JP2005538987A - Methods and compositions for treating diabetes mellitus - Google Patents

Abstract

Disclosed are methods for modulating diabetes, impaired glucose tolerance, gestational diabetes and glucose resistance in mammals, particularly humans. In one embodiment, the method includes administering a gallotannin composition to a mammal in need of the composition. The gallotannin composition contains one or more selectively hydrolyzable gallotannins. In another embodiment, the method comprises administering to the subject a gallotannin modifying composition containing one or more selective gallotannin modifying compounds. Disclosed are methods for preventing or treating weight gain in a subject.

Description

(Field of Invention)
The present invention relates to methods and compositions for modulating diabetes mellitus and other disorders associated with abnormal glucose and / or insulin levels in a mammalian subject. The methods of the invention use compositions that do not induce adipogenesis or hypoglycemia.

(Background of the Invention)
Diabetes mellitus (commonly referred to as diabetes) refers to a disease process derived from multiple causative factors and characterized by increased levels of plasma glucose (called hyperglycemia). For example, LeRoith, D.M. (Eds.), DIABETES MELLITUS (Lippincott-Raven Publishers, Philadelphia, Pa. USA 1996). All references are cited herein. According to the American Diabetes Association, diabetes mellitus is estimated to affect approximately 6% of the world population. Uncontrolled hyperglycemia is an increased risk for microvascular and macrovascular diseases, including nephropathy, neuropathy, retinopathy, hypertension, cerebrovascular disease and coronary heart disease Associated with increased premature mortality due to sex.

  There are two main forms of diabetes: type 1 diabetes (formerly called insulin-dependent diabetes or IDDM); and type 2 diabetes (formerly called non-insulin-dependent diabetes or NIDDM). ) Type 1 diabetes is the result of an absolute deficiency of insulin, a hormone that regulates glucose utilization. This insulin deficiency is usually characterized by the destruction of beta cells in the pancreatic islets of Langerhans and an absolute insulin deficiency. Type 2 diabetes is a disease characterized by insulin resistance with relative but not absolute insulin deficiency. Type 2 diabetes can range from predominant insulin resistance with relative insulin deficiency to dominant insulin deficiency with some insulin resistance. Insulin resistance is a reduction in the ability of insulin to exert biological effects over a wide range of concentrations. In an insulin resistant individual, the body secretes an abnormally large amount of insulin to replenish this deficiency. The state of impaired glucose tolerance develops when there is an inappropriate amount of insulin to complement insulin resistance and adequately control glucose. In many individuals, insulin secretion further declines and plasma glucose levels rise, resulting in a clinical condition of diabetes.

  The majority of patients with type 2 diabetes are treated with either a hypoglycemic agent that acts by stimulating the release of insulin from beta cells, or an agent that enhances the patient's tissue sensitivity to insulin, or with insulin . Sulfonylurea is an example of an agent that stimulates the release of insulin from beta cells. Of the drugs applied to enhance tissue sensitivity to insulin, metformin is a representative example. Despite the wide use of sulfonylureas in the treatment of type II diabetes, this therapy is in most cases unsatisfactory. In many type II diabetic patients, sulfonylureas are not sufficient to normalize blood glucose levels and therefore patients are at risk of developing diabetic complications. Also, many patients gradually lose the ability to respond to treatment with sulfonylureas and are therefore increasingly forced to take insulin treatment. This patient transition from oral hypoglycemic agents to insulin treatment is usually due to pancreatic β-cell depletion in type II diabetic patients.

  Insulin stimulates glucose uptake by skeletal muscle and adipose tissue, primarily through translocation of glucose transporter 4 (GLUT4) from intracellular storage sites on the cell surface (Saltiel, AR and Kahn, CR). (2001) Nature 414: 799-806; Saltiel, A. and Pessin, JE (2002 Trends in Cell Biol. 12: 65-71; White, MF (1998) Mol. Cell. Biochem. 182. : 3-11) In response to insulin, the fraction of GLUT4 present in the intracellular membrane is redistributed to the plasma membrane, resulting in an increase in GLUT4 on the cell surface and enhanced glucose uptake by these cells. Mainly GLUT4 translocation is It is mediated through the insulin receptor (IR).

  In addition to glucose transport, insulin is deeply involved in adipogenesis. Adipogenesis is a process involved in the proliferation of preadipocytes and the differentiation of preadipocytes into adipocytes associated with the accumulation of fat in adipocytes. Studies using the adipocyte cell line 3T3-L1 suggest that the role of insulin in adipogenesis is primarily division (43). Prior to differentiation, 3T3-L1 cells are fibroblast-like preadipocytes that contain more IGF-1 receptors than IR. In vitro, preadipocyte adipogenesis can be induced by MDI, a commonly used differentiation-inducing mixture. MDI consists of methylisobutylxanthine (MIX), which enhances cAmp; dexamethasone (DEX), a glucocorticoid; and insulin (or IGF-1), which interacts with the IGF-1 receptor in preadipocytes (Tong, Q Hotisligil, GS (2001) Rev. in Endoc. & Metabolic Disorders. 2: 349-355; Rosen, ED et al. (2000) Genes Dev. 14: 1293-1307). When treated with MDI, confluent preadipocytes reenter the cell cycle, resulting in approximately two rounds of mitosis (Moden-Moses, D. et al. (1998) Biochem. J. 333: 825-831; Tong. , Q., Hotamisligil, GS (2001) Rev. in Endoc. & Metabolic Disorders. 2: 349-355; Rosen, ED, et al. (2000) Genes Dev. 14: 1293-1307). This is a process commonly referred to as clonal propagation. After clonal expansion, preadipocytes exit the cell cycle and express adipocyte genes, including C / EBP-α, C / EBP-β, C / EBP-γ, and PPAR-γ. Initiates differentiation into adipocytes.

  As a result of its adipogenic effect, insulin has an undesirable effect that promotes obesity in patients with type 2 diabetes (see Moller, DE (2001) Nature 4141: 821-827). Unfortunately, other anti-diabetic drugs recently used to facilitate glucose transport in patients with type 2 diabetes also have adipogenic activity. Thus, recent drug treatments can provide a reduction in blood glucose, but often promote obesity. Therefore, it would be highly desirable to develop a new generation of anti-diabetic drugs that correct hyperglycemia without the accompanying side effects of adipogenesis. Compounds that induce glucose intake in diabetic patients without causing hyperglycemia are also desirable.

(Summary of the Invention)
The present invention provides methods for modulating diabetes, impaired glucose tolerance, gestational diabetes and glucose resistance in mammals, particularly humans. In one embodiment, the method comprises administering to a mammal in need thereof a composition referred to hereinafter as a “gallotannin composition”. The gallotannin composition is substantially pure and 1,2,3,4-tetra-O-galloyl-α-D-glucose, 1,2,3,6-tetra-O-galloyl-α- D-glucose, 1,3,4,6-tetra-O-galloyl-α-D-glucose, 1,2,3,4,6-penta-O-galloyl-α-D-glucose, 1,2, 3,4,6-penta-O-galloyl-β-D-glucose, 1,2,3,4,6-hexa-galloyl-α-D-glucose, 1,2,3,4,6-hexa- O-galloyl-β-D-glucose, 1,2,3,4,6-hepta-O-galloyl-α-D-glucose, 1,2,3,4,6-hepta-O-galloyl-β- D-glucose, 1,2,3,4,6-octa-O-galloyl-α-D-glucose, 1,2,3,4, -Octa-O-galloyl-β-D-glucose, 1,2,3,4,6-nona-O-galloyl-α-D-glucose, 1,2,3,4,6-nona-O-galloyl -Β-D-glucose, 1,2,3,4,6-deca-O-galloyl-α-D-glucose, and 1,2,3,4,6-deca-O-galloyl-β-D- Contains one or more hydrolyzable gallotannins selected from the group consisting of glucose. As used herein, the term “substantially pure” means that the gallotannin composition contains at least 95% by dry weight of one or a combination of the listed gallotannins, and the following compound: Mono-O-galloyl-β-D-glucose, di-O-galloyl-β-D-glucose, tri-O-galloyl-β-D-glucose, tetra-O-galloyl-β-D-glucose, undeca- Means containing less than 5% dry weight by weight of one or a combination of O-galloyl-β-D-glucose, dodeca-O-galloyl-β-D-glucose or mixtures thereof.

  In another embodiment, the method comprises administering to a subject a composition referred to hereinafter as a “gallotannin modified composition”. The gallotannin modifying composition contains one or more gallotannin modifying compounds or salts thereof. This gallotannin modifying compound is:

Having the structure of
Here, R is D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose, L-altrose, D- Growth, L-growth, D-idose, L-idose, D-talose, L-talose, D-fructose, L-fructose, α-D-xylose, α-D lyxose, β-D lyxose, α-D arabinose , Β-D arabinose, α-D ribose, β-D ribose, D-trehalose, D-maltose, D-cellobiose, myo-inositol, D-glucitol,
X is an ester bond or an ether bond,
A is trihydroxybenzoic acid selected from the group consisting of 3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid, Or dihydroxybenzoic acid selected from the group consisting of 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, or 3-hydroxybenzoic acid and 4-hydroxybenzoic acid Monohydroxybenzoic acid selected from the group of
R is D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose, L-altrose, D-gulose, L- In the case of growth, D-idose, L-idose, D-talose, L-talose, D-fructose, L-fructose, n is 5 and q is 0, 1, 2, 3, 4, or 5 And z is 0;
When R is α-D-xylose, α-D lyxose, β-D lyxose, α-D arabinose, β-D arabinose, α-D ribose, β-D ribose, n is 4 and q is Is 0, 1, 2, 3, or 4 and z is 0, 1, or 2;
When R is D-glucitol or myo-inositol, n is 6, q is 0, 1, 2, 3, 4, 5, or 6 and z is 0; and R is D When trehalose, D-maltose, or D-cellobiose, n is 8, q is 0, 1, 2, 3, 4, 5, 6, 7, or 8 and z is 0 is there. Each compound in the modified gallotannin composition comprises tetra-O-galloyl-β-D-glucose, 1,2,3,4,6-penta-O-galloyl-β-D-glucose, 1,2, 3,4,6-hexa-O-galloyl-β-D-glucose, 1,2,3,4,6-hepta-O-galloyl-β-D-glucose, 1,2,3,4,6- Octa-O-galloyl-β-D-glucose, 1,2,3,4,6-nona-O-galloyl-β-D-glucose, and 1,2,3,4,6-deca-O-galloyl It has a structure other than the structure of -β-D-glucose.

