Methylated phloretin analogues as SGLT2 inhibitors

Abstract

The present invention relates to compositions and methods for improving metabolic health. In particular, the present disclosure relates to the use of methylated phloretin analogs as sodium glucose transporter 2 (SGLT2) inhibitors to improve metabolic health.

Description

[Technical Field]

[0001] [Background technology]

[0001] This disclosure relates generally to compositions and methods for improving metabolic health. Specifically, this disclosure relates to the use of methylated phloretin analogs as sodium glucose transporter 2 (SGLT2) inhibitors to improve metabolic health. For example, it has been discovered for the first time that methylated phloretin analogs can be used both as foods or supplements and as sodium glucose transporter 2 (SGLT2) inhibitors to effectively inhibit SGLT2 and improve metabolic health.

[0002]

[0002] The SGLT family of proteins includes active glucose transporters that play an important role in maintaining blood glucose balance. SGLT2 is expressed in the epithelial cells lining the first segment of the proximal tubule in the kidney and is responsible for approximately 90% of renal glucose reabsorption. An ideal inhibitor would inhibit SGLT2, thereby preventing glucose reuptake from the glomerular filtrate in the kidney, subsequently lowering blood glucose levels and promoting glucose excretion in the urine.

[0003]

[0003] Phlorizin is a natural compound and a known inhibitor of SGLT1 and SGLT2. SGLT1 is expressed in the intestine as well as the kidney. Inhibition of SGLT1 in the intestine is associated with adverse gastrointestinal effects, such as diarrhea. Because phlorizin is associated with adverse gastrointestinal effects, it cannot be added to food as a supplement and is not approved for use as a pharmaceutical. The present invention does not inhibit SGLT1 in the intestine and therefore, unlike phlorizin, may be added to food.

[0004]

[0004] Known SGLT2 inhibitors are a class of oral antihyperglycemic drugs that are insulin-independent and used by clinicians in the treatment of type 2 diabetes. Since 2013, four SGLT2 inhibitors have been approved by the Food and Drug Administration: canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin.

[0005]

[0005] Known SGLT2 inhibitors are a class of FDA-approved prescription medications for lowering blood glucose in adults with type 2 diabetes, in combination with diet and exercise. Pharmacological agents in the SGLT2 inhibitor class include canagliflozin, dapagliflozin, and empagliflozin. These compounds are available as single-ingredient products and in combination with other diabetes medications, such as metformin. SGLT2 inhibitors cause the kidneys to remove sugar from the body through urine, lowering blood glucose. However, none of these known SGLT2 inhibitors are natural compounds and can only be used as drugs, not as foods or supplements.

[0006] There is a clear unmet need for novel compounds and compositions as SGLT2 inhibitors for improving metabolic health. There is a need in the art for novel compounds and compositions as SGLT2 inhibitors that can be used as foods or supplements for improving metabolic health. [Summary of the Invention]

[0007]

[0007] The present disclosure includes the recognition that administering a methylated phloretin analog compound or related composition to a subject as a sodium glucose transporter 2 (SGLT2) inhibitor can effectively inhibit SGLT2 and, as a result, improve metabolic health.

[0008] The methylated phloretin analogue is selected from the group consisting of phloretin-4-methyl-ether, 4'-O-methylphloretin, calomelanone and flavocavain A.

[0009]

[0009] Methylated phloretin analogs or related compositions have improved bioavailability compared to phloretin and can be used in subjects as foods or supplements to effectively inhibit SGLT2 and thereby improve metabolic health.

[0010] Furthermore, such compounds and related compositions are less potent (2-3 times less potent) at inhibiting GLUT.

[0011] Another advantage of the present compounds and related compositions is that fewer glycosides can be produced in vivo compared to phloretin, resulting in improved biotransformation. [Brief explanation of the drawings]

[0012] [Figure 1] Levels of internalized [H]2-deoxyglucose, a GLUT transporter substrate, are shown following treatment with (A) phloretin, (B) calomelanone, (C) phloretin 4-methyl ether, (D) acepogenin, and (E) flavocabain A. Data are shown relative to the effects of the pharmacological GLUT inhibitors BAY-876 (100% effect) and DMSO (0% effect). [Figure 2-1] The indicated individual methyl-phloretin compounds: calomelanone, were incubated with UDP-glucose in the presence of rat microsomes and analyzed by uHPLC-MS after solid-phase extraction as described. For the compounds: i) the extracted mass chromatogram shows the expected monoisotopic masses for the parent and glycosidic forms from LC-MS analysis of the SPE elution fraction containing both the parent and the resulting glycosidic metabolites (A). ii) For each peak observed in the extracted mass chromatogram, the sum of MS-level spectra acquired over the peak duration (>5 distinct detection events) is shown, to which Waters Lockmass correction was applied to determine the accurate monoisotopic mass (B). [Figure 2-2]For the indicated individual methyl-phloretin compounds: calomelanone, incubation with UDP-glucose was performed in the presence of rat microsomes and analyzed by uHPLC-MS after solid-phase extraction as described. For compounds: iii) For each major peak of the parent and derived glycoside species observed in the extracted mass chromatogram, selected parent ion and glycoside species MS / MS spectra are shown (C). For glycoside species, selected ions fragment with a characteristic loss of 162 mu, consistent with the loss of the glucosyl moiety, generating fragment ions at the m / z expected for the parent methylphloretin compound. iv) For each methylphloretin species, the location of the predicted nonequivalent site of glycosylation is shown (D). [Figure 3-1] The indicated individual methyl-phloretin compound: phloretin-4 methyl ether (FIG. 3) was incubated with UDP-glucose in the presence of rat microsomes and analyzed by uHPLC-MS after solid-phase extraction as described. For the compound: i) The extracted mass chromatogram shows the expected monoisotopic masses for the parent and glycosidic forms from LC-MS analysis of the SPE elution fraction containing both the parent and the resulting glycosidic metabolite (A). ii) For each peak observed in the extracted mass chromatogram for the parent and glycosidic species, the sum of the MS-level spectra obtained over the peak duration (>5 distinct detection events) is shown, to which Waters Lockmass correction was applied to determine the measured monoisotopic accurate mass (B). [Figure 3-2]The indicated individual methyl-phloretin compound: phloretin-4 methyl ether (FIG. 3) was incubated with UDP-glucose in the presence of rat microsomes and analyzed by uHPLC-MS after solid-phase extraction as described. For the compound: iii) For each major peak of the parent and derived glycoside species observed in the extracted mass chromatogram, selected parent ion and glycoside species MS / MS spectra are shown (C). For glycoside species, selected ions fragment with a characteristic loss of 162 mu, consistent with the loss of the glucosyl moiety, and generate fragment ions at the m / z expected for the parent methylphloretin compound. iv) For each methylphloretin species, the location of the predicted non-equivalent site of glycosylation is shown (D). [Figure 4-1] The indicated individual methyl-phloretin compound: phloretin-4' methyl ether (acepogenin) (FIG. 4) was incubated with UDP-glucose in the presence of rat microsomes and analyzed by uHPLC-MS after solid-phase extraction as described. For the compound: i) The extracted mass chromatogram shows the expected monoisotopic masses for the parent and glycosidic forms from LC-MS analysis of the SPE elution fraction containing both the parent and the resulting glycosidic metabolites (A). ii) For each peak observed in the extracted mass chromatogram, the sum of MS-level spectra acquired over the peak duration (>5 distinct detection events) is shown, to which Waters Lockmass correction was applied to determine the measured monoisotopic accurate mass (B). [Figure 4-2]The indicated individual methyl-phloretin compound: phloretin-4' methyl ether (acepogenin) (FIG. 4) was incubated with UDP-glucose in the presence of rat microsomes and analyzed by uHPLC-MS after solid-phase extraction as described. For the compound: iii) For each major peak of the parent and derived glycoside species observed in the extracted mass chromatogram, selected parent ion and glycoside species MS / MS spectra are shown (C). For glycoside species, selected ions fragment with a characteristic loss of 162 mu, consistent with the loss of the glucosyl moiety, and generate fragment ions at the m / z expected for the parent methylphloretin compound. iv) For each methylphloretin species, the location of the predicted non-equivalent site of glycosylation is shown (D). [Figure 5-1] The indicated individual methyl-phloretin compound, flavocavain (FIG. 5), was incubated with UDP-glucose in the presence of rat microsomes and analyzed by uHPLC-MS after solid-phase extraction as described. For the compound: i) The extracted mass chromatogram shows the expected monoisotopic masses for the parent and glycosidic forms from LC-MS analysis of the SPE elution fraction containing both the parent and the resulting glycosidic metabolites (A). ii) For each peak observed in the extracted mass chromatogram, the sum of MS-level spectra acquired over the peak duration (>5 distinct detection events) is shown, to which Waters Lockmass correction was applied to determine the measured monoisotopic accurate mass (B). [Figure 5-2]The indicated individual methyl-phloretin compounds, including flavocavain (FIG. 5), were incubated with UDP-glucose in the presence of rat microsomes and analyzed by uHPLC-MS after solid-phase extraction as described. For compounds: iii) For each major peak of the parent and derived glycoside species observed in the extracted mass chromatogram, selected parent ion and glycoside species MS / MS spectra are shown (C). For glycoside species, selected ions fragment with a characteristic loss of 162 mu, consistent with the loss of the glucosyl moiety, and generate fragment ions at the m / z expected for the parent methylphloretin compound. iv) For each methylphloretin species, the location of the predicted non-equivalent sites of glycosylation is shown (D). [Figure 6] 1 is a graph and chemical formula showing the normalized SGLT1 and SGLT2 inhibitory activity of 4'-methylphlorizin (NI00046322). [Figure 7] 1 is a graph and chemical formula showing the normalized SGLT1 and SGLT2 inhibitory activity of 4-methylphlorizin (NI00046291) and (NI00001380). [Figure 8] The chemical formulas of the methylated phloretin analogs and glycosides corresponding to the letters shown in Table 1 are shown. DETAILED DESCRIPTION OF THE INVENTION

