Urethins for improving cardiac function and health

Abstract

Methods and compositions for improving cardiac function and health are disclosed. The methods and compositions are based on urolithin.

Description

Related Applications

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

[0002] Today's rapidly growing and aging population is faced with cardiac dysfunction, such as myocardial infarction, coronary artery disease (CAD), congestive heart failure (CHF), angina, stroke, arrhythmia, fibrillation, peripheral arterial disease (PAD), cardiac disease, arterial disease and post-cardiac transplantation. These cardiac diseases and conditions require "polymedication" to allow sick individuals, especially middle-aged and elderly individuals, to continue to function optimally and have an improved quality of life, with a reduction in the total symptoms associated with cardiac dysfunction. However, many commonly used drugs prescribed and used to improve or treat cardiac dysfunction can cause side effects or are associated with side effects. There is still an unmet need to improve or treat individuals with cardiac dysfunction and reduce the side effects of drugs.

[0003] Urolithins are ellagitannins and ellagic acid derived metabolites produced, for example, by mammalian colonic microflora, including human colonic microflora. Urolithins have potent effects on improving many health conditions, and they have been shown to be highly bioactive in vitro and in vivo. Urolithins have been proposed as treatments for a variety of conditions, including diseases and conditions associated with cardiac dysfunction. Summary of the invention

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

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

[0006] In some embodiments, the urolithin is administered after the ischemia-reperfusion injury (e.g., at least about one day, about two days, about three days, about four days, about five days, or about one week after the ischemia-reperfusion injury).

[0007] In certain embodiments, cardiac hypertrophy induced by myocardial infarction is reduced in a mammal (eg, cardiac hypertrophy in the left ventricle is reduced).

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

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

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

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

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

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

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

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

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

[0017] In some embodiments, the mammal has an increased left ventricular ejection fraction (LVEF).

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

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

[0020] In certain embodiments, the mammal has an increased right ventricular ejection fraction (RVEF).

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

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

[0023] 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.

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

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

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

[0027] In certain embodiments, the cardiac systolic function of the mammal is improved by about 56%.

[0028] In some embodiments, the cardiac systolic function of the mammal is improved by about 64%.

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

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

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

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

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

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

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

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

[0037] In some embodiments, myocardial contractility is increased.

[0038] In some embodiments, cardiac fibrosis is reduced.

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

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

[0041] In some embodiments, 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 reduced.

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

[0043] In some embodiments, heart rate variability is increased.

[0044] In some embodiments, circulation (eg, blood flow) is improved.

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

[0046] In some embodiments, mitochondrial damage is ameliorated.

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

[0048] In some embodiments, the urolithin is administered in the form of a tablet or capsule.

[0049] In some embodiments, the 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 elderly.

[0052] In some embodiments, the human is at least about 40 years old (eg, 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, the urolithin is selected from urolithin A, urolithin B, urolithin C, urolithin D, and any combination thereof.

[0055] In some embodiments, the urolithin is selected from urolithin A, urolithin B, and a combination of urolithin A and urolithin B.

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

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

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

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

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

[0061] In certain embodiments, the additional agent is a therapeutic agent (e.g., angiotensin converting enzyme (ACE) inhibitors, angiotensin receptor-neprilysin inhibitors (ARNi), beta-blockers, mineralocorticoid receptor antagonists (MRA), sodium-glucose cotransporter 2 (SGLT2) inhibitors (e.g., dapagliflozin, empagliflozin), angiotensin-2 receptor blockers (ARBs), diuretics, If channel blockers, aldosterone antagonists, aspirin, P2Y12 inhibitors, calcium channel blockers, cholesterol-lowering drugs (e.g., statins), antiarrhythmic drugs, hydralazine with nitrates, digoxin, and anticoagulants.

[0062] In some embodiments, the additional agent is a nutritional supplement (e.g., omega-3 fatty acids, nitrate-rich beets, 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 phosphate salts). BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 is a schematic diagram depicting the study design in Example 1.

[0064] Figure 2A Shown are the effects of urolithin A (UA) on cardiac contractile function measured as ejection fraction and fractional shortening in mice subjected to myocardial infarction (MI). Sham-operated animals were used as controls. *p<0.05 unpaired Student's t-test.

[0065] Figure 2B Shown is the effect of urolithin A (UA) on cardiac diastolic function measured as isovolumetric relaxation time in mice subjected to myocardial infarction (MI). Sham-operated animals were used as controls. *p<0.05 unpaired Student's t-test.

[0066] Figure 2C Shown is the effect of urolithin A (UA) on cardiac hypertrophy measured as isovolumetric relaxation time in mice subjected to myocardial infarction (MI). Sham-operated animals were used as controls. *p<0.05 unpaired Student's t-test.

[0067] Figure 3 Gene set analysis is shown, namely several gene sets that are simultaneously repressed by the development of MI in diseased animals (MI vs. sham, grey bars) and also induced by urolithin A (MI+UA, red bars).

[0068] Figure 4 is a schematic diagram depicting the study design in Example 2.

[0069] Figure 5A Shown that in aged mice, urolithin A increased fractional shortening both at basal (+32%) and after high workload (+39%). *p<0.05 Left: Two-way ANOVA (effect of treatment).

[0070] Figure 5B Urolithin A significantly prevents age-related decline in skeletal muscle function. Unpaired Student's t-test.

[0071] Fig. 6A Shown is the effect of urolithin A on mitochondrial area (expressed in mm2).

[0072] Figure 6B Images showing the ultrastructure of the cristae.

[0073] Figure 7 Images showing cristae ultrastructure of cardiac mitochondria and the effects of urolithin A on cristae volume density and cristae number. Administration of urolithin A significantly increased cristae volume density and number in aged mice.

[0074] Figure 8 Shows the effect of urolithin A on the number of mitochondrial autophagic lysosomes.

[0075] Fig. 9 Data are shown for several gene sets that are simultaneously repressed by senescence and also induced by urolithin A in aged animals. Normalized enrichment scores indicate downregulation of a gene set when values ​​are negative and upregulation of a gene set when values ​​are positive.

[0076] Fig.10 The enzymatic activity of the mitochondrial complex II enzyme SDH is shown, demonstrating that administration of UA to aged mice significantly increased SHD activity in the heart compared to aged vehicle control mice. DETAILED DESCRIPTION

[0077] Substantial innovation is needed to develop approaches to improve cardiac function and health with urolithins. Quantitative analysis of cardiac systolic function, diastolic function, hypertrophy, and skeletal muscle function was performed to demonstrate that urolithin A has protective effects against heart failure and cardioprotective effects during natural aging. definition

[0078] The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0079] As used herein in the specification and claims, the phrase "and / or" should be understood to refer to "either or both" of the elements so combined, i.e., elements that are present in combination in some cases and separately in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so combined. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open language such as "comprising", may refer to A only (optionally including elements other than B) in one embodiment; may refer to B only (optionally including elements other than A) in another embodiment; may refer to both A and B (optionally including other elements) in yet another embodiment; and so on.

[0080] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when items are separated in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one of a plurality or a series of elements, but also including more than one, and optionally, additional unlisted items. Only terms that clearly indicate the opposite, such as "only one" or "exactly one", or "consisting of" when used in the claims will refer to including exactly one element in a plurality or a series of elements. Generally, as used herein, when there is an exclusive term after the term "or", such as "either", "one of them", "only one of them" or "exactly one of them", it should only be interpreted as indicating exclusive substitution (i.e., "one or the other, but not both"). When used in the claims, "consisting essentially of" should have the conventional meaning as used in the field of patent law.

[0081] As used herein in the specification and claims, the phrase "at least one" with respect to a series of one or more elements should be understood to refer to at least one element selected from any one or more elements in a list of elements, but does not necessarily include at least one of each and every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows that elements other than the elements specifically identified in the list of elements referred to by the phrase "at least one" may optionally be present, whether related or unrelated to those elements specifically identified. Thus, as a 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 refer to at least one, optionally including more than one A without B (and optionally including elements other than B) in one embodiment; may refer to at least one, optionally including more than one B without A (and optionally including elements other than A) in another embodiment; may refer to at least one (optionally including more than one) A and at least one (optionally including more than one) B (and optionally including other elements) in yet another embodiment; and so on.

[0082] It should also be understood that in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order of the steps or actions of the method as recited unless explicitly indicated to the contrary.

[0083] In the claims and the description above, all transitional phrases such as "comprising", "including", "with", "having", "containing", "involving", "holding", "composed of", etc. shall be understood as open-ended, i.e., meaning including but not limited to. As provided in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedures, only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively.

[0084] As used herein, the term "prodrug" includes compounds that are converted to therapeutically active agents under physiological conditions. Common methods for preparing prodrugs include hydrolysis under physiological conditions to reveal selected portions of the desired molecule. In other embodiments, the prodrug is converted by enzymatic activity of the host animal.

[0085] As used herein, the phrase "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, which is involved in carrying or transporting the subject chemical 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 non-pyrogenic. Some examples of materials 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 carboxymethylcellulose, 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, safflower oil, sesame oil, olive oil, corn starch, and safflower oil. Rice oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical formulations. In certain embodiments, the pharmaceutical compositions of the present invention are pyrogen-free, i.e., do not induce a significant temperature increase when administered to a patient.