  In a third embodiment, the method includes administering to a patient a combination of a gallotannin composition of the present invention and a gallotannin modified composition of the present invention.

  The present invention also provides a method for preventing or treating weight gain in a patient. This method comprises administering to a subject a gallotannin composition of the present invention, a gallotannin modified composition of the present invention, or a combination of a gallotannin composition of the present invention and a gallotannin modified composition of the present invention.

  The present invention also provides a method for inhibiting the differentiation of preadipocytes into adipocytes. This method contacts preadipocytes with a natural gallotannin composition of the invention, a gallotannin modified composition of the invention, or a combination of a natural gallotannin composition of the invention and a gallotannin modified composition of the invention. Process. The preadipocytes can be present in culture or in a mammalian subject.

  The present invention also includes:

The present invention relates to a modified gallotannin composition of the present invention, which is a compound having the structure:

(Detailed description of the invention)
(Definition)
The term “diabetes mellitus” or “diabetes” generally refers to a disease or condition characterized by a metabolic deficiency in glucose production and utilization that results in failure to maintain adequate blood glucose levels in the body To do. These deficiencies result in an increase in blood glucose called “hyperglycemia”. The two major types of diabetes are type 1 diabetes and type 2 diabetes. As noted above, type 1 diabetes is generally the result of a complete deficiency of insulin, a hormone that regulates glucose utilization. Type 2 diabetes often occurs despite normal levels of insulin, or even elevated levels of insulin, and can result from the inability of the tissue to respond properly to insulin. Most patients with type 2 diabetes are insulin resistant and have a relative deficiency of insulin, in which insulin secretion cannot complement the resistance of peripheral tissues to response to insulin. Furthermore, many patients with type 2 diabetes are obese. Other types of disorders of glucose homeostasis include glucose tolerance disorder, a metabolic stage intermediate between normal glucose homeostasis and diabetes, and glucose during pregnancy in women who do not have a history of type 1 or type 2 diabetes And gestational diabetes mellitus, which is resistant.

  Guidance on diagnosis for type 2 diabetes, impaired glucose tolerance, and gestational diabetes has been reviewed by the Diabetes Association (eg, The Expert Committee on the Diagnosis and Classification of Diabetes Melitus, Diabetes Care, (1999) V 1): S5-19).

  As used herein, the term “symptoms” of diabetes includes polyuria, polyposis, and bulimia, hyperinsulinemia, and hyperglycemia (these common Usage is incorporated), but is not limited thereto. For example, “polyuria” means excretion of a large amount of urine in a given period; “polyplegia” means chronic excessive thirst; “polyphagia” By hyperinsulinemia is meant elevated blood levels of insulin. Other symptoms of diabetes include, for example, increased susceptibility to certain infections (particularly fungal and staphylococcal infections), nausea, and ketoacidosis (enhanced production of ketone bodies in the blood) .

  The term “complication” of diabetes includes, but is not limited to, microvascular complications and macrovascular complications. Microvascular complications are those complications that generally cause microvascular damage. These complications include, for example, retinopathy (visual impairment or loss due to vascular damage in the eye); neuropathy (nerve damage and foot problems due to vascular damage to the nervous system) and nephropathy ( Kidney disease caused by vascular damage in the kidney). Macrovascular complications are complications that generally result from macrovascular injury. Examples of these complications include cardiovascular disease and peripheral vascular disease. Cardiovascular disease refers to diseases of the blood vessels of the heart. For example, Kaplan, R .; M.M. Et al., “Cardiovascular diseases” in HEALTH AND HUMAN BEHAVIOR, pp. 206-242 (McGraw-Hill, New York 1993). Cardiovascular disease is generally one of several forms listed below; for example, hypertension, coronary heart disease, stroke, and rheumatic heart disease. Peripheral vascular disease refers to diseases of any blood vessel outside the heart. This is often a stenosis of a blood vessel that transports blood to leg and arm muscles.

  As used herein, “hydrolyzable gallotannin” refers to a gallotannin glucose compound that is an ester of glucose having one or more trihydroxybenzoic acids. A substantially pure hydrolyzable gallotannin composition of the invention comprises one or more alpha anomers of hydrolyzable gallotannins having 5, 6, 7, 8, 9, or 10 galloyl groups or Contains a beta anomer, or a pharmaceutically acceptable salt thereof. The hexa-, hepta-, octa-, nona-, and deca-forms of hydrolyzable gallotannins are each a galloyl group linked to carbon 1, carbon 2, carbon 3, carbon 4, and carbon 6 of the glucose nucleus, respectively. As well as a secondary set of galloyl groups containing 1-5 additional galloyl groups. A secondary set of galloyl groups is linked to a separate galloyl group in the primary set. The substantially pure gallotannin composition of the present invention also comprises 1,2,3,4-tetra-O-galloyl-α-D-glucose, 1,2,3,6-tetra-O-galloyl-α-. It may contain D-glucose, 1,3,4,6-tetra-O-galloyl-α-D-glucose.

  The hydrolyzable gallotannin β-anomer is found in many plant-based foods (common fruits (fruits, bananas, grapes, apples); cereals (barley, sorghum); plant-derived beverages such as tea and wine) To be found. Typically, the hydrolyzable gallotannin β-anomer is also found in commercial tannic acid mixtures. Commercial tannic acid mixtures also contain varying amounts of methyl galloyl and galloyl glucose compounds, including 1, 2, 3, 11, and 12 galloyl groups. The substantially pure hydrolyzable gallotannin composition of the present invention comprises the following compounds: mono-O-galloyl-β-D-glucose, di-O-galloyl-β-D-glucose, tri-O-galloyl- β-D-glucose, tetra-O-galloyl-β-D-glucose, undeca-O-galloyl-β-D-glucose, dodeca-O-galloyl-β-D-glucose or one of these mixtures Or the mixture comprises less than 5% by dry weight, preferably less than 3% by dry weight, more preferably less than 1% by dry weight. Thus, the substantially pure hydrolyzable gallotannin composition used in the method of the present invention is different from a commercially available mixture of gallotannins.

  As used herein, “gallotannin variant” refers to tetra-O-galloyl-β-D-glucose, 1,2,3,4,6-penta-O-galloyl-β-D- Glucose, hexa-O-galloyl-β-D-glucose, hepta-O-galloyl-β-D-glucose, octa-O-galloyl-β-D-glucose, nona-O-galloyl-β-D-glucose, Compounds that are structurally similar but not identical to deca-O-galloyl-β-D-glucose, undeca-O-galloyl-β-D-glucose, and dodeca-O-galloyl-β-D-glucose Say.

  This variant is:

Having the structure of
Here, R is D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose, L-altrose, D- Growth, L-growth, D-idose, L-idose, D-talose, L-talose, D-fructose, L-fructose, α-D-xylose, α-D lyxose, β-D lyxose, α-D arabinose , Β-D arabinose, α-D ribose, β-D ribose, D-trehalose, D-maltose, D-cellobiose, myo-inositol, D-glucitol,
X is an ester bond or an ether bond,
A is trihydroxybenzoic acid selected from the group consisting of 3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid, Or dihydroxybenzoic acid selected from the group consisting of 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, or 3-hydroxybenzoic acid and 4-hydroxybenzoic acid Monohydroxybenzoic acid selected from the group of
R is D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose, L-altrose, D-gulose, L- In the case of growth, D-idose, L-idose, D-talose, L-talose, D-fructose, L-fructose, n is 5 and q is 0, 1, 2, 3, 4, or 5 And z is 0;
When R is α-D-xylose, α-D lyxose, β-D lyxose, α-D arabinose, β-D arabinose, α-D ribose, β-D ribose, n is 4 and q is Is 0, 1, 2, 3, or 4 and z is 0, 1, or 2;
When R is D-glucitol or myo-inositol, n is 6, q is 0, 1, 2, 3, 4, 5, or 6 and z is 0; and R is D When trehalose, D-maltose, or D-cellobiose, n is 8, q is 0, 1, 2, 3, 4, 5, 6, 7, or 8 and z is 0 is there.

  As used herein, “adipocyte” refers to a fat cell. Morphologically, adipocytes are round and are cells containing triglyceride (fat) vesicles. Biochemically, adipocytes express high levels of insulin receptors on their cell surface and display a highly active insulin-mediated glucose transport signaling pathway with respect to glucose transporter 4 (GLUT4). In vivo, adipocytes are involved in the synthesis and storage of fat (triglycerides) and glucose metabolism (intake of glucose from the blood and conversion of glucose into fat).

  As used herein, “preadipocytes” refers to adipocyte precursor cells that divide and differentiate into adipocytes under the action of hormones (eg, insulin and glucocorticoids). Morphologically, preadipocytes are like fibroblasts (thin, spindle type) and lack triglyceride (fat) vesicles in their cytoplasm. Compared to adipocytes, preadipocytes contain low levels of insulin receptor and relatively high levels of insulin-like growth factor 1 (IGF-1) receptor to receive mitogenic and differentiation signals. Without induction or complete differentiation, preadipocytes do not express GLUT4 or other differentiation-related genes (eg, PPAR-γ, C / EBP-α or C / EBP-γ). The intracellular glucose transport activity of preadipocytes is lower than that of adipocytes.

  As used herein, “adipogenesis” refers to the process by which preadipocytes divide and differentiate into adipocytes.

  As used herein, “lipogenesis” refers to the process by which fat is synthesized and accumulated in adipocytes.

  The term “mammal” includes, but is not limited to, humans, domestic animals (eg, dogs or cats), farm animals (bovines, horses, or pigs), monkeys, rabbits, mice, and laboratory animals. It is done.

  In one aspect, the present invention provides a method for stimulating glucose uptake in cells of a mammal, particularly a mammal having diabetes, glucose intolerance disorder, insulin resistance or gestational diabetes. In another aspect, the present invention relates to preadipocyte adipocytes in mammals, particularly those that are obese, overweight, or exhibit symptoms of diabetes mellitus, glucose intolerance, or gestational diabetes. A method for inhibiting the differentiation of is provided. The present invention is based in part on the fact that certain hydrolyzable gallotannins and certain gallotannin variants can stimulate glucose transport into adipocytes and inhibit the differentiation of preadipocytes into adipocytes. Based on the discovery of the inventors. The methods of the invention are also based, in part, on the inventors' discovery that certain hydrolyzable gallontannins reduce blood glucose levels and blood insulin levels in mammals. Thus, the methods of the invention are useful for treating or preventing diabetes, impaired glucose tolerance, insulin resistance and gestational diabetes in mammals.