[0013]

[0017] definition

[0018] Some definitions are provided below. However, definitions may be found in the "Embodiments" section below, and the heading "Definitions" above does not imply that such disclosure in the "Embodiments" section is not a definition.

[0014]

[0019] All percentages set forth herein are by weight of the total composition unless otherwise specified. As used herein, "about," "approximately," and "substantially" are understood to refer to numbers within a range of numerical values, e.g., within -10% to +10% of the referenced number, preferably within -5% to +5% of the referenced number, more preferably within -1% to +1% of the referenced number, and most preferably within -0.1% to +0.1% of the referenced number. All numerical ranges herein should be understood to include all integers or fractions within that range. Furthermore, these numerical ranges should be interpreted as supporting claims directed to any number or subset of numbers within that range. For example, a disclosure of 1 to 10 should be interpreted as corresponding to ranges of 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0015]

[0020] As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise. Thus, for example, reference to "a component" or "the component" includes two or more components.

[0016]

[0021] The terms "comprise," "comprises," and "comprising" should be interpreted as inclusive rather than exclusive. Similarly, the terms "include," "including," and "or" should all be interpreted as inclusive unless such interpretation is clearly prohibited by context. However, the compositions disclosed herein may not include elements not specifically disclosed herein. Thus, disclosure of embodiments using the term "comprising" includes disclosure of embodiments "consisting essentially of" the specified components, as well as embodiments "consisting of" the specified components.

[0017]

[0022] The term "and / or" when used in the context of "X and / or Y" shall be interpreted as "X" or "Y" or "X and Y". Similarly, "at least one of X or Y" shall be interpreted as "X" or "Y" or "X and Y". For example, "at least one dithionite or a functionally equivalent reducing agent" shall be interpreted as "dithionite" or "a functionally equivalent reducing agent" or "both dithionite and a functionally equivalent reducing agent".

[0018]

[0023] As used herein, the terms "examples" and "such as," particularly when followed by a list of terms, are merely exemplary and illustrative and should not be considered exclusive or inclusive. As used herein, a condition "associated with" or "linked with" another condition means that the conditions occur simultaneously, preferably that the conditions are caused by the same underlying condition, and most preferably that one of the specified conditions is caused by the other specified condition.

[0019]

[0024] As used herein, the term "sodium glucose transporter 2 inhibitor" or "SGLT2 inhibitor" refers to a compound or substance that exhibits an inhibitory effect on SGLT2 (sodium-sugar carboxy-2), particularly human SGLT2. The inhibitory effect on hSGLT2, as measured by IC50, is preferably 1000 nM or less, more preferably 100 nM or less, and most preferably 50 nM or less. The IC50 value of an SGLT2 inhibitor is typically greater than 0.01 nM, or even greater than 0.1 nM. The inhibitory effect on hSGLT2 can be measured by methods known in the literature, particularly the methods described in WO 2005 / 092877 or WO 2007 / 093610 (pp. 23 / 24), which are incorporated herein by reference. The term "SGLT2 inhibitor" also includes pharmaceutically acceptable salts, hydrates, and solvates, including their respective crystalline forms.

[0020]

[0025] In one embodiment, the known SGLT2 inhibitor is selected from the group consisting of dapagliflozin, canagliflozin, empagliflozin, ertugliflozin, remogliflozin, sergliflozin, and derivatives thereof.

[0021]

[0026] For example, known SGLT2 inhibitors may comprise a class of FDA-approved prescription drugs for lowering blood sugar in adults with type 2 diabetes in combination with diet and exercise. Known pharmaceuticals in the SGLT2 inhibitor class include canagliflozin, dapagliflozin, and empagliflozin. These compounds are available as single-ingredient products and in combination with other diabetes medications, such as metformin. SGLT2 inhibitors cause the kidneys to remove sugar from the body through urine, lowering blood sugar.

[0022]

[0027] Curr Opin Endocrinol Diabetes Obes. 2017 Feb;24(1):73-79.doi;10.1097 / MED to Hsia et al provide a list of other compounds that are SGLT2 inhibitors.

[0028] However, none of these known SGLT2 inhibitors are natural compounds and can only be used as drugs, not as foods or supplements.

[0023]

[0029] As used herein, the term "nutritional composition" or "nutritional supplement" refers to a nutritional product that provides an individual with nutrients that the individual may not otherwise consume in sufficient amounts. For example, a nutritional composition or nutritional supplement of the present invention can include vitamins, minerals, fiber, fatty acids, or amino acids. A nutritional composition or nutritional supplement of the present invention can be provided in the form of, for example, a pill, tablet, lozenge, chewable capsule or tablet, tablet or capsule, or a powdered supplement that can be dissolved in water or sprinkled on food, for example.

[0024]

[0030] In one embodiment, the nutritional composition or dietary supplement of the present invention may provide selected nutrients but not contribute to a significant portion of a subject's overall nutritional needs. Typically, the nutritional composition or dietary supplement of the present invention does not represent more than 0.1%, 1%, 5%, 10%, or 20% of a subject's daily energy needs. The nutritional composition or dietary supplement of the present invention may be used in any subject, such as a pregnant subject, for example, as a maternal supplement.

[0025]

[0031] As used herein, an "effective amount" or "pharmaceutically effective amount" is an amount that prevents a deficiency, treats a disease or medical condition in an individual, or more generally, alleviates symptoms, manages the progression of a disease, or provides a nutritional, physiological, or medical benefit to the individual.

[0026]

[0032] The term "unit dosage form" as used herein refers to a physically discrete unit suitable as a unit of administration for human and animal subjects, each unit containing a predetermined amount of the nutritional compositions disclosed herein in an amount sufficient to produce the desired effect, preferably together with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage form depend on the particular compound used, the effect to be achieved, and the pharmacodynamics associated with each compound in the host. In one embodiment, the unit dosage form may be a predetermined amount of powder in a sachet.

[0027]

[0033] As used herein, the term "nutritional product" refers to any product that can be used to provide nutrition to a subject. Typically, a nutritional product contains a protein source, a carbohydrate source, and a lipid source.

