Methylated phloretin analogs as inhibitors of SGLT2

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 for improving metabolic health.

Description

Background Art

[0001] The present disclosure as a whole relates to compositions and methods for improving metabolic health. Specifically, the present disclosure relates to the use of methylated phloretin analogs as sodium-glucose transporter-2 (SGLT2) inhibitors for improving metabolic health. For example, it is the first time to discover that methylated phloretin analogs can be used as both food or supplements and sodium-glucose transporter-2 (SGLT2) inhibitors that effectively inhibit SGLT2, thereby improving metabolic health.

[0002] The SGLT protein family includes active glucose transporters that play an important role in maintaining glucose balance in the blood. SGLT2 is expressed in the kidney on epithelial cells lining the first segment of the proximal convoluted tubule and is responsible for approximately 90% of renal glucose reabsorption. By inhibiting SGLT2, an ideal inhibitor would prevent glucose reuptake by the glomerular filtrate in the kidney, subsequently reducing glucose levels in the blood and promoting glucose excretion in the urine.

[0003] Phlorizin is a natural compound and is a known inhibitor of SGLT1 and SGLT2. SGLT1 is expressed in the intestine in addition to being expressed in the kidney. Inhibition of SGLT1 in the intestine is associated with negative gastrointestinal effects including dysentery. Phlorizin is associated with negative gastrointestinal effects and cannot be added to food as a supplement and is not approved for use as a drug. The present invention does not result in SGLT1 inhibition in the intestine and is therefore different from phlorizin in this manner and can therefore be added to food.

[0004] Known as SGLT2 inhibitors, they are a class of insulin-independent oral antihyperglycemic drugs used by clinicians to treat type 2 diabetes. Since 2013, the Food and Drug Administration has approved four SGLT2 inhibitors: canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin.

[0005] The known SGLT2 inhibitors are a class of prescription drugs approved by the FDA for use with diet and exercise to lower blood sugar in adults with type 2 diabetes. Drugs in the SGLT2 inhibitor class include canagliflozin, dapagliflozin, and empagliflozin. They are available as single ingredient products and are also combined with other diabetes drugs such as metformin. SGLT2 inhibitors lower blood sugar by causing the kidneys to remove sugar from the body through urine. However, none of these known SGLT2 inhibitors are natural compounds, and they can only be used as drugs and not as food or supplements.

[0006] There is a clear unmet need for new compounds and compositions that are SGLT2 inhibitors to improve metabolic health. There is a need in the art for new compounds and compositions that are SGLT2 inhibitors that can be used as foods or supplements to improve metabolic health. Summary of the invention

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

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

[0009] The methylated phloretin analogs or related compositions have improved bioavailability compared to phloretin and can be used as food or supplements for subjects to effectively inhibit SGLT2, thereby improving metabolic health.

[0010] Additionally, such compounds and related compositions have reduced potency (2- to 3-fold) for GLUT inhibition.

[0011] Another advantage of the presented compounds and related compositions is improved biotransformation, since they may produce less glycosides in vivo compared to phloretin. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 (AE) shows the level of [3H]2-deoxyglucose (a GLUT transporter substrate) incorporated after treatment with (A) phloretin, (B) calometron, (C) phloretin 4-methyl ether, (D) asebogenin, and (E) calometron A. The data show the effect relative to the pharmacological GLUT inhibitor BAY-876 (100% effect) and DMSO (0% effect).

[0013] Figures 2 to 5 (AD): Cultivation of specific individual methyl-phloretin compounds: carlosinone ( Figure 2 ); Phloretin-4-methyl ether ( Figure 3 ); Phloretin-4′ methyl ether (Phloretinin) ( Figure 4 ); yellow kava with UDP-glucose after solid phase extraction as described ( Figure 5 ) were analyzed by uHPLC-MS in the presence of rat microsomes. For each compound:

[0014] i) LC-MS analysis of fractions eluted from the SPE containing both the parent and the produced glycosidic metabolites (A) showing the extracted mass chromatograms of the expected monoisotopic masses of the parent and glycosidic forms.

[0015] ii) Each peak observed in the extracted mass chromatograms of the parent and glycoside species is shown as the sum of the MS-level spectra acquired over the duration of the peak (over 5 separate detection events), to which a Waters Lockmass calibration was applied to determine the exact monoisotopic mass (B).

[0016] iii) For each major peak observed in the extracted mass chromatogram for the parent and derived glycoside species, the selected parent ion and MS / MS spectra of the glycoside species are shown (C). For the glycoside species, the selected ion fragments fragmented with a characteristic loss of 162mu consistent with the loss of the glucosyl moiety, thereby generating fragmented ions at the expected m / z of the parent methyl-phloretin compound.

[0017] iv) For each methylphloretin species, the positions of the expected non-equivalent glycosylation sites are shown (D).

[0018] Figure 6 is a graph showing the normalized SGLT1 and SGLT2 inhibitory activity of 4'methylphlorizin (NI00046322) and chemical formula.

[0019] Figure 7 is a graph showing the normalized SGLT1 and SGLT2 inhibitory activities of 4-methylphlorizin (NI00046291) and (NI00001380) with chemical formula.

[0020] Figure 8 The chemical formulas of methylated phloretin analogs and glycosides corresponding to the letters shown in Table 1 are shown. DETAILED DESCRIPTION

[0021] definition

[0022] Some definitions are provided below. However, definitions may be located in the "Implementation" section below, and the above heading "Definitions" does not indicate that such disclosure in the "Implementation" section is not a definition.

[0023] All percentages expressed herein are by weight of the total weight of the composition, unless otherwise indicated. As used herein, "about", "approximately" and "substantially" should be understood to refer to numbers within a certain numerical range, such as within the range of -10% to +10% of the mentioned number, preferably -5% to +5% of the mentioned number, more preferably -1% to +1% of the mentioned number, and most preferably -0.1% to +0.1% of the mentioned number. All numerical ranges herein should be understood to include all integers or fractions within the range. In addition, these numerical ranges should be understood to provide support for claims involving any number or subset of numbers within the range. For example, the disclosure of 1 to 10 should be understood to support ranges of 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0024] As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a component" or "the component" includes two or more components.

[0025] The words "including / comprising" will all be interpreted as inclusive rather than exclusive. Likewise, the terms "including / comprising" and "or" should all be considered inclusive unless the context clearly prohibits such an interpretation. However, the compositions disclosed herein may not contain any elements not specifically disclosed herein. Thus, disclosures of embodiments using the term "including / comprising" include disclosures of embodiments "consisting essentially of the specified components" and embodiments "consisting of the specified components."

