day, 2 mg / kg / day to 10 mg / kg / day, 2 mg / kg / day to 7.5 mg / kg / day, or 2 mg / kg / day to 5 mg / kg / day.

[0188] In one embodiment, urolithin is administered at a dosage in the range of about 0.25 milligrams (mg) / kg / day to about 25 mg / kg / day. For example, the dosage can be 0.25 mg / kg / day, 0.5 mg / kg / day, 0.75 mg / kg / day, 1.0 mg / kg / day, 1.25 mg / kg / day, 1.5 mg / kg / day, 1.75 mg / kg / day, 2.0 mg / kg / day, 2.25 mg / kg / day, 2.5 mg / kg / day, 2.75 mg / kg / day, 3.0 mg / kg / day, 3.25 mg / kg / day, 3.5 mg / kg / day, 3.75 mg / kg / day, 4.0 mg / kg / day, 4.25 mg / kg / day, 4.5 mg / kg / day, 4.75 mg / kg / day, 5 mg / kg / day, 5.5 mg / kg / day, 6.0 mg / kg / day, 6.5 mg / kg / day, 7.0 mg / kg / day, 7.5 mg / kg / day, 8.0 mg / kg / day, 8.5 mg / kg / day, 9.0 mg / kg / day, 9.5 mg / kg / day, 10 mg / kg / day, 11 mg / kg / day, 12 mg / kg / day, 13 mg / kg / day, 14 mg / kg / day, 15 mg / kg / day, 16 mg / kg / day, 17 mg / kg / day, 18 mg / kg / day, 19 mg / kg / day, 20 mg / kg / day, 21 mg / kg / day, 22 mg / kg / day, 23 mg / kg / day, 24 mg / kg / day, 25 mg / kg / day, 26 mg / kg / day, 27 mg / kg / day, 28 mg / kg / day, 29 mg / kg / day, 30 mg / kg / day, 31 mg / kg / day, 32 mg / kg / day, 33 mg / kg / day, 34 mg / kg / day, 35 mg / kg / day, 36 mg / kg / day, 37 mg / kg / day, 38 mg / kg / day, 39 mg / kg / day, 40 mg / kg / day, 41 mg / kg / day, 42 mg / kg / day, 43 mg / kg / day, 44 mg / kg / day, 45 mg / kg / day, 46 mg / kg / day, 47 mg / kg / day, 48 mg / kg / day, 49 mg / kg / day, or 50 mg / kg / day.

[0189] In another embodiment, urolithin is administered at a concentration in the range of 0.01 micromolar (μM) to 500 μM or more. For example, the dosage can be 0.01 μM, 0.02 μM, 0.05 μM, 0.1 μM, 0.15 μM, 0.2 μM, 0.5 μM, 0.7 μM, 1.0 μM, 3.0 μM, 5.0 μM, 7.0 μM, 10.0 μM, 15.0 μM, 20.0 μM, 25.0 μM, 30.0 μM, 35.0 μM, 40.0 μM, 45.0 μM, 50.0 μM, 60.0 μM, 70.0 μM, 80.0 μM, 90.0 μM, 100.0 μM, 150.0 μM, 200.0 μM, 250.0 μM, 300.0 μM, 350.0 μM, 400.0 μM, 450.0 μM, greater than about 500.0 μM from these, or any incremental value thereof. It is to be understood that all values and ranges between these values and ranges are intended to be encompassed by the present invention.

[0190] In yet another embodiment, urolithin is administered at a concentration in the range of 0.10 μg / mL to 500.0 μg / mL. For example, the concentration can be 0.10 μg / mL, 0.50 μg / mL, 1 μg / mL, 2.0 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, 50 μg / mL, 60.0 μg / mL, 70.0 μg / mL, 80.0 μg / mL, 90.0 μg / mL, 100.0 μg / mL, 150.0 μg / mL, 200.0 μg / mL, 250.0 μg / mL, 250.0 μg / mL, 300.0 μg / mL, 350.0 μg / mL, 400.0 μg / mL, 450.0 μg / mL, greater than about 500.0 μg / mL from these, or any incremental value thereof. It is to be understood that all values and ranges between these values and ranges are intended to be encompassed by the present invention.

[0191] Table 1 provides further non-limiting exemplary examples of urolithin dosages that can be used.

Table 1

[0192] Table 2 provides further non-limiting exemplary examples of the range of urolithin dosages that can be used. [Table 2] TIFF2025521186000005.tif46115

[0193] Any dosage can be given as a single dose or divided doses.

[0194] In one embodiment, urolithin is administered in a dosage sufficient to achieve peak serum levels of at least 0.001 micromolar concentration (μM) of urolithin and its known metabolites (such as glucoronide, sulfate, etc.). In one embodiment, urolithin is administered in a dosage sufficient to achieve a peak serum level of at least 0.01 μM of urolithin. In one embodiment, urolithin is administered in a dosage sufficient to achieve a peak serum level of at least 0.1 μM of urolithin. In one embodiment, urolithin is administered in a dosage sufficient to achieve a peak serum level of at least 1 μM of urolithin. In various embodiments, urolithin is administered in a dosage sufficient to achieve a peak serum level of at least 10 μM, at least 20 μM, at least 30 μM, at least 40 μM, at least 50 μM, at least 60 μM, at least 70 μM, at least 80 μM, at least 90 μM, at least 100 μM, or at least 200 μM of urolithin.