[0028]

[0034] As used herein, the term "food product" refers to any type of product that can be safely consumed by humans or animals. Such food products may be in solid, semi-solid, or liquid form and may contain one or more nutrients, foods, or dietary supplements. For example, food products may further contain the following nutrients and micronutrients: a protein source, a lipid source, a carbohydrate source, vitamins, and minerals. Food products may also contain antioxidants, stabilizers (if provided in solid form), or emulsifiers (if provided in liquid form).

[0029]

[0035] As used herein, the term "functional food product" refers to a food product that provides an individual with additional health-promoting or disease-preventing functions.

[0030]

[0036] As used herein, the term "healthy aging product" refers to a product that provides an individual with additional health-promoting or disease-preventing functions associated with healthy aging.

[0031]

[0037] The term "dairy product" as used herein refers to food products made from milk or milk fractions of animals such as cows, goats, sheep, yaks, horses, camels, and other mammals. Examples of dairy products are low-fat milk (e.g., 0.1%, 0.5%, or 1.5% fat), non-fat milk, milk powder, whole milk, whole milk products, butter, buttermilk, buttermilk products, skim milk, skim milk products, high milkfat products, condensed milk, crème fraîche, cheese, ice cream, and confectionery products, probiotic drinks or probiotic yogurt-type drinks.

[0032]

[0038] As used herein, the term "dairy substitute product" refers to a product that is similar to a dairy product but is made without milk.

[0033]

[0039] The term "milk" as used herein is defined by the Codex Alimentarius as the normal mammary secretion of a lactating animal obtained by one or more milkings, without additions or extractions, intended for consumption as liquid milk or for further processing.

[0034]

[0040] As used herein, the term "beverage product" refers to a nutritional product in liquid or semi-liquid form that can be safely consumed by an individual.

[0035]

[0041] As used herein, the term "diet product" refers to a food product that has restricted and / or reduced calorie content.

[0036]

[0042] The term "pet food product" as used herein refers to a nutritional product intended for consumption by pets. Pet or companion animals referred to herein should be understood as animals selected from dogs, cats, birds, fish, rodents, e.g., mice, rats, etc.

[0037]

[0043] As used herein, an "effective amount" is an amount that prevents a deficiency, treats a disease or medical condition in an individual, or more generally, alleviates symptoms, manages the progression of a disease, or provides a nutritional, physiological, or medical benefit to the individual.

[0038]

[0044] All components of the composition can be mixed together, or the composition can be provided in the form of a kit of parts, where components or groups of components are provided separately, and these separate compositions can be intended to be taken separately or together.

[0039]

[0045] As used herein, the term "analog" refers to a compound that has a similar structure to a compound but differs from the compound with respect to certain constituents. A "derivative" is a compound that can be imagined or actually synthesized to result from a parent compound by replacing one or more atoms with another atom or group of atoms.

[0040] [Table 1]

[0041]

[0046] As used herein, the term "calomelanone" refers to 2',6'-dihydroxy-4,4'-dimethoxydihydrochalcone, CAS number 35241-54-4.

[0042]

[0047] As used herein, the term "flavocabain A" refers to 2'-hydroxy-4,4',6'-trimethoxychalcone, CAS number 37951-13-6.

[0043]

[0048] The term "phloretin-4-methyl ether" refers to 1-(2,6-dihydroxy-4-methoxyphenyl)-3-(4-hydroxyphenyl)propan-1-one, CAS number 520-42-3.

[0044]

[0049] The term "4'-O-methylphloretin" refers to acevogenin or 1-(2,6-dihydroxy-4-methoxyphenyl)-3-(4-hydroxyphenyl)propan-1-one, CAS number 520-42-3.

[0045]

[0050] It is understood that, according to certain embodiments, the compounds of the present invention or compositions thereof may be a nutraceutical composition, a pharmaceutical composition, a functional food, a functional nutritional product, a medical food, a medical nutritional product, or a dietary supplement.

[0046]

[0051] The term "nutraceutical" is a combination of the words "nutrition" and "pharmaceutical." These are foods or food products that provide health and medical benefits, including the prevention and treatment of conditions, disorders, or diseases. Nutraceuticals are commonly sold in pharmaceutical forms that are not normally associated with food and are products isolated or purified from foods. Nutraceuticals have been demonstrated to have physiological benefits or to provide protection against conditions, disorders, or diseases. Such products can range from isolated nutrients, dietary supplements, and special diets to genetically modified foods, herbal products, and processed foods, such as cereals, soups, and beverages.

[0047]

[0052] The term "nutraceutical" as used herein means useful in both the nutritional and pharmaceutical fields of application. Thus, the novel nutraceutical compositions can be used as supplements to foods and beverages, as well as pharmaceutical preparations for enteral or parenteral administration, which can be solid preparations, such as capsules or tablets, or liquid preparations, such as solutions or suspensions.

[0048]

[0053] The nutraceutical compositions according to the present invention may further contain protective hydrocolloids (gums, proteins, modified starches, etc.), binders, film-forming agents, encapsulants / encapsulants, wall / shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizers (oils, fats, waxes, lecithin, etc.), adsorbents, carriers, fillers, co-compounds, dispersants, wetting agents, processing aids (solvents), flow agents, taste masking agents, weighting agents, jelling agents, gel-forming agents, antioxidants and antimicrobial agents.

[0049]

[0054] Additionally, multivitamin and mineral supplements can be added to the nutraceutical compositions of the present invention to provide adequate amounts of essential nutrients that are lacking in some diets. Multivitamin and mineral supplements can also be useful in disease prevention and preventing nutritional deficiencies and malnutrition due to lifestyle patterns.

[0050]

[0055] The nutraceutical compositions of the present invention may be in any galenic form suitable for administration to the body, in particular any form conventionally used for oral administration, such as food or feed, food or feed premixes, fortified food or feed, tablets, pills, granules, dragees, capsules, and effervescent preparations (such as powders and tablets), or in liquid form, such as solutions, emulsions, or suspensions (e.g., as beverages, pastes, and oily suspensions). Pastes may be incorporated into hard- or soft-shell capsules, characterized, for example, by a matrix consisting of (fish, porcine, poultry, or bovine) gelatin, plant protein, or lignin sulfonate. Other application forms include those for transdermal, parenteral, or injectable administration. Nutritional and pharmaceutical compositions may be in the form of controlled (delayed) release formulations.

[0051]

[0056] Beverages include non-alcoholic and alcoholic beverages, as well as liquid preparations added to drinking water and liquid food.Non-alcoholic beverages include, for example, soft drinks, sports drinks, fruit juices, tea, and milk-based drinks.Liquid food includes, for example, soup and dairy products.The nutraceutical composition comprising the compound of the present invention can be added to soft drinks, energy bars, or candy.

[0052]

[0057] When the nutraceutical composition is a pharmaceutical preparation and the composition further contains a pharmaceutically acceptable excipient, diluent or adjuvant, standard techniques may be used for its formulation, for example, as disclosed in Remington's Pharmaceutical Sciences, 20th edition, Williams & Wilkins, PA, USA. For oral administration, it is preferred to use tablets and capsules containing a suitable binder, such as gelatin or polyvinylpyrrolidone, a suitable filler, such as lactose or starch, a suitable lubricant, such as magnesium stearate, and optionally further additives.

[0053]

[0058] As used herein, the terms "functional food," "functional nutritional product," "medical food," or "medical nutritional product" refer to any health food that claims to have health-promoting or disease-preventing properties beyond its basic function of providing nutrients. The general category of functional food includes processed foods or foods that have been enriched with health-promoting additives, such as "vitamin-fortified" products.

[0054]

[0059] The terms "food," "food product," and "food composition" or "diet food" refer to a product or composition intended for consumption by an individual, such as a human, and which provides at least one nutrient to such an individual. The compositions of the present disclosure, including the many embodiments described herein, may comprise, consist of, or essentially consist of the elements disclosed herein, as well as any additional or optional ingredients, components, or elements described or not described herein that are useful in a diet.