[0026] The term "and / or" used in the context of "X and / or Y" should be interpreted as "X" or "Y" or "X and Y". Similarly, "at least one of X or Y" should be interpreted as "X" or "Y" or "X and Y". For example, "at least one dithionite or a functionally similar reducing agent" should be interpreted as "dithionite" or "functionally similar reducing agent" or "both dithionite and a functionally similar reducing agent".

[0027] As used herein, the terms "example" and "such as" (especially when followed by a list of terms) are exemplary and illustrative only and should not be considered exclusive or comprehensive. As used herein, one condition is "associated with" or "associated with" another condition means that the conditions occur at the same time, preferably means that the conditions are caused by the same underlying condition, and most preferably means that one of the identified conditions is caused by the other identified condition.

[0028] 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 sodium-glucose carboxyl-2 (SGLT2), in particular on human SGLT2. The inhibitory effect on hSGLT2 measured by IC50 is preferably 1,000 nM or less, more preferably 100 nM or less and most preferably 50 nM or less. The IC50 value of SGLT2 inhibitors is generally 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, in particular as described in applications WO 2005 / 092877 or WO 2007 / 093610 (pages 23 / 24); the documents are hereby incorporated by reference. The term "SGLT2 inhibitor" also includes pharmaceutically acceptable salts, hydrates and solvates thereof, including corresponding crystalline forms.

[0029] In one embodiment, the known SGLT2 inhibitor is selected from dapagliflozin, canagliprofine, empagliflozin, artigliprozine, resmogliflozin, sergliprozine and derivatives thereof.

[0030] For example, known SGLT2 inhibitors may include a class of prescription drugs that are approved by the FDA for diet and exercise to lower blood sugar in adults with type 2 diabetes. Known drugs in the SGLT2 inhibitor class may include canagliflozin, dapagliflozin, and empagliflozin. They are available as single ingredient products and are also combined with other diabetes drugs such as metformin. SGLT2 inhibitors lower blood sugar by causing the kidneys to remove sugar from the body through urine.

[0031] Hsia et al. Curr Opin Endocrinol Diabetes Obes. 2017 Feb;24(1):73-79. doi:10.1097 / MED provides a series of other compounds that act as SGLT2 inhibitors.

[0032] However, none of these known SGLT2 inhibitors are natural compounds, and they can only be used as drugs and not as food or supplements.

[0033] 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, the nutritional composition or nutritional supplement of the present invention may include vitamins, minerals, fiber, fatty acids, or amino acids. The nutritional composition or nutritional supplement of the present invention may be provided, for example, in the form of a pill, tablet, lozenge, chewable capsule or tablet, tablet or capsule, or a powder supplement that can be dissolved in water or sprinkled on food.

[0034] In one embodiment, the nutritional composition or nutritional supplement of the present invention can provide selected nutrients without representing a significant portion of the overall nutritional requirements of the subject. Typically, they do not provide more than 0.1%, 1%, 5%, 10% or 20% of the daily energy requirements of the subject. The nutritional composition or nutritional supplement of the present invention can be used for any subject, such as a subject during pregnancy, for example as a maternal supplement.

[0035] As used herein, "effective amount" or "pharmaceutically effective amount", as used herein, refers to an amount that prevents a defect, treats a disease or medical condition in a subject, or more generally, refers to an amount that alleviates symptoms, manages disease progression, or provides a nutritional, physiological or medical benefit to a subject.

[0036] As used herein, the term "unit dosage form" refers to physically discrete units suitable as unit doses for human and animal subjects, each unit containing a predetermined amount of the nutritional composition disclosed herein, in an amount sufficient to produce the desired effect, preferably in combination with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications of the unit dosage form depend on the specific compound used, the effect to be achieved, and the pharmacodynamics associated with each compound in the host. In one embodiment, the unit dosage form can be a predetermined amount of powder in a sachet.

[0037] 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.

[0038] As used herein, the term "food product" refers to any type of product that can be safely consumed by humans or animals. A food product can be in solid, semisolid or liquid form and can contain one or more nutrients, foods or nutritional supplements. For example, a food product can additionally contain the following nutrients and micronutrients: a protein source, a lipid source, a carbohydrate source, vitamins and minerals. The food product can also contain an antioxidant, a stabilizer (when provided in solid form) or an emulsifier (when provided in liquid form).

[0039] As used herein, the term "functional food product" refers to a food product that provides additional health-promoting or disease-preventing functions to an individual.

[0040] 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.

[0041] As used herein, the term "dairy product" refers to a food product produced from milk or milk components from 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), fat-free milk, milk powder, whole milk, whole milk products, butter, buttermilk, buttermilk products, skim milk, skim milk products, high milkfat products, condensed milk, crème brûlée, cheese, ice cream and dessert products, probiotic beverages or probiotic yogurt-type beverages.

[0042] As used herein, the term "dairy alternative product" refers to a product that is similar to dairy products but is not produced with milk.

[0043] As used herein, the term "milk" is defined by the Codex Alimentarius Commission as the normal mammary secretion of a dairy animal obtained from one or more milkings, without additions or extractions, intended for consumption as liquid milk or for further processing.

[0044] 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.

[0045] As used herein, the term "diet product" refers to a food product having a restricted and / or reduced caloric content.

[0046] As used herein, the term "pet food product" refers to a nutritional product intended for consumption by a pet. Pets or companion animals as referred to herein are understood to be animals selected from dogs, cats, birds, fish, rodents (such as mice, rats).

[0047] As used herein, an "effective amount" is an amount that prevents a defect, treats a disease or medical condition in an individual, or more generally, reduces symptoms, manages disease progression, or provides a nutritional, physiological or medical benefit to the individual.

[0048] All ingredients of the composition may be mixed together, or alternatively the composition may be provided in the form of a kit of parts, wherein the ingredients or groups of ingredients are provided separately. These separate compositions may be intended to be consumed separately or together.

[0049] As used herein, the term "analog" refers to a compound that has a structure similar to another structure, but differs therefrom in certain components. A "derivative" is a compound that can be conceivably produced or actually synthesized from a parent compound by replacing one or more atoms with another atom or group of atoms.

[0050]

[0051] Table 1: List of phloretin analogs, common names and expected monoisotopic accurate masses of the [MH]-ions of the aglycones and glycosides. The chemical formulas of the compounds in Table 1 are shown in Figure 8 middle.

[0052] https: / / www.molbase.com / cas / 76172-68-4.html

[0053] https: / / www.molbase.com / cas / 520-42-3.html

[0054] As used herein, the term "carometone" refers to 2',6'-dihydroxy-4,4'-dimethoxydihydrochalcone, CAS No. 35241-54-4.

[0055] As used herein, the term "Cavaline A" refers to 2'-hydroxy-4,4',6'-trimethoxychalcone, CAS No. 37951-13-6.