  In one embodiment, the method of the invention for treating or preventing diabetes, impaired glucose tolerance, insulin resistance and gestational diabetes in a mammal comprises a therapeutically effective amount of a substantially pure hydrolyzable gallotannin composition. Administering a product to the mammal. The substantially pure gallotannin composition comprises 1,2,3,4-tetra-O-galloyl-α-D-glucose, 1,2,3,6-tetra-O-galloyl-α-D-glucose, 1,3,4,6-tetra-O-galloyl-α-D-glucose, 1,2,3,4,6-penta-O-galloyl-α-D-glucose, 1,2,3,4, 6-penta-O-galloyl-β-D-glucose, 1,2,3,4,6-hexa-galloyl-α-D-glucose, 1,2,3,4,6-hexa-O-galloyl- β-D-glucose, 1,2,3,4,6-hepta-O-galloyl-α-D-glucose, 1,2,3,4,6-hepta-O-galloyl-β-D-glucose, 1,2,3,4,6-octa-O-galloyl-α-D-glucose, 1,2,3,4,6-octa-O— Royl-β-D-glucose, 1,2,3,4,6-nona-O-galloyl-α-D-glucose, 1,2,3,4,6-nona-O-galloyl-β-D- From the group consisting of glucose, 1,2,3,4,6-deca-O-galloyl-α-D-glucose, and 1,2,3,4,6-deca-O-galloyl-β-D-glucose Contains one or more hydrolyzable gallotannins selected or pharmaceutically acceptable salts of these hydrolyzable gallotannins. The substantially pure hydrolyzable gallotannin composition comprises the following compounds: mono-O-galloyl-β-D-glucose, di-O-galloyl-β-D-glucose, tri-O-galloyl-β-D. 1 or more of glucose, tetra-O-galloyl-β-D-glucose, undeca-O-galloyl-β-D-glucose, dodeca-O-galloyl-β-D-glucose or mixtures thereof Contains less than dry weight.

  In another embodiment, a method for treating or preventing diabetes, impaired glucose tolerance, insulin resistance and gestational diabetes in a mammal comprises a therapeutically effective amount of a gallotannin modifying composition comprising one or more gallotannin modifying compounds. In a patient. In a further embodiment, the method includes administering to the patient both the substantially pure hydrolyzable gallotannin composition of the present invention and the gallotannin modified composition.

  Other substances (insulin, sulfonylurea, meglitinide, biguanide (Glucophage or metformin), thiazolidinedione (TZD), and α-glucosidase used to treat or prevent diabetes, as appropriate Inhibitors) are administered to a mammal in combination with the hydrolyzable gallotannin or gallotannin modified composition of the present invention. For these mammals with type I diabetes mellitus, it is preferred that insulin be administered in combination with a gallotannin composition and / or a gallotannin modifying composition.

(Subject)
The methods of the present invention are useful for treating mammals that have been diagnosed with diabetes, gestational diabetes, insulin resistance or glucose tolerance disorders. The methods of the present invention also include mammals exhibiting symptoms of diabetes, gestational diabetes, insulin resistance or impaired glucose tolerance, or mammals having a genetic predisposition to diabetes, gestational diabetes, insulin resistance or impaired glucose tolerance. Useful for treating animals. The methods of the present invention are also useful for preventing or treating weight gain in patients, particularly those who are obese or overweight.

(Dosage form)
The compositions of the invention are administered to a subject by injection (subcutaneous injection, parenteral injection, and intravenous injection) or by oral administration. Because of its ease of administration, the preferred route of administration is oral administration.

(Prescription)
Gallotannin compositions and gallotannin modified compositions for use in accordance with the methods of the present invention are formulated into pharmaceutical compositions using conventional methods. Such pharmaceutical preparations contain one or more hydrolyzable gallotannins of the present invention and / or one or more gallotannin modified compositions of the present invention. Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or diluent. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the gallotannin composition or the modified gallotannin composition. Many such carriers are routinely used and can be identified by reference to the pharmaceutical text. The characteristics of the carrier will depend on the route of administration and the particular compound or combination of compounds in the composition. The preparation of such formulations is within the level of ordinary skill in the art. The preparation further contains other substances that either enhance the activity of gallotannins or gallotannin variants or supplement their activity. The preparation may further include excipients, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.

  The pharmaceutical composition can be conveniently provided in unit dosage form and can be prepared by any method well known in the pharmaceutical arts. The term “unit dosage” means a predetermined amount of a gallotannin composition or gallotannin modified composition sufficient to be effective in treating a target disease or disorder. All methods include the step of contacting either or both of the gallotannin composition, the gallotannin modified composition, or a carrier or diluent, and any other accessory ingredients.

  For oral administration, the pharmaceutical composition comprises, for example, a pharmaceutically acceptable excipient (eg, a binder (eg, pregelatinized corn starch, polyvinyl pyrrolidone, or hydroxypropyl methylcellulose); a filler (eg, , Lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (eg, magnesium stearate, or talc); disintegrants (eg, potato starch, or sodium starch glycolate); or wetting agents (eg, lauryl) Along with sodium sulfate)), it may take the form of tablets or capsules prepared by conventional means. The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, aqueous solutions, syrups or suspensions, or can be provided as a dry product for constitution with water or other suitable vehicle before use . Such liquid preparations include pharmaceutically acceptable additives (eg, suspensions (eg, sorbitol syrup, cellulose derivatives, or hardened edible fats); emulsifiers (eg, lecithin, or acacia); non-aqueous vehicles. (E.g., almond oil, oily ester, ethyl alcohol, or fractionated vegetable oil); and preservatives (e.g., methyl-p-hydrobenzoate or propyl-p-hydrobenzoate, or sorbic acid)) and prepared by conventional methods Can be done. The preparation may also contain buffer salts, flavoring, coloring and sweetening agents as desired. The preparation can also take the form of a nutritional formulation. Preparations for oral administration can be formulated so that the gallotannin composition is sustained release. Due to the presence of proteins with high levels of proline in saliva, it is desirable that the preferred oral formulation be in the form of a capsule that includes a coating to protect the gallotannin composition from interaction with saliva.

  The gallotannin composition and gallotannin modified composition may be formulated for parenteral administration by injection (eg, bolus injection, or continuous injection). Formulations for injection can be provided in unit dosage forms (eg ampoules or multiple dose containers) with added preservatives. The compositions can take such forms as suspensions, oily vehicles or solutions or emulsions in aqueous vehicles, and can contain formulating agents (eg, suspending, stabilizing and / or dispersing agents). Alternatively, the active ingredient can be in powder form, tablets or capsules for constitution with a suitable vehicle (eg, sterile pyrogen-free water) before use.

  Gallotannin compositions and gallotannin modified compositions are also formulated in rectal compositions such as suppositories or retention enemas, eg, containing the main components of conventional suppositories (eg, cocoa butter, other glycerides or carbowaxes). Is done.

  In addition to the above formulations, gallotannin compositions and gallotannin modified compositions can also be formulated as storage preparations. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compound may be a suitable polymeric or hydrophobic material (eg, as an emulsion in a suitable oil) or an on-exchange resin, or a slightly insoluble derivative (eg, a slightly insoluble salt). Can be prescribed.

(Dosage)
The gallotannin composition and the gallotannin modified composition are administered to the subject in a therapeutically effective amount. As used herein, the term “therapeutically effective amount” means a significant benefit (eg, reduction of hyperglycemia (reduction of blood glucose level), reduction of hyperinsulinemia (reduction of blood insulin levels). ), Improved glucose tolerance, prevention of weight gain and weight loss) means the total amount that is sufficient to show. The dosage of the gallotannin composition or gallotannin modified composition required to obtain meaningful results can be determined by routine experimentation with appropriate controls by those skilled in the art in view of the present disclosure. Comparison of the appropriate treatment group relative to the control indicates whether a particular dosage is effective in lowering the subject's blood glucose level or inhibiting adipogenesis.

  The amount of gallotannin composition required will depend on the nature and severity of the condition to be treated and the nature of the previous treatment that the subject has received. Ultimately, dosage is determined using clinical trials. First, the clinician administers a dose derived from animal studies. An effective amount can be achieved by a single administration of the composition. Alternatively, an effective amount is achieved by multiple administrations of the composition to the subject. In vitro, a biologically effective amount (ie, an amount sufficient to induce glucose uptake) is administered in 2-fold increments to determine the full range of activity. The efficacy of oral, subcutaneous and intravenous administration is determined by clinical studies. While a single administration of a gallotannin composition can be beneficial, multiple doses are expected to be preferred.

(Method of determining dosage to stimulate glucose uptake in cells)
Glucose uptake activity in cells can be analyzed by measuring uptake of 2-deoxy-D- [ ³ H] glucose using standard assays. Confluent 3T3-L1 adipocytes grown in 12-well plates are washed twice with serum-free DMEM and incubated with 1 mL of the same medium for 2 hours at 37 ° C. The cells are washed 3 times with Krebs-Ringer-Hepes (KRP) buffer and incubated with 0.9 mL KRP buffer at 37 ° C. for 30 minutes. Insulin (positive control) or gallotannin or a gallotannin variant (experiment) is then added at a predetermined concentration and the adipocytes are incubated at 37 ° C. for 15 minutes. Glucose intake is initiated by the addition of 0.1 mL KRP buffer, 37 MBq / L 2-deoxy-D- [ ³ H] glucose, and 1 mmol / L glucose as the final concentration. After 10 minutes, glucose uptake is terminated by washing the cells three times with cold PBS. The cells are lysed with 0.7 mL of 1% Triton X-100 at 37 ° C. for 20 minutes. Radioactivity retained by the cell lysate is determined by scintillation counter. The dosage that induces maximum glucose uptake can be selected between experimental samples.

(Method of determining dosage to stimulate glucose intake in animals)
Eight week old male db / db (leptin receptor deficient) mice can be used to determine dosages to stimulate glucose uptake in vivo. The mice are divided into 3-4 groups depending on how many doses are analyzed. 10 μl of test solution with a predetermined concentration of gallotannin composition is orally administered to test mice. Negative control mice receive the same amount of water. After administration, blood is collected from the tail of the mouse at various times after oral administration. The blood glucose level of the mouse at a predetermined time after administration is measured by applying 6 μl of blood to One Touch Basic Complete Diabetes Monitoring System (manufactured by Lifescan). Effective dosage ranges and optimal dosages can be determined by comparing the reduction in blood glucose levels with various dosages against the glucose level of the negative (water) control group.