[0055]

[0060] Dietary supplements, also known as food supplements or nutritional supplements, are preparations intended to supplement the diet and provide nutrients, such as vitamins, minerals, fiber, fatty acids, or amino acids, that may be deficient or insufficiently consumed in an individual's diet. Some countries define dietary supplements as foods, while others define them as drugs or natural health products. Dietary supplements containing vitamins or dietary minerals are included as a category of foods in the Codex Alimentarius, a collection of internationally recognized standards, codes of practice, guidelines, and other recommendations on food, food production, and food safety. Codex Alimentarius standards are developed by the Codex Alimentarius Commission, an organization sponsored by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO).

[0056]

[0061] Veterinary compositions include food compositions, animal treats (e.g., biscuits), and / or dietary supplements for providing necessary dietary requirements to animals. The compositions may be dry compositions (e.g., kibble), semi-moist compositions, wet compositions, or any mixture thereof. In one embodiment, the composition is a dietary supplement such as gravy, drinking water, beverage, yogurt, powder, granules, paste, suspension, chew, morsel, treat, snack, pellet, pill, capsule, tablet, or other suitable delivery form. The dietary supplement may contain high concentrations of UFAs and NORCs, as well as B vitamins and antioxidants. This allows such dietary supplements to be administered in small amounts to animals or can be diluted before administration to the animal. The dietary supplement may need to be or can be mixed with water or other diluents before administration to the animal.

[0057]

[0062] A "pet food" or "pet treat composition" comprises about 15% to about 50% crude protein. The crude protein material may include vegetable proteins such as soybean meal, soy protein concentrate, corn gluten meal, wheat gluten, cottonseed, and peanut meal, or animal proteins such as casein, albumin, and meat protein. Examples of meat proteins useful herein include pork, lamb, horse, poultry, fish, and mixtures thereof. The composition may further comprise about 5% to about 40% fat. The composition may further comprise a carbohydrate source. The composition may comprise about 15% to about 60% carbohydrate. Examples of such carbohydrates include grains or cereals such as rice, corn, milo, sorghum, alfalfa, barley, soybeans, canola, oats, wheat, and mixtures thereof. The composition may also optionally contain other ingredients such as dried whey and other dairy by-products.

[0058]

[0063] As used herein, the term "diabetes" includes insulin-dependent diabetes mellitus (i.e., IDDM, also known as type 1 diabetes), non-insulin-dependent diabetes mellitus (i.e., NIDDM, also known as type 2 diabetes), and pre-diabetes. Type 1 diabetes is the result of an absolute deficiency of insulin, the hormone that regulates glucose utilization. Type 2 diabetes often occurs with normal or even elevated insulin levels and is thought to be the result of a loss of tissues' ability to respond appropriately to insulin. This condition is called "insulin resistance." Most type 2 diabetes patients are also overweight or obese. One of the criteria for diagnosing diabetes is fasting plasma glucose levels. A diabetic subject has a fasting plasma glucose level of 126 mg / dL or higher. A pre-diabetic subject is one who is suffering from pre-diabetes. A subject with pre-diabetes is one who has impaired fasting glucose (fasting plasma glucose concentration ≥ 100 mg / dL and < 126 mg / dL); or impaired glucose tolerance (2-hour plasma glucose concentration ≥ 140 mg / dL and < 200 mg / dL); or insulin resistance, thereby being at increased risk of developing diabetes. Prevention of type 2 diabetes includes treatment of pre-diabetes.

[0059]

[0064] As used herein, the term "dyslipidemia" encompasses abnormalities in any lipid fraction and specific lipoprotein abnormalities. For example, dyslipidemia refers to elevated plasma cholesterol and / or elevated triglycerides and / or elevated free fatty acids and / or low high-density lipoprotein (HDL) levels and / or high low-density lipoprotein (LDL) levels and / or high very-low-density lipoprotein (VLDL) levels. Dyslipidemia can contribute to the development of symptomatic vascular diseases, including, for example, atherosclerosis and ultimately coronary heart disease. Dyslipidemia may or may not be associated with diabetes.

[0060]

[0065] As used herein, the term "metabolic disorder" encompasses any abnormal chemical or enzymatic reaction caused by environmental and genetic factors (environmental factors include physical activity and nutrition) that disrupts normal metabolism, resulting in an excess or deficiency of a particular substance, and dysfunction of energy homeostasis. Non-limiting examples of metabolic disorders include diabetes, dyslipidemia, hypertension, overweight, obesity, and any combination thereof.

[0061]

[0066] As used herein, the term "prevention" or "preventing" refers to the reduction of the risk and / or severity of a condition, disorder, or disease.

[0062]

[0067] As used herein, the terms "treatment," "treating," "treat," "alleviate," and "alleviate" include both preventative or prophylactic treatment (treatment that prevents and / or delays the onset of a targeted pathological condition or disorder) and curative, therapeutic, or disease-modifying treatment, including, for example, therapeutic measures to cure, delay, alleviate symptoms, and / or halt the progression of a diagnosed pathological condition or disorder, as well as treatment of patients at risk of or suspected of having a disease, and patients who are unwell or diagnosed with a disease or medical condition. The terms do not necessarily imply that a subject is treated to the point of complete recovery. These terms also refer to the maintenance and / or promotion of health in subjects who are not diseased but are susceptible to an ill-health condition. These terms are also intended to include synergism or potentiation of one or more primary prophylactic or therapeutic measures. The terms "treatment," "treat," "alleviate," and "alleviate" are also intended to include treatment of a disease or condition or for the prophylaxis or prevention of a disease or condition. Treatment may be patient-related or physician-related.

[0063]

[0068] Obesity, which is an excess of body fat relative to lean body mass, is a very common chronic disease in modern society. Obesity not only carries social stigma, but is also associated with a shortened lifespan and numerous medical problems, including adverse mental development, coronary artery disease, hypertension, stroke, diabetes, hyperlipidemia, and some cancers (see, for example, Nishina, et al., Metab. 43:554-558, 1994; Grundy and Barnett, Dis. Mon. 36:641-731, 1990; Rissanen, et al., British Medical Journal, 301:835-837, 1990).

[0064]

[0069] As used herein, the term "obesity-related disease" refers to a disease or condition in which excess body weight or a high "body mass index (BMI)" contributes to the progression or suppression of the disease or condition. Representative examples of obesity-related diseases include, but are not limited to, diabetes, diabetic complications, insulin sensitivity, polycystic ovarian disease, hyperglycemia, dyslipidemia, insulin resistance, metabolic syndrome, obesity, weight gain, inflammatory diseases, digestive disorders, angina pectoris, myocardial infarction, sequelae of angina pectoris or myocardial infarction, senile dementia, and vascular dementia. See Harrison's Principles of Internal Medicine, 13th Ed., McGraw Hill Companies Inc., New York (1994). Non-limiting examples of inflammatory conditions include diseases of the digestive tract (ulcerative colitis, Crohn's disease, pancreatitis, gastritis, benign tumors of the digestive tract, digestive polyps, hereditary polyposis syndromes, colon cancer, rectal cancer, gastric cancer, and digestive ulcer disease), angina pectoris, myocardial infarction, sequelae of angina pectoris or myocardial infarction, senile dementia, cerebrovascular dementia, immune diseases, and cancer in general.

[0065]

[0070] As used herein, the terms "subject," "individual," or "patient" refer to any animal, including a human, that can benefit from one or more of the compounds, compositions, or methods disclosed herein. Generally, a subject is a human or an animal, such as a bird, cow, dog, horse, cat, goat, wolf, mouse, sheep, or pig. As used herein, the term "companion animal" refers to any domesticated animal, including, but not limited to, cats, dogs, rabbits, guinea pigs, ferrets, hamsters, mice, gerbils, horses, cows, goats, sheep, donkeys, pigs, etc. Preferably, the subject is a human or a companion animal, such as a dog or cat. The term "elderly" in the human context means an age of at least 60 years, preferably greater than 63 years, more preferably greater than 65 years, and most preferably greater than 70 years. The term "elderly," in the human context, refers to an age of at least 45 years, preferably greater than 50 years, more preferably greater than 55 years, and includes geriatric subjects. In the case of other animals, an "elderly individual" refers to an animal that has exceeded 50% of the average lifespan of its respective species and / or strain within a species. An animal is considered "elderly" if it has exceeded 66% of its average expected lifespan, preferably greater than 75% of its average expected lifespan, and more preferably greater than 80% of its average expected lifespan. An elderly cat or dog is at least about 7 years old.