[0056] The term "phloretin-4-methyl ether" refers to 1-(2,6-dihydroxy-4-methoxyphenyl)-3-(4-hydroxyphenyl)propan-1-one, CAS No. 520-42-3.

[0057] The term "4'-O-methylphloretin" refers to phloretin or 1-(2,6-dihydroxy-4-methoxyphenyl)-3-(4-hydroxyphenyl)propan-1-one, CAS No. 520-42-3.

[0058] It should be understood that according to certain embodiments, the compounds of the present invention or compositions thereof can be nutraceutical compositions, pharmaceutical compositions, functional foods, functional nutritional products, medical foods, medical nutritional products, or dietary supplements.

[0059] The term "nutraceutical" combines the words "nutrition" and "medicine" together. It is a food or food product that provides health and medical benefits, including the prevention and treatment of conditions, disorders, or diseases. Nutrients are products separated or purified from foods that are generally sold in the form of medicines that are not usually associated with food. Nutrients are proven to have physiological benefits or provide prevention of chronic conditions, disorders, or diseases. Such products can range from isolated nutrients, dietary supplements, and specific diets to genetically engineered foods, herbal products, and processed foods such as cereals, soups, and beverages.

[0060] As used herein, the term "nutraceutical" indicates usefulness in the fields of nutrition and pharmaceutical applications. Thus, the novel nutritional composition can be used as a supplement to food and beverages, and can be used as a pharmaceutical preparation for enteral or parenteral administration, which can be a solid preparation such as a capsule or tablet, or a liquid preparation such as a solution or suspension.

[0061] The nutraceutical composition according to the present invention may also comprise protective hydrophilic colloids (such as gums, proteins, modified starches), binders, film formers, encapsulating agents / materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizers (oils, fats, waxes, lecithins, etc.), adsorbents, carriers, fillers, co-compounds, dispersants, wetting agents, processing aids (solvents), flow agents, taste masking agents, weighting agents, gelling agents, gel forming agents, antioxidants and antimicrobial agents.

[0062] In addition, multivitamin and mineral supplements can be added to the nutritional composition of the present invention to obtain sufficient amounts of essential nutrients that are missing in some diets. Multivitamin and mineral supplements can also be used to prevent disease and prevent nutritional losses and deficiencies due to lifestyle patterns.

[0063] The nutraceutical composition of the invention may be in any galenic dosage form suitable for administration to the body, in particular any form conventionally used for oral administration, for example in solid form, such as food or feed, food or feed premix, fortified food or feed, tablet, pill, granule, dragee, capsule and effervescent preparations such as powder and tablet; or in liquid form, such as solution, emulsion or suspension, such as, for example, beverage, paste and oily suspension. The paste may be incorporated into a hard or soft shell capsule, whereby the capsule is characterized by a matrix of, for example, (fish, pig, poultry, cattle) gelatin, vegetable protein or lignin sulfonate. Examples of other application forms are those for transdermal, parenteral or injection administration. Dietary and pharmaceutical compositions may be in the form of controlled (slow release) preparations.

[0064] Beverages include non-alcoholic beverages and alcoholic beverages and liquid preparations to be added to drinking water and liquid foods. Non-alcoholic beverages are, for example, soft drinks, sports drinks, fruit juices, teas, and milk-based beverages. Liquid foods are, for example, soups and dairy products. The nutritional composition comprising the compounds of the present invention can be added to soft drinks, energy bars, or candies.

[0065] If the nutraceutical composition is a pharmaceutical preparation, and the composition further comprises a pharmaceutically acceptable excipient, diluent or adjuvant, then standard techniques may be used for its formulation, as disclosed, for example, in Remington's Pharmaceutical Sciences, 20th edition, Williams & Wilkins, PA, USA. For oral administration, tablets and capsules containing suitable binders (e.g., gelatin or polyvinyl pyrrolidone), suitable fillers (e.g., lactose or starch), suitable lubricants (e.g., magnesium stearate), and optionally other additives are preferably used.

[0066] 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 the basic function of providing nutrients. The general category of functional foods includes processed foods or foods fortified with additives that promote health, such as "vitamin-enriched" products.

[0067] The terms "food," "food product," and "food composition" or "diet product" mean a product or composition that is intended for ingestion by an individual (such as a human) and that provides at least one nutrient to the individual. The compositions of the present disclosure (including the various embodiments described herein) may comprise, consist of, or consist essentially of the elements disclosed herein, as well as any additional or optional ingredients, components, or elements described herein or that may be used in a diet.

[0068] Dietary supplements, also known as food supplements or nutritional supplements, are preparations designed to supplement the diet and provide nutrients (such as vitamins, minerals, fiber, fatty acids or amino acids) that may be missing or may be inadequately consumed in a person's diet. Some countries define dietary supplements as foods, while other countries define dietary supplements as medicines or natural health products. Supplements containing vitamins or dietary minerals are included as a category of food in the Codex Alimentarius, which is an internationally recognized collection of standards, codes of practice, guidelines and other recommendations related to food, food production and food safety. These texts are developed by the Codex Alimentarius Commission, an organization funded by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO).

[0069] Compositions intended for animals include food compositions that provide necessary dietary needs for animals, animal rewards (e.g., biscuits) and / or dietary supplements. Compositions can be dry compositions (e.g., coarsely ground food), semi-wet compositions, wet compositions, or any mixture thereof. In one embodiment, the composition is a dietary supplement, such as gravy, drinking water, beverage, yogurt, powder, granule, paste, suspension, chew, small pieces of food, rewards, snacks, pellets, pills, capsules, tablets, or any other suitable delivery form. Dietary supplements can include high concentrations of UFA and NORC, as well as B vitamins and antioxidants. This allows supplements to be administered to animals in small amounts, or can be diluted before being administered to animals in an alternative. Dietary supplements may need to be mixed, or can be mixed with water or other diluents before being administered to animals.

[0070] A "pet food" or "pet treat composition" contains about 15% to about 50% crude protein. The crude protein material may include plant proteins such as soy flour, 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 that can be used in the present invention include pork, lamb, horse, poultry, fish and mixtures thereof. The composition may also contain about 5% to about 40% fat. The composition may also contain a carbohydrate source. The composition may contain about 15% to about 60% carbohydrates. Examples of such carbohydrates include grains or cereals such as rice, corn, millet, sorghum, alfalfa, barley, soybeans, canola, oats, wheat and mixtures thereof. The composition may also optionally contain other materials such as dry whey and other dairy by-products.