[0086] The term "pharmaceutically acceptable salt" refers to relatively nontoxic, inorganic and organic acid addition salts of a compound. These salts can be prepared in situ during the final separation and purification of the compound, or by reacting the purified compound in its free base form with a suitable organic or inorganic acid alone, and separating the salt thus formed to prepare. Representative salts include hydrobromate, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, mesylate, glucoheptonate, lactobionate and lauryl sulfonate, etc. (see, e.g., Berge et al., (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19.).

[0087] In other cases, the compound used in the method of the invention may contain one or more acidic functional groups, and therefore can form a pharmaceutically acceptable salt with a pharmaceutically acceptable base. In these cases, the term "pharmaceutically acceptable salt" refers to a relatively nontoxic, inorganic and organic base addition salt of a compound. These salts can be similarly prepared in situ during the final separation and purification of the compound, or prepared by reacting the purified compound in its free acid form with a suitable base (e.g., a pharmaceutically acceptable metal cation hydroxide, carbonate or bicarbonate), ammonia or a pharmaceutically acceptable organic primary amine, secondary amine or tertiary amine separately. Representative alkali metal or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium and aluminum salts, etc. Representative organic amines that can be used to form base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, etc. (see, for example, Berge et al., the same).

[0088] A "therapeutically effective amount" (or "effective amount") with respect to a compound used in treatment means that amount of the compound in a formulation which, when administered (to a mammal, preferably a human) as part of a desired dosing regimen, alleviates symptoms, improves the condition, or slows 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.

[0089] The terms "preventative" or "therapeutic" treatment are art-recognized and include administering one or more of the subject compositions to a host. If administered prior to clinical manifestation of an undesirable condition (e.g., a disease or other undesirable state of the host animal), the treatment is preventative (i.e., it protects the host from developing the undesirable condition), while if administered after manifestation of the undesirable condition, the treatment is therapeutic (i.e., it is intended to reduce, ameliorate, or stabilize an existing undesirable condition or its side effects).

[0090] The term "patient" or "subject" refers to a mammal in need of a particular treatment. In certain embodiments, the patient is a primate, canine, feline, or equine. In certain embodiments, the patient is a human.

[0091] An "effective amount" is an amount sufficient to achieve a beneficial or desired result. For example, a therapeutic amount is an amount that achieves a desired therapeutic effect. This amount may be the same or different from a prophylactic effective amount, which is the amount necessary to prevent the onset of a disease or disease symptom. An effective amount may be administered in one or more administrations, applications, or dosings. The therapeutically effective amount of the composition depends on the composition selected. The composition may be administered from one or more times per day to one or more times per week; including once every other day. Those skilled in the art will appreciate that certain factors may affect the dosage and time required to effectively treat a subject, including but not limited to the severity of the disease or condition, previous treatment, the overall health and / or age of the subject, and other diseases present. In addition, treating a subject with a therapeutically effective amount of a composition described herein may include a single treatment or a series of treatments.

[0092] The terms "reduce", "reduce", "reduced", "reduction", "reduce" and "inhibit" are all used herein generally to refer to a reduction by a statistically significant amount relative to a reference. However, for the avoidance of doubt, "reduce", "reduction" or "reduce" or "inhibit" generally refers to a reduction of at least 10% compared to a reference level, and can include, for example, a reduction of 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%, up to and including, for example, the complete absence of a given entity or parameter, or any reduction between 10-99% compared to the absence of a given treatment, compared to a reference level.

[0093] The terms "increased", "increase" or "enhance" or "activate" are all used herein generally to refer to an increase by a statistically significant amount; for the avoidance of any doubt, the terms "increased", "increase" or "enhance" or "activate" refer to an increase of at least 10% compared to a reference level, such as an increase of 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 and including an increase of 100% or any increase between 10-100% compared to a reference level, 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-fold and 10-fold or greater compared to a reference level.

[0094] As used herein, the term "modulate" includes up-regulation and down-regulation, eg, enhancing or inhibiting a response.

[0095] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances 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 substituted aryl groups and aryl groups having no substitution.

[0096] As used herein, the term "treating" used in connection with a disease, disorder or condition of a subject refers to reducing at least one clinical or objective manifestation of the disease, disorder or condition of the subject by a detectable amount. In one embodiment, the term "treating" used in connection with a disease, disorder or condition of a subject refers to curing the disease, disorder or condition of the subject.

[0097] As used herein, "food" refers to products prepared from natural foods. Non-limiting examples of food include juices, wines, concentrates, jams, jellies, preserves, pastes, and extracts.

[0098] As used herein, "nutritional supplement" refers to a product suitable for consumption or other administration primarily for its health-promoting properties rather than for its caloric content.

[0099] As used herein, the term "alkyl" refers to a straight or branched, non-cyclic 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.; while saturated branched chain alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, etc. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.; while 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 alkenyl groups include ethenyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like; while representative straight chain and branched alkynyl groups include ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

[0100] As used herein, the term "aryl" refers to a hydrocarbon ring system radical containing hydrogen, 6-18 carbon atoms and at least one aromatic ring. For the purposes of the present invention, an aryl group can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems. Aryl groups include, but are not limited to, aryl groups derived from: aceanthrene, acenaphthylene, acephenanthrene, anthracene, azulene, benzene, Fluoranthene, fluorene, as-indane, s-indane, indane, indene, naphthalene, phenalene, phenanthrene, heptahedralene, pyrene and triphenylene. Unless otherwise specifically stated in the specification, the term "aryl" or the prefix "ar-" (for example, in "aralkyl") is intended to include optionally substituted aryl groups.

[0101] As used herein, the term "monosaccharide" refers to a simple sugar of formula (CH2O)n. Monosaccharides can be straight chains or ring systems, and can include sucrose units of formula—CH(OH)—C(═O)—. Examples of monosaccharides include erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, erythulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, erythropentulose, threopentulose, (R)-1,3,4-trihydroxybutan-2-one (glycerotetrulose), pyranose glucose, furanose fructose. In certain embodiments, monosaccharides refer to pyranose glucose.

[0102] As used herein, the term "oligosaccharide" refers to a sugar consisting of at least two and up to 10 glycosidically linked monosaccharide units, preferably 2 to 8 monosaccharide units, more preferably 2 to 7 monosaccharide units, and even more preferably 2 to 6 monosaccharide units or 2 to 5 monosaccharide units.

[0103] As used herein (eg, in the case of a substituted heterocyclyl or a substituted aryl), the term "substituted" means that at least one hydrogen atom is replaced with 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, as well as -NRaRb, -NRaC(=O)Rb, -NRaC(=O)NRaNRb, -NRaC(=O)ORb, -NRaS02Rb, -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 and -SO2NRaRb. In the above, Ra and Rb in this case may be the same or different, and are independently hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, aralkyl, heterocyclic. In addition, the above substituents may be further substituted by one or more of the above substituents. Method of the present invention

[0104] According to the present invention, it has been found that urolithin unexpectedly improves cardiac systolic function, diastolic function, hypertrophy and skeletal muscle function. The method of the present invention can be used to improve and / or protect cardiac function, reduce the rate of cardiac function decline, prevent cardiac function decline, control or treat peripheral arterial disease (PAD).

[0105] In certain embodiments according to this and other aspects of the invention, the urolithin is urolithin A. In certain embodiments according to this and other aspects of the invention, the urolithin is urolithin B. In certain embodiments according to this and other aspects of the invention, the urolithin is urolithin C. In certain embodiments according to this and other aspects of the invention, the urolithin is urolithin D.

[0106] In certain aspects, provided herein is a method of improving cardiac function, the method comprising administering an effective amount of a urolithin to a mammal in need thereof; wherein the effective amount of the urolithin is about 10 mg / kg to about 30 mg / kg; and the mammal suffers from myocardial infarction, coronary artery disease (CAD), congestive heart failure (CHF), angina, stroke, arrhythmia, fibrillation, peripheral arterial disease (PAD), or a heart or artery disorder.

[0107] Ischemia / reperfusion (I / R) injury is a causal factor leading to morbidity and mortality. The vulnerability of the liver to I / R injury is a major obstacle to liver resection and transplantation surgery, where reperfusion after prolonged ischemia is inevitable during liver resection and revascularization. Mitochondrial dysfunction is known to be one of the key downstream events leading to I / R-mediated cell death.

[0108] In some embodiments, the urolithin is administered after the ischemia-reperfusion injury (e.g., at least about one day, about two days, about three days, about four days, about five days, or about one week after the ischemia-reperfusion injury).

[0109] In certain embodiments, cardiac hypertrophy induced by myocardial infarction is reduced in a mammal (eg, cardiac hypertrophy in the left ventricle is reduced).

[0110] In certain aspects, provided herein are methods of improving cardiac function, the methods comprising administering an effective amount of a urolithin to a mammal in need thereof; wherein the effective amount of the urolithin is from about 10 mg / kg to about 30 mg / kg.