[0066]

[0071] In one embodiment, the term "subject" as used herein refers to a mammal. Mammals include, but are not limited to, rodents, aquatic mammals, domestic animals such as dogs and cats, livestock such as sheep, pigs, cows and horses, and humans. In one embodiment, the mammal may be a cat, dog, or human. The human may be a female, for example, a female who is trying to conceive or is pregnant. In one embodiment of the present invention, the subject is a mammal selected from the group consisting of a cat, a dog, and a human. For example, the subject may be an elderly human.

[0067]

[0072] As used herein, the terms "aglycone," "aglycon," or "genin" refer to the compound that remains after the glycosyl group on a glycoside is replaced with hydrogen. For example, the aglycone of a cardiac glycoside is a steroid molecule.

[0068]

[0073] Embodiment

[0074] Compounds and Compositions

[0075] One aspect of the present disclosure is compounds for use as sodium glucose transporter 2 (SGLT2) inhibitors, and compositions comprising such compounds.

[0069]

[0076] In one embodiment, the compounds and related compositions comprising such compounds can be used both as foods or supplements and as SGLT2 inhibitors to effectively inhibit SGLT2 and thereby improve the metabolic health of a subject.

[0070]

[0077] Applicants have surprisingly found that administering a methylated phloretin analog or related composition as an SGLT2 inhibitor to a subject in need thereof can effectively inhibit SGLT2 and result in improved metabolic health. Such compounds have many advantages over phloretin and SGLT2 inhibitors, in particular, their reduced potency (2-3 times lower) in GLUT inhibition allows them to avoid the side effects of known SGLT2 inhibitors.

[0071]

[0078] Known SGLT2 inhibitors (e.g., canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin) have been previously reported to have many side effects. For example, genital infections appear to be the most common adverse effect of these known SGLT2 inhibitors. Other side effects include fungal infections, urinary tract infections, and osmotic diuresis, the risk of diabetic ketoacidosis (DKA), decreased bone mineral density and the resulting increased risk of fractures, an increased risk of lower limb amputation, or an increased risk of Fournier's gangrene.

[0072]

[0079] In one embodiment, the methylated phloretin analogues or related compositions as SGLT2 inhibitors in the present invention can reduce or eliminate all of the above side effects.

[0073]

[0080] Furthermore, methylated phloretin analogs or related compositions have improved bioavailability as well as improved solubility compared to phloretin and can be used in subjects as foods or supplements to effectively inhibit SGLT2 and thereby improve metabolic health.

[0074]

[0081] Another advantage of the present compounds and related compositions is that fewer glycosides can be produced in vivo compared to phloretin, resulting in improved biotransformation.

[0075]

[0082] In one aspect, the present disclosure relates to a composition for use in inhibiting SGLT2 in a subject, comprising a methylated phloretin analog. The subject is a mammal, such as a cat, a dog, or a human. In one embodiment, the subject is a female human or a male human. In a preferred embodiment, the subject is an elderly female human or an elderly male human.

[0076]

[0083] In one embodiment, the methylated phloretin analog is selected from the group consisting of phloretin-4-methyl-ether, 4'-O-methylphloretin, calomelanone, and flavocabain A.

[0077]

[0084] In one embodiment, the methylated phloretin analogs disclosed herein are for use in treating or preventing type 2 diabetes and / or obesity-related conditions, disorders, or diseases in a subject.

[0078]

[0085] In one embodiment, the methylated phloretin analogs disclosed herein are for use in the treatment or prevention of cardiovascular, weight management, renal, kidney, or brain-related conditions, disorders, or diseases.

[0079]

[0086] In one embodiment, the methylated phloretin analogs disclosed herein are used as SGLT2 inhibitors through either a direct or an indirect inhibitory mechanism.

[0080]

[0087] In one embodiment, the methylated phloretin analogs disclosed herein are used as SGLT2 inhibitors via a direct inhibitory mechanism.

[0081]

[0088] In one embodiment, inhibition of SGLT2 by using a methylated phloretin analog can occur at any site in a subject.

[0082]

[0089] In one embodiment, the inhibition of SGLT2 by using the methylated phloretin analogs disclosed herein is in muscle tissue and / or kidney tissue.

[0083]

[0090] In one embodiment, the methylated phloretin analogs disclosed herein may be derived from plants or plant extracts. In a preferred embodiment, the methylated phloretin analogs can be obtained from unripe apples, for example, by deglycosylation followed by methylation. The methylated phloretin analogs can also be obtained from orange peels by deglycosylation.

[0084]

[0091] In one embodiment, the methylated phloretin analogs disclosed herein are for use in the preparation of a medicament for treating or preventing a condition, disorder, or disease responsive to SGLT2 inhibition.

[0085]

[0092] In one aspect, the present invention relates to compositions comprising the methylated phloretin analogs disclosed herein.

[0086]

[0093] For example, compositions comprising the methylated phloretin analogs disclosed herein can be used both as foods or supplements and as effective SGLT2 inhibitors.

[0087]

[0094] Thus, in one embodiment, the composition is a food, beverage, or dietary supplement that includes a methylated phloretin analog as one of its ingredients.

[0088]

[0095] In another aspect, the present invention relates to a pharmaceutical composition for use in inhibiting SGLT2, comprising a therapeutically effective amount of a methylated phloretin analog disclosed herein, or a pharmaceutically acceptable salt or solvate thereof, as an active ingredient, and a pharmaceutically acceptable carrier.

[0089]

[0096] The composition according to the present invention can be administered in any pharmaceutically effective amount. Typically, the pharmaceutically effective amount will depend on the type, age, size, health condition, lifestyle and / or genetic traits of the subject. The pharmaceutically effective amount may be divided into several small amounts and administered throughout the day, so that the total daily intake is an effective amount. Those skilled in the art will be able to suggest the appropriate amount of methylated phloretin analog to be consumed per day.

[0090]

[0097] In one embodiment, the methylated phloretin analog disclosed herein may be provided in an amount of about 0.001 mg to about 100 mg per daily dose, about 0.005 mg to about 50 mg per daily dose, about 0.01 mg to about 20 mg per daily dose, or about 0.015 mg to about 10 mg per daily dose.

[0091]

[0098] In one embodiment, the compositions comprising the methylated phloretin analogs disclosed herein can be used as nutritional compositions or dietary supplements. For example, the compositions comprising the methylated phloretin analogs disclosed herein can be beneficial to a human subject (such as an elderly person or a patient in need of improved metabolic health) by inhibiting SGLT2 and thereby improving his / her metabolic health.

[0092]

[0099] In one embodiment, the composition comprising the methylated phloretin analog disclosed herein can be used for daily nutritional supplementation or routine administration. For example, the composition can be administered to elderly people or patients in need of improved metabolic health. In one embodiment, the composition can be administered to human subjects with metabolic disorders such as obesity, type 2 diabetes, cardiovascular disease, or obesity-related diseases such as diabetic complications, insulin sensitivity, polycystic ovarian disease, hyperglycemia, dyslipidemia, insulin resistance, metabolic syndrome, obesity, weight gain, inflammatory diseases, gastrointestinal diseases, angina pectoris, myocardial infarction, sequelae of angina pectoris or myocardial infarction, senile dementia, and vascular dementia.

[0093]

[0100] In some embodiments, the composition may further comprise at least one of an excipient, a diluent, or a carrier.

[0094]

[0101] Non-limiting examples of suitable excipients include buffering agents, preservatives, stabilizers, binders, compression agents, lubricants, chelating agents, dispersion enhancers, disintegrants, flavoring agents, sweetening agents, and coloring agents.

[0095]

[0102] In some embodiments, the excipient may be a buffer. Non-limiting examples of suitable buffers include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate. Buffers such as sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium gluconate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide, and calcium salts, or combinations thereof, may be used in the pharmaceutical composition.