[0071] As used herein, the term "diabetes" includes insulin-dependent diabetes (i.e., IDDM, also known as type 1 diabetes), non-insulin-dependent diabetes (i.e., NIDDM, also known as type 2 diabetes) and prediabetes. Type 1 diabetes is the result of an absolute lack of insulin, a hormone that regulates glucose utilization. Type 2 diabetes usually occurs in the face of normal or even elevated insulin levels, and appears to be the result of tissues being unable to respond appropriately to insulin. This is called "insulin resistance." Most patients with type 2 diabetes are also overweight or obese. One of the criteria for diagnosing diabetes is the fasting plasma glucose level. A diabetic subject has a fasting plasma glucose level greater than or equal to 126 mg / dl. A prediabetic subject is a person with prediabetes. A prediabetic subject is a subject with impaired fasting glucose (a fasting plasma glucose level greater than or equal to 100 mg / dl and less than 126 mg / dl); or a subject with impaired glucose tolerance (a 2-hour plasma glucose level ≥140 mg / dl and less than 200 mg / dl); or a subject with increased risk of developing diabetes due to insulin resistance. Preventing type 2 diabetes includes treating prediabetes.

[0072] As used herein, the term "dyslipidemia" encompasses abnormal levels of any lipid fraction as well as specific lipoprotein abnormalities. For example, it refers to a rise in plasma cholesterol and / or a rise in triglycerides and / or a rise in 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, for example, lead to the development of atherosclerosis and ultimately symptomatic vascular disease (including coronary heart disease). Dyslipidemia may or may not be associated with diabetes.

[0073] As used herein, the term "metabolic disorder" encompasses any abnormal chemical and enzymatic reactions that disrupt normal metabolism due to environmental and genetic factors (environmental factors include physical activity, nutrition), resulting in excessive levels or deficiencies of certain substances and dysfunction of energy homeostasis. Non-limiting examples of metabolic disorders include diabetes, dyslipidemia, hypertension, overweight, obesity, and any combination thereof.

[0074] As used herein, the term "preventing" or "preventing" refers to reducing the risk and / or severity of a condition, disorder or disease.

[0075] As used herein, the terms "treat", "mitigate" or "mitigate" refer to preventive or prophylactic treatment (preventing and / or slowing the development of a target pathological condition or disorder) and also include curative, therapeutic or disease-modifying treatments, including therapeutic measures that cure, slow down, alleviate the symptoms of a diagnosed confirmed pathological condition or disorder and / or interrupt its progress; and include treating patients who are at risk of contracting a disease or suspected of having contracted a disease, as well as treating patients who are sick or have been diagnosed with a disease or medical condition. The term does not necessarily mean that the subject is treated until full recovery. These terms also refer to health maintenance and / or promotion in subjects who do not have a disease but may be prone to developing an unhealthy condition. These terms are also intended to include strengthening or otherwise enhancing one or more primary preventive or therapeutic measures. The terms "treat", "mitigate" and "mitigate" are also intended to include dietary management of a disease or condition or dietary management that prevents or prevents a disease or condition. Treatment can be patient-related or doctor-related.

[0076] Obesity is an excess of body fat relative to lean body mass and is a highly prevalent chronic disease in modern societies. It is associated not only with social stigma but also with shortened lifespan and numerous medical problems, including adverse psychological development, coronary artery disease, hypertension, stroke, diabetes, hyperlipidemia, and some cancers (see, e.g., 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).

[0077] As used herein, the term "obesity-related conditions" refers to those diseases or conditions that are associated with excessive weight or a high "body mass index (BMI)" that is associated with the progression or inhibition of a disease or condition. Representative examples of obesity-related conditions 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 organ diseases, angina pectoris, myocardial infarction, sequelae of angina pectoris or myocardial infarction, senile dementia, and cerebrovascular dementia. See, Harrison's Principles of Internal Medicine, 13th Edition, McGraw Hill Companies Inc., New York (1994). Examples of inflammatory disorders include, but are not limited to, diseases of the digestive organs (such as ulcerative colitis, Crohn's disease, pancreatitis, gastritis, benign tumors of the digestive organs, digestive polyps, hereditary polyposis syndromes, colon cancer, rectal cancer, gastric cancer, and ulcerative diseases of the digestive organs), angina pectoris, myocardial infarction, sequelae of angina pectoris or myocardial infarction, senile dementia, cerebrovascular dementia, immune diseases, and general cancers.

[0078] As used herein, the terms "subject", "individual" or "patient" refer to any animal, including humans, that can benefit from one or more of the compounds, compositions or methods disclosed herein. Generally speaking, the subject is a human or an avian, bovine, canine, equine, feline, caprine, wolf, murine, ovine and porcine animal. As used herein, "companion animal" refers to any domesticated animal, and includes, but is not limited to, cats, dogs, rabbits, guinea pigs, ferrets, hamsters, mice, gerbils, horses, cattle, goats, sheep, donkeys, pigs, etc. Preferably, the subject is a human or a companion animal, such as a dog or a cat. In the case of humans, the term "elderly" refers to an age of at least 60 years old since birth, preferably over 63 years old, more preferably over 65 years old, and most preferably over 70 years old. In the case of humans, the term "middle-aged and elderly" refers to an age of at least 45 years old since birth, preferably over 50 years old, more preferably over 55 years old, and includes elderly subjects. For other animals, "old age" means having exceeded 50% of the average lifespan for its particular species and / or breed within a species. An animal is considered "old" if it has exceeded 66% of the average life expectancy, preferably exceeded 75% of the average life expectancy, and more preferably exceeded 80% of the average life expectancy. A senior cat or dog is at least about 7 years old since birth.

[0079] In one embodiment, as used herein, the term "subject" refers to a mammal. Mammals include, but are not limited to, rodents, aquatic mammals, domestic animals (such as dogs and cats), farm animals (such as sheep, pigs, cattle and horses), and humans. In one embodiment, the mammal can be a cat, a dog, or a human. The human can be a female, for example, a female who is trying to become pregnant or is already pregnant. In one embodiment of the invention, the subject is a mammal selected from a cat, a dog, and a human. For example, the subject can be an elderly person.

[0080] As used herein, the term "aglycone", "aglycone" or "genin" refers to the remaining compound after the sugar group on a glycoside is replaced by a hydrogen atom. For example, the aglycone of a cardiac glycoside is a steroid molecule.

[0081] Implementation

[0082] Compounds and compositions

[0083] One aspect of the present disclosure is compounds useful as sodium-glucose transporter-2 (SGLT2) inhibitors and compositions comprising such compounds.

[0084] In one embodiment, the compounds and related compositions comprising such compounds can be used as a food or supplement and as a SGLT2 inhibitor to effectively inhibit SGLT2, thereby improving the metabolic health of a subject.