(Inhibition of adipogenesis and weight gain)
In another aspect, the present invention provides a method for inhibiting preadipocyte differentiation into adipocytes. Adipocytes can be present in culture or in a mammalian subject. In one embodiment, the method includes contacting preadipocytes with a biologically effective amount of a hydrolyzable gallotannin composition. The gallotannin composition is substantially pure. And this gallotannin composition is 1,2,3,4-tetra-O-galloyl-α-D-glucose, 1,2,3,6-tetra-O-galloyl-α-D-glucose, 3,4,6-tetra-O-galloyl-α-D-glucose, 1,2,3,4,6-penta-O-galloyl-α-D-glucose, 1,2,3,4,6- Penta-O-galloyl-β-D-glucose, 1,2,3,4,6-hexa-galloyl-α-D-glucose, 1,2,3,4,6-hexa-O-galloyl-β- D-glucose, 1,2,3,4,6-hepta-O-galloyl-α-D-glucose, 1,2,3,4,6-hepta-O-galloyl-β-D-glucose, 1, 2,3,4,6-octa-O-galloyl-α-D-glucose, 1,2,3,4,6-octa-O-ga Yl-β-D-glucose, 1,2,3,4,6-nona-O-galloyl-α-D-glucose, 1,2,3,4,6-nona-O-galloyl-β-D- From the group consisting of glucose, 1,2,3,4,6-deca-O-galloyl-α-D-glucose, and 1,2,3,4,6-deca-O-galloyl-β-D-glucose Contains one or more hydrolyzable gallotannins selected.

  In another embodiment, the method comprises contacting the preadipocytes with a biologically effective amount of a gallotannin modifying composition containing one or more gallotannin variants. In a further embodiment, the method includes contacting the patient with preadipocytes with both the hydrolyzable gallotannin composition and the modified gallotannin composition of the present invention.

(Procedure for determining dosage in vitro)
To determine the effective concentration of gallotannin or a gallotannin variant used to prevent adipogenesis in vitro, undifferentiated preadipocytes are obtained from 3-isobutyl-1-methylxanthine, dexamethasone, and insulin (MDI). A differentiating cocktail; or MDI and either gallotannin or a gallotannin variant. After about 10 days, MDI induces clearly visible differentiation as a change from fibroblast-like preadipocytes to round fat vesicle-containing adipocytes. The extent of cell differentiation is assessed by microscopic observation of qualification and oil red O staining (only vesicles containing triglycerides can be stained red) and glucose uptake activity exhibited by the treated cells at the end of the incubation period. The glucose uptake assay is used to determine the extent of adipocyte differentiation based on the observation that differentiated adipocytes can be induced by insulin and take up glucose, but not preadipocytes. Selected and implemented.

(Procedure for determining dosage in vivo)
Genetically diabetic 5-week-old female mice (type II, KK-A ^y ) are used to determine the anti-lipogenic effect in vivo and the effective amount of gallotannin or modified gallotannin composition. The gallotannin or gallotannin variant is delivered orally or IP injected to mice daily at various concentrations for 6-10 weeks. At the end of the experiment, parameter adipose tissue is removed from the treated and control mice, weighed and compared. In addition, livers of treated and control mice are also removed and the fat content of the liver is measured. Dosages that produce the greatest reduction in parameter adipose tissue and liver lipid content without significantly changing food intake are related to the anti-adipogenic activity of the gallotannin or gallotannin-modified compositions of the present invention. Consider the optimal dosage.

Exemplary method for producing hydrolyzable gallotannins
A. Isolation Method The beta form of hydrolyzable gallotannin can be isolated from commercial tannic acid preparations using HPLC. The HPLC system is a Beckman System Gold consisting of a 125 solvent module, a 168 PDA detector, and a 508 autosampler. For the separation, a Beckman Ultrasphere C-18 reverse phase Semi-Prep column (10.0 mm × 250 mm ID, 5 μm) is used. The detection wavelength is set to 320 nm or 330 nm. The eluent A is water to which 0.1% trifluoroacetic acid is added, and the eluent B is acetonitrile containing 0.1% trifluoroacetic acid. ISCO's Foxy Jr. Using a fraction collector, individual peaks are collected in a timed window. Separation is achieved using a constant composition eluent (A: B 82:18) for 40 minutes at a flow rate of 3 mL / min. Under these conditions, suitable retention times for the beta form of gallotannin are as follows:
Penta-O-galloyl-β-D-glucose 13.5 minutes Hexa-O-galloyl-β-D-glucose 20.2 minutes Octa-O-galloyl-β-D-glucose 22.0 minutes Nona-O-galloyl -Β-D-glucose 30.0 minutes Deca-O-galloyl-β-D-glucose 36.3 minutes Undeca-O-galloyl-β-D-glucose 39.8 minutes.

B. Synthetic Method The method for chemical synthesis of PGG alpha and beta forms consists of four steps:
(I) Starting with the methyl ester of gallic acid and reacting with benzyl chloride in the presence of potassium carbonate and potassium iodide in acetone, the protected gallic acid derivative methyl 3,4,5-tri -O-benzyl gallate is obtained.

  (Ii) Ester cleavage with sodium hydroxide in aqueous ethanol leads to the structure of benzyl 3,4,5-tri-O-benzyl gallate.

  (Iii) Use of benzyl 3,4,5-tri-O-benzyl gallate in dicyclohexylcarbodiimide-mediated esterification of D-glucose in the presence of 4- (dimethylamino) pyridine in 1,2-dichloroethane . Penta-O- (3,4,5-tri-O-benzylgalloyl) -D-glucopyranose is obtained as a mixture of α and β anomers.

  (Iv) Penta-O- (3,4,5-tri-O-benzylgalloyl) -D-glucopyranose is deprotected by hydrogenation in the presence of a palladium catalyst in tetrahydrofuran. Chromatotron separation of the α / β mixture of PGG gives the individual pure anomers.

The identity and purity of naturally occurring intermediates and products is determined by ¹ H-NMR spectroscopy and ¹³ C { ¹ H} NMR spectroscopy, electrospray mass spectrometry, and UV-visible spectra.

  Using as a starting material a mixture of α-penta-O-galloylglucose and β-penta-O-galloylglucose, α-hexa-O-galloylglucose, β-hexa-O-galloylglucose , Α-hepta-O-galloyl glucose, β-hepta-O-galloyl glucose, α-octa-O-galloyl glucose, β-octa-O-galloyl glucose, α-nona-O-galloyl glucose Β-nona-O-galloyl glucose, α-deca-O-galloyl glucose, β-deca-O-galloyl glucose can be produced. The further reaction of one or more gallic acids to α / β-penta-O-galloylglucose is identical to or very similar to the reaction steps for the synthesis of α / β-penta-O-galloylglucose as described above. Similar to. This reaction synthesizes a mixture of galloylglucose with 6, 7, 8, 9, or 10 gallic acids. These different compounds can be separated into single species by HPLC, and the identity of their individual structures can be confirmed by mass spectra and NMR analysis.

  α-Tetra-O-galloylglucose and β-tetra-O-galloylglucose protect one of the hydroxyl groups in glucose before the addition of gallic acid, and deoxidize the hydroxyl group after the addition reaction. It can be generated by protecting.

Exemplary method for generating gallotannin variants
1) Synthesis of pentakis-O- (3,4-dibenzyloxybenzoyl) -β-D-glucopyranose Step 1: Ethyl 3,4-dibenzyloxybenzoate

procedure:
A mixture of ethyl 3,4-dihydroxybenzoate (10 g, 54.3 mmol), potassium iodide (4 g, 24 mmol) and anhydrous powdered potassium carbonate (40 g, 289 mmol) in acetone (500 mL) was stirred at room temperature for 20 minutes. Benzyl chloride (14.85 g, 117 mmol) dissolved in 100 mL acetone was added. This suspension was refluxed for 18 hours. The solid was removed by filtration and the filtrate was evaporated. The residue was redissolved in 300 mL dichloromethane and filtered again. The solvent was evaporated. A dark yellow oil was obtained. This was used in the next step without further purification.

  Step 2: 3,4-dibenzyloxybenzoic acid

procedure:
The crude product from the above step was suspended in 95% ethanol and sodium hydroxide pellets (3.54 g, 88.5 mmol) were added. The mixture was heated under reflux for 3 hours. This warm solution was poured into a mixture of water (500 mL) and concentrated hydrochloric acid (25 mL). After the flask was swirled for 10 minutes, the product was filtered off and washed successively with water (100 mL), 95% ethanol (100 mL), methanol (100 mL), and methyl tert-butyl ether (100 mL). The white solid was dried overnight at room temperature in an oil pump vacuum (approximately 0.1 bar).

  Yield over two steps: 18.6 g (93%).

  Step 3: pentakis-O- (3,4-dibenzyloxybenzoyl) -β-D-glucopyranose

Procedure D-Glucose (0.2 g, 1.11 mmol), 3,4-dibenzyloxybenzoic acid (2.78 g, 8.31 mmol), dicyclohexylcarbodiimide (DCC, 1.84 g, 8.92 mmol), and N, N-dimethylaminopyridine (DMAP, 1.08 g, 8.84 mmol) was added to dry dichloromethane (130 mL). This suspension was refluxed for 2.5 days. After cooling to room temperature, the urea byproduct was filtered off. 8 g of silica gel was added to the filtrate and evaporated to dryness. The residue was applied to a silica gel column (solvent system dichloromethane: toluene: ethyl acetate = 300: 100: 4). The clean fractions were combined and evaporated.

  Yield: 1.72 g (88%) very viscous clear oil.

  Step 4: pentakis-O- (3,4-dihydroxybenzoyl) -β-D-glucopyranose

procedure:
The benzyl protected starting material (392 mg, 0.222 mmol) was dissolved in dry THF (50 mL). The solution was degassed by applying about 30 seconds to a vacuum with a water aspirator with magnetic stirring. The flask was then flushed with argon gas. Degassing and flushing were repeated two more times. 10% palladium on charcoal (287 mg, 0.27 mmol) was added. The mixture was degassed and then flushed with hydrogen gas. Degassing and flushing were repeated two more times. The suspension was then stirred for 5 hours at maximum pressure at 40 ° C. under a hydrogen gas atmosphere. The mixture was cooled, filtered through celite, and the filtrate was evaporated.

  Yield: 190 mg (99%) foamy amorphous solid.

  Other gallotannin variants containing a dihydroxybenzyloxybenzoyl moiety are generated as described above except that various ethyl dihydroxybenzoates are used as the starting material in Step 1.