[0195] In one embodiment, urolithin is administered at a dose sufficient to achieve a sustained serum level of urolithin of at least 0.001 micromolar (μM). In one embodiment, urolithin is administered at a dose sufficient to achieve a sustained serum level of urolithin of at least 0.01 μM. In one embodiment, urolithin is administered at a dose sufficient to achieve a sustained serum level of urolithin of at least 0.1 μM. In one embodiment, urolithin is administered at a dose sufficient to achieve a sustained serum level of urolithin of at least 1 μM. In one embodiment, urolithin is administered at a dose sufficient to achieve a sustained serum level of urolithin of at least 10 μM, and urolithin is administered at a dose sufficient to achieve a sustained serum level of urolithin of at least 50 μM. The sustained serum level can be measured using any suitable method, such as high performance liquid chromatography (HPLC) or HPLC-MS.

Example

[0196] Although the present invention has been generally described above, it can be more easily understood by referring to the following, which are included merely for the purpose of exemplifying specific aspects and embodiments of the present invention and are not intended to limit the present invention.

[0197] Example 1: Administration of urolithin A after myocardial infarction The goal of this study was to evaluate the treatment effect of urolithin A on cardiac function after myocardial infarction. Figure 1 shows a schematic of the study design, and Table 3 shows the experimental design. Myocardial infarction (MI) was induced by permanent occlusion of the left anterior descending coronary artery in 6-week-old male Wistar rats in groups 1, 2, and 3 (Table 3) (ligation was performed at D0).

Table 3

[0198] Rats were anesthetized by intraperitoneal (IP) injection of medetomidine (0.5 mg / kg) and ketamine (50 mg / kg), intubated, and ventilated at a tidal volume of 10 mL / kg and 70 - 80 cycles per minute. Body temperature was maintained at 36.5°C - 37.5°C using a temperature-controlled heating pad connected to a rectal probe. The rats were placed in the supine position, the chest was shaved, and standard surgical conditions were prepared. A left thoracotomy was carefully performed to expose the heart. Next, to obtain an infarct size (IS) of nearly 40% of the total left ventricle (LV) area, a suture (4 / 0 silk, Ethicon) was placed around the left anterior descending artery (LAD) near the interventricular junction, approximately 2 mm below the left atrium. The chest was closed, air was evacuated from the chest cavity to avoid pneumothorax, and rapid resuscitation was performed using atipamezole hydrochloride (IM, 0.5 mg / ml, Zoetis).

[0199] Perioperative care of the animals during surgery was performed as follows: · Preoperative care with a single injection of carprofen (SC, 5 mg / kg, Pfizer) and a single injection of lidocaine (SC, 4 mg / kg, Vetoquinol) at the incision site. · Postoperative care: Three injections of buprenorphine (SC, 0.01 mg / kg, Axience) were administered after awakening and within the first 24 hours after surgery.

[0200] SHAM (Group 1) Animals in Group 1 were subjected to the same protocol as the MI group. However, after exposing the heart by left thoracotomy, the chest cavity was closed without passing a suture around the LAD.

[0201] Urolithin A or vehicle was started one week after myocardial infarction and force-fed orally at 25 mg / kg. Heart and plasma samples were collected at each time point. Ejection fraction (EF), fractional shortening (FS) of the left ventricular internal diameter, and isovolumic relaxation time (IVRT) were measured by echocardiography at 1 and 2 months to evaluate cardiac function.

[0202] Echocardiography To non-invasively evaluate the cardiac morphology and function, transthoracic echocardiographic examination was performed on rats. Echocardiography was performed using a digital ultrasound system (Vivid 7, GE Medical Systems) equipped with a 10 MHz phased array and a 13 MHz linear array transducer. All image acquisition, measurements, and calculations were performed according to the American Society of Echocardiography (Schiller et al., J Am Soc Echocardiogr 1989) and past validation of this method in rats (Litwin et al., Circulation 1994).

[0203] Rats were anesthetized with 4% isoflurane in a 50% oxygen - 50% air mixture and maintained with 2% isoflurane during the procedure. Rats were placed supine on a heating pad and the chest hair was shaved. Body temperature was monitored with a rectal probe connected to a temperature-controlled heating pad and maintained at 36.5 - 37.5 °C.

[0204] Standard B-mode (brightness mode) and M-mode (motion mode) images of the heart were acquired in the two-dimensional (2D) parasternal long-axis view (PSLA). LV parameters were measured and calculated as the mean of three consecutive cardiac cycles by a single blinded trained operator.