[0096]

[0103] In some embodiments, the excipient may contain a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as α-tocopherol and ascorbate, and antimicrobial agents, such as parabens, chlorobutanol, and phenol. Antioxidants may also include, but are not limited to, EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, p-aminobenzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol, and N-acetylcysteine. In some examples, the preservative may include validamycin A, TL-3, sodium orthovanadate, sodium fluoride, N-α-tosyl-Phe-chloromethylketone, N-α-tosyl-Lys-chloromethylketone, aprotinin, phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, kinase inhibitors, phosphatase inhibitors, caspase inhibitors, granzyme inhibitors, cell adhesion inhibitors, cell division inhibitors, cell cycle inhibitors, lipid signaling inhibitors, protease inhibitors, reducing agents, alkylating agents, antibacterial agents, oxidase inhibitors, or other inhibitors.

[0097]

[0104] In one embodiment, the composition may further comprise a binder. Non-limiting examples of suitable binders include starch, pregelatinized starch, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyloxoazolidone, polyvinyl alcohol, C 12 ~C 18 Included are fatty acid alcohols, polyethylene glycols, polyols, sugars, oligosaccharides, and combinations thereof.

[0098]

[0105] The composition may be administered by any method recognized by those skilled in the art. For example, the methylated phloretin analog or composition thereof of the present invention is preferably administered orally. In some embodiments, the methylated phloretin analog or composition thereof may be administered intravenously, topically, parenterally, intraperitoneally, intramuscularly, intrathecally, intralesionally, intracranially, intranasally, intraocularly, intracardially, intravitreally, intraosseously, intracerebrally, intraarterially, intraarticularly, intradermally, transdermally, transmucosally, sublingually, enterally, sublabially, by insufflation, by suppository, by inhalation, or subcutaneously.

[0099]

[0106] In one embodiment, the compositions of the present invention may have an acute effect observed in less than one month. Additionally or alternatively, the compositions may have a long-term effect, which in various embodiments involves administering the composition (e.g., by oral administration) to an individual for a period of at least one month, preferably at least two months, more preferably at least three, four, five, or six months, and most preferably at least one year. During this period, the composition may be administered to the individual at least one day per week, preferably at least two days per week, more preferably at least three, four, five, or six days per week, and most preferably seven days per week. The composition may be administered in a single dose once per day or in multiple divided doses per day. In one embodiment, the single dose is about 100 mg or more. In one embodiment, the single dose is about 1000 mg or less. In one embodiment, the single dose is about 100 mg to about 1000 mg.

[0100]

[0107] In one embodiment, the composition may be in liquid or solid form (eg, a solid dosage form).

[0101]

[0108] In some embodiments, solid dosage forms of the composition for oral administration may include capsules, tablets, caplets, pills, troches, lozenges, powders, and granules.

[0102]

[0109] For example, the capsule may comprise a core material comprising a composition including a methylated phloretin analog, and a shell wall encapsulating the core material. In some embodiments, the core material may comprise at least one of a solid, a liquid, and an emulsion. In some embodiments, the shell wall material may comprise at least one of soft gelatin, hard gelatin, and a polymer. Suitable polymers include, but are not limited to, cellulose-based polymers, such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose (HPMC), methylcellulose, ethylcellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose succinate, and sodium carboxymethylcellulose; acrylic acid polymers and copolymers, such as those formed from acrylic acid, methacrylic acid, methyl acrylate, ammoniomethyl acrylate, ethyl acrylate, methyl methacrylate, and / or ethyl methacrylate (for example, copolymers sold under the trade name "Eudragit"); vinyl polymers and copolymers, such as polyvinylpyrrolidone, polyvinyl acetate, polyvinyl acetate phthalate, vinyl acetate crotonic acid copolymer, and ethylene-vinyl acetate copolymer; and shellac (purified lac). In some embodiments, at least one polymer can function as a taste-masking agent.

[0103]

[0110] In some embodiments, tablets, pills, and the like may be compressed, multi-compressed, multi-layered, and / or coated, for example, the coating may be single or multiple.

[0104]

[0111] Liquid formulations of compositions comprising methylated phloretin analogs include syrups (e.g., oral formulations), intravenous formulations, intranasal formulations, ophthalmic formulations (e.g., for treating eye infections), otic formulations (e.g., for treating ear infections), ointments, creams, aerosols, and the like. In some cases, combinations of various formulations may be administered. In some embodiments, tablets, pills, and the like may be formulated for a sustained-release profile. In some embodiments, the compositions may be formulated to increase shelf stability when stored in a sealed container under standard ambient conditions.

[0105]

[0112] Each component in the compositions of the present invention may be used in any amount effective to achieve the objectives of the present invention (i.e., increasing the breast milk micronutrient levels of a subject after the subject has given birth). For example, one skilled in the art would be able to determine the appropriate dosage depending on the age, size, and health of each particular subject, the subject's lifestyle, and the subject's genetic makeup.

[0106]

[0113] In one embodiment, the amount used in this application is the amount per daily dose.The amount of each component can be used as disclosed, or can be changed (for example, increased or decreased) according to the age, physique and health condition of each specific subject, the lifestyle of the subject, and the genetic traits of the subject.In one embodiment, the nutritional composition or dietary supplement of the present invention can be administered periodically, for example, twice a day, once a day, once every two days, or once a week.

[0107]

[0114] In one aspect, the compositions of the present invention may be in any form suitable for administering all of the ingredients, for example, the compositions of the present invention may be in the form of a powdered nutritional composition to be reconstituted in milk or water, a food product, a beverage, a dietary supplement, or a nutraceutical.

[0108]

[0115] When the composition of the present invention is in the form of a powdered nutritional composition to be reconstituted with milk or water, the nutritional composition or dietary supplement may preferably contain a protein source, a carbohydrate source, and a lipid source, preferably together with lecithin. The composition may also contain soy lecithin and / or a bulking agent. The protein source may be dry milk or dry skim milk. Sucrose and / or maltodextrin may be used as a carbohydrate source. The lipid source may be vegetable oil. Alternatively or additionally, the formulation may also contain glucose syrup, milk fat, magnesium citrate, choline salts and esters, prebiotic fiber, and / or ascorbyl palmitate. Flavoring compounds such as cocoa powder or honey may be added to provide a variety of flavors.

[0109]

[0116] In another aspect, the composition of the present invention may be a product selected from the group consisting of a nutritional product, a functional food product, a healthy aging product, a dairy product, a dairy alternative product, a beverage product, a dietary product, and a pet food product.

[0110]

[0117] In another embodiment, compositions comprising the methylated phloretin analogs disclosed herein can be used to treat or prevent a condition, disorder, or disease associated with type 2 diabetes, non-alcoholic fatty liver disease, and / or obesity in a subject.

[0111]

[0118] In one embodiment, the condition may be a metabolic disorder such as obesity, type 2 diabetes, cardiovascular disease, or an obesity-related disease such as diabetic complications, insulin sensitivity, polycystic ovarian disease, hyperglycemia, dyslipidemia, insulin resistance, metabolic syndrome, obesity, weight gain, inflammatory disease, gastrointestinal disease, angina pectoris, myocardial infarction, sequelae of angina pectoris or myocardial infarction, senile dementia, and vascular dementia.

[0112]

[0119] In another embodiment, compositions comprising the methylated phloretin analogs disclosed herein may be used to treat or prevent a condition selected from the group consisting of obesity, type 2 diabetes, cardiovascular disease, diabetic complications, insulin sensitivity, polycystic ovary disease, hyperglycemia, dyslipidemia, insulin resistance, metabolic syndrome, obesity, weight gain, inflammatory disease, gastrointestinal disease, angina pectoris, myocardial infarction, sequelae of angina pectoris or myocardial infarction, senile dementia, and vascular dementia.

[0113]

[0120] method

[0121] Another aspect of the present disclosure is a method for improving metabolic health (e.g., treating or preventing type 2 diabetes, and / or obesity, cardiovascular, weight management, renal, kidney or brain-related conditions, disorders, or diseases, metabolic disorders, or obesity-related diseases) by inhibiting SGLT2. In one embodiment, the method comprises administering to a subject in need thereof a composition comprising a methylated phloretin analog of the present invention as an SGLT2 inhibitor.