2) Synthesis of tetrakis-O- (3,4-dihydroxybenzoyl) -β-D-glucopyranose Step 1: Methyl 3,4,5-tribenzyloxybenzoate

procedure:
Methyl 3,4,5-trihydroxybenzoate (10 g, 54.3 mmol), potassium iodide (4 g, 24 mmol), and anhydrous powdered potassium carbonate (44 g, 318 mmol) in acetone (500 mL) were stirred at room temperature for 20 minutes. did. Benzyl chloride (22 g, 174 mmol) dissolved in 100 mL acetone was added. This suspension was refluxed for 18 hours. The solid was removed by filtration and the filtrate was evaporated. The residue was collected in 400 mL dichloromethane. The suspension was filtered through celite and the filtrate was evaporated. The residue was dried in an oil pump vacuum for 45 minutes at room temperature.

  Yield: 26.528 g (107%) (This product contains some benzyl chloride as an impurity).

  Step 2: 3,4,5-tribenzyloxybenzoic acid

procedure:
The crude product from the above step was suspended in 95% ethanol (300 mL) and sodium hydroxide pellets (3.54 g, 88.5 mmol) were added. The mixture was heated at reflux for 3 hours. This warm solution was poured into a mixture of water (500 mL) and concentrated hydrochloric acid (25 mL). After spinning the flask for 10 minutes, the product was filtered off and washed successively with water (100 mL), 95% ethanol (100 mL), methanol (100 mL), and methyl tert-butyl ether (100 mL). The white solid was dried overnight at room temperature in an oil pump vacuum (about 0.1 bar).

Yield over two steps: 22.42 g (94%)
Step 3: Tetrakis-O- (3,4,5-tribenzyloxybenzoyl) -D-xylopyranose

procedure:
D-xylose (0.2 g, 1.33 mmol), 3,4,5-tribenzyloxybenzoic acid (3.515 g, 7.98 mmol), dicyclohexylcarbodiimide (DCC, 1.77 g, 8.57 mmol), and N , N-dimethylaminopyridine (DMAP, 1.04 g, 8.49 mmol) was added to dry dichloromethane (130 mL). This suspension was refluxed for 2.5 days. After cooling to room temperature, the urea byproduct was filtered off. 9 g of silica gel was added to the filtrate and evaporated to dryness. The residue was applied to a silica gel column (solvent system dichloromethane: toluene: ethyl acetate = 300: 100: 4). The clean fractions were combined and evaporated.

  Yield: 208 mg β isomer, 220 mg α isomer, 770 mg mixed fraction (α + β).

  Step 4: Tetrakis-O- (3,4,5-trihydroxybenzoyl) -β-D-glucopyranose

procedure:
The benzyl protected starting material (150 mg, 0.0815 mmol) was dissolved in dry THF (20 mL). The solution was degassed by applying a water aspirator vacuum for about 30 seconds with magnetic stirring. The flask was then flushed with argon gas. Degassing and flushing were repeated two more times. 10% palladium on charcoal (120 mg, 0.113 mmol) was added. The mixture was degassed and then flushed with hydrogen gas. Degassing and flushing were repeated two more times. The suspension was then stirred for 5 hours at maximum pressure at 40 ° C. under a hydrogen gas atmosphere. The mixture was cooled, filtered through celite, and the filtrate was evaporated.

  Yield: 62 mg (100%) foamy amorphous solid.

  Other gallotannin variants containing sugar nuclei other than glucose (e.g., galactose, mannose, trehalose, maltose, cellobiose, inositol, and glucitol) except that the xylose added in step 3 is replaced with another sugar. Generate as above.

  3) Replacing the ester bond between gallic acid and glucose with an ether bond

  The first three steps of this synthesis consist of literature procedures (E. Eich, H. Pertz, M. Kaloga, J. Schulz, MR Fesen, A. Mazumder, Y. Pommier, (-)-Arcigenin. as a Lead Structure for Inhibitors of Human Immunofidelity Virus Type-1 Integrate, J. Med. Chem. 1996, 39, 86-95).

  Subsequent steps are similar to the standard benzyl protection / deprotection chemistry of carbohydrates. The final hydrogenolysis is much faster for phenolic benzyl groups than for trihydroxybenzyl groups with carbohydrate attached. The reduced sensitivity of the ether bond to acid hydrolysis is expected to increase the stability (half-life) of the molecule and therefore increase the duration of action and overall apparent activity in vivo.

  The following examples are for illustrative purposes only and are not intended to limit the scope of the appended claims. All references cited herein are specifically incorporated herein in their entirety.

Example 1: Stimulation of glucose uptake in cells by penta-O-galloyl-D-glucose (PGG)
A 50:50 mixture of α-PGG and β-PGG was synthesized as described above. The α and β anomers were separated as described above. The glucose transport stimulating activity of these two anomers was compared with the glucose transport stimulating activity of β-PGG derived from a standard plant. Chemically synthesized PGG and standard plant-derived PGG are spectrally identical.

3T3-L1 adipocytes were purchased and maintained from ATCC and recognized as preadipocytes in DMEM supplemented with 10% calf serum in a 37 ° C. incubator containing 10% CO ₂ as required by the cells. It was. The cells are referred to as Liu, F. et al. Kim, J .; Li, Y .; Liu, X .; Li, J .; & Chen, X .; (2001) were induced to differentiate into adipocytes by the addition of an MDI induction cocktail. Lagerstromia speciosa L. Extract has insulin-like glucose uptake stimulating activity and adipocyte differentiation inhibiting activity in 3T3-L1 cells. J. et al. Nutrition 131: 2242-224 is specifically incorporated herein by reference. Various amounts of α-PGG and β-PGG are then added to the medium and the glucose intake by control and treated cells is used using standard glucose uptake assays as described in Liu et al. Decided. As shown in FIG. 2A, chemically synthesized β-PGG and plant-derived β-PGG have similar glucose transport stimuli (GTS). The GTS activity of α-PGG is consistently 10-20% higher than that of β-PGG (FIG. 2).

To investigate the mechanism by which PGG induces GTS activity, ¹⁴ C-labeled PGG was synthesized and used in cell studies. To determine whether PGG acts on the cell membrane or intracellularly, radioactive PGG was incubated with 3T3-L1 adipocytes at either 37 ° C or 4 ° C for various times. After incubation, excess radioactive PGG was removed by washing and cells were collected and sorted into cell membrane and intracellular fractions using published protocols. Radioactivity greater than 90-95% is associated with the membrane fraction, suggesting that PGG interacts with 3T3-L1 cells by binding to the cell membrane.

Ligand displacement studies were performed using 3T3-L1 cells incubated overnight at 4 ° C. with ¹⁴ C-PGG and increasing amounts of unlabeled PGG. Radioactive insulin was used as a receptor binding control. In the concentration range tested, PGG appeared to function as a ligand that binds to the target with an apparent receptor binding constant (Kd) of approximately 10 ⁻⁵ M (FIG. 3). It is noteworthy that the estimated Kd is very similar to the EC50 of PGG for both GTS and AD activity (FIG. 2). Since PGG has insulin-like GTS activity and seems to induce GTS activity in the same manner as insulin, it is speculated that PGG induces GTS activity in 3T3-L1 cells by binding to insulin receptor It was done.

  To determine whether PGG and insulin have a common target, 3T3-L1 cells are combined with a constant concentration of radioactive insulin plus increasing amounts of cold PGG or with increasing amounts of cold insulin containing radioactive PGG. Incubated. The results of the binding assay show that PGG does not compete with insulin for the insulin binding site located on the IR (FIG. 4). Conversely, insulin was also not found to compete with PGG for PGG binding sites located on the cell membrane. Thus, PGG appears to bind to sites on the IR other than the insulin binding site, or membrane receptors other than IP.

  A competitive binding assay using radiolabeled bovine serum albumin (BSA) as a tracer was used to determine whether PGG selectively binds to the protein. The relative affinity of the three standard proteins for PGG differs from each other by at least 10-fold (FIG. 5), and the difference in binding affinity of PGG for gelatin and ovalbumin is over 100-fold. This indicates that the PGG-protein interaction has specificity and that PGG can selectively act on a single biochemical target such as IR.

Molecular targets of PGG were further identified using chemical inhibitors that selectively inhibit proteins / enzymes involved in the insulin-mediated signaling pathway. Three inhibitors were tested: Hydroxy-2-naphthalenylmethylphosphonic acid triacetoxymethyl ester (HNMPA- (AM) ₃ ) (Qiu, Z. et al. (2001) J. Biol), a chemical that inhibits IR tyrosine kinases. Chem. 276: 11988-11995); wortmannin, a compound that specifically inhibits PI-3K (Superstein, R. et al. (1989) Biochemistry 28: 5694-5701); and specific to GLUT4 mediated glucose transport Cytochalasin B (Tomiyama, K. et al. (1995) Biochem. Biophys. Res. Commu. 212: 263-269; Kletzien, RF et al. (1972) J. Biol Chem.247: 2964-2966). All three inhibitors completely inhibited GTS activity mediated by either insulin or PGG (FIG. 6). This suggests that all GTS activities mediated by PGG are via the insulin-mediated signal pathway and only via the insulin-mediated signal pathway. The fact that HNMPA- (AM) ₃ , an inhibitor of the primary enzyme (IR tyrosine kinase) in the insulin signaling pathway, also completely abolishes the GTS activity of PGG suggests that the molecular target of PGG is IR. The idea that PGG uses the insulin-mediated GTS pathway is further dictated by our Western blot analysis, which shows that Akt, an important protein kinase for this pathway (zz), is It is phosphorylated (FIG. 7).

(Example 2: Effect of PGG on adipogenesis)
3T3-L1 adipocytes were purchased and maintained from ATCC and recognized as preadipocytes in DMEM supplemented with 10% calf serum in a 37 ° C. incubator containing 10% CO ₂ as required by the cells. It was. In order to test the effect of PGG on adipogenesis, this preadipocyte was differentiated into a differentiation-inducing cocktail consisting of 3-isobutyl-1-methylxanthine, dexamethasone (MDI), 3-isobutyl-1-methylxanthine, dexamethasone (MD). And a cocktail consisting of and PGG; or incubated with MDI + PGG. When insulin in MDI was replaced by PGG, the new cocktail failed to induce preadipocyte differentiation (see FIG. 2B). These results indicate that PGG cannot replace insulin due to induction of preadipocyte differentiation into adipocytes.