[0111] In certain aspects, provided herein are methods of protecting cardiac function, the methods comprising administering to a mammal in need thereof an effective amount of a urolithin; wherein the effective amount of the urolithin is from about 10 mg / kg to about 30 mg / kg.

[0112] In certain aspects, provided herein is a method of reducing the rate of decline in cardiac function, the method comprising administering an effective amount of a urolithin to a mammal in need thereof; wherein the effective amount of the urolithin is from about 10 mg / kg to about 30 mg / kg.

[0113] In certain aspects, provided herein is a method for preventing cardiac function decline, the method comprising administering an effective amount of a urolithin to a mammal in need thereof; wherein the effective amount of the urolithin is about 10 mg / kg to about 30 mg / kg.

[0114] In certain aspects, provided herein is a method of managing or treating peripheral arterial disease (PAD), the method comprising administering an effective amount of a urolithin to a mammal in need thereof; wherein the effective amount of the urolithin is about 10 mg / kg to about 30 mg / kg.

[0115] In certain aspects, provided herein are methods of controlling or improving tissue oxygenation, the methods comprising administering an effective amount of a urolithin to a mammal in need thereof; wherein the effective amount of the urolithin is from about 10 mg / kg to about 30 mg / kg.

[0116] In certain aspects, provided herein are methods of improving cardiac output (e.g., heart rate, stroke volume, heart rate variability), the methods comprising administering to a mammal in need thereof an effective amount of a urolithin; wherein the effective amount of the urolithin is about 10 mg / kg to about 30 mg / kg.

[0117] In some embodiments, the cardiac output is heart rate.

[0118] In certain aspects, provided herein is a method of increasing ventricular ejection fraction, the method comprising administering an effective amount of a urolithin to a mammal in need thereof; wherein the effective amount of the urolithin is about 10 mg / kg to about 30 mg / kg.

[0119] In some embodiments, the mammal has an increased left ventricular ejection fraction (LVEF).

[0120] In certain embodiments, prior to administration of the urolithin, the mammal has an LVEF of about less than 60%, about less than 55%, about less than 50%, about less than 45%, about less than 40%, about less than 35%, about less than 30%, or about less than 25%, about less than 20%.

[0121] In some embodiments, prior to administration of the urolithin, the mammal has an LVEF of about 5-75% (e.g., about 15-50%, about 15-40%, about 15-35%, about 20-45%, about 40-49%, or about 41-49%).

[0122] In certain embodiments, the mammal has an increased right ventricular ejection fraction (RVEF).

[0123] In certain embodiments, prior to administration of the urolithin, the mammal has an RVEF of about less than 60%, about less than 55%, about less than 50%, about less than 45%, about less than 40%, about less than 35%, about less than 30%, or about less than 25%, about less than 20%.

[0124] In some embodiments, prior to administration of the urolithin, the mammal has an RVEF of about 5-75% (e.g., about 15-50%, about 15-40%, about 15-35%, about 20-45%, about 40-49%, or about 41-49%).

[0125] 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.

[0126] In some embodiments, cardiac systolic function is improved.

[0127] In certain aspects, provided herein is a method of improving cardiac contractile function, the method comprising administering an effective amount of a urolithin to a mammal in need thereof; wherein the effective amount of the urolithin is about 10 mg / kg to about 30 mg / kg.

[0128] In some embodiments, the cardiac contractile function of the mammal is improved by about 20-90% (eg, about 30-80%, or 50-70%).

[0129] In certain embodiments, the cardiac systolic function of the mammal is improved by about 56%.

[0130] In some embodiments, the cardiac systolic function of the mammal is improved by about 64%.

[0131] In certain embodiments, the mammal has reduced diastolic dysfunction.

[0132] In some embodiments, the diastolic dysfunction in the mammal is reduced by about 5-10%.

[0133] The myocardium undergoes a progressive decline in mitochondrial function similar to that observed in skeletal muscle, leading to an increase in reactive oxygen species and an increased accumulation of defective organelles. Clearance of these damaged organelles by autophagy is important for maintaining myocardial function. Since autophagy decreases with age, promoting autophagy could be used to protect myocardial function.

[0134] The myocardium is also intensely exposed to ischemic episodes during MI. The level of myocardial damage produced by these ischemic episodes strongly depends on the ability of the cells to mount an effective autophagic response to clear damaged organelles. In aged animals, a defective autophagic response leads to increased myocardial damage after ischemic events. Therefore, promoting autophagy during these acute events could be used to protect the myocardium from damage.

[0135] In certain aspects, provided herein is a method for improving cardiac contractile function and skeletal muscle function, the method comprising administering an effective amount of a urolithin to a mammal in need thereof; wherein the effective amount of the urolithin is about 10 mg / kg to about 30 mg / kg; and the mammal is an aging mammal.

[0136] In some embodiments, the cardiac systolic function of the mammal is improved by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%.

[0137] In certain embodiments, skeletal muscle function of the mammal is improved by about 1-50% (eg, about 2-5%).

[0138] In certain aspects, provided herein is a method of maintaining, supporting or improving blood circulation, the method comprising administering an effective amount of a urolithin to a mammal in need thereof; wherein the effective amount of the urolithin is about 10 mg / kg to about 30 mg / kg.

[0139] In certain aspects, provided herein are methods of maintaining, supporting or improving cardiac function, the methods comprising administering an effective amount of a urolithin to a mammal in need thereof; wherein the effective amount of the urolithin is from about 10 mg / kg to about 30 mg / kg.

[0140] In certain aspects, provided herein is a method of maintaining, supporting or improving myocardial function, the method comprising administering an effective amount of a urolithin to a mammal in need thereof; wherein the effective amount of the urolithin is about 10 mg / kg to about 30 mg / kg.

[0141] In some embodiments, myocardial contractility is increased.

[0142] In some embodiments, cardiac fibrosis is reduced.

[0143] In some embodiments, cardiac mitochondrial function is improved.

[0144] In some embodiments, cardiac mitochondrial mass is improved.

[0145] In some embodiments, cardiac mitochondrial area is increased.

[0146] In some embodiments, expression of genes associated with mitochondrial function in cardiac tissue is upregulated.

[0147] In some embodiments, the gene is a mitochondrial protein complex-containing gene or a respiratory gene.

[0148] In some embodiments, cardiac mitophagy is improved.

[0149] In some embodiments, 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 reduced.

[0150] In some embodiments, heart rate is improved.

[0151] In some embodiments, heart rate variability is increased.

[0152] In some embodiments, circulation is improved.

[0153] In certain embodiments, the method further comprises transplanting the mitochondria into a mammal.

[0154] In some embodiments, mitochondrial damage is ameliorated.

[0155] In some embodiments, the urolithin is administered orally.

[0156] In some embodiments, the urolithin is administered in the form of a tablet or capsule.

[0157] In some embodiments, the 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).

[0158] In some embodiments, the mammal is a human.

[0159] In some embodiments, the human is elderly.

[0160] 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).

[0161] In some embodiments, the effective amount of urolithin is about 25 mg / kg.

[0162] In some embodiments, the urolithin is selected from urolithin A, urolithin B, urolithin C, urolithin D, and any combination thereof.

[0163] In some embodiments, the urolithin is selected from urolithin A, urolithin B, and a combination of urolithin A and urolithin B.

[0164] In certain embodiments, the urolithin is urolithin A.

[0165] In certain embodiments, the urolithin is urolithin B.

[0166] In certain embodiments, the urolithin is urolithin C.

[0167] In certain embodiments, the urolithin is urolithin D.

[0168] In some embodiments, the methods described herein further comprise co-administering an additional pharmaceutical agent to the mammal.

[0169] In certain embodiments, the additional agent is a therapeutic agent (e.g., angiotensin converting enzyme (ACE) inhibitors, angiotensin receptor-neprilysin inhibitors (ARNi), beta-blockers, mineralocorticoid receptor antagonists (MRA), sodium-glucose cotransporter 2 (SGLT2) inhibitors (e.g., dapagliflozin, empagliflozin), angiotensin-2 receptor blockers (ARBs), diuretics, If channel blockers, aldosterone antagonists, aspirin, P2Y12 inhibitors, calcium channel blockers, cholesterol-lowering drugs (e.g., statins), antiarrhythmic drugs, hydralazine with nitrates, digoxin, and anticoagulants.

[0170] In some embodiments, the additional agent is a nutritional supplement (e.g., fish oil (e.g., omega-3 fatty acids)), nitrate-rich beetroot, nitric oxide, red yeast rice, beta-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). Exemplary compounds of the present invention

[0171] Urolithins can be used to practice any of the methods herein, including but not limited to improving and / or protecting cardiac function, reducing the rate of cardiac function decline, preventing cardiac function decline, and controlling or treating peripheral arterial disease (PAD).

[0172] As used herein, "urolithin" refers to a compound of Formula I: in R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are independently selected from H and OR; and For each occurrence, R is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted monosaccharide, or substituted or unsubstituted oligosaccharide.

[0173] In one embodiment, "urolithin" refers to any one or any combination of urolithin A, urolithin B, urolithin C, and urolithin D.