[0085] The applicant surprisingly found that administering a methylated phloretin analog or related composition to a subject in need thereof as an SGLT2 inhibitor can effectively inhibit SGLT2, thereby improving metabolic health. Such compounds have many advantages over phloretin and SGLT2 inhibitors, especially reduced potency (2 to 3 times) in GLUT inhibition, thereby avoiding the side effects of known SGLT2 inhibitors.

[0086] Previously recorded known SGLT2 inhibitors (e.g., canagliflozin, dapagliflozin, empagliflozin and ergliflozin) have many side effects. For example, genital infection seems to be the most common adverse effect of these known SGLT2 inhibitors. Other side effects may also include fungal infection, urinary tract infection and osmotic diuresis, diabetic ketoacidosis (DKA) risk, reduced bone mineral density and thus increased risk of fracture, increased risk of lower limb amputation or increased risk of Fournier's disease.

[0087] In one embodiment, the methylated phloretin analogs or related compositions of the present invention as SGLT2 inhibitors can reduce or eliminate all of these side effects.

[0088] Additionally, the methylated phloretin analogs or related compositions have improved bioavailability and improved solubility compared to phloretin and can be used as food or supplements for subjects to effectively inhibit SGLT2, thereby improving metabolic health.

[0089] Another advantage of the presented compounds and related compositions is improved biotransformation, since they may produce less glycosides in vivo compared to phloretin.

[0090] In one aspect, the present disclosure relates to a composition for the inhibition of SGLT2 in a subject, the composition 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.

[0091] In one embodiment, the methylated phloretin analog is selected from the group consisting of phloretin-4-methyl-ether, 4'-O-methyl phloretin, calometron and calometron A.

[0092] In one embodiment, the methylated phloretin analogs disclosed herein are used to treat or prevent a condition, disorder or disease associated with type 2 diabetes and / or obesity in a subject.

[0093] In one embodiment, the methylated phloretin analogs disclosed herein are used to treat or prevent cardiovascular, weight management, renal, kidney or brain related conditions, disorders or diseases.

[0094] In one embodiment, the methylated phloretin analogs disclosed herein act as SGLT2 inhibitors via a direct inhibition mechanism or an indirect inhibition mechanism.

[0095] In one embodiment, the methylated phloretin analogs disclosed herein act as SGLT2 inhibitors via a direct inhibition mechanism.

[0096] In one embodiment, inhibition of SGLT2 by use of a methylated phloretin analog can occur at any site in the subject.

[0097] In one embodiment, inhibition of SGLT2 by use of a methylated phloretin analog disclosed herein is performed in muscle and / or kidney tissue.

[0098] In one embodiment, the methylated phloretin analogs disclosed herein can be obtained from plants or plant extracts. In a preferred embodiment, it can be obtained from unripe apples, for example, by deglycosylation followed by methylation. It can also be obtained from orange peels by deglycosylation.

[0099] In one embodiment, the methylated phloretin analogs disclosed herein are used in the preparation of a medicament for the treatment or prevention of a condition, disorder or disease responsive to SGLT2 inhibition.

[0100] In one aspect, the present invention is directed to a composition comprising a methylated phloretin analog disclosed herein.

[0101] For example, the compositions comprising the methylated phloretin analogs disclosed herein can be used as food or supplements as well as being effective SGLT2 inhibitors.

[0102] Thus, in one embodiment, the composition is a food, a beverage or a dietary supplement comprising a methylated phloretin analog as one of the ingredients.

[0103] In another aspect, the present invention relates to a pharmaceutical composition for the inhibition of SGLT2, comprising as an active ingredient a therapeutically effective amount of a methylated phloretin analog as disclosed herein or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.

[0104] It can be administered in any pharmaceutically effective amount according to the present invention. Generally, the pharmaceutically effective amount will depend on the type, age, size, health condition, lifestyle and / or genetic heritage of the subject. The pharmaceutically effective amount can be divided into several smaller 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 come up with an appropriate amount of methylated phloretin analogs to consume each day.

[0105] In one embodiment, the methylated phloretin analogs disclosed herein in the present invention can be provided in an amount between about 0.001 mg and about 100 mg / day, between about 0.005 mg and about 50 mg / day, between about 0.01 mg and about 20 mg / day, between about 0.015 mg and about 10 mg / day.

[0106] In one embodiment, the composition comprising the methylated phloretin analog disclosed herein can be used as a nutritional composition or nutritional supplement. For example, a composition comprising the methylated phloretin analog disclosed herein or its analogs may be beneficial to a human subject (such as an elderly person or a patient whose metabolic health needs to be improved) for inhibiting SGLT2, thereby improving his / her metabolic health.

[0107] In one embodiment, the composition comprising the methylated phloretin analog disclosed herein can be used for daily supplementation or routine administration. For example, it can be administered to the elderly or patients whose metabolic health needs to be improved. 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 disorders such as diabetic complications, insulin sensitivity, polycystic ovary disease, hyperglycemia, dyslipidemia, insulin resistance, metabolic syndrome, obesity, weight gain, inflammatory diseases, digestive organ diseases, angina pectoris, myocardial infarction, sequelae of angina pectoris or myocardial infarction, senile dementia, and cerebrovascular dementia.

[0108] In some embodiments, the composition may further comprise at least one of an excipient, a diluent, or a carrier.

[0109] Non-limiting examples of suitable excipients may include buffers, preservatives, stabilizers, binders, compacting agents, lubricants, chelating agents, dispersion enhancers, disintegrants, flavoring agents, sweeteners, coloring agents.

[0110] In some embodiments, the excipient can be a buffer. The non-limiting example of a suitable buffer can include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate and calcium bicarbonate. 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 other calcium salts or their combinations can be used as buffers in the pharmaceutical composition.

[0111] In some embodiments, the excipient may include a preservative. Non-limiting examples of suitable preservatives may include antioxidants such as alpha-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 cases, the preservative can include validamycin A, TL-3, sodium orthovanadate, sodium fluoride, Na-tosyl-Phe-chloromethyl ketone, Na-tosyl-Lys-chloromethyl ketone, 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.

[0112] In one embodiment, the composition may further include a binder. Non-limiting examples of suitable binders may include starch, pregelatinized starch, gelatin, polyvinyl pyrrolidone, cellulose, methyl cellulose, sodium carboxymethyl cellulose, ethyl cellulose, polyacrylamide, polyvinyl oxazolidinone, polyvinyl alcohol, C 12 -C 18 Fatty acid alcohols, polyethylene glycols, polyols, sugars, oligosaccharides and combinations thereof.