[0205] - Left ventricular (LV) function LV function was calculated using the LV dimensions parameter described above. 〇 FS = fractional shortening of left ventricular internal diameter = [(LVID d - LVID s ) / LVID d *100 This is a calculation based on the difference in dimensions between systole and diastole measured in the M-mode. This is a reflection of LV contractility. 〇 EF = Ejection fraction = [(End-diastolic volume - End-systolic volume) / End-diastolic volume] * 100 This is a calculation based on the difference in volume between systole and diastole measured in B-mode and M-mode. This is a reflection of LV contractility.

[0206] Additional echocardiographic measurements in rats included analysis of IVRT, which is the period from aortic valve closure to mitral valve opening and is an indicator of diastolic dysfunction. A total of three echocardiographic examinations were performed on all animals.

[0207] The first examination was performed 5 days after surgery (Table 4). This was used as a surgical control to exclude rats with small infarcts and limited reduction in EF. Two exclusion criteria were used: - Akinetic IVS (infarct too small) - EF > 45%

[0208] Table 4 represents the theoretical schedule for the first 2 weeks of the experiment, with days dedicated to surgery and the first echocardiographic examination 5 days after surgery.

Table 4

[0209] MI rats (Groups 2 and 3) were randomly assigned to three homogeneous groups based on the following parameters: - LVID d - EF - Body weight

[0210] Sham animals, having no LAD ligation, showed the same parameters of LV dimensions and function but should have the same body weight. After the first echocardiographic examination, randomization was performed for each session. After randomization, a longitudinal follow-up study by echocardiography was conducted on rats in the four groups.

[0211] The second and third echocardiograms were performed at 2 months after the start of the diet (M2). Using these, cardiac remodeling after MI was evaluated based on the aforementioned echocardiogram parameters.

[0212] For the planned number of rats for the study, at least three surgical sessions were performed consecutively for 4 weeks. Therefore, cardiac evaluation at M2 became available only after the analysis of the three sessions was completed.

[0213]

Table 5

[0214] The required amount of urolithin A was determined based on the dose being tested, the total number of animals to which the compound was administered, and an average final body weight of approximately 400 g.

[0215] Preparation of candidate compounds: Vehicle: 0.5% carboxymethyl cellulose Preparation: 0.5% carboxymethyl cellulose was used as the vehicle, and one solution per test compound and per concentration was prepared weekly. Each solution was sonicated at 37°C for 15 minutes to allow optimal dissolution of the compound. Preparation frequency: Weekly Storage: Each formulation was aliquoted into seven tubes and stored at -80°C. For compounds expected to form suspensions (due to low solubility), the formulation was stirred using a magnet during the aliquoting process. One aliquot was thawed daily and the amount for gavage was adjusted based on the most recently measured body weight.

[0216] PO administration PO administration was performed in the animals of groups 1 - 3 according to a forced standard operating procedure (Table 3). PO administration was performed in non - anesthetized animals using an orogastric feeding probe.

[0217] The final dosage was set at 5 ml / kg. According to the inventors' procedure, the dosage was adjusted weekly based on the average body weight of the animals in the corresponding group. If an animal had a body weight exceeding 20% compared to the average weight of its group, the dosage was specifically adjusted for that animal based on its own body weight. On the day of the final record, the animals were given the dosage corresponding to their own body weight. The vehicle as well as candidate compounds 1 and 2 were orally administered to the rats once a day, 7 days a week. The administration (treatment) started 1 week after MI and continued for 2 months.

[0218] Mortality Surgery (MI) may be related to the mortality within the first 24 hours. To ensure that 70 animals were included in the experiment, the use of preliminary animals was envisaged. Over time, MI may be related to mortality, which depends on the model.

[0219] After the first echocardiogram and randomization, deviations were notified as soon as possible. After the first echocardiogram, animals that died were not replaced.

[0220] Euthanasia and collection of heart tissue At the end of the procedure, after the last echocardiogram, after buprenorphine injection (0.003 mg / kg, SC), the animals were deeply anesthetized with 2.5% isoflurane in a 50% O2 / 50% air mixture. At the end of the procedure, one blood sample was collected through the jugular vein before sacrifice. At least 0.4 ml of blood was collected into a K3-EDTA coated tube and centrifuged for 10 minutes (10000 rpm, 20 °C). 200 μL of plasma was frozen and stored at approximately -80 °C until shipped to the sponsor.

[0221] Subsequently, the heart was arrested in diastole for subsequent histological analysis by jugular vein injection (2M KCl, 1 ml / kg). The atria were removed, the heart was washed with cold phosphate-buffered saline (PBS, 4°C), quickly blotted with a sponge to remove the liquid from the ventricles. Subsequently, the right ventricle (RV) wall was separated from the rest of the heart (LV + septum), and LV+S was carefully weighed using an analytical balance (AX205 (METTLER TOLEDO) or equivalent). Finally, the RV was removed, and LV+S was collected and processed as follows: - Fixed LV+S: They were placed in 10% NBF for 48 hours and transferred to 70% ethanol before paraffin embedding. (Group 1, n = 10; Groups 2 - 4, n = 20).