[0174] In some embodiments, "urolithin" is, or when taken as a combination may include, a glucuronidated, methylated or sulfated urolithin.

[0175] In some embodiments, the urolithin is urolithin A.

[0176] In some embodiments, the urolithin is urolithin B.

[0177] In some embodiments, the urolithin is urolithin C.

[0178] In some embodiments, the urolithin is urolithin D. Preparation and clinical use

[0179] Urolithin can be administered to a subject (e.g., a mammal) alone or in combination with another agent in various ways. For example, urolithin can be administered orally or parenterally. Parenteral includes, but is not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, intraarticular, intrasynovial, intraocular, intrathecal, topical, or by inhalation. Thus, urolithin dosage forms can be in various forms, including natural foods, processed foods, natural juices, concentrates and extracts, injectable solutions, microcapsules, nanocapsules, liposomes, plasters, inhalation forms, nasal sprays, nasal drops, eye drops, sublingual tablets, and sustained-release preparations.

[0180] The compounds of the present invention can be provided in an isolated form. As used herein, the term "isolated" refers to being substantially removed from other compounds or components that are otherwise found with the compound of interest (e.g., as found together in nature). In one embodiment, a compound is isolated when it is substantially completely removed from other compounds or components that are otherwise found with the compound of interest. In one embodiment, a compound is isolated when it is pure.

[0181] The compound of the present invention can be incorporated into various preparations for therapeutic administration. More particularly, the compound of the present invention can be formulated into a pharmaceutical composition by combining with a suitable pharmaceutically acceptable carrier or diluent, and can be formulated into a preparation in solid, semisolid, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. Therefore, the use of the compound can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal and intratracheal administration. Active agent can be systemic after administration, or can be positioned by using topical administration, internal administration (intramuraladministration) or using an implant for maintaining active dose at the implantation position.

[0182] The compounds of the present invention may also be formulated as food additives, food ingredients, functional foods, dietary supplements, medical foods, nutraceuticals or food supplements.

[0183] In pharmaceutical dosage forms, the compounds can be administered in the form of their pharmaceutically acceptable salts. They can also be used in combination with other pharmaceutically active compounds as appropriate. The following methods and excipients are exemplary only and are not intended to be limiting.

[0184] For oral preparations, the compound can be used alone or in combination with appropriate additives to prepare tablets, powders, granules or capsules, for example, in combination with conventional additives such as lactose, mannitol, corn starch or potato starch; in combination with binders such as crystalline cellulose, cellulose derivatives, gum arabic, 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 necessary, with diluents, buffers, wetting agents, preservatives and flavoring agents.

[0185] The compound can be formulated into preparations for injection by dissolving, suspending or emulsifying it in an aqueous or non-aqueous solvent, such as vegetable oil or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids or propylene glycol; and, if necessary, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

[0186] The compounds may be used in an aerosol formulation for administration via inhalation. The compounds of the present invention may be formulated into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0187] In addition, the compound can be made into suppositories by mixing with various bases such as emulsifying bases or water-soluble bases. The compound of the present invention can be administered via suppositories rectum. Suppositories can include vehicles, such as cocoa butter, carbowaxes and polyethylene glycol, which melt at body temperature and solidify at room temperature.

[0188] Unit dosage forms for oral or rectal administration, such as syrups, elixirs and suspensions, can be provided, wherein each dosage unit, such as a teaspoon, a tablespoon, 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 a compound of the present invention in a composition as a solution in sterile water, saline or another pharmaceutically acceptable carrier, wherein each dosage unit, such as mL or L, contains a predetermined amount of a composition comprising one or more compounds of the present invention.

[0189] Implants for sustained release formulations are well known in the art. Implants are formulated with biodegradable or non-biodegradable polymers as microspheres; slabs, etc. 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 near the site of interest so that the local concentration of the active agent is increased relative to the rest of the body.

[0190] As used herein, the term "unit dosage form" refers to physically discrete units suitable as unit dosages for human and animal subjects, each unit containing a predetermined amount of the compound of the present invention, the predetermined amount being calculated in an amount sufficient to produce the desired effect in combination with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications of the novel unit dosage forms of the present invention depend on the specific compound used and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.

[0191] Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. In addition, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, etc., are readily available to the public.

[0192] For clinical use, urolithins are administered in therapeutically effective amounts.

[0193] The compound of the present invention can also be formulated as a food additive, a food ingredient, a functional food, a dietary supplement, a medical food, a nutraceutical or a food supplement. In certain embodiments, the compound of the present invention can be included in a nutraceutical beverage of different volumes to allow a daily dose to be administered in a convenient form. As a non-limiting example, a beverage can deliver an effective dose in a final volume ranging from 5mL to 1,000mL, which is delivered as a single dose or multiple doses. In certain embodiments, the compositions and methods of the present invention are used for and used in non-human animals. Therefore, the compounds and compositions of the present invention can be formulated as veterinary products. Compounds and compositions can also be formulated into functional foods for use in animals, such as dogs, cats, horses, etc.

[0194] Dosing will generally be daily to weekly. In one embodiment, dosing is at least weekly. For example, a subject may receive a dose once a week, twice a week, three times a week, or every other day. In one embodiment, dosing is at least daily. For example, a subject may receive one or more doses per day.

[0195] It is believed that the most efficient dosing in humans involves extended daily administration. Extended use is contemplated including use for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or even longer.

[0196] For clinical use, urolithins can be administered to treat chronic or acute conditions. When autophagy in cells is observed to increase in less than a day, for example, after 8 hours, urolithins can be administered in an acute manner to treat acute conditions requiring induction of autophagy. Such conditions can include conditions caused by reperfusion injury (e.g., organ transplantation), heart attack, stroke, ischemic injury, during surgery (e.g., during angioplasty procedures or heart valve replacement), or after traumatic injury.

[0197] For clinical use, the urolithin will generally be administered in an amount equal to or equivalent to 0.2-2000 milligrams (mg) of urolithin per kilogram (kg) of subject body weight per day. In one embodiment, the urolithin is administered at a dose equal to or equivalent to 2-2000 mg of urolithin per kg of subject body weight per day. In one embodiment, the urolithin is administered at a dose equal to or equivalent to 20-2000 mg of urolithin per kg of subject body weight per day. In one embodiment, the urolithin is administered at a dose equal to or equivalent to 50-2000 mg of urolithin per kg of subject body weight per day. In one embodiment, the urolithin is administered at a dose equal to or equivalent to 100-2000 mg of urolithin per kg of subject body weight per day. In one embodiment, the urolithin is administered at a dose equal to or equivalent to 200-2000 mg of urolithin per kg of subject body weight per day.

[0198] The preparation of urolithin can be administered to a human subject in a therapeutically effective amount. Typical dosage ranges are from about 0.01 micrograms / kg body weight to about 2 mg / kg body weight per day. The dosage of the drug to be administered may depend on variables such as the type and extent of the condition, the overall health status of the particular subject, the particular compound being administered, the excipient used to formulate the compound, and its route of administration. Routine experimentation can be used to optimize the dosage and dosing frequency for any particular compound.

[0199] In one embodiment, urolithin is administered at a concentration ranging from about 0.001 microgram / kg to greater than about 500 mg / kg. For example, the concentration can be 0.001 microgram / kg, 0.01 microgram / kg, 0.05 microgram / kg, 0.1 microgram / kg, 0.5 microgram / kg, 1.0 microgram / kg, 10.0 microgram / kg, 50.0 microgram / kg, 100.0 microgram / kg, 500 microgram / 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 In some embodiments, the present invention relates to an amount of at least about 500 mg / kg of the present invention, or an amount of at least about 200 mg / kg of the present invention, or an amount of at least about 300 mg / kg of the present invention, or an amount of at least about 400 mg / kg of the present invention, or an amount of at least about 500 mg / kg of the present invention, or an amount of at least about 200 mg / kg of the present invention, or an amount of at least about 300 mg / kg of the present invention, or an amount of at least about 400 mg / kg of the present invention, or an amount of at least about 200 mg / kg of the present invention, or an amount of at least about 300 mg / kg of the present invention, or an amount of at least about 400 mg / kg of the present invention, or an amount of at least about 500 mg / kg of the present invention,

[0200] In one embodiment, urolithin is administered at a dosage ranging from about 0.2 mg / kg / day to greater 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 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 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 100 mg / kg / day. day to 10mg / kg / day, 0.25mg / kg / day to 7.5mg / kg / day, 0.25mg / kg / day to 5mg / kg / day, 0.5mg / kg / day to 50mg / kg / day, 0.5mg / kg / day to 25mg / kg / day, 0.5mg / kg / day to 20mg / kg / day, 0.5mg / kg / day to 15mg / kg / day, 0.5mg / kg / day to 10mg / kg / day, 0.5mg / kg / day to 7.5mg / kg / day, 0.5mg / kg / day to 5mg / kg / day, 0.75mg / kg / day day to 50mg / kg / day, 0.75mg / kg / day to 25mg / kg / day, 0.75mg / kg / day to 20mg / kg / day, 0.75mg / kg / day to 15mg / kg / day, 0.75mg / kg / day to 10mg / kg / day, 0.75mg / kg / day to 7.5mg / kg / day, 0.75mg / kg / day to 5mg / kg / day, 1.0mg / kg / day to 50mg / kg / day, 1.0mg / kg / day to 25mg / kg / day, 1.0mg / kg / day to 20mg / kg / day, 1.0mg / kg / day to 15mg / kg / day, 1.0mg / kg / day to 10mg / kg / day, 1.0mg / kg / day to 7.5mg / kg / day, 1.0mg / kg / day to 5mg / kg / day, 2mg / kg / day to 50mg / kg / day, 2mg / kg / day to 25mg / kg / day, 2mg / kg / day to 20mg / kg / day, 2mg / kg / day to 15mg / kg / day, 2mg / kg / day to 10mg / kg / day, 2mg / kg / day to 7.5mg / kg / day, or 2mg / kg / day to 5mg / kg / day.