  A cell proliferation assay was used to determine whether PGG inhibits adipocyte differentiation by blocking clonal growth. This assay showed that the first round of clonal expansion was not inhibited by either α-PGG or β-PGG. The second round of clonal expansion is partially inhibited by α-PGG and completely inhibited by β-PGG (FIG. 10). The basis for differences between anomers in clonal expansion and differentiation is not known. However, our results indicate that clonal growth inhibition or apoptosis (programmed cell death) in preadipocytes is not responsible for the observed differentiation inhibition. This is because α-PGG is associated with slightly limited inhibition of clonal growth and abrogating differentiation without significantly increased cell death compared to MDI-derived cells (data shown). ) Thus, it appears that the critical step of inhibition is at a different step from clonal propagation.

  In order to study the mechanism by which PGG inhibits 3T3-L1 preadipocyte differentiation, we utilized gene expression studies. This strategy makes it possible to determine whether PGG acts specifically on genes involved in the differentiation process. Furthermore, this gene expression study overcomes the difficulty of studying insulin receptors in preadipocytes that express 1/10 less IR on the cell surface (Moden-Moses et al. (1998) Biochem. J.333: 825-831). To examine the genes affected by PGG during AD, preadipocytes were incubated with a differentiation-inducing cocktail consisting of 3-isobutyl-1-methylxanthine, dexamethasone, and insulin; or with either MDI + PGG. After about 10 days, MDI induces distinct differentiation as a change from fibroblast-like preadipocytes to round fat vesicle-containing adipocytes (FIG. 8, middle). In contrast, preadipocytes treated with MDI + PGG retain their fibroblast-like morphology and remain free of fat vesicles (FIG. 8, right).

  Cells treated at various times were collected, total RNA was isolated and expression was analyzed by Northern blot using probes complementary to various genes involved in this differentiation process. Northern blot analysis revealed that the expression of genes PPAR-γ, c / EBP-α required for differentiation and induced by MDI was completely abolished by PGG (FIG. 9). The relatively consistent β-actin level in differentially treated cells indicates that other cellular processes such as β-actin expression are not significantly affected by PGG (FIG. 9).

(Example 3: Effect of PGG in lowering blood glucose level in diabetic animals)
To determine whether α-PGG can exhibit anti-diabetic activity in vivo, α-PGG in aqueous form was administered orally to fasting diabetic 8-week-old male db / db mice. Delivered to. A single dose of α-PGG at a concentration of 25 mg / kg body weight significantly increased blood glucose levels in db / db mice compared to db / db mice given vehicle (identical aqueous solution without α-PGG). Was found to decrease (Figure 11A). The decrease in glucose level is approximately 15-20%, depending on the time after α-PGG administration (P <0.01, FIG. 11A).

  To determine if α-PGG is also effective in improving glucose tolerance in diabetic and obese mice, a glucose tolerance test was performed using ob / ob mice. Glucose, or glucose + α-PGG, was delivered orally to male ob / ob mice, and blood glucose levels were measured at various times after glucose / PGG administration. Mice receiving glucose + α-PGG have significantly lower blood glucose levels compared to mice treated with glucose alone (P <0.001, FIG. 11B). Interestingly, improved blood glucose levels can still be observed 24 hours after α-PGG treatment, indicating that the effect of α-PGG is persistent.

  A single large dose of glucose in ob / ob mice is either because of very high blood glucose levels around 24 hours after glucose challenge, or abnormally low glucose levels 2-3 days after glucose challenge. Can be lethal. We not only protected α / PGG from ob / ob mice from extremely high glucose levels immediately after glucose challenge (FIG. 11B), but also these mice at extremely low glucose levels after 2-5 days. (Fig. 12). The protection mechanism is currently unknown, but it is likely that PGG protected the ob / ob mice not only by its GTS activity but also by some other activity such as AD-related activity. No toxic effects associated with PGG were observed in these mice.

  These animal studies clearly show that α-PGG not only works in 3T3-L1 cells but is also effective in an in vivo diabetes / obesity model.

(Example 4: Effect of modified gallotannin on glucose uptake by cells)
We tested several polyphenolic polymer classes for GTS in the 3T3-L1 cell line (Table 1). Banaba extract and gallotannin preparation (mixture of tannic acid, galloyl glucose; and purified PGG) had high activity. Proanthocyanidins have limited activity, and tea catechin EGCG has no activity. This supports the idea that certain protein-PGG interactions are responsible for GTS activity. This is because only PGG and closely related compounds were active. If GTS activity is simply the result of general protein binding by polyphenol polymers, flavonoid-based proanthocyanidins and tea catechin preparations are active.

  Difference in reproducibility in GTS activity and AD activity between α-PGG and β-PGG (FIG. 2), and difference in reproducibility in GTS activity and AD activity between PGG and hexa-GG (Table 1) Was obtained as specified. The orientation of gallic acid at the sugar carbon 1 is responsible for the observed difference in activity between α-PGG and β-PGG. Pentagalloylgalactose (PGGal), a modified gallotannin of the present invention, and pentagalloyldeoxynojirimycin (PG-DJM) (FIG. 13) were synthesized. We have found that PGGal is 60-70% of the activity of PGG, but PG-DJM has no detectable GTS activity.

Example 5: Effect of PGG on plasma insulin levels
Diabetic and obese ob / ob mice were injected intraperitoneally with either water or α-PGG. Plasma from each mouse was isolated at various times after injection and measured for insulin levels. As shown in FIG. 15, ob / ob diabetic and obese mice treated with a single injection of α-PGG have lower plasma insulin levels than ob / ob diabetic mice treated with water alone (negative control). did. Based on these results and the effect of PGG on glucose uptake, it is believed that PGG can enhance the glucose transport stimulating activity of insulin in vivo. Thus, it is expected that PGG can be used for therapy to improve insulin resistance in mammalian subjects.

Example 6: Pentax-O- (3,4-dihydroxybenzoyl) -β-D-glucopyranose and tetrakis-O- (3,4,5, trihydroxybenzoyl) -α-D- in glucose uptake by adipocytes Effect of xylopyranose)
Confluent 3T3-L1 adipocytes grown in 12-well plates were washed twice with serum-free DMEM and incubated for 2 hours at 37 ° C. with 1 mL of the same medium. The cells were washed 3 times with Krebs-Ringer-Hepes (KRP) buffer and incubated with 0.9 mL KRP buffer at 37 ° C. for 30 minutes. The compounds listed in the table below were then added at a concentration of 20-40 μM (final concentration?) And the adipocytes were incubated at 37 ° C. for 15 minutes. Glucose uptake, 0.1 mL KRP buffer, and 37 MBq / L ^2-deoxy-D-[3 H] glucose, and were initiated by the addition of glucose 1 mmol / L as a final concentration. After 10 minutes, glucose uptake was terminated by washing the cells three times with cold PBS. The cells were lysed with 0.7 mL of 1% Triton X-100 at 37 ° C. for 20 minutes. Radioactivity retained by cell lysates was determined by scintillation counter. As shown in the table, pentakis-O- (3,4 dihydroxybenzoyl) -β-D-glucopyranose and tetrakis-O- (3,4,5, trihydroxybenzoyl-) α- which are gallotannin variants D-xylopyranose increased glucose uptake by cells.

(Example 7: PGG does not cause hyperglycemia)
Normal (healthy) mice were fed orally with 40 mg glucose at time zero. After 2 hours (120 minutes), if blood glucose is at a standard or basal level, mice are fed orally with either various concentrations of PGG or water (negative control) or insulin Were injected intraperitoneally. At various time intervals before and after administration of PGG or insulin, blood was collected from control and treated animals and blood glucose levels were determined. As shown in FIG. 15, insulin injection caused hyperglycemia in mice but not with PGG administration. Therefore, PGG lowers blood glucose levels when higher than normal. However, PGG does not lower blood glucose levels beyond normal or basal values.

FIG. 1 shows the structure of penta-O-galloyl-D-glucose (PGG). PGG consists of a glucose nucleus that is covalently bonded to five gallic acids via ester bonds. There are two anomers of PGG for the two possible configurations of glucose at carbon 1 ( ^* ). The computer-simulated PGG conformation shows that α-PGG can be more symmetric (and therefore less polar) than β-PGG. FIG. 2 shows the glucose transport stimulating activity and antilipogenic activity of PGG. PGG (either mixture or single isomer (α or β)) was added to either adipocytes (A) or preadipocytes (B). The GTS activity (A) and AD activity (B) of PGG were measured by ³ H-glucose intake. GLUT4 is not expressed in preadipocytes, so glucose uptake can be used as an indirect measure of adipocyte differentiation. FIG. 3 shows that PGG binds to IR with a Kd of about 10 ⁻⁵ M. Each 3T3-L1 adipocyte in a 24-well plate was increased in cold PGG concentration in the presence of 8 pM ¹²⁵ I-insulin or in the presence of 20 μM ¹⁴ C-PGG, respectively. Incubate overnight at 4 ° C. The isotope that did not bind to the cells was then removed and then measured for ¹²⁵ I-insulin or ¹⁴ C-PGG bound to the cells. FIG. 4 shows that PGG does not displace insulin bound to insulin receptor IR in 3T3-L1 adipocytes. 3T3-L1 adipocytes were incubated overnight at 4 ° C. with increasing concentrations of ¹⁴ C-PGG in the presence of 8 pM ¹²⁵ I-insulin, and then ¹²⁵ I-insulin and ¹⁴ C-bound to the cells. Counted for PGG. An approximately constant number of insulin between 0.1 μM and 20 μM PGG indicates that PGG cannot displace insulin from IR in this concentration range. Insulin binding increased at higher PGG concentrations. FIG. 5 shows the protein binding selectivity of PGG. A fixed amount of ¹²⁵ I BSA was incubated with PGG in the presence of varying amounts of cold gelatin or either BSA or ovalbumin. Bound was separated from unbound by precipitation and expressed as% binding in the absence of competitor. Three competition curves indicate that PGG selectivity binds to various proteins with significantly different affinities. FIG. 6 shows that three insulin signaling pathway specific inhibitors also abolish PGG-induced glucose transport in 3T3-L1 adipocytes. Adipocytes were induced by insulin or PGG in the presence or absence of various inhibitors. The glucose transport activity of the treated cells was measured by ³ H glucose uptake by the cells. HNMPA- (AM) 3 inhibits IR Tyr kinase activity, cytochalasin B inhibits GLUT4, and wortmannin inhibits PI-3K, respectively. FIG. 7 shows that PGG induces phosphorylation of Akt in 3T3-L1 adipocytes. Differentially treated adipocytes were lysed and cellular proteins were analyzed by SDS-PAGE. 1 = no treatment; 2 = insulin; 3 = pGG, 15 μM; 4 = PGG, 30 μM; M = protein size marker. FIG. 8 shows oil red O staining of 3T3-L1 cells induced by MDI or insulin and 30 μM PGG. Ten days after induction, the cells were stained with Oil Red O and photographed at a magnification of × 200. Only cells that contain fat vesicles (triglycerides) can be stained. FIG. 9 shows Northern blot analysis of PPARγ gene expression and C / EBPα gene expression in 3T3-L1 preadipocytes. Differentially treated preadipocytes were analyzed for their mRNA expression at various times after treatment. Lane 1 = no treatment; Lane 2 = MDI; Lane 3 = 30 μM PGG + MDI; Lane 4 = 30 μM PGG alone. Time = time after treatment when mRNA was isolated. FIG. 10 shows clonal expansion in α-PGG and β-PGG treated with 3T3-L1 preadipocytes. Preadipocytes were induced for clonal expansion to occur with MDI, or α-PGG, or β-PGG in the presence of MDI. At 24 or 48 hours after induction, the media was removed and the cells were lysed. The lactose dehydrogenase (LDH) activity of the cell lysate was measured by the LDH kit. Cell growth media was also collected and their LDH activity from dead cells was also measured (not shown in this graph). LDH activity is constant per cell in a given cell type, and the measured LDH activity is proportional to the number of cells in the sample. FIG. 11 shows the effect of PGG on blood glucose levels in db / db and ob / ob mice. Various doses of α-PGG were delivered orally without glucose for db / db mice (A) or with glucose for ob / ob mice (B). At various times after delivery, glucose in the sample was determined from tail blood. FIG. 12 shows that PGG protects ob / ob mice from hyperglycemia immediately after the glucose challenge and from hyperglycemia a few days after the challenge. Ob / ob mice were tested for glucose tolerance as shown in FIG. 11B. Blood glucose levels in the tail blood were measured at various times after the glucose challenge. FIG. 13 shows the chemical structure of a selective gallotannin variant. G represents trihydroxybenzoic acid. FIG. 14 shows the effect of PGG on plasma insulin levels in ob / ob mice. FIG. 15 shows the effect of PGG on blood glucose levels in mice with normal blood glucose levels.