[0222] The results of the study are shown in Figures 2A - 2C. Urolithin A significantly improved the systolic dysfunction measured as the ejection fraction and fractional shortening of the left ventricular internal diameter (Figure 2A), improved the diastolic function measured as the isovolumic relaxation time (Figure 2B), and decreased the cardiac hypertrophy calculated as the heart weight / tibia length (Figure 2C). In conclusion, urolithin A has a protective effect against heart failure.

[0223] Example 1A: Effect of urolithin A on gene sets related to the improvement of mitochondrial function RNA extraction and quality control Heart sections from the rats of the groups described in Figure 1 (Example 1) were prepared using a microtome, deparaffinized, and RNA was extracted using the following kit (RNAeasy FFPE 76504, Qiagen). After extraction, the RNA was quality controlled using an Agilent Fragment Analyzer System.

[0224] Library preparation and sequencing Library preparation was performed using a strand-specific cDNA library according to the procedure described below: · rRNA depletion · mRNA fragmentation · Random-primed cDNA synthesis (strand-specific) · Adapter ligation and adapter-specific PCR amplification RNA-seq was performed using NovaSeq6000 with S4 flow cells at 2 × 150 bp.

[0225] Bioinformatics analysis QC and preprocessing of raw sequencing reads were performed using an in-house QC pipeline. The pipeline utilized fastp version 0.23.2 (Chen, A. et al. Bioinformatics 2018, 34(17), i884-i890), an all-in-one preprocessing tool for FastQ files, to automate quality control, quality trimming, and adapter clipping procedures. High-quality RNA-seq read pairs were mapped to the Ensembl rat reference genome.

[0226] Reads with a minimum PHRED score of 15, a minimum length of 15 bp, and a minimum unqualified percent limit of 40 (40%) were retained for downstream analysis.

[0227] Prior to DE analysis, pre-filtering steps were applied to remove low-abundance mRNA measurements by excluding genes with a total count of less than 10 across the entire sample. Additionally, the independent filtering procedure of DESeq2 was enabled to filter out genes with very low counts that are unlikely to show significant changes in gene expression. Gene symbols, descriptions, and biotypes were matched to Ensembl GTF IDs using the R package biomaRt.v.2.46.3 (Durink, S. et al. Nature Protocol 2009, 4, 1184-1191). p-values were corrected for multiple testing using the Benjamini-Hochberg (BH) method (Benjamini, Y. and Hochberg, Y. Journal of the Royal Statistical Society, Series B, 1995, 57, 289-300). Genes showing an adjusted (corrected) p-value of less than 0.05 and an absolute log2 fold change greater than 1 were considered differentially expressed (differentially expressed genes - DEGs).

[0228] Gene Set Enrichment Analysis (GSEA) (Subramanian, et al. Proc Natl Acad Sci USA 2005, 102(43), 15545-50) was performed to characterize the complex relationship between gene expression changes under different biological conditions and gene ontology (cellular component). GSEA used all processed genes and sorted them according to the log2 fold change. Gene ranking information was converted to the pathway level, and statistical significance was assigned to each analyzed gene set. Each gene set was analyzed independently, and the direction of gene regulation (upregulation / downregulation) was inferred using the biological comparisons examined.

[0229] Results Gene set enrichment analysis was performed focusing on gene sets (i.e., biological pathways) belonging to the "Cellular Components" category. Specifically, which biological pathways are: (i) significantly downregulated when rats with myocardial infarction are compared with healthy control rats (MI vs sham); (ii) significantly upregulated when MI rats treated with urolithin A (MI+UA) are compared with MI rats not treated with urolithin A (MI); were investigated.

[0230] Figure 3 shows several gene sets that are suppressed with the development of MI (MI vs Sham, gray bars) and simultaneously induced by urolithin A in diseased animals (MI+UA, dark gray bars). The x-axis represents the normalized enrichment score, where negative values indicate downregulation of the gene set; positive values indicate upregulation of the gene set.

[0231] Some of these gene sets are related to mitochondrial function, such as mitochondrial protein-containing complexes that contain genes essential for optimal mitochondrial function; and respirasomes that contain genes necessary for proper mitochondrial respiration (i.e., energy production).

[0232] These data indicate that myocardial infarction is associated with a decrease in the expression of genes related to mitochondrial function in heart tissue. And it is also shown that administration of urolithin A can restore the expression of these gene sets to positive.

[0233] Additional gene sets that are suppressed by the development of MI (MI vs sham, gray bars) and induced by urolithin A in diseased animals (MI+UA, red bars) are related to nucleoid and peroxisome biology.