[0201] In one embodiment, urolithin is administered at a dosage ranging from about 0.25 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, 4.0 ...25 mg / kg / day, 4.5 mg / kg / day, 4.75 mg / kg / day, 4.0 mg / kg / day, 4.25 mg / kg / day, 4.5 mg / kg / day, 4.75 mg / kg / day / kg / day, 4.25mg / kg / day, 4.5mg / kg / day, 4.75mg / kg / day, 5mg / kg / day, 5.5mg / kg / day, 6.0mg / kg / day, 6.5mg / kg / day, 7.0mg / kg / day, 7.5mg / kg / day, 8.0mg / kg / day, 8.5mg / kg / day, 9.0mg / kg / day, 9.5mg / kg / day, 10mg / kg / day, 11mg / kg / day, 12mg / kg / day, 13mg / kg / day, 14mg / kg / day, 15mg / kg / day, 16mg / kg / day, 17mg / kg / day, 18mg / kg / day, 19mg / kg / day, 20mg / kg / day, 21mg / kg / day, 22mg / kg / day, 23mg / kg / day, 24mg / kg / day, 25mg / kg / day, 26mg / kg / day, 27mg / kg / day, 28mg / kg / day, 29mg / kg / day, 30mg / kg / day, 31mg / kg / day, 32mg / kg / day, 33mg / kg / day, 34mg / kg / day, 35mg / kg / day, 36mg / kg / day, 37mg / kg / day, 38mg / kg / day, 39mg / kg / day, 40mg / kg / day, 41 mg / kg / day, 42mg / kg / day, 43mg / kg / day, 44mg / kg / day, 45mg / kg / day, 46mg / kg / day, 47mg / kg / day, 48mg / kg / day, 49mg / kg / day, or 50mg / kg / day.

[0202] In another embodiment, the urolithin is administered at a concentration ranging from 0.01 micromolar to greater than or equal to 500 micromolar. For example, the dosage can be 0.01 micromolar, 0.02 micromolar, 0.05 micromolar, 0.1 micromolar, 0.15 micromolar, 0.2 micromolar, 0.5 micromolar, 0.7 micromolar, 1.0 micromolar, 3.0 micromolar, 5.0 micromolar, 7.0 micromolar, 10.0 micromolar, 15.0 micromolar, 20.0 micromolar, 25.0 micromolar, 30.0 micromolar, 35.0 micromolar, 40.0 micromolar, 45.0 micromolar, 46.0 micromolar, 47.0 micromolar, 48.0 micromolar, 49.0 micromolar, 50.0 micromolar, 51.0 micromolar, 52.0 micromolar, 53.0 micromolar, 54.0 micromolar, 56.0 micromolar, 57.0 micromolar, 58.0 micromolar, 59.0 micromolar, 60.0 micromolar, 61.0 micromolar, 62.0 micromolar, 63.0 micromolar, 64.0 micromolar, 65.0 micromolar, 66.0 micromolar, 67.0 micromolar, 68.0 micromolar, 69.0 micromolar, 70.0 micromolar, 71.0 micromolar, 72.0 micromolar, 73.0 micromolar, 74.0 micromolar, 75.0 micromolar, 76.0 micromolar, 77.0 micromolar, 78.0 micromolar, 79.0 micro In some embodiments, the present invention relates to a method for producing a micromolar mass of at least about 500 micromolar mass, or a micromolar mass of at least about 200 micromolar mass, or a micromolar mass of at least about 300 micromolar mass, or a micromolar mass of at least about 400 micromolar mass, or a micromolar mass of at least about 500 micromolar mass, or a micromolar mass of at least about 600 micromolar mass, or a micromolar mass of at least about 700 micromolar mass, or a micromolar mass of at least about 800 micromolar mass, or a micromolar mass of at least about 900 micromolar mass, or a micromolar mass of at least about 100 micromolar mass, or a micromolar mass of at least about 15 ...100 micromolar mass, or a micromolar mass of at least about 100 micromolar mass, or a micromolar mass of at least about 150 micromolar mass, or a micromolar mass of at least about 100 micromolar mass, or a micromolar mass of at least about 100 micromolar mass

[0203] In yet another embodiment, urolithin is administered at a concentration ranging from 0.10 micrograms / mL to 500.0 micrograms / mL. For example, the concentration can be 0.10 micrograms / mL, 0.50 micrograms / mL, 1 microgram / mL, 2.0 micrograms / mL, 5.0 micrograms / mL, 10.0 micrograms / mL, 20 micrograms / mL, 25 micrograms / mL. 30 micrograms / mL, 35 micrograms / mL, 40 micrograms / mL, 45 micrograms / mL, 50 micrograms / mL, 60.0 micrograms / mL, 70.0 micrograms / mL, 80.0 micrograms / mL, 90.0 micrograms / mL, 100.0 micrograms / mL, 150.0 micrograms / mL, 200.0 micrograms / mL, 250.0 g / mL. 250.0 micrograms / mL, 300.0 micrograms / mL, 350.0 micrograms / mL, 400.0 micrograms / mL, 450.0 micrograms / mL, to greater than about 500.0 micrograms / mL or any incremental value thereof. It should be understood that all values ​​and ranges between these values ​​and ranges are intended to be included by the present invention.

[0204] Additional non-limiting illustrative examples of urolithin dosages that may be used are provided in Table 1. Table 1 Urolithin dosage in mice and humans

[0205] Additional non-limiting illustrative examples of urolithin dosage ranges that may be used are provided in Table 2. Table 2 Urolithin dosage in humans

[0206] Any dose may be administered as a single dose or as divided doses.

[0207] In one embodiment, urolithin is administered at a dose sufficient to achieve a peak serum level of at least 0.001 micromolar (μM) of urolithin and its known metabolites (glucoronides, sulfonates, etc.). In one embodiment, urolithin is administered at a dose sufficient to achieve a peak serum level of at least 0.01 μM of urolithin. In one embodiment, urolithin is administered at a dose sufficient to achieve a peak serum level of at least 0.1 μM of urolithin. In one embodiment, urolithin is administered at a dose sufficient to achieve a peak serum level of at least 1 μM of urolithin. In various embodiments, urolithin is administered at a dose 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.

[0208] In one embodiment, urolithin is administered at a dose sufficient to achieve a sustained serum level of at least 0.001 micromolar (μM) of urolithin. In one embodiment, urolithin is administered at a dose sufficient to achieve a sustained serum level of at least 0.01 μM of urolithin. In one embodiment, urolithin is administered at a dose sufficient to achieve a sustained serum level of at least 0.1 μM of urolithin. In one embodiment, urolithin is administered at a dose sufficient to achieve a sustained serum level of at least 1 μM of urolithin. In one embodiment, urolithin is administered at a dose sufficient to achieve a sustained serum level of at least 10 μM of urolithin, and urolithin is administered at a dose sufficient to achieve a sustained serum level of at least 50 μM of urolithin. Sustained serum levels can be measured using any suitable method, for example, high pressure liquid chromatography (HPLC) or HPLC-MS. Example

[0209] Having now generally described the invention, the invention will be more readily understood by reference to the following, which is included for the purpose of illustrating certain aspects and embodiments of the invention only and is not intended to be limiting of the invention.

[0210] Example 1: Administration of Urolithin A after Myocardial Infarction The purpose of the study was to evaluate the therapeutic effect of urolithin A on cardiac function after myocardial infarction. Figure 1 A schematic diagram showing the study design is shown and the experimental design is shown in Table 3. Myocardial infarction (MI) was induced in 6-week-old Wistar male rats in Groups 1, 2 and 3 by permanent occlusion of the left anterior descending coronary artery (ligation performed at D0) (Table 3). Table 3

[0211] Rats were anesthetized by intraperitoneal (IP) injection of medetomidine (0.5 mg / kg) and ketamine (50 mg / kg), intubated and ventilated with 10 mL / kg tidal volume and 70-80 cycles / min. Body temperature was maintained between 36.5°C and 37.5°C via a thermoregulatory heating pad connected to a rectal probe. Rats were placed in a supine position, the chest was shaved, and prepared for standard surgical conditions. A left thoracotomy was carefully performed to expose the heart. Sutures were then placed around the LAD (4 / 0 Silk, Ethicon), approximately 2 mm below the left atrium and near the interventricular junction, so as to obtain an infarct size (IS) close to 40% of the total left ventricular (LV) area. The chest cavity was closed, air was evacuated from the thorax to avoid pneumothorax, and rapid resuscitation was performed using atipamezole hydrochloride (IM, 0,5 mg / ml, Zoetis).