[0113] The composition can be administered by any method understood by those skilled in the art. For example, the methylated phloretin analogs of the present invention or compositions thereof are preferably administered orally. In some embodiments, it can be administered by intravenous administration, topical administration, parenteral administration, intraperitoneal administration, intramuscular injection administration, intrathecal administration, intralesional administration, intracranial administration, intranasal administration, intraocular administration, intracardiac administration, intravitreal administration, intraosseous administration, intracerebral administration, intraarterial administration, intraarticular administration, intradermal administration, transdermal administration, transmucosal administration, sublingual administration, enteral administration, sublabial administration, insufflation administration, suppository administration, inhalation administration or subcutaneous administration.

[0114] In some embodiments, the composition of the present invention may have an acute effect that can be observed in less than one month. In addition or alternatively, the composition may have a long-term effect, and therefore various embodiments include administering the composition to an individual (e.g., orally) for a period of at least one month; preferably at least two months, more preferably at least three months, four months, five months, or six months; most preferably a period of at least one year. During this period, the composition may be administered to an individual at least once a week; preferably at least two days a week, more preferably at least three days, four days, five days, or six days a week; most preferably seven days a week. The composition may be administered daily in a single dose or daily in a variety of separate doses. In one embodiment, a single dose is not less than about 100 mg. In one embodiment, a single dose is no more than about 1000 mg. In one embodiment, a single dose is between about 100 mg and about 1000 mg.

[0115] In one embodiment, the composition can be in liquid form or in solid form (eg, a solid dosage form).

[0116] In some embodiments, solid dosage forms of the compositions for oral administration may include capsules, tablets, caplets, pills, troches, lozenges, powders, and granules.

[0117] For example, the capsule may comprise a core material and a shell wall encapsulating the core material, the core material comprising a composition comprising a methylated phloretin analog. 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 a soft gelatin, a hard gelatin, and a polymer. Suitable polymers may include, but are not limited to, cellulosic polymers such as hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose (HPMC), methylcellulose, ethylcellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose succinate, and sodium carboxymethylcellulose; acrylic polymers and copolymers such as those formed from acrylic acid, methacrylic acid, methyl acrylate, ammonium methacrylate, ethyl acrylate, methyl methacrylate, and / or ethyl methacrylate (e.g., those copolymers sold under the trade name "Eudragit"); vinyl polymers and copolymers such as polyvinyl pyrrolidone, polyvinyl acetate, polyvinyl acetate phthalate, vinyl acetate crotonic acid copolymer, and ethylene-vinyl acetate copolymer; and shellac (purified shellac). In some embodiments, at least one polymer may be used as a taste masking agent.

[0118] In some embodiments, tablets, pills, etc. can be compressed, compressed multiple times, multi-layered and / or coated. For example, the coating can be single-layer or multi-layer.

[0119] Liquid formulations of compositions comprising methylated phloretin analogs may 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, etc. In some cases, combinations of various formulations may be administered. In some embodiments, tablets, pills, etc. may be formulated for extended release profiles. In some embodiments, the compositions may be formulated to increase shelf stability when stored in a closed container under standard ambient conditions.

[0120] Each component in the composition of the present invention can be used in any amount effective to achieve the purpose of the present invention (i.e., to increase the micronutrient level of the subject's breast milk after the subject gives birth). For example, a skilled person will be able to determine the appropriate dosage depending on the age, size and health of each specific subject, their lifestyle and their genetic inheritance.

[0121] In one embodiment, the amount used in the present application is the amount of a daily dose. Depending on the age, body shape and health status of each specific subject, its lifestyle and its genetic inheritance, the amount of each component can be used or changed (e.g., increased or decreased) according to the disclosed amount. In one embodiment, the nutritional composition or nutritional supplement of the present invention can be regularly applied, such as twice a day, every day, every two days or weekly.

[0122] In one aspect, the composition of the invention can be in any form suitable for administration of all ingredients. For example, the composition of the invention can be in the form of a powdered nutritional composition for reconstitution in milk or water, a food product, a beverage, a nutritional supplement or a nutraceutical.

[0123] When the composition of the present invention is in the form of a powdered nutritional composition to be reconstituted in milk or water, the nutritional composition or nutritional supplement may preferably include a protein source, a carbohydrate source and a lipid source, preferably together with lecithin. The composition may also include soy lecithin and / or a leavening agent. The protein source may be milk powder or skim milk powder. Sucrose and / or maltodextrin may be used as a carbohydrate source. The lipid source may be a vegetable oil. Alternatively or in addition thereto, the preparation may also include glucose syrup, milk fat, magnesium citrate, choline salts and choline esters, prebiotic fiber and / or ascorbyl palmitate. For example, flavor compounds (e.g., cocoa powder or honey) may be added to provide different tastes.

[0124] 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 substitute product, a beverage product, a dietary product and a pet food product.

[0125] In one embodiment, a composition comprising a methylated phloretin analog 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.

[0126] In one embodiment, the condition can be a metabolic disorder such as obesity, type 2 diabetes, cardiovascular disease or obesity-related conditions such as diabetic complications, insulin sensitivity, polycystic ovary disease, hyperglycemia, dyslipidemia, insulin resistance, metabolic syndrome, obesity, weight gain, inflammatory diseases, digestive organ diseases, angina pectoris, myocardial infarction, sequelae of angina pectoris or myocardial infarction, senile dementia and cerebrovascular dementia.

[0127] In another embodiment, the compositions comprising methylated phloretin analogs as disclosed herein can be used in 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 ovary disease, hyperglycemia, dyslipidemia, insulin resistance, metabolic syndrome, obesity, weight gain, inflammatory diseases, digestive organ diseases, angina pectoris, myocardial infarction, sequelae of angina pectoris or myocardial infarction, senile dementia, and cerebrovascular dementia.

[0128] method

[0129] Another aspect of the present disclosure is a method for improving metabolic health (e.g., treating or preventing conditions, disorders or diseases associated with type 2 diabetes and / or obesity, cardiovascular, weight management, kidney, renal or brain, metabolic disorders or obesity) by inhibiting SGLT2. In one embodiment, the method comprises administering a composition comprising a methylated phloretin analog of the present invention to a subject in need thereof as an SGLT2 inhibitor.

[0130] In one embodiment, the method for improving metabolic health includes treating or preventing a metabolic disorder. In one embodiment, the metabolic disorder includes obesity, type 2 diabetes, cardiovascular disease. In one embodiment, the metabolic disorder is selected from obesity, type 2 diabetes and cardiovascular disease.