[0234] Example 2: Preclinical in vivo study to evaluate the improvement of both myocardial and skeletal muscle functions The goal of this study was to evaluate the effect of urolithin treatment on age-related heart dysfunction. Figure 3 shows a schematic diagram of the study design. Aged C57BL / 6 mice (26 months) were used as a model to reproduce the natural decline in cardiac function associated with aging. Solid diets with and without urolithin A were administered at 50 mg / kg for 2 months. Postmortem myocardial, skeletal muscle, and postmortem plasma samples were collected at the end of the study. Ejection fraction (EF) and fractional shortening (FS) of the left ventricular internal diameter were measured by echocardiography. Muscle contractility was also measured. Analysis of the functions of both myocardial and skeletal muscle was performed at the end of the study.

[0235] The results are shown in Figures 5A - 5B. Urolithin A significantly improves the functions of both myocardial and skeletal muscle during natural aging. Figure 5A shows that urolithin A increases the fractional shortening of the left ventricular internal diameter in aged mice (Old) both at baseline and after high workload, and Figure 5B shows that urolithin A significantly prevents the age-related decline in skeletal muscle function. As a conclusion, urolithin A has a cardioprotective effect during natural aging.

[0236] Example 3: The effect of urolithin A on mitochondrial morphology related to the improvement of mitochondrial function The goal of this study was to evaluate the treatment effect of urolithin on age-related cardiac dysfunction. Aged C57BL / 6 mice (24 months at the end of the study) were used as a model to reproduce the natural decline in cardiac function associated with aging, and either 50 mg / kg of urolithin A (aged UA) or vehicle (aged vehicle) was force-fed orally for 2 months. Vehicle was administered to young mice (18 weeks old at the end of the study) for 2 months. Postmortem myocardium was collected at the end of the study.

[0237] Transmission electron microscopy and analysis Muscle samples from the animals described in Example 2 were fixed in a 2% glutaraldehyde solution in 0.1M Sorenson buffer (pH 7.3). These samples were then post-fixed in 1% osmium tetroxide for 8 minutes [pulse microwave (MW), 100 watts (W)] and rinsed in distilled water (3×150W, 40 seconds / step). After dehydrating the samples in a series of acetone at stepwise concentrations (25%; 50%; 75%; 3×100%; 150W, 40 seconds / step), they were impregnated with a gradually increasing concentration of epoxy resin (TAAB medium viscosity resin) in acetone (25%; 50%; 75%; 3×100%; 300W, 3 minutes / step). The samples were then embedded in 100% fresh resin and polymerized at 60°C for at least 24 hours in a normal oven.

[0238] All resin blocks were trimmed using a razor blade to form a trapezoidal block face. Sections were cut longitudinally or transversely on an ultramicrotome (Wolf et al., 2019) using a diamond knife. Semi-thin sections (0.5μm) were stained with toluidine blue and observed with an optical microscope (LM) to confirm the tissue orientation. Ultra-thin sections (70nm) were then cut out and picked up onto copper grids. Sections from the blocks were stained with 1% uranyl acetate (30 minutes) and 3% lead citrate (7 minutes). All sections were examined using an HT7800 120kV TEM (Hitachi). Digital micrographs were taken using an EMSIS Xarosa CMOS camera and Radius software.

[0239] Subsequently, these samples were post-fixed in 1% osmium tetroxide for 1 hour and dehydrated with gradually increasing concentrations of acetone. To confirm the orientation of the muscle tissue, 1-μm thick sections were stained with toluidine blue before ultramicrotomy. Ultrathin sections were cut longitudinally or transversely with an Ultracut ultramicrotome (Leica). These sections were then stretched to remove compression, placed on copper grids, and stained with 2% aqueous uranyl acetate and lead citrate (Leica). The sections were then imaged using a Philips CM100 electron microscope. Digital micrographs were taken at a magnification of x7900 using an AMT XR80 CCD digital camera.

[0240] Mitochondria from the individual hearts of 3 young mice vehicle (young vehicle), 3 old mice vehicle (old vehicle), and 3 old mice UA (old UA) O-AL mice were manually traced in the transverse orientation using ImageJ software (NIH) (https: / / imagej.nih.gov / ij / ) and the following morphological and shape descriptors were quantified: area (μm 2 ), perimeter (μm), circularity (4π×(area / perimeter 2 ), aspect ratio ((major axis) / (minor axis)): The aspect ratio is a measure of the length-to-width ratio, form factor ((perimeter) / (4π×surface area)): a measure sensitive to the complexity and branched appearance of mitochondria, Feret's diameter (the longest distance (μm) between two points of the mitochondria tested) and minimum Feret's diameter (corresponding to the minimum diameter of the mitochondria) (Picard et al., 2012). Here, it is important to emphasize that n = 3 - 4 is the sample size commonly used in TEM-based studies to investigate mitochondrial morphology and ultrastructure.

[0241] RNA-seq RNA-seq analysis from the samples derived from Example 3 was performed as described for Example 1.