[0114]

[0122] In one embodiment, the method of improving metabolic health comprises treating or preventing a metabolic disorder. In one embodiment, the metabolic disorder comprises obesity, type 2 diabetes, and cardiovascular disease. In one embodiment, the metabolic disorder is selected from the group consisting of obesity, type 2 diabetes, and cardiovascular disease.

[0115]

[0123] In another embodiment, the method of improving metabolic health comprises treating or preventing an obesity-related disorder. In one embodiment, the obesity-related disorder comprises diabetic complications, insulin sensitivity, polycystic ovarian disease, hyperglycemia, dyslipidemia, insulin resistance, metabolic syndrome, obesity, weight gain, inflammatory disorders, gastrointestinal disorders, angina pectoris, myocardial infarction, sequelae of angina pectoris or myocardial infarction, senile dementia, and vascular dementia. In one embodiment, the obesity-related disorder is selected from the group consisting of diabetic complications, insulin sensitivity, polycystic ovarian disease, hyperglycemia, dyslipidemia, insulin resistance, metabolic syndrome, obesity, weight gain, inflammatory disorders, gastrointestinal disorders, angina pectoris, myocardial infarction, sequelae of angina pectoris or myocardial infarction, senile dementia, and vascular dementia.

[0116]

[0124] In one embodiment, the disclosure is a method of treating or preventing a condition selected from the group consisting of obesity, type 2 diabetes, cardiovascular disease, diabetic complications, insulin sensitivity, polycystic ovarian disease, hyperglycemia, dyslipidemia, insulin resistance, metabolic syndrome, obesity, weight gain, inflammatory disease, gastrointestinal disease, angina pectoris, myocardial infarction, sequelae of angina pectoris or myocardial infarction, senile dementia, and vascular dementia.

[0117]

[0125] In another embodiment, the disclosure is a method of treating or preventing type 2 diabetes, non-alcoholic fatty liver disease, and / or obesity-related conditions, disorders, or diseases in a subject by inhibiting SGLT2.

[0118]

[0126] As disclosed above, the method includes administering to a subject in need thereof a composition comprising at least one of phloretin-4-methyl-ether, 4'-O-methylphloretin, calomelanone, or flavocabain A.

[0119]

[0127] In one embodiment, the method comprises administering to a subject in need thereof a composition comprising at least phloretin-4-methyl-ether.

[0120]

[0128] In one embodiment, the method comprises administering to a subject in need thereof a composition comprising at least 4'-O-methylphloretin.

[0121]

[0129] In one embodiment, the method comprises administering to a subject in need thereof a composition comprising at least calomelanone.

[0122]

[0130] In one embodiment, the method comprises administering a composition comprising at least flavocabain A to a subject in need thereof.

[0123]

[0131] In one embodiment, the method comprises administering to a subject in need thereof a composition comprising at least one of phloretin-4-methyl-ether, 4'-O-methylphloretin, calomelanone, or flavocabain A to inhibit SGLT2.

[0124]

[0132] In one embodiment, the inhibition of SGLT2 is by either a direct or an indirect inhibitory mechanism.

[0125]

[0133] In one embodiment, the inhibition of SGLT2 is by a direct inhibitory mechanism.

[0126]

[0134] In one embodiment, the inhibition of SGLT2 occurs in any tissue of the subject that is associated with the condition, disorder, or disease.

[0127]

[0135] In one embodiment, the inhibition of SGLT2 is in muscle tissue, liver tissue, and / or kidney tissue.

[0128]

[0136] In one embodiment, the methylated phloretin analog is derived from a plant or plant extract.

[0129]

[0137] In one embodiment, the methylated phloretin analogs of the present invention can be used to inhibit glucose reabsorption in the kidney, thus lowering blood glucose.

[0130]

[0138] In one embodiment, the binding of various sugars to the glucose moiety of the compounds of the present invention affects the orientation of the aglycone in the access vestibule. Thus, when the aglycone of the compounds of the present invention binds, it affects the entire inhibitor. The applicants anticipate that these mechanisms together may result in synergistic interactions. Therefore, changes in the structure of both the sugar and the aglycone of the compounds of the present invention are important to the pharmacophore of SGLT inhibitors.

[0131]

[0139] In one embodiment, the compounds of general formula (I) and related compositions of the present invention may reduce or eliminate at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 11 , 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, and 80% reductions can be achieved.

[0132]

[0140] In one embodiment, the reduction in glucose levels may persist for up to 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 36 months, or 48 months.

[0133]

[0141] In another aspect, the present invention relates to an in vitro method of inhibiting SGLT2, comprising contacting a methylated phloretin analog with SGLT2 and assessing the inhibition of SGLT2.

[0134]

[0142] In one embodiment, direct inhibition of SGLT2 can be assessed by the ability to bind to SGLT2.

[0135]

[0143] In yet another aspect, the present invention relates to a method for treating or preventing a cardiovascular, weight management, renal, kidney, brain, type 2 diabetes, and / or obesity-related condition, disorder, or disease comprising administering a composition comprising a methylated phloretin analog disclosed herein. [Example]

[0136]

[0145] Example 1: Methylated analogs of phloretin exhibit a reduced degree of GLUT inhibition.

[0146] protocol

[0147] CHO-K1 cells were maintained in DMEM growth medium containing 4.5 g / L glucose, Glutamax, sodium pyruvate (Thermo, 10569010), and 10% fetal bovine serum (Thermo, 10270106) supplemented with non-essential amino acids (Thermo, 11140050) at 37°C under 5% CO. Cells were seeded at 80% confluence in 24-well tissue culture test plates (TPP, 92424). To assess GLUT inhibition, wells were washed four times with Krebs-Ringers-Hepes (140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl, 1.25 mM MgSO, 1.2 mM KHPO, 10 mM HEPES, 2 mM sodium pyruvate, pH 7.4) to remove extracellular glucose, followed by the addition of 250 μL of Krebs-Ringers-Hepes solution containing 0.1% (v / v) DMSO, vehicle, the indicated concentrations of phloretin or specific methylphloretin analogs, or 10 μM of the GLUT inhibitor BAY-876 (Merck, SML1774-5MG) for 30 min. After the treatment period, 100 μM 2-deoxyglucose mixed with 10 μCi [3H]2-deoxyglucose (Perkin Elmer, NET328A001MC) was added to the wells and incubated at 37°C for 5 minutes. The reaction was stopped by adding 5 mM glucose to the wells, which were then washed four times with Krebs-Ringers-Hepes buffer to remove extracellular [3H]2-deoxyglucose. Cells were lysed with 0.1 M NaOH, and the relative level of incorporated radioactivity was determined as counts per minute per well, measured using a TriCarb scintillation counter equipped with UltimaGold Scintillant (Perkin Elmer, 6013329). All other reagents were from Sigma.

[0137]

[0148] result

[0149] The data shown in Figure 1 demonstrate the levels of internalized [H]2-deoxyglucose, a GLUT transporter substrate, following treatment with (A) phloretin, (B) calomelanone, (C) phloretin 4-methyl ether, (D) acepogenin, and (E) flavocabain A. The data are shown relative to the effects of the pharmacological GLUT inhibitors BAY-876 (100% effect) and DMSO (0% effect). The data demonstrate that methylated analogs of phloretin exhibit a reduced degree of GLUT inhibition compared to phloretin.