[0131] In another embodiment, the method for improving metabolic health includes treating or preventing obesity-related disorders. In one embodiment, obesity-related disorders include diabetic complications, insulin sensitivity, polycystic ovarian disease, hyperglycemia, dyslipidemia, insulin resistance, metabolic syndrome, obesity, weight gain, inflammatory diseases, digestive organ diseases, angina pectoris, myocardial infarction, sequelae of angina pectoris or myocardial infarction, senile dementia and cerebrovascular dementia. In one embodiment, obesity-related disorders are selected from diabetic complications, insulin sensitivity, polycystic ovarian disease, hyperglycemia, dyslipidemia, insulin resistance, metabolic syndrome, obesity, weight gain, inflammatory diseases, digestive organ diseases, angina pectoris, myocardial infarction, sequelae of angina pectoris or myocardial infarction, senile dementia and cerebrovascular dementia.

[0132] In one embodiment, the present disclosure is a method for treating or preventing 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 diseases, digestive organ diseases, angina pectoris, myocardial infarction, sequelae of angina pectoris or myocardial infarction, senile dementia, and cerebrovascular dementia.

[0133] In another embodiment, the present disclosure is a method of treating or preventing a condition, disorder or disease associated with type 2 diabetes, non-alcoholic fatty liver disease and / or obesity in a subject by inhibiting SGLT2.

[0134] As disclosed above, the method comprises administering a composition comprising at least one of phloretin-4-methyl-ether, 4'-O-methylphloretin, calometron, or calometron A to a subject in need thereof.

[0135] In one embodiment, the method comprises administering to a subject in need thereof a composition comprising at least phloretin-4-methyl-ether.

[0136] In one embodiment, the method comprises administering to a subject in need thereof a composition comprising at least 4'-O-methylphloretin.

[0137] In one embodiment, the method comprises administering to a subject in need thereof a composition comprising at least carometron.

[0138] In one embodiment, the method comprises administering a composition comprising at least yellow kava A to a subject in need thereof.

[0139] 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, calometron, or calometron A to inhibit SGLT2.

[0140] In one embodiment, inhibition of SGLT2 is by a direct inhibition mechanism or an indirect inhibition mechanism.

[0141] In one embodiment, inhibition of SGLT2 is by a direct inhibition mechanism.

[0142] In one embodiment, inhibition of SGLT2 occurs at any tissue of the subject associated with the condition, disorder or disease.

[0143] In one embodiment, inhibition of SGLT2 is performed in muscle, liver, and / or kidney tissue.

[0144] In one embodiment, the methylated phloretin analog is obtained from a plant or a plant extract.

[0145] In one embodiment, the methylated phloretin analogs of the invention can be used to inhibit the reabsorption of glucose in the kidney and thereby lower blood sugar.

[0146] In one embodiment, the combination of different sugars of the compounds of this invention with glucose sites affects the orientation of the aglycone in the pathway vestibule. Therefore, when the aglycone of the compounds of this invention binds, it affects the entire inhibitor. Applicants imagine that these mechanisms together may lead to synergistic interactions. Therefore, the structural changes of the sugars of the compounds of this invention and the aglycone are crucial for the pharmacophore of SGLT inhibitors.

[0147] In one embodiment, the compounds of formula (I) and related compositions of the present invention can cause a decrease in glucose levels in a subject by 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%, 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%, 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 %, 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%.

[0148] In one embodiment, the reduction in glucose levels may continue 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.

[0149] In another aspect, the invention relates to an in vitro method of inhibiting SGLT2. The method comprises contacting a methylated phloretin analog with SGLT2 and determining inhibition of SGLT2.

[0150] In one embodiment, direct inhibition of SGLT2 can be determined by the ability to bind SGLT2.

[0151] In another aspect, the invention relates to a method of treating or preventing a condition, disorder or disease associated with cardiovascular, weight management, renal, kidney, brain, type 2 diabetes and / or obesity comprising administering a composition comprising a methylated phloretin analog disclosed herein.

[0152] Example

[0153] Example 1: Methylated analogs of phloretin show reduced GLUT inhibition

[0154] process

[0155] CHO-K1 cells were grown in DMEM growth medium containing 4.5 g / L glucose, L-alanyl-L-glutamine (Glutamax), sodium pyruvate (Thermo, 10569010), supplemented with 10% fetal bovine serum (Thermo, 10270106) and supplemented with non-essential amino acids (Thermo, 11140050), and maintained at 37° C. with 5% CO 2. Cells were plated in 24-well tissue culture assay plates (TPP, 92424) at 80% confluence. To determine GLUT inhibition, wells were washed four times with krebs-ringers-hepes (140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.25 mM MgSO4, 1.2 mM KH2PO4, 10 mM 4-hydroxyethylpiperazineethanesulfonic acid (hepes), 2 mM sodium pyruvate, pH 7.4) solution to remove extracellular glucose, and then 250 μL of krebs-ringers-hepes solution containing the indicated concentrations of vehicle, phloretin or specific methyl phloretin analogs or 10 μM 0.1% (v / v) DMSO ultimate GLUT inhibitor BAY-876 (Merck, SML1774-5MG) was transferred for a treatment duration of 30 minutes. After the treatment phase, 100 μM 2-deoxyglucose spiked with 10 μCi [3H]2-deoxyglucose (Perkin Elmer, NET328A001MC) was added to the wells for 5 minutes at 37°C. The reaction was stopped by adding 5 mM glucose to the wells before washing 4 times with krebs-ringers-hepes buffer to remove extracellular [3H]2-deoxyglucose. The cells were lysed with 0.1 M NaOH and the relative level of incorporated radioactivity was determined by counting per well per minute using a TriCarb scintillation counter with UltimaGold Scintillant (Perkin Elmer, 6013329). All other reagents were from Sigma.

[0156] result

[0157] Figure 1 The data presented in Figure 2 show the level of [3H]2-deoxyglucose (a GLUT transporter substrate) incorporated after treatment with (A) phloretin, (B) calometron, (C) phloretin 4-methyl ether, (D) scutellarin, (E) calometron A. The data show the effect relative to the pharmacological GLUT inhibitor BAY-876 (100% effect) and DMSO (0% effect). The data show that the methylated analogs of phloretin exhibit a reduced degree of GLUT inhibition compared to phloretin.

[0158] Example 2

[0159] process

[0160] In the presence of 1 mg / ml rat liver microsomes (M9066-1VL, Sigma), the methyl phloretin compound was incubated in 50mM Tris-HCl pH 8.0 containing 5mM UDP-glucose, 25mM sucrose, 10mM MgCl2 and 1mM DTT for 4 hours while shaking at 150rpm at 37°C. Using a benchtop vacuum manifold, a Waters Oasis HLB SPE column was used to prepare samples for LC-MS analysis by solid phase extraction. In brief, the SPE column was activated with three column volumes of methanol, balanced with reaction buffer, sample applied and washed with deionized water. The analyte was eluted in fractions containing increasing methanol concentrations. The SPE eluted fractions were dried under a N2 stream and reconstructed in 20% methanol. The samples were run on a Waters Acquity uHPLC system with a 10 cm Waters Aquity HSS T3 1.8 μM column maintained at 40 °C, connected to a Synapt G2-S high-resolution mass spectrometer operated in negative ion time-of-flight mode. The analysis was performed using MassLynx 4.2 software. A linear gradient from 0% acetonitrile to 100% acetonitrile was applied from 0 min to 16 min. Mobile phases A and B were water and acetonitrile containing 0.1% formic acid, respectively. The Waters Lockspray technique was applied to determine the accurate mass values. The expected monoisotopic mass was calculated from the molecular formula using a molecular mass calculator.

