CN1668311A - Method and composition for treating diabetes - Google Patents

Abstract

Methods for modulating diabetes, impaired glucose tolerance, gestational diabetes and glucose resistance in a mammal, particularly a human. In one embodiment the method comprises administering a gallotannin composition to a mammal in need of the same. The gallotannin composition comprises one or more select hydrolysable gallotannins. In another embodiment the method comprises administering a gallotannin variant composition comprising one ore more select gallotannin variant compounds to the subject. Methods of preventing or treating weight gain in a subject. The method comprises administering the gallotannin composition of the present invention, the gallotannin variant composition of the present invention, or a combination of the gallotannin composition of the present invention and the gallotannin variant composition of the present invention to the subject. The present invention also relates a gallotannin variant compound or a salt thereof, and a pharmaceutical composition comprising such compound or the salt thereof.

Description

Methods and compositions for treating diabetes

field of invention

The present invention relates to methods and compositions for modulating diabetes and other diseases associated with abnormal glucose and/or insulin levels in mammals. The methods use compositions that do not induce adipogenesis or hypoglycemia.

Background technique

Diabetes mellitus, commonly called diabetes, refers to a multifactorial disease process characterized by elevated levels of plasma glucose, known as hyperglycemia. See, eg, LeRoith, D. et al. (eds.), DIABETES MELLITUS (Lippincott-Raven Publishers, Philadelphia, Pa. U.S.A., 1996), and all references cited therein. According to the American Diabetes Association, diabetes is estimated to affect approximately 6% of humans worldwide. Uncontrolled hyperglycemia is associated with increased and premature mortality due to an increased risk of microvascular and macrovascular disease, including nephropathy, neuropathy, retinopathy, hypertension, cerebrovascular disease, and coronary heart disease. Therefore, control of glucose homeostasis is an important approach in the treatment of diabetes.

There are two main forms of diabetes, type 1 diabetes (formerly known as insulin-dependent diabetes or IDDM) and type 2 diabetes (formerly known as non-insulin-dependent diabetes or NIDDM). Type 1 diabetes is caused by a complete lack of insulin, the hormone that regulates glucose utilization. Insulin deficiency is usually characterized by the destruction of islet β-cells in the pancreas, with complete insulin deficiency. Type 2 diabetes is a disease characterized by insulin resistance with relative, rather than absolute, insulin deficiency. Type 2 diabetes can range from prominent insulin resistance with relative insulin deficiency, to prominent insulin deficiency with some insulin resistance. Insulin resistance is the reduced ability of insulin to exert its biological activity over a broad concentration range. In insulin resistant individuals, the body secretes abnormally high amounts of insulin to compensate for this deficiency. A state of impaired glucose tolerance occurs when insufficient amounts of insulin are present to compensate for insulin resistance and adequately control glucose. In a large number of individuals, insulin secretion further decreases and plasma glucose levels rise, leading to clinical diabetes.

Most patients with type 2 diabetes are treated with hypoglycemic drugs that work by stimulating the release of insulin from beta cells, or with drugs that increase the patient's tissue sensitivity to insulin, or insulin. An example is sulfonylureas, which stimulate the release of insulin from beta cells. Metformin is a representative example of drugs that enhance tissue sensitivity of patients to insulin. Even though sulfonylureas are widely used in the treatment of type 2 diabetes, their treatment is unsatisfactory in most cases. In a large number of patients with type 2 diabetes, sulfonylureas are insufficient to normalize blood glucose levels, and patients are therefore at high risk of developing diabetic complications. Also, many patients gradually lose their response to sulfonylurea therapy and, thus, are gradually forced to insulin therapy. The transition of patients from oral hypoglycemic drug therapy to insulin therapy is often attributed to the depletion of pancreatic β-cells in type 2 diabetes.

Insulin stimulates glucose uptake by skeletal muscle and adipose tissue, which is translocated from intracellular storage sites to the cell surface primarily by glucose transporter 4 (GLUT4) (Saltiel, A.R. & Kahn, C.R. (2001) Nature 414:799-806; Saltiel , A. & Pessin, J.E. (2002) Trends in Cell Biol. 12: 65-71; White, M.F. (1998) Mol. Cell. Biochem. 182: 3-11). In response to insulin, GLUT4 components present in the inner cell membrane are redistributed to the plasma membrane, resulting in an increase of GLUT4 on the cell surface and enhanced glucose uptake by the cell. GLUT4 translocation is mainly mediated through the insulin receptor (IR).

In addition to glucose transport, insulin is closely related to adipogenesis, a process that includes the proliferation and differentiation of preadipocytes (pre-adipocytes) into adipocytes (adipocytes) as fat accumulates within the cell. Studies with the adipocyte cell line 3T3-L1 revealed that the role of insulin in adipogenesis is predominantly mitogenic (43). Before differentiation, 3T3-L1 cells are fibroblast-like preadipocytes that contain more IGF-1 receptors than IR. In vitro, adipogenesis of preadipocytes can be induced by a cocktail (MDI) commonly used to induce differentiation, which consists of a cAmp-raising agent methylisobutylxanthine (MIX); a glucocorticoid Dexamethasone (DEX); and insulin (or IGF-1) composition in response to IGF-1 receptors on preadipocytes (Tong, Q., Hotamisligil, G.S. (2001) Rev. in Endoc. & Metabolic Disorders.2 : 349-355; Rosen, E.D. et al., (2000) Genes Dev. 14: 1293-1307). When treated with MDI, confluent preadipocytes re-enter the cell cycle and undergo approximately 2 rounds of mitosis (Modan-Moses, D. et al. (1998) Biochem. J. 333:825-831; Tong, Q. Hotamisligil , G.S. (2001) Rev.in Endoc. & Metabolic Disorders.2: 349-355; Rosen, E.D., etc. (2000) Genes Dev. 14: 1293-1307), this process usually refers to clonal expansion. Following clonal expansion, preadipocytes exit the cell cycle and begin to differentiate into adipocytes by expressing adipocyte genes including C/EBP-α, β, δ, and PPAR-γ.