[0242] Succinate dehydrogenase activity The hearts in OCT were cut into 8-μm sections using a cryostat. Subsequently, the sections were incubated at room temperature for 6 minutes in a solution containing nitroblue tetrazolium (1.5 mM), sodium succinate (130 mM), phenazine methosulfate (0.2 mM), and sodium azide (0.1 mM). The reaction was stopped by washing twice in PBS. The slides were mounted using Fluoromont mounting medium (Thermofisher, 00-4958-02). All samples were processed continuously using the same incubation solution to ensure that they were subjected to exactly the same experimental conditions. Images of the tissues were taken with a bright-field microscope. The intensity of the staining was analyzed with Image J.

[0243] Results Older mice showed a significant decrease in the area of mitochondria, expressed as mm 2 Administration of UA for 2 months in older mice significantly increased the mitochondrial area to levels comparable to those of younger animals (Figure 6A). A decrease in mitochondrial area has also been reported in skeletal muscle of elderly human subjects compared to younger counterparts (Crane, J.D. et al. J Gerontol A Biol Sci Med Sci 2010, 65(2). 119-128). This decline is associated with a decrease in mitochondrial function (Crane, J.D. et al. 2010). This data indicates that UA can improve the mitochondrial area associated with the improvement of mitochondrial function in the aging heart.

[0244] Furthermore, the ultrastructure of the cristae of cardiac mitochondria was analyzed. These represent intricate invaginations of the inner mitochondrial membrane, where mitochondrial respiration occurs, and maintaining their correct structure is important for maintaining optimal mitochondrial bioenergetic capacity (Baker, N. et al. Mitochondrion 2019, 49, 259-268). Aged mice were observed to have disrupted cristae structure, quantified as cristae volume and number, compared to younger animals (Figure 7). UA administration significantly increased cristae volume density and number in aged mice. This indicates that UA has the ability to improve mitochondrial quality in the aging heart.

[0245] In the hearts of the same animals as in this example, the inventors quantified mitophagy events, shown as the number of mitochondria that are engulfed by autophagosome vesicles and thus form mitolysosomes. Aged mice were observed to have a decrease in mitophagy events quantified as the number of mitolysosomes compared to younger animals (Figure 8). UA administration significantly increased mitophagy events in aged mice. Since mitophagy has been shown to be essential for cardiac function in experimental models (Kubli, D.A. et al. J Biol Chem 2013, 288(2), 915-926), this is clinically important. Also, mitophagy is decreased in human patients with heart failure (Billia, F. et al. Proc Natl Acad Sci USA 2011, 108(23), 9572-7). This indicates that UA has the ability to increase mitophagy in the aging heart.

[0246] Gene set enrichment analysis was performed focusing on gene sets (i.e., biological pathways) belonging to the "Cellular Components" category. In particular, we investigated which biological pathways were: (i) significantly downregulated when old mice were compared to young mice; and (ii) significantly upregulated when old mice treated with UA were compared to old mice treated with vehicle.

[0247] Figure 9 lists several gene sets that are suppressed with aging and at the same time induced by urolithin A in old animals. The normalized enrichment score indicates downregulation of the gene set when the value is negative and upregulation of the gene set when the value is positive.

[0248] Some of these gene sets are related to mitochondrial functions such as the respirasome, which contain genes necessary for proper mitochondrial respiration (i.e., energy production). Additional gene sets are related to the improvement of heart cell health by UA treatment, such as the sarcolemma.

[0249] These data indicate that myocardial infarction is associated with a decrease in the expression of genes related to mitochondrial function in heart tissue. And administration of urolithin A can restore the expression of these gene sets to positive.

[0250] To further verify the positive effect of UA on mitochondrial function in health, the enzyme activity of mitochondrial complex II enzyme SDH was measured. This immunoenzymatic assay is an indicator of mitochondrial respiratory activity and thus also an indicator of mitochondrial function. Figure 10 shows that administration of UA to old mice significantly increases SHD activity in the heart compared to old vehicle control mice.

[0251] Incorporation by reference All U.S. patents and U.S. and PCT published patent applications referred to in the above description are hereby incorporated by reference in their entirety.

[0252] equivalents For purposes of clarity of understanding, the invention has been described in some detail by way of illustration and example, but it will be apparent to those skilled in the art that the invention may be practiced within the broad and equivalent conditions, formulations, and other parameters without affecting the scope of the invention or any particular embodiment thereof. Also, such modifications or changes are intended to be encompassed within the scope of the appended claims.

Claims

**Claim 1** A method for improving cardiac function, comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg, and the mammal has myocardial infarction, coronary artery disease (CAD), congestive heart failure (CHF), angina pectoris, stroke, arrhythmia, fibrillation, peripheral arterial disease (PAD), or a heart or arterial disorder, said method. **Claim 2** The method according to claim 1, wherein urolithin is administered after ischemic reperfusion injury (e.g., at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days or about 1 week after ischemic reperfusion injury). **Claim 3** The method according to claim 1 or 2, wherein cardiac hypertrophy caused by myocardial infarction is reduced in the mammal. **Claim 4** The method according to claim 3, wherein cardiac hypertrophy in the left ventricle is reduced. **Claim 5** A method for improving cardiac function, comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg, said method. **Claim 6** A method for protecting cardiac function, comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg, said method. **Claim 7** A method for reducing the rate of decline of cardiac function, comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg, said method. **Claim 8** A method for preventing decline of cardiac function, comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg, said method. **Claim 9** A method for managing or treating peripheral arterial disease (PAD), comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg, said method. **Claim 10** A method for managing or improving tissue oxygenation, comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg, said method. **Claim 11** A method for improving cardiac output, comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg, said method.