[0138]

[0150] Example 2

[0151] protocol

[0152] Methylphloretin compounds were incubated in the presence of 1 mg / mL rat liver microsomes (M9066-1VL, Sigma) in 50 mM Tris-HCl pH 8.0 containing 5 mM UDP-glucose, 25 mM sucrose, 10 mM MgCl2, and 1 mM DTT for 4 hours at 37°C with shaking at 150 rpm. Samples for LC-MS analysis were prepared by solid-phase extraction using a Waters Oasis HLB SPE cartridge using a benchtop vacuum manifold. Briefly, the SPE cartridge was activated with three column volumes of methanol, equilibrated with reaction buffer, the sample was applied, and washed with deionized water. Analytes were eluted in fractions containing increasing concentrations of methanol. The SPE eluted fractions were dried under a stream of N2 and reconstituted in 20% methanol. Samples were run on a Waters Acquity uHPLC system equipped with a 10 cm Waters Aquity HSS T3 1.8 μM column maintained at 40 °C, coupled to a Synapt G2-S high-resolution mass spectrometer operated in negative ion time-of-flight mode. Analyses were performed and analyzed using MassLynx 4.2 software. A linear gradient from 0% acetonitrile to 100% acetonitrile was applied over 0–16 min. Mobile phases A and B were water and acetonitrile containing 0.1% formic acid, respectively. Waters Lockspray technology was applied to determine calculated accurate mass values. The expected monoisotopic mass was calculated from the molecular formula using a molecular weight calculator.

[0139]

[0153] The results are shown in Figures 2-5.

[0154] For the indicated individual methyl-phloretin compounds (calomelanone (FIG. 2); phloretin-4 methyl ether (FIG. 3); phloretin-4' methyl ether, acepogenin (FIG. 4); flavocavain (FIG. 5)), incubations with UDP-glucose were performed in the presence of rat microsomes and analyzed by uHPLC-MS after solid-phase extraction as described.

[0140]

[0155] For each of the above compounds:

[0156] Extracted mass chromatograms from LC-MS analysis of SPE elution fractions containing both parent and generated glycosidic metabolites show the expected monoisotopic masses for the parent methylphloretin and glycosidic forms, except for flavocabain, whose aglycone form is not readily observable in negative ion time-of-flight mode, and which only shows the glycosidic form (A).

[0141]

[0157] For each parent and glycosidic peak observed in the extracted mass chromatogram, the sum of MS-level spectra acquired over the peak duration (at least five scans) is displayed, to which Waters Lockmass correction was applied to determine the measured accurate mass (B).

[0142] For each major peak of the parent and derived glycoside species observed in the extracted mass chromatogram, the MS / MS spectra of the parent ion and glycoside species are shown (C). For the glycoside species, the selected ion produces a fragment with a loss of 162 mu, consistent with the loss of a glucose molecule, and produces a fragment ion at the m / z expected for the parent methylphloretin compound.

[0143] For each methylphloretin species, the location of predicted nonequivalent sites for glycosylation is shown (D).

[0144]

[0158] Example 3

[0159] Assay Protocol:

[0160] Briefly, CHO-K1 cells stably transfected with SGLT1 or SGLT2 were seeded at 20,000 cells / well in a 384-well plate (black clear bottom, Greiner Bio-One #781091) and incubated in 50 μL of culture medium (RPMI 1640 + 5% serum) for 24 h. Prior to the experiment, cells were starved for 3 h by removing the culture medium and replacing it with 20 μL of glucose- and serum-free medium. The experiment was initiated by adding 20 μL of assay buffer containing 320 μM 1NBDG (a fluorescent glucose analog) in glucose- and serum-free medium supplemented with 1% DMSO (final concentration) and with or without compound, and incubating at 37°C for 2 h. Cells were then washed three times with PBS to completely remove extracellular 1NBDG. The fluorescence (RFU) corresponding to 1NBDG uptake in transfected cells was then measured with a multimode reader (Bioteck synergy neo signal exc 485nm em 535nm).

[0145]

[0161] Data were finally normalized: DMSO was used as the maximum uptake signal (100%) and 10 μM dapagliflozin as the minimum signal (0%, complete inhibition of uptake), and the percentage of activity was calculated using the formula: (x-min) / (max-min)*100.

[0146]

[0162] result

[0163] For these graphs, six concentrations of each compound were tested in duplicate on both SGLT1 and SGLT2 transfected cell lines, with 3-fold serial dilutions starting at 10 μM. In the absence of inhibition (DMSO control), 1NBDG uptake is maximal, resulting in a high RFU signal that is used to determine 100% normalized activity; conversely, in the presence of 10 μM dapagliflozin, there is complete inhibition of active transport of 1NBDG, and the fluorescence observed to be generated under these conditions gives 0% normalized activity: the following formula is applied for normalization: Normalized activity (cpd) = (RFU(cpd) - RFU(DAPA) / (RFU(DMSO) - RFU(DAPA))

[0147]

[0164] Figure 6 shows the normalized SGLT1 and SGLT2 inhibitory activity of 4'-methylphlorizin (NI00046322). Thus, the decline in both curves represents increasing inhibition of SGLT1 and SGLT2 with increasing test concentrations of 4'-methylphlorizin (NI00046322).

[0148]

[0165] We observed that 4'-methylphlorizin (NI00046322) is active against both SGLT1 and SGLT2 with different IC50s. 4'-methylphlorizin (NI00046322) is more effective against SGLT2 than SGLT1, as complete inhibition was observed for SGLT2 at 1 μM (−6 on the log scale) with an IC50 of approximately 33 nM (−7.5 on the log scale), whereas complete inhibition was observed for SGLT2 at 1 μM (−6 on the log scale) with an IC50 of 300 nM to 1 μM (−6.5 to −6 on the log scale).

[0149]

[0166] Figure 7 shows the normalized SGLT1 and SGLT2 inhibitory activity of 4-methylphlorizin (NI00046291). Thus, a decline in either curve represents increased inhibition of SGLT1 and SGLT2 with increasing test concentrations of 4-methylphlorizin (NI00046291).

[0150]

[0167] We observed that 4-methylphlorizin (NI00046291) is active against both SGLT1 and SGLT2 with different IC50s. 4-methylphlorizin (NI00046291) is more effective against SGLT2 than SGLT1, as complete inhibition was observed at 3 μM (−5.5 on the log scale) with an IC50 of 300 nM to 1 μM (−6.5 to −6 on the log scale) compared to 1 μM (−6 on the log scale) with an IC50 of approximately 33 nM (−7.5 on the log scale) for SGLT2.

Claims

1. A composition for use in inhibiting SGLT2, comprising an effective amount of at least one methylated phloretin analog.

2. 2. The composition of claim 1, wherein the methylated phloretin analog is selected from the group consisting of phloretin-4-methyl-ether, 4'-O-methylphloretin, calomelanone, and flavocabain A.

3. The composition of claim 1 or 2, wherein the methylated phloretin analog is derived from a plant or plant extract.

4. The composition of any one of claims 1 to 3, wherein the methylated phloretin analog is derived from unripe apple or orange peel.

5. The composition according to any one of claims 1 to 4, wherein the inhibition of SGLT2 is in muscle tissue and / or kidney tissue.

6. 6. The composition of any one of claims 1 to 5 for use in the treatment and / or prevention of type 2 diabetes and / or obesity related conditions, disorders or diseases in a subject in need thereof.

7. 6. The composition of any one of claims 1 to 5 for use in the treatment and / or prevention of a cardiovascular, weight management, renal, kidney, brain-related condition, disorder or disease in a subject in need thereof.

8. 6. A composition according to any one of claims 1 to 5 for use in the treatment and / or prevention of a condition, disorder or disease responsive to SGLT2 inhibition in a subject in need thereof.

9. The composition of any one of claims 1 to 8, wherein the composition is a food, beverage, or dietary supplement.

10. The composition of any one of claims 1 to 9, wherein the composition further comprises a pharmaceutically acceptable carrier.

11. 11. A method for treating and / or preventing a condition, disorder, or disease associated with type 2 diabetes and / or obesity in a subject in need thereof, comprising administering a composition comprising a compound according to any one of claims 1 to 10.

12. 11. A method for treating and / or preventing a condition, disorder, or disease responsive to SGLT2 inhibition in a subject in need thereof, comprising administering a composition comprising a compound according to any one of claims 1 to 10.

13. A pharmaceutical composition for inhibiting SGLT2, comprising a therapeutically effective amount of a compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt or solvate thereof, as an active ingredient, and a pharmaceutically acceptable carrier.

14. 11. An in vitro method of inhibiting SGLT2, said in vitro method comprising contacting a compound according to any one of claims 1 to 10 with SGLT2 and assessing the inhibition of SGLT2.

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