[0212] Perioperative care of animals during surgical procedures: • Preoperative care was performed with one injection of carprofen (SC, 5 mg / kg, Pfizer) and one injection of lidocaine (SC, 4 mg / kg, Vetoquinol) at the incision site. • Postoperative Care: Three injections of buprenorphine (SC, 0.01 mg / kg, Axience) will be administered upon awakening and during the first 24 hours post-surgery.

[0213] Sham operation (Group 1) Animals from Group 1 were subjected to the same protocol as the MI group. However, after a left thoracotomy to expose the heart, the thorax was closed without passing sutures around the LAD.

[0214] Urolithin A or vehicle was administered by gavage at 25 mg / kg starting one week after myocardial infarction. Heart and plasma samples were collected at each time point. Ejection fraction (EF), fractional shortening (FS), isovolumetric relaxation time (IVRT) were measured by echocardiography at 1 month and 2 months to assess cardiac function.

[0215] Echocardiography Rats were subjected to transthoracic echocardiography examinations to assess cardiac morphology and function in a non-invasive manner. Echocardiography was performed using a digital ultrasound system (Vivid 7, GE Medical Systems) equipped with a 10 MHz phased array and 13 MHz linear array transducer. All images were collected, measured, and calculated according to the American Society of Echocardiography (Schiller et al. J Am Soc Echocardiogr 1989) and previously validated methods in rats (Litwin et al., Circulation 1994).

[0216] During the procedure, rats were anesthetized with 4% isoflurane in a 50% oxygen-50% air mixture and maintained with 2% isoflurane. They were placed in a supine position on a heating pad and the chest was shaved. Body temperature was monitored via a rectal probe connected to a temperature regulating heating pad and maintained between 36.5-37.5°C.

[0217] Standard B-mode (brightness mode) and M-mode (motion mode) images of the heart will be obtained in a two-dimensional (2D) parasternal long axis view (PSLA). LV parameters will be measured by a blinded trained operator and calculated as the average of 3 consecutive cardiac cycles.

[0218] -Left ventricular function LV function was calculated using the LV dimension parameters described previously. oFS = Fractional Shortening = [(LVID d -LVID s ) / LVID d ]*100. It is a calculation based on the difference in size between systole and diastole measured in M-mode. It is a reflection of LV contractility. oEF = Ejection Fraction = [(end diastolic volume - end systolic volume) / end diastolic volume] * 100. It is a calculation based on the volume difference between systole and diastole measured in B-mode and M-mode. It is a reflection of LV contractility.

[0219] Additional echocardiographic measurements for the rats include analysis of IVRT, which is the time from aortic valve closure to mitral valve opening, and is an indicator of diastolic dysfunction. A total of four (3) echocardiographic examinations will be performed on all animals. The first examination will be performed 5 days after surgery (Table 4). It will serve as a control for the surgery to exclude rats with small infarcts and limited reduced EF. Two exclusion criteria will be used: -IVS without akinesia (infarction is too small) -EF>45% Table 4 shows the theoretical schedule for the initial two week experiment with the days of surgery and the days of the first echocardiography 5 days after surgery. Table 4. Theoretical timetable D, day; Echo, echocardiography

[0220] The included MI rats (Groups 2 and 3) were randomized into three homogeneous groups based on the following parameters: -LVID d -EF -weight Sham-operated animals present the same parameters of LV dimensions and function due to the absence of LAD ligation, but should present the same body weight. Randomization is achieved by session after the first echocardiography. After randomization, longitudinal follow-up studies by echocardiography are performed on the 4 groups of rats.

[0221] The 2nd and 3rd echocardiographic examinations were performed in the second month after the start of the diet (M2).Based on the echocardiographic parameters described previously, they were used to assess cardiac remodeling after MI.

[0222] Due to the number of rats planned for the study, at least three surgical sessions were performed over four consecutive weeks. Therefore, cardiac assessments at M2 were only available at the end of the analysis of three sessions. Table 5. Candidate compounds

[0223] The required amount of urolithin A is defined based on the dose to be tested, the total number of animals receiving compound, and an average final body weight of approximately 400 g.

[0224] Preparation of candidate compounds: Vehicle: 0.5% carboxymethyl cellulose

[0225] Preparation: One solution per test compound and per concentration was prepared weekly using 0.5% carboxymethylcellulose as vehicle. Each solution was sonicated for 15 minutes at 37°C to allow for optimal dissolution of the compound.

[0226] Preparation frequency: weekly

[0227] Storage: Each formulation was then aliquoted into 7 tubes which were 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 will be thawed each day and the volume of the gavage will be adjusted based on the last measured body weight.

[0228] PO administration PO administration was performed in animals of Groups 1 to 3 (Table 3) according to standard operating procedures performed at .PO administration was performed in non-anesthetized animals using an oral gavage probe.

[0229] The final volume of administration was set at 5 ml / kg. According to our procedure, the volume of administration was adjusted weekly based on the average body weight of the animals in the corresponding group. If an animal has a weight that is more than 20% higher than the average body weight of its group, the volume of administration will be adjusted specifically for that animal based on its own body weight. On the day of the last recording, the animal received an administration volume corresponding to its own body weight. Rats were treated orally with vehicle and candidate compounds 1 and 2 once a day, 7 days a week. Treatment started one week after MI and will continue for 2 months.

[0230] mortality rate Surgery (MI) may be associated with mortality during the first 24 hours. Additional animals were considered to ensure that 70 animals were included for the experiment. MI may be associated with mortality over time, which is model dependent.

[0231] Deviations were reported as soon as possible after the first echocardiogram and randomization. Animals that died after the first echocardiogram were not replaced.

[0232] Euthanasia and collection of cardiac tissue At the end of the procedure, after the last echocardiography, the animals were deeply anesthetized with 2,5% isoflurane in a 50% O2 / 50% air mixture after a buprenorphine injection (0,03 mg / kg, SC). Before sacrifice, a blood sample was collected at the end of the procedure through the jugular vein. Blood was collected in a volume of at least 0.4 ml on a K3-EDTA coated tube and centrifuged for 10 minutes (10000 rpm, 20°C). 200 microliters of plasma were frozen and stored at about -80°C until transported to the sponsor.

[0233] Then, the heart was stopped in diastole (KCl 2M, 1 ml / kg) for subsequent histological analysis by jugular vein injection. The atria were removed and the heart was washed in cold phosphate buffered saline (PBS, 4°C) and quickly sponged to remove fluid from the ventricles. Then, the right ventricle (RV) wall was separated from the rest of the heart (LV+septum) and the LV+S was carefully weighed using an analytical balance (AX 205 (Mettler Toledo) or equivalent). Finally, the RV was removed and the LV+S was collected and processed as follows: - Fixation of LV+S: they were placed in NBF 10% during 48 hours and transferred in ethanol 70% before paraffin inclusion. (Group 1, n=10; Groups 2 to 4, n=20).

[0234] The results of the study are shown in Figures 2A-2C Urolithin A significantly improved systolic dysfunction as measured by ejection fraction and fractional shortening ( Figure 2A ), improved diastolic function as measured by isovolumetric relaxation time ( Figure 2B ), and reduced cardiac hypertrophy calculated as heart weight / tibia length ( Figure 2C ). In conclusion, urolithin A has a protective effect against heart failure.

[0235] Example 1A: Effects of Urolithin A on Gene Sets Associated with Improved Mitochondrial Function RNA extraction and quality control From Figure 1 Heart sections of rats in the group described in (Example 1) were prepared using a microtome, dewaxed and RNA was extracted using the following kit (RNAeasy FFPE 76504, Qiagen). After extraction, RNA quality control was performed using an Agilent FragmentAnalyzer System.

[0236] Library preparation and sequencing Use a strand-specific cDNA library and follow the steps indicated here for library preparation: rRNA depletion mRNA fragmentation Random primed cDNA synthesis (strand-specific) Adapter ligation and adapter-specific PCR amplification RNA sequencing runs were performed with NovaSeq 6000 using an S4 flow cell with 2×150 bp.

[0237] Bioinformatics analysis The QC and preprocessing of the original sequencing reads were performed using an internal QC pipeline. The pipeline uses fastp, version 0.23.2 (Chen, A et al., Bioinformatics 2018, 34 (17), i884-i890), which is an integrated preprocessing tool for FastQ files, automating quality control, quality trimming, and adapter clipping procedures. High-quality RNA sequencing reads were mapped to the Ensembl rat reference genome.

[0238] Reads with a minimum PHRED score of 15, a minimum length of 15 bp, and a minimum ineligible percentage limit of 40 (40%) were retained for downstream analysis.