[0161] The results are shown as Figures 2 to 5 .

[0162] Cultivation of specific individual methyl-phloretin compounds (carometrones ( Figure 2 ); Phloretin-4-methyl ether ( Figure 3 ); phloretin-4′ methyl ether, ... Figure 4 ); yellow kava with UDP-glucose after solid phase extraction as described ( Figure 5 ) were analyzed by uHPLC-MS in the presence of rat microsomes.

[0163] For each of the above compounds:

[0164] - LC-MS analysis of fractions eluted from the SPE containing both the parent and the produced glycoside metabolites showed extracted mass chromatograms of the expected monoisotopic mass of the parent methylphloretin and the glycoside forms, with the exception of flavopiridol where the aglycone form was not readily observable in the negative ion time-of-flight mode and only the glycoside form was shown (A).

[0165] - For each peak observed in the extracted mass chromatograms of the parent and glycoside species, the sum of the MS-level spectra acquired over the duration of the peak (at least five scans) is shown, to which a Waters Lockmass calibration was applied to determine the accurate mass (B).

[0166] - For each major peak observed in the extracted mass chromatogram for the parent and derived glycoside species, the parent ion and MS / MS spectra of the glycoside species are shown (C). For the glycoside species, the selected ion fragments fragmented at a loss of 162 mu, consistent with the loss of a glucose molecule, resulting in fragmented ions at the expected m / z for the parent methyl-phloretin compound.

[0167] - For each methylphloretin species, the positions of the expected non-equivalent glycosylation sites are shown (D).

[0168] Example 3

[0169] Assay protocol:

[0170] In brief, the CHO-K1 cells of stable transfection SGLT1 or SGLT2 were plated with 20000 cells / well in 384 plates (black transparent bottom Greiner Bio-One #781091) in 50ul culture medium (RPMI1 640+5% serum) for 24 hours. Before the experiment, by removing the culture medium, and replacing the culture medium with 20 μl without glucose and serum to make the cells starved for 3 hours. In the culture medium without glucose and serum, 20 μl were added to the assay buffer (containing or not containing the compound of 1% DMSO final solution) containing 320 μM 1NBDG (fluorescent glucose analog) and incubated at 37°C for 2 hours, triggering the experiment. Then PBS was used to wash the cells 3 times to remove any extracellular 1NBDG traces. Then the fluorescence (RFU) corresponding to the 1NBDG uptake in the transfected cells was measured on a multi-mode reader (Biotecksynergy neo signal exc 485nm em 535nm).

[0171] Finally, the data were normalized: using DMSO as the maximum uptake signal (100%) and 10 μM dapagliflozin as the minimum signal (0% complete uptake inhibition), the percentage activity was determined using the formula: (x-min) / (max-min)*100

[0172] result

[0173] In these figures, each compound was tested at 6 different concentrations, starting from 10 μM, with serial 1 / 3 step dilutions in duplicate for SGLT1 and SGLT2 transfected cell lines. In the absence of inhibition (DMSO control), the uptake of 1NBDG was maximal, resulting in a high RFU signal, which was used to determine the 100% normalized activity, whereas in the presence of 10 μM dapagliflozin, the active transport of 1NBDG was completely inhibited, and the resulting fluorescence observed under such conditions gave a 0% normalized activity: The following formula was applied for normalization:

[0174] Normalized activity (cpd) = (RFU (cpd) - RFU (dapa)) / (RFU (DMSO) - RFU (dapa)).

[0175] Figure 6 The normalized SGLT1 and SGLT2 inhibitory activities of 4'methyl phlorizin (NI00046322) are shown. Thus, the downward direction of both curves indicates that the inhibition of SGLT1 and SGLT2 increases with increasing tested concentrations of 4'methyl phlorizin (NI00046322).

[0176] We observed that 4'Methyl Phlorizin (NI00046322) has different IC50 activities against both SGLT1 and SGLT2. 4'Methyl Phlorizin (NI00046322) is more potent against SGLT2 than SGLT1 as we observed complete inhibition of SGLT2 at 1 μM (-6 on a log scale) with an IC50 of approximately 33 nM (-7.5 on a log scale), while complete inhibition was observed at 10 μM (-5 on a log scale) with an IC50 between 300 nM and 1 μM (between -6.5 and -6 on a log scale).

[0177] Figure 7 The normalized SGLT1 and SGLT2 inhibitory activities of 4-methyl phlorizin (NI00046291) are shown. Thus, the downward direction of both curves indicates that the inhibition of SGLT1 and SGLT2 increases with increasing tested concentrations of 4-methyl phlorizin (NI00046291).

[0178] We observed that 4-methyl phlorizin (NI00046291) has different IC50 activities against both SGLT1 and SGLT2. 4-methyl phlorizin (NI00046291) is more potent against SGLT2 than SGLT1 as we observed complete inhibition of SGLT2 at 1 μM (-6 on a log scale) with an IC50 of approximately 33 nM (-7.5 on a log scale), while complete inhibition was observed at 3 μM (-5.5 on a log scale) with an IC50 between 300 nM and 1 μM (between -6.5 and -6 on a log scale).

Claims

1. A composition comprising an effective amount of at least one methylated phloretin analogue for inhibition of SGLT2.

2. The composition according to claim 1, wherein the methylated phloretin analog is selected from the group consisting of phloretin-4-methyl-ether, 4'-O-methyl phloretin, calometron and yellow kavalerin A.

3. The composition according to claim 1 or 2, wherein the methylated phloretin analogue is obtained from a plant or a plant extract.

4. The composition according to any one of claims 1 to 3, wherein the methylated phloretin analogue 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 carried out in muscle and / or kidney tissue.

6. A composition according to any one of claims 1 to 5 for use in treating and / or preventing a condition, disorder or disease associated with type 2 diabetes and / or obesity in a subject in need thereof.

7. The composition according to 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. A composition according to any one of claims 1 to 5 for use in treating and / or preventing a condition, disorder or disease responsive to SGLT2 inhibition in a subject in need thereof.

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

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

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

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

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

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

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