12. The method according to claim 11, wherein the cardiac output is heart rate.

13. The method according to claim 11, wherein the cardiac output is stroke volume.

14. The method according to claim 11, wherein the cardiac output is heart rate variability.

15. A method for increasing ventricular ejection fraction, comprising administering to a mammal in need thereof an effective amount of urolithin, wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg, said method.

16. The method according to claim 15, wherein the left ventricular ejection fraction (LVEF) of the mammal increases.

17. The method according to claim 16, wherein the LVEF of the mammal is less than about 50% before administration of urolithin.

18. The method according to claim 16, wherein the LVEF of the mammal is less than about 40% before administration of urolithin.

19. The method according to claim 16, wherein the LVEF of the mammal is less than about 35% before administration of urolithin.

20. The method according to claim 16, wherein the LVEF of the mammal is less than about 30% before administration of urolithin.

21. The method according to claim 16, wherein the LVEF of the mammal is from about 15 to 50% before administration of urolithin.

22. The method according to claim 16, wherein the LVEF of the mammal is from about 15 to 40% before administration of urolithin.

23. The method according to claim 16, wherein the LVEF of the mammal is from about 15 to 35% before administration of urolithin.

24. The method according to claim 16, wherein the LVEF of the mammal is from about 20 to 45% before administration of urolithin.

25. The method according to claim 16, wherein the LVEF of the mammal is from about 40 to 49% before administration of urolithin.

26. The method according to claim 16, wherein the LVEF of the mammal is from about 41 to 49% before administration of urolithin.

27. The method according to any one of claims 15 to 26, wherein the right ventricular ejection fraction (RVEF) of the mammal increases.

28. The method according to claim 27, wherein the RVEF of the mammal is less than about 50% before administration of urolithin.

29. The method according to claim 27, wherein the RV EF of the mammal is less than about 40% before the administration of urolithin.

30. The method according to claim 27, wherein the RV EF of the mammal is less than about 35% before the administration of urolithin.

31. The method according to claim 27, wherein the RV EF of the mammal is less than about 30% before the administration of urolithin.

32. The method according to claim 27, wherein the RV EF of the mammal is about 15-50% before the administration of urolithin.

33. The method according to claim 27, wherein the RV EF is about 15-40% before the administration of urolithin.

34. The method according to claim 27, wherein the RV EF is about 15-35% before the administration of urolithin.

35. The method according to claim 27, wherein the RV EF of the mammal is about 20-45% before the administration of urolithin.

36. The method according to claim 27, wherein the RV EF of the mammal is about 40-49% before the administration of urolithin.

37. The method according to claim 27, wherein the RV EF of the mammal is about 41-49% before the administration of urolithin.

38. The method according to any one of claims 15-37, wherein the cardiac function is improved.

39. The method according to claim 38, wherein the cardiac function (HFpEF) with preserved ejection fraction is improved.

40. The method according to claim 38, wherein the cardiac function (HFrEF) with decreased ejection fraction is improved.

41. The method according to any one of claims 1-40, wherein the cardiac contractile function is improved.

42. A method for increasing cardiac contractile function, comprising administering an effective amount of urolithin to a mammal in need thereof, wherein the effective amount of urolithin is about 10 mg / kg to about 30 mg / kg, said method.

43. The method according to claim 41 or 42, wherein the cardiac contractile function of the mammal increases by about 30-80%.

44. The method according to claim 41 or 42, wherein the cardiac contractile function of the mammal increases by about 50-70%.

45. The method according to claim 41 or 42, wherein the cardiac contractile function of the mammal increases by about 56%.

46. The method according to claim 41 or 42, wherein the cardiac contractile function of the mammal increases by about 64%.

47. The method according to any one of claims 1-46, wherein the cardiac diastolic dysfunction of the mammal is reduced.

48. The method according to claim 47, wherein the cardiac diastolic dysfunction of the mammal is reduced by about 5 to 10%.

49. A method for increasing cardiac contractile function and skeletal muscle function, comprising administering an effective amount of urolithin to a mammal in need thereof, wherein the effective amount of urolithin is about 10 mg / kg to about 30 mg / kg, and the mammal is an aged mammal, said method

50. The method according to claim 49, wherein the cardiac contractile function of the mammal is increased by at least about 20%, 25%, 30%, or 35%.

51. The method according to claim 49, wherein the cardiac contractile function of the mammal is increased by at least about 30%.