[0239] Before DE analysis, a pre-filtering step was applied to remove low abundance mRNA measurements by excluding genes with a total count of less than 10 in the sample. In addition, an independent filtering program of DESeq2 was enabled to filter out genes with very low counts that were unlikely to show significant changes in gene expression. Gene symbols, descriptions, and biotypes were matched with Ensembl GTF ids using the R package biomaRt.v.2.46.3 (Durink, S et al., Nature Protocol 2009, 4, 1184-1191). The P value of multiple tests was corrected using the Benjamini and Hochberg (BH) method (Benjamini, Y and Hochberg, Y, Journal of Royal Statistical Society, Series B, 1995, 57, 289-300). Genes showing a p value of correction (adjustment) less than 0.05 and an absolute log2 fold change greater than 1 were considered to be differentially expressed (differentially expressed genes-DEGs).

[0240] 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 and gene ontology (cellular components) under different biological conditions. GSEA uses all treated genes and classifies them according to log2 fold changes. Gene ranking information is converted to pathway level to assign statistical significance to each analyzed gene set. Each gene set is analyzed independently, and the direction of gene regulation (up / down regulation) is inferred by using the biological comparison examined.

[0241] result Gene set enrichment analysis was performed by looking at the gene sets (i.e., biological pathways) that belonged to the category of "cellular components." In particular, we investigated which biological pathways were: (i) significantly downregulated when comparing rats with myocardial infarction to healthy control rats (Mi vs. sham); and (ii) significantly upregulated when comparing MI rats treated with urolithin A (MI+UA) to our rats with MI that were not treated with urolithin A (MI).

[0242] Figure 3Several gene sets are shown that are both suppressed by the development of MI in diseased animals (MI vs. sham, grey bars) and also induced by urolithin A (MI+UA, red bars). The X-axis represents the normalized enrichment score indicating downregulation of a gene set when values ​​are negative and upregulation of a gene set when values ​​are positive.

[0243] Several of these gene sets are associated with mitochondrial function, such as the mitochondrial protein-containing complex, which contains genes essential for optimal mitochondrial function, and the respirosome, which contains genes required for proper mitochondrial respiration (ie, energy production).

[0244] These data indicate that myocardial infarction is associated with decreased expression of genes related to mitochondrial function in cardiac tissue and that administration of urolithin A can positively reverse the expression of these gene sets.

[0245] Additional gene sets that were suppressed by the development of MI in diseased animals (MI vs. sham, grey bars) and also induced by urolithin A (MI+UA, red bars) were related to nucleoid and peroxisome biology.

[0246] Example 2: Preclinical in vivo studies to evaluate improvements in both cardiac and skeletal muscle function The aim of the study was to evaluate the effects of urolithin treatment on age-related cardiac dysfunction. Figure 3 Schematic diagram showing the study design. Aged C57BL / 6 mice (26 months) were used as a model to reproduce the natural decline of cardiac function with aging. Chow diet with and without urolithin A was 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), fractional shortening (FS) were measured by echocardiography. Muscle contractility was also measured. Analysis of both myocardial and skeletal muscle function was performed at the end of the study.

[0247] The results are shown in Figure 4 A-4B. Urolithin A significantly improves both cardiac and skeletal muscle function during natural aging. Figure 4 A shows that urolithin A increased fractional shortening both at basal and after high workload in aged mice, and Figure 4 B shows that urolithin A significantly prevented the age-related decline in skeletal muscle function. In conclusion, urolithin A has a cardioprotective effect during natural aging.

[0248] Example 3: Effect of Urolithin A on Mitochondrial Morphology Associated with Improved Mitochondrial Function The purpose of the study was to evaluate the effect of urolithin treatment 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 of cardiac function with aging and were administered urolithin A (aged UA) or vehicle (aged vehicle) at 50 mg / kg by gavage for 2 months. Young mice (18 weeks of age at the end of the study) were administered vehicle for 2 months. Myocardium was collected postmortem at the end of the study.

[0249] Transmission electron microscopy methods and analysis Muscle samples from animals as described in Example 2 were fixed in 2% glutaraldehyde solution in 0.1 M Sorenson buffer (pH 7.3). These samples were then post-fixed in 1% osmium tetroxide for 8 minutes [pulsed microwave (MW), 100 watts (W)] and cleaned in distilled H2O (3×150 W for 40 s each step). The samples were dehydrated in a series of graded acetone (25%; 50%; 75%; 3×100%; 150 W for 40 s each step) before being impregnated with increasing concentrations of epoxy resin (TAAB medium resin) in acetone (25%; 50%; 75%; 3×100%; 300 W for 3 minutes each step). The samples were then embedded in 100% fresh resin and polymerized in a conventional oven at 60°C for a minimum of 24 hours.

[0250] All resin blocks were trimmed using a razor blade to form a trapezoidal block face. Sections were cut in longitudinal or transverse orientation on an ultramicrotome (Wolf et al., 2019) using a diamond knife. Semi-thin sections (0.5 μm) were stained with toluidine blue and viewed on a light microscope (LM) to verify the orientation of the tissue. Ultrathin sections (70 nm) were then cut and picked up onto a copper grid. Sections from the blocks were stained with 1% uranyl acetate (30 min) and 3% lead citrate (7 min). All sections were examined using an HT7800 120kVTEM (Hitachi). Digital micrographs were captured using an EMSIS Xarosa CMOS camera with Radius software.

[0251] These samples were then post-fixed in 1% osmium tetroxide for 1 hour and dehydrated in increasing concentrations of acetone. To verify the orientation of muscle tissue, 1 μm thick sections were stained with toluidine blue before ultrathin sectioning. Ultrathin sections were cut in longitudinal or transverse orientation on an Ultracut ultramicrotome (Leica). These sections were then stretched to remove compression and fixed on a copper grid and then stained with 2% aqueous uranyl acetate and lead citrate (Leica). The sections were then imaged using a Philips CM 100 electron microscope. Digital micrographs were captured using an AMT XR80 CCD digital camera at × 7900 magnification.

[0252] Individual cardiac mitochondria from 3 young vehicle mice, 3 aged vehicle mice, and 3 aged UAO-AL mice were manually traced in transverse orientation using ImageJ software (NIH) (https: / / imagej.nih.gov / ij / ) and the following morphological and shape descriptors were quantified: area (μm2), perimeter (μm), circularity 4π×(area / perimeter2), aspect ratio ((major axis) / (minor axis)): aspect ratio is a measure of the ratio of length to width, shape factor ((perimeter) / (4π×surface)): a measure sensitive to the complexity and branching appearance of mitochondria, Feret diameter (the longest distance (μm) between two points of the studied mitochondria), and minimum Feret diameter (corresponding to the smallest diameter of the mitochondria) (Picard et al., 2012). It is important to emphasize here that n = 3–4 is a commonly used sample size in TEM-based studies investigating mitochondrial morphology and ultrastructure.

[0253] RNA sequencing As described in Example 1, RNA sequencing analysis of the samples derived from Example 3 has been performed.

[0254] Succinate dehydrogenase activity The heart in OCT was cut into 8 μM slices using a cryostat. The slices were then incubated at room temperature for 6 minutes in a solution containing tetrazolium nitro blue (1.5 mM), sodium succinate (130 mM), phenazine methosulphate (0.2 mM) and sodium azide (0.1 mM). The reaction was stopped by washing twice in PBS. The slides were fixed 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 using a bright field microscope. The staining intensity was analyzed using Image J.

[0255] result Compared with young mice, old mice showed significantly reduced mitochondrial area (expressed in mm 2 ). UA administration for 2 months significantly increased mitochondrial area in aged mice compared with young animals to a certain level ( Fig. 6A ). It has been reported that mitochondrial area also declines in skeletal muscle of elderly subjects compared to young subjects (Crane, JD et al., J Gerontol A Biol Sci Med Sci 2010, 65(2). 119-128). This decline has been associated with reduced mitochondrial function (Crane, JD et al., 2010). This data indicates that UA can improve mitochondrial area, which is associated with improved mitochondrial function in the aging heart.

[0256] In addition, the cristae ultrastructure of cardiac mitochondria was analyzed. These represent complex invaginations of the inner mitochondrial membrane. This is where mitochondrial respiration occurs, and therefore maintaining its correct structure is key to maintaining optimal mitochondrial bioenergetic capacity (Baker, N. et al., Mitochondrion 2019, 49, 259-268). It was observed that aged mice had impaired cristae structure compared to young animals, which was quantified as cristae volume and number ( Figure 7 UA administration significantly increased the volume density and number of cristae in aged mice. This indicates the ability of UA to improve mitochondrial quality in the heart during aging.

[0257] In the hearts of the same animals as in this example, we quantified mitophagic events expressed as the number of mitochondria engulfed into autophagosomal vesicles and thus forming mitophagolysosomes. It was observed that aged mice had reduced mitophagic events compared to young animals, which was quantified as the number of mitophagolysosomes ( Figure 8 ). Administration of UA significantly increased mitophagic events in aged mice. This is clinically relevant because mitophagy has been shown to be essential for cardiac function in experimental models (Kubli, DA et al., J Biol Chem 2013, 288(2), 915-926). In addition, mitophagy is impaired in human patients with heart failure (Billia, F. et al. Proc Natl Acad Sci USA 2011, 108(23), 9572-7). This indicates the ability of UA to increase mitophagy in the heart during aging.