Claims (24)

A method for treating or preventing diabetes in a mammalian subject, the method comprising:
Administering a therapeutically effective amount of a gallotannin composition to a subject in need of the composition;
Here, the gallotannin composition comprises 1,2,3,4-tetra-O-galloyl-α-D-glucose, 1,2,3,6-tetra-O-galloyl-α-D-glucose, , 3,4,6-Tetra-O-galloyl-α-D-glucose, 1,2,3,4,6-penta-O-galloyl-α-D-glucose, 1,2,3,4,6 -Penta-O-galloyl-β-D-glucose, 1,2,3,4,6-hexa-galloyl-α-D-glucose, 1,2,3,4,6-hexa-O-galloyl-β -D-glucose, 1,2,3,4,6-hepta-O-galloyl-α-D-glucose, 1,2,3,4,6-hepta-O-galloyl-β-D-glucose, , 2,3,4,6-octa-O-galloyl-α-D-glucose, 1,2,3,4,6-octa-O-gallo -Β-D-glucose, 1,2,3,4,6-nona-O-galloyl-α-D-glucose, 1,2,3,4,6-nona-O-galloyl-β-D- Glucose, 1,2,3,4,6-deca-O-galloyl-α-D-glucose, and 1,2,3,4,6-deca-O-galloyl-β-D-glucose, or these One or more hydrolyzable gallotannins selected from the group consisting of salts, and the gallotannin composition comprises the following compounds: mono-O-galloyl-β-D-glucose, di-O-galloyl-β- D-glucose, tri-O-galloyl-β-D-glucose, tetra-O-galloyl-β-D-glucose, undeca-O-galloyl-β-D-glucose, dodeca-O-galloyl-β-D- One or more of glucose or a mixture thereof , Containing less than 5% by dry weight, the composition. 2. The method of claim 1, wherein the gallotannin composition comprises at least 50% by dry weight of 1,2,3,4,6-penta-O-galloyl-α-D-glucose, or 1,2, 3,4,6-penta-O-galloyl-β-D-glucose or 1,2,3,4,6-penta-O-galloyl-α-D-glucose and 1,2,3,4,6 -A method comprising a combination with penta-O-galloyl-β-D-glucose. The method of claim 1, wherein the gallotannin composition contains at least 50% by dry weight of 1,2,3,4,6-penta-O-galloyl-α-D-glucose. The method of claim 1, wherein the gallotannin composition contains at least 50% by dry weight of 1,2,3,4,6-penta-O-galloyl-β-D-glucose. 2. The method of claim 1, wherein the gallotannin composition is administered to the subject orally or by injection. 2. The method of claim 1, further comprising administering insulin to the subject. A method for treating or preventing diabetes in a subject comprising the following:
Administering a gallotannin modified composition to the subject,
Here, the gallotannin modifying composition contains one or more gallotannin modifying compounds or salts thereof, and each of the gallotannin modifying compounds is:

Having the structure of
Here, R is D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose, L-altrose, D- Growth, L-growth, D-idose, L-idose, D-talose, L-talose, D-fructose, L-fructose, α-D-xylose, α-D lyxose, β-D lyxose, α-D arabinose , Β-D arabinose, α-D ribose, β-D ribose, D-trehalose, D-maltose, D-cellobiose, myo-inositol, D-glucitol,
X is an ester bond or an ether bond,
A is trihydroxybenzoic acid selected from the group consisting of 3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid, Or dihydroxybenzoic acid selected from the group consisting of 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, or 3-hydroxybenzoic acid and 4-hydroxybenzoic acid Monohydroxybenzoic acid selected from the group of
R is D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose, L-altrose, D-gulose, L- In the case of growth, D-idose, L-idose, D-talose, L-talose, D-fructose, L-fructose, n is 5 and q is 0, 1, 2, 3, 4, or 5 And z is 0;
When R is α-D-xylose, α-D lyxose, β-D lyxose, α-D arabinose, β-D arabinose, α-D ribose, β-D ribose, n is 4 and q is Is 0, 1, 2, 3, or 4 and z is 0, 1, or 2;
When R is D-glucitol or myo-inositol, n is 6, q is 0, 1, 2, 3, 4, 5, or 6 and z is 0; and R is D When trehalose, D-maltose, or D-cellobiose, n is 8, q is 0, 1, 2, 3, 4, 5, 6, 7, or 8 and z is 0 There is a way. 8. The method of claim 7, wherein X is an ether bond. 8. The method according to claim 7, wherein R is L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose, L-alt. Loin, D-gulose, L-gulose, D-idose, L-idose, D-talose, L-talose, D-fructose, L-fructose, α-D-xylose, α-D lyxose, β-D lyxose, A method, which is α-D arabinose, β-D arabinose, α-D ribose, β-D ribose, D-trehalose, D-maltose, D-cellobiose, myo-inositol, or D-glucitol. 5. The method of claim 4, wherein A is trihydroxybenzoic acid selected from the group consisting of 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid, Or dihydroxybenzoic acid selected from the group consisting of 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, or 3-hydroxybenzoic acid and 4-hydroxybenzoic acid A process which is a monohydroxybenzoic acid selected from the group consisting of: 5. The method of claim 4, wherein each gallotannin modifying compound is tetra-O-galloyl-β-D-glucose, 1,2,3,4,6-penta-O-galloyl-β-D. Glucose, 1,2,3,4,6-hexa-O-galloyl-β-D-glucose, 1,2,3,4,6-hepta-O-galloyl-β-D-glucose, 1,2 , 3,4,6-octa-O-galloyl-β-D-glucose, 1,2,3,4,6-nona-O-galloyl-β-D-glucose, and 1,2,3,4, A method having a structure other than that of 6-deca-O-galloyl-β-D-glucose. In a subject in need of decreasing increased blood glucose level, a method of decreasing increased blood glucose level without causing hypoglycemia in the subject, the method comprising the following compositions (a) and ( b) administering to the subject one or both of:
Here, the composition (a) is a gallotannin composition, which is 1,2,3,4-tetra-O-galloyl-α-D-glucose, 1,2,3,6-tetra-O-galloyl. -Α-D-glucose, 1,3,4,6-tetra-O-galloyl-α-D-glucose, 1,2,3,4,6-penta-O-galloyl-α-D-glucose, 1 , 2,3,4,6-penta-O-galloyl-β-D-glucose, 1,2,3,4,6-hexa-galloyl-α-D-glucose, 1,2,3,4,6 Hexa-O-galloyl-β-D-glucose, 1,2,3,4,6-hepta-O-galloyl-α-D-glucose, 1,2,3,4,6-hepta-O-galloyl -Β-D-glucose, 1,2,3,4,6-octa-O-galloyl-α-D-glucose, 1,2,3,4, -Octa-O-galloyl-β-D-glucose, 1,2,3,4,6-nona-O-galloyl-α-D-glucose, 1,2,3,4,6-nona-O-galloyl -Β-D-glucose, 1,2,3,4,6-deca-O-galloyl-α-D-glucose, and 1,2,3,4,6-deca-O-galloyl-β-D- Glucose, or one or more hydrolyzable gallotannins selected from the group consisting of pharmaceutically acceptable salts thereof, and the gallotannin composition comprises the following compound: mono-O-galloyl-β-D -Glucose, di-O-galloyl-β-D-glucose, tri-O-galloyl-β-D-glucose, tetra-O-galloyl-β-D-glucose, undeca-O-galloyl-β-D-glucose , Dodeca-O-galloyl-β-D-glu One or more of over scan, or mixtures thereof, containing less than 5% by dry weight, a gallotannins composition, and the composition (b) is a gallotannin modifying composition comprising:

One or more compounds having the structure: or a pharmaceutically acceptable salt thereof,
Here, R is D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose, L-altrose, D- Growth, L-growth, D-idose, L-idose, D-talose, L-talose, D-fructose, L-fructose, α-D-xylose, α-D lyxose, β-D lyxose, α-D arabinose , Β-D arabinose, α-D ribose, β-D ribose, D-trehalose, D-maltose, D-cellobiose, myo-inositol, D-glucitol,
X is an ester bond or an ether bond,
A is trihydroxybenzoic acid selected from the group consisting of 3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid, Or dihydroxybenzoic acid selected from the group consisting of 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, or 3-hydroxybenzoic acid and 4-hydroxybenzoic acid Monohydroxybenzoic acid selected from the group of
R is D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose, L-altrose, D-gulose, L- In the case of growth, D-idose, L-idose, D-talose, L-talose, D-fructose, L-fructose, n is 5 and q is 0, 1, 2, 3, 4, or 5 And z is 0;
When R is α-D-xylose, α-D lyxose, β-D lyxose, α-D arabinose, β-D arabinose, α-D ribose, β-D ribose, n is 4 and q is Is 0, 1, 2, 3, or 4 and z is 0, 1, or 2;
When R is D-glucitol or myo-inositol, n is 6, q is 0, 1, 2, 3, 4, 5, or 6 and z is 0; and R is D When trehalose, D-maltose, or D-cellobiose, n is 8, q is 0, 1, 2, 3, 4, 5, 6, 7, or 8 and z is 0 A method, which is a modified gallotannin composition. A method of treating a subject exhibiting one or more of obesity, type II diabetes, glucose intolerance, insulin resistance, hyperglycemia, or hyperinsulinemia, the method comprising:
Administering a gallotannin composition to the subject,
Here, the gallotannin composition comprises 1,2,3,4-tetra-O-galloyl-α-D-glucose, 1,2,3,6-tetra-O-galloyl-α-D-glucose, , 3,4,6-Tetra-O-galloyl-α-D-glucose, 1,2,3,4,6-penta-O-galloyl-α-D-glucose, 1,2,3,4,6 -Penta-O-galloyl-β-D-glucose, 1,2,3,4,6-hexa-galloyl-α-D-glucose, 1,2,3,4,6-hexa-O-galloyl-β -D-glucose, 1,2,3,4,6-hepta-O-galloyl-α-D-glucose, 1,2,3,4,6-hepta-O-galloyl-β-D-glucose, , 2,3,4,6-octa-O-galloyl-α-D-glucose, 1,2,3,4,6-octa-O-gallo -Β-D-glucose, 1,2,3,4,6-nona-O-galloyl-α-D-glucose, 1,2,3,4,6-nona-O-galloyl-β-D- Glucose, 1,2,3,4,6-deca-O-galloyl-α-D-glucose, and 1,2,3,4,6-deca-O-galloyl-β-D-glucose, or these One or more hydrolyzable gallotannins selected from the group consisting of salts, and the gallotannin composition comprises the following compounds: mono-O-galloyl-β-D-glucose, di-O-galloyl-β- D-glucose, tri-O-galloyl-β-D-glucose, tetra-O-galloyl-β-D-glucose, undeca-O-galloyl-β-D-glucose, dodeca-O-galloyl-β-D- One or more of glucose or a mixture thereof , Containing less than 5% by dry weight method. 14. The method of claim 13, wherein administration of the gallotannin composition inhibits differentiation of preadipocytes into adipocytes in the subject. 14. The method of claim 13, wherein the gallotannin composition is administered to the subject orally or by injection. A method of inhibiting the differentiation of preadipocytes into adipocytes in vitro or in vivo, comprising:
Contacting the preadipocyte and the gallotannin composition,
Here, the gallotannin composition comprises 1,2,3,4-tetra-O-galloyl-α-D-glucose, 1,2,3,6-tetra-O-galloyl-α-D-glucose, , 3,4,6-Tetra-O-galloyl-α-D-glucose, 1,2,3,4,6-penta-O-galloyl-α-D-glucose, 1,2,3,4,6 -Penta-O-galloyl-β-D-glucose, 1,2,3,4,6-hexa-galloyl-α-D-glucose, 1,2,3,4,6-hexa-O-galloyl-β -D-glucose, 1,2,3,4,6-hepta-O-galloyl-α-D-glucose, 1,2,3,4,6-hepta-O-galloyl-β-D-glucose, , 2,3,4,6-octa-O-galloyl-α-D-glucose, 1,2,3,4,6-octa-O-gallo -Β-D-glucose, 1,2,3,4,6-nona-O-galloyl-α-D-glucose, 1,2,3,4,6-nona-O-galloyl-β-D- Glucose, 1,2,3,4,6-deca-O-galloyl-α-D-glucose, and 1,2,3,4,6-deca-O-galloyl-β-D-glucose, or these A method comprising one or more hydrolyzable gallotannins selected from the group consisting of salts. 17. The method of claim 16, wherein the gallotannin composition comprises the following compounds: mono-O-galloyl-β-D-glucose, di-O-galloyl-β-D-glucose, tri-O-galloyl. One or more of β-D-glucose, tetra-O-galloyl-β-D-glucose, undeca-O-galloyl-β-D-glucose, dodeca-O-galloyl-β-D-glucose or mixtures thereof Containing less than 5% by dry weight. 17. The method of claim 16, wherein the gallotannin composition comprises at least 50% by dry weight of 1,2,3,4,6-penta-O-galloyl-α-D-glucose, or 1,2, 3,4,6-penta-O-galloyl-β-D-glucose or 1,2,3,4,6-penta-O-galloyl-α-D-glucose and 1,2,3,4,6 -A method comprising a combination with penta-O-galloyl-β-D-glucose. 17. The method of claim 16, wherein the gallotannin composition contains at least 50% by dry weight of 1,2,3,4,6-penta-O-galloyl-β-D-glucose. A method of inhibiting the differentiation of preadipocytes into adipocytes in vitro or in vivo, comprising:
Following preadipocytes:

Contacting one or more compounds having the structure: a composition containing these salts,
Here, R is D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose, L-altrose, D- Growth, L-growth, D-idose, L-idose, D-talose, L-talose, D-fructose, L-fructose, α-D-xylose, α-D lyxose, β-D lyxose, α-D arabinose , Β-D arabinose, α-D ribose, β-D ribose, D-trehalose, D-maltose, D-cellobiose, myo-inositol, D-glucitol,
X is an ester bond or an ether bond,
A is trihydroxybenzoic acid selected from the group consisting of 3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid, Or dihydroxybenzoic acid selected from the group consisting of 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, or 3-hydroxybenzoic acid and 4-hydroxybenzoic acid Monohydroxybenzoic acid selected from the group of
R is D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose, L-altrose, D-gulose, L- In the case of growth, D-idose, L-idose, D-talose, L-talose, D-fructose, L-fructose, n is 5 and q is 0, 1, 2, 3, 4, or 5 And z is 0;
When R is α-D-xylose, α-D lyxose, β-D lyxose, α-D arabinose, β-D arabinose, α-D ribose, β-D ribose, n is 4 and q is Is 0, 1, 2, 3, or 4 and z is 0, 1, or 2;
When R is D-glucitol or myo-inositol, n is 6, q is 0, 1, 2, 3, 4, 5, or 6 and z is 0; and R is D When trehalose, D-maltose, or D-cellobiose, n is 8, q is 0, 1, 2, 3, 4, 5, 6, 7, or 8 and z is 0 There is a way. A gallotannin modifying composition, wherein the gallotannin modifying composition contains one or more gallotannin modifying compounds, or pharmaceutically acceptable salts thereof, wherein each of the gallotannin modifying compounds is:

Having the structure of
Here, R is D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose, L-altrose, D- Growth, L-growth, D-idose, L-idose, D-talose, L-talose, D-fructose, L-fructose, α-D-xylose, α-D lyxose, β-D lyxose, α-D arabinose , Β-D arabinose, α-D ribose, β-D ribose, D-trehalose, D-maltose, D-cellobiose, myo-inositol, D-glucitol,
X is an ester bond or an ether bond,
A is trihydroxybenzoic acid selected from the group consisting of 3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid, Or dihydroxybenzoic acid selected from the group consisting of 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, or 3-hydroxybenzoic acid and 4-hydroxybenzoic acid Monohydroxybenzoic acid selected from the group of
R is D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose, L-altrose, D-gulose, L- In the case of growth, D-idose, L-idose, D-talose, L-talose, D-fructose, L-fructose, n is 5 and q is 0, 1, 2, 3, 4, or 5 And z is 0;
When R is α-D-xylose, α-D lyxose, β-D lyxose, α-D arabinose, β-D arabinose, α-D ribose, β-D ribose, n is 4 and q is Is 0, 1, 2, 3, or 4 and z is 0, 1, or 2;
When R is D-glucitol or myo-inositol, n is 6, q is 0, 1, 2, 3, 4, 5, or 6 and z is 0; and R is D When trehalose, D-maltose, or D-cellobiose, n is 8, q is 0, 1, 2, 3, 4, 5, 6, 7, or 8 and z is 0 Yes,
And each said gallotannin modification compound is tetra-O-galloyl-D-glucose, penta-O-galloyl-D-glucose, hexa-galloyl-α-D-glucose, hepta-O-galloyl-D-glucose, A modified gallotannin composition that is not the beta anomeric form of octa-O-galloyl-D-glucose, nona-O-galloyl-D-glucose, or deca-O-galloyl-D-glucose. 22. The composition of claim 21, wherein the composition comprises pentakis-O- (3,4-dihydroxybenzoyl) -β-D-glucopyranose, or tetrakis-O- (3,4,5-tri A composition comprising hydroxybenzoyl) -α-D-xylopyranose. A compound for treating diabetes, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia or obesity in a subject, or a pharmaceutically acceptable salt of the compound, the compound comprising: Less than:

Having the structure of
Here, R is D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose, L-altrose, D- Growth, L-growth, D-idose, L-idose, D-talose, L-talose, D-fructose, L-fructose, α-D-xylose, α-D lyxose, β-D lyxose, α-D arabinose , Β-D arabinose, α-D ribose, β-D ribose, D-trehalose, D-maltose, D-cellobiose, myo-inositol, D-glucitol,
X is an ester bond or an ether bond,
A is trihydroxybenzoic acid selected from the group consisting of 3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid, Or dihydroxybenzoic acid selected from the group consisting of 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, or 3-hydroxybenzoic acid and 4-hydroxybenzoic acid Monohydroxybenzoic acid selected from the group of
R is D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose, L-altrose, D-gulose, L- In the case of growth, D-idose, L-idose, D-talose, L-talose, D-fructose, L-fructose, n is 5 and q is 0, 1, 2, 3, 4, or 5 And z is 0;
When R is α-D-xylose, α-D lyxose, β-D lyxose, α-D arabinose, β-D arabinose, α-D ribose, β-D ribose, n is 4 and q is Is 0, 1, 2, 3, or 4 and z is 0, 1, or 2;
When R is D-glucitol or myo-inositol, n is 6, q is 0, 1, 2, 3, 4, 5, or 6 and z is 0; and R is D When trehalose, D-maltose, or D-cellobiose, n is 8, q is 0, 1, 2, 3, 4, 5, 6, 7, or 8 and z is 0 A compound. 24. A pharmaceutical composition for treating insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia or obesity in a subject, said pharmaceutical composition being an effective amount of claim 23. Or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.