52. The method according to any one of claims 49 to 51, wherein the skeletal muscle function of the mammal is increased by about 2 to 5%.

53. A method for maintaining, supporting, or improving blood circulation, comprising administering an effective amount of urolithin to a mammal in need thereof, wherein the effective amount of urolithin is about 10 mg / kg to about 30 mg / kg, said method.

54. A method for maintaining, supporting, or improving cardiac function, comprising administering an effective amount of urolithin to a mammal in need thereof, wherein the effective amount of urolithin is about 10 mg / kg to about 30 mg / kg, said method.

55. A method for maintaining, supporting, or improving myocardial function, comprising administering an effective amount of urolithin to a mammal in need thereof, wherein the effective amount of urolithin is about 10 mg / kg to about 30 mg / kg, said method.

56. The method according to any one of claims 1 to 55, wherein the contractility of the myocardium is increased.

57. The method according to any one of claims 1 to 56, wherein cardiac fibrosis is reduced.

58. The method according to any one of claims 1 to 57, wherein mitochondrial function of the heart is improved.

59. The method according to any one of claims 1 to 57, wherein the quality of the mitochondria in the heart is improved.

60. The method according to any one of claims 1 to 57, wherein the area of the mitochondria in the heart is increased.

61. The method according to any one of claims 1 to 60, wherein the expression of a gene related to mitochondrial function in heart tissue is upregulated.

62. The method according to claim 61, wherein the gene is a gene of a mitochondrial protein-containing complex or a gene of a respirasome.

63. The method according to any one of claims 1 to 62, wherein cardiac mitophagy is improved.

64. The method according to any one of claims 1 to 63, wherein the plasma ceramide level, acylcarnitine level, brain natriuretic peptide (BNP) level, creatine kinase (CK) level, C-reactive protein (CRP) level, troponin level, or galectin-2 level is decreased.

65. The method according to any one of claims 1 to 64, wherein the heart rate is improved.

66. The method according to any one of claims 1 to 65, wherein the heart rate variability is increased.

67. The method according to any one of claims 1 to 66, wherein the circulation is improved.

68. The method according to any one of claims 1 to 67, further comprising transplanting mitochondria into the mammal.

69. The method according to any one of claims 1 to 68, wherein the mitochondrial damage is improved.

70. The method according to any one of claims 1 to 69, wherein urolithin is administered orally.

71. The method according to claim 70, wherein urolithin is administered in the form of tablets or capsules.

72. The method according to any one of claims 1 to 71, wherein urolithin is administered over a period of at least about one month or at least about two months.

73. The method according to any one of claims 1 to 72, wherein the mammal is a human.

74. The method according to claim 73, wherein the human is an elderly person.

75. The method according to claim 73, wherein the human is at least about 50 years old.

76. The method according to claim 73, wherein the human is at least about 80 years old.

77. The method according to any one of claims 1 to 76, wherein the effective amount of urolithin is about 25 mg / kg.

78. The method according to any one of claims 1 to 77, wherein urolithin is selected from the group consisting of urolithin A, urolithin B, urolithin C, urolithin D, and any combination thereof.

79. The method according to any one of claims 1 to 77, wherein urolithin is selected from the group consisting of urolithin A, urolithin B, and a combination of urolithin A and urolithin B.

80. The method according to claims 1 to 77, wherein urolithin is urolithin A.

81. The method according to any one of claims 1 to 77, wherein urolithin is urolithin B.

82. The method according to any one of claims 1 to 77, wherein urolithin is urolithin C.

83. The method according to any one of claims 1 to 77, wherein urolithin is urolithin D.

84. The method according to any one of claims 1 to 83, further comprising co-administering an additional agent to the mammal.

85. The method according to claim 84, wherein the additional agent is a therapeutic agent.

86. The therapeutic agent is selected from the group consisting of an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin receptor-neprilysin inhibitor (ARNi), a β-blocker, a mineralocorticoid receptor antagonist (MRA), a sodium-glucose cotransporter 2 (SGLT2) inhibitor, an angiotensin-2 receptor antagonist (ARB), a diuretic, an If channel blocker, an aldosterone antagonist, aspirin, a P2Y12 inhibitor, a calcium channel blocker, a cholesterol-lowering agent (e.g., statin), an antiarrhythmic agent, hydralazine containing nitrate, digoxin, and an anticoagulant. The method according to claim 85.

87. The therapeutic agent is an SGLT2 inhibitor, and the SGLT2 inhibitor is dapagliflozin or empagliflozin. The method according to claim 86.

88. The method according to claim 84, wherein the additional agent is a nutritional supplement.

89. The nutritional supplement is selected from the group consisting of fish oil, nitrate-rich beetroot, nitric oxide, red yeast rice, β-glucan, vitamin A, vitamin C, vitamin E, vitamin K, potassium salts, magnesium salts, calcium salts, iron salts, manganese salts, copper salts, zinc salts and phosphates, coenzyme q10 (Coq10), carnitine, grape seed extract and cocoa. The method according to claim 88.

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