[0258] Gene set enrichment analysis was performed by looking at gene sets (i.e., biological pathways) that fell into the category of "cellular components." In particular, we investigated which biological pathways were: (i) significantly downregulated when comparing old mice to young mice; and (ii) significantly upregulated when comparing old mice treated with UA to old mice treated with vehicle.

[0259] Fig. 9 Several gene sets are listed that are both repressed by senescence and also induced by urolithin A in aged animals. The normalized enrichment score indicates downregulation of a gene set when the value is negative; and upregulation of a gene set when the value is positive.

[0260] Several of these gene sets were associated with mitochondrial function, such as the respirosome, which contains genes required for proper mitochondrial respiration (ie, energy production). Additional gene sets were also associated with heart cell health that was improved by UA treatment, such as the sarcolemma.

[0261] These data indicate that myocardial infarction is associated with decreased expression of genes related to mitochondrial function in cardiac tissue and that administration of urolithin A can positively reverse the expression of these gene sets.

[0262] To further confirm the positive effects of UA on mitochondrial function in health, we measured the enzyme activity of the mitochondrial complex II enzyme SDH. This immunoenzymatic assay is an indicator of mitochondrial respiratory activity and therefore mitochondrial function. Fig.10 It was shown that administration of UA to aged mice significantly increased SHD activity in the heart compared with aged vehicle control mice.

[0263] Incorporated by Reference All U.S. patents and U.S. and PCT published patent applications mentioned in the above specification are incorporated herein by reference in their entirety.

[0264] Equivalent Having now described the invention in detail by way of illustration and example for purposes of clear understanding, it will be apparent to one skilled in the art that the same may be practiced by modification or alteration of the invention within a broad and equivalent range of conditions, formulations, and other parameters without affecting the scope of the invention or any specific embodiment thereof, and such modifications or alterations are intended to be included within the scope of the appended claims.

Claims

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

2. The method of claim 1, wherein the urolithin is administered after ischemia-reperfusion injury (e.g., at least about one day, about two days, about three days, about four days, about five days, or about one week after ischemia-reperfusion injury).

3. The method of claim 1 or 2, wherein cardiac hypertrophy induced by myocardial infarction is reduced in the mammal. The method of claim 3 , wherein cardiac hypertrophy in the left ventricle is reduced.

5. A method for improving cardiac function, the method 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.

6. A method for protecting cardiac function, the method 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.

7. A method for reducing the rate of decline in cardiac function, the method comprising administering an effective amount of a urolithin to a mammal in need thereof; wherein the effective amount of the urolithin is from about 10 mg / kg to about 30 mg / kg.

8. A method for preventing cardiac function decline, the method comprising administering an effective amount of urolithin to a mammal in need thereof; wherein the effective amount of urolithin is from about 10 mg / kg to about 30 mg / kg.

9. A method of controlling or treating peripheral arterial disease (PAD), the method comprising administering an effective amount of a urolithin to a mammal in need thereof; wherein the effective amount of the urolithin is from about 10 mg / kg to about 30 mg / kg.

10. A method of controlling or improving tissue oxygenation, the method comprising administering an effective amount of a urolithin to a mammal in need thereof; wherein the effective amount of the urolithin is from about 10 mg / kg to about 30 mg / kg.

11. A method of improving cardiac output, the method comprising administering an effective amount of a urolithin to a mammal in need thereof; wherein the effective amount of the urolithin is from about 10 mg / kg to about 30 mg / kg.

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

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

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

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

16. The method of claim 15, wherein the mammal has an increased left ventricular ejection fraction (LVEF).

17. The method of claim 16, wherein prior to administration of the urolithin, the mammal has a LVEF of less than about 50%.

18. The method of claim 16, wherein prior to administration of the urolithin, the mammal has a LVEF of less than about 40%.

19. The method of claim 16, wherein prior to administration of the urolithin, the mammal has a LVEF of less than about 35%.

20. The method of claim 16, wherein prior to administration of the urolithin, the mammal has a LVEF of less than about 30%.

21. The method of claim 16, wherein prior to administration of the urolithin, the mammal has a LVEF of about 15-50%.

22. The method of claim 16, wherein prior to administration of the urolithin, the mammal has a LVEF of about 15-40%.

23. The method of claim 16, wherein prior to administration of the urolithin, the mammal has a LVEF of about 15-35%.

24. The method of claim 16, wherein prior to administration of the urolithin, the mammal has a LVEF of about 20-45%.

25. The method of claim 16, wherein prior to administration of the urolithin, the mammal has a LVEF of about 40-49%.

26. The method of claim 16, wherein prior to administration of the urolithin, the mammal has a LVEF of about 41-49%.

27. The method of any one of claims 15-26, wherein the mammal has an increased right ventricular ejection fraction (RVEF).

28. The method of claim 27, wherein prior to administration of the urolithin, the mammal has a RVEF of less than about 50%.

29. The method of claim 27, wherein prior to administration of the urolithin, the mammal has a RVEF of less than about 40%.

30. The method of claim 27, wherein prior to administration of the urolithin, the mammal has a RVEF of less than about 35%.

31. The method of claim 27, wherein prior to administration of the urolithin, the mammal has a RVEF of less than about 30%.

32. The method of claim 27, wherein prior to administration of the urolithin, the mammal has a RVEF of about 15-50%.

33. The method of claim 27, wherein prior to administration of the urolithin, RVEF is about 15-40%.

34. The method of claim 27, wherein prior to administration of the urolithin, RVEF is about 15-35%.

35. The method of claim 27, wherein prior to administration of the urolithin, the mammal has a RVEF of about 20-45%.

36. The method of claim 27, wherein prior to administration of the urolithin, the mammal has a RVEF of about 40-49%.

37. The method of claim 27, wherein prior to administration of the urolithin, the mammal has a RVEF of about 41-49%.

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

39. The method of claim 38, wherein heart function with preserved ejection fraction (HFpEF) is improved.

40. The method of claim 38, wherein heart function with reduced ejection fraction (HFrEF) is improved.

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

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

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

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

45. The method of claim 41 or 42, wherein cardiac systolic function of the mammal is improved by about 56%.

46. ​​The method of claim 41 or 42, wherein cardiac systolic function of the mammal is improved by about 64%.

47. The method of any one of claims 1-46, wherein the mammal has reduced diastolic dysfunction.

48. The method of claim 47, wherein the mammal's diastolic dysfunction is reduced by about 5-10%.

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

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

51. The method of claim 49, wherein the cardiac systolic function of the mammal is improved by at least about 30%.

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

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

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

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

56. The method of any one of claims 1-55, wherein myocardial contractility is increased.

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

58. The method of any one of claims 1-57, wherein cardiac mitochondrial function is improved.

59. The method of any one of claims 1-57, wherein cardiac mitochondrial mass is improved.

60. The method of any one of claims 1-57, wherein cardiac mitochondrial area is increased.

61. The method of any one of claims 1-60, wherein expression of genes associated with mitochondrial function in cardiac tissue is upregulated.

62. The method of claim 61, wherein the gene is a gene containing a mitochondrial protein complex or a gene of a respiratory body.

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

64. The method of any one of claims 1-63, wherein 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 reduced.

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

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

67. The method of any one of claims 1-66, wherein circulation is improved.

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

69. The method of any one of claims 1-68, wherein mitochondrial damage is ameliorated.

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

71. The method of claim 70, wherein the urolithin is administered in the form of a tablet or capsule.

72. The method of any one of claims 1-71, wherein the urolithin is administered over a period of at least about 1 month or at least about 2 months.

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

74. The method of claim 73, wherein the human is elderly.

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

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

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

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

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

80. The method of any one of claims 1-77, wherein the urolithin is urolithin A.

81. The method of any one of claims 1-77, wherein the urolithin is urolithin B.

82. The method of any one of claims 1-77, wherein the urolithin is urolithin C.

83. The method of any one of claims 1-77, wherein the urolithin is urolithin D.

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

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

86. The method of claim 85, wherein the therapeutic agent is selected from angiotensin converting enzyme (ACE) inhibitors, angiotensin receptor-neprilysin inhibitors (ARNi), beta-blockers, mineralocorticoid receptor antagonists (MRA), sodium-glucose cotransporter 2 (SGLT2) inhibitors, angiotensin-2 receptor blockers (ARBs), diuretics, I f channel blockers, aldosterone antagonists, aspirin, P2Y12 inhibitors, calcium channel blockers, cholesterol-lowering drugs (e.g. statins), antiarrhythmic drugs, hydralazine with nitrates, digoxin, and anticoagulants.

87. The method of claim 86, wherein the therapeutic agent is a SGLT2 inhibitor; and the SGLT2 inhibitor is dapagliflozin or empagliflozin.

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

89. The method of claim 88, wherein the nutritional supplement is selected from the group consisting of fish oil, nitrate-rich beets, nitric oxide, red yeast rice, beta-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.

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