Due to its adipogenic effects, insulin has the adverse effect of promoting obesity in type 2 diabetic patients (see Moller, D.E. (2001) Nature 414:821-827). Unfortunately, other anti-diabetic drugs currently used to stimulate glucose transport in type 2 diabetic patients also have adipogenic activity. Thus, while current drug treatments can lower blood sugar, they often promote obesity. Therefore, it is highly desirable to develop a new anti-diabetic drug capable of correcting hyperglycemia without the attendant lipogenic side effects. Compounds capable of inducing glucose uptake in diabetic patients without causing hypoglycemia are also needed.

Contents of the invention

The present invention provides methods of modulating diabetes, impaired glucose tolerance, gestational diabetes and glucose resistance in mammals, especially humans. In one embodiment of the method comprising administering to a mammal in need thereof a composition, referred to herein as a "gallotannin composition". The gallotannin composition is substantially pure, comprising one or more hydrolyzable gallotannins selected from the group consisting of: 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-galloyl-β-D-glucose, 1,2,3,4,6-nona-O-galloyl-α-D-glucose, 1,2,3, 4,6-Nine-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. As used herein, the term "substantially pure" means that the gallotannin composition comprises at least 95% by dry weight of one or a combination of the listed gallotannins, and less than 5% by dry weight of one or more of the following Compounds: Mono-O-galloyl-β-D-glucose, Di-O-galloyl-β-D-glucose, Tris-O-galloyl-β-D-glucose, Tetra-O-galloyl-β-glucose D-glucose, undecyl-O-galloyl-β-D-glucose, dodecanoyl-O-galloyl-β-D-glucose, or mixtures thereof.

In another embodiment of the method comprising administering to the individual a composition, referred to herein as a "gallotannin variant composition". The modified gallotannin composition comprises one or more modified gallotannin compounds or salts thereof. The gallotannin variant composition has the following structure:

RXA( _n )-XA( _q )-XA( _z ),

Wherein R is selected from: D-glucose, L-glucose, D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-alpha Lulose, D-altrose, L-altrose, 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, α-D-arabinose, β-D-arabinose, α-D- Ribose, β-D-ribose, D-trehalose, D-maltose, D-cellobiose, inositol, D-glucitol,

X is an ester or ether linkage,

A is a 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: 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid; or monohydroxybenzoic acid, selected from 3-hydroxybenzoic acid and 4-hydroxybenzoic acid ,

where n is 5, q is 0, 1, 2, 3, 4 or 5, and, when 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-gulose, D-idose, L-idose , D-talose, L-talose, D-fructose, L-fructose, z is 0;

where n is 4, q is 0, 1, 2, 3 or 4, and, when R is: α-D-xylose, α-D-lyxose, β-D-lyxose, α-D -arabinose, β-D-arabinose, α-D-ribose, β-D-ribose, z is 0, 1 or 2.

wherein n is 6, q is 0, 1, 2, 3, 4, 5 or 6, and, when R is: D-glucitol or inositol, z is 0, and

wherein n is 8, q is 0, 1, 2, 3, 4, 5, 6, 7 or 8, and, when R is D-trehalose, D-maltose or D-cellobiose, z is 0 .

Each of the gallotannin variant compositions has a structure other than: 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-β-D-glucose.

In a third embodiment of the method comprising administering to a patient a combination of a gallotannin composition of the invention and a gallotannin variant composition of the invention.

The present invention also provides methods of preventing or treating weight gain in an individual. The method comprises administering to an individual a gallotannin composition of the invention, a gallotannin variant composition of the invention, or a combination of a gallotannin composition of the invention and a gallotannin variant composition of the invention.

The invention also provides methods of inhibiting the differentiation of preadipocytes into adipocytes. The method comprises combining pre-adipocytes with a natural gallotannin composition of the invention, a gallotannin variant composition of the invention, or a natural gallotannin composition of the invention and a gallotannin variant of the invention combination of objects. The pre-adipocytes may be in culture or in a mammalian subject.

The present invention also relates to the present gallotannin variant composition, the compound having the structure: RXA( _n )-XA( _q )-XA( _z ), or a salt thereof, and a pharmaceutical composition comprising the compound or a salt thereof.

Description of drawings

Figure 1. Structure of penta-O-galloyl-D-glucose (PGG).

PGG consists of a glucose core covalently linked to five gallic acids via ester bonds. With two possible conformations at carbon 1 ( ^* ) of glucose, two PGG anti-forms exist. Computer-simulated conformations of PGG show that α-PGG is more symmetrical (and thus less polar) than β-PGG.

Figure 2. Glucose transport stimulating activity and anti-adipogenic activity of PGG.

PGG, either as a mixture or as a single isomer (α or β), was added to adipocytes (A) or preadipocytes (B). GTS activity (A) and AD activity (B) of PGG measured by ³ H-glucose uptake. GLUT4 is not expressed in preadipocytes, so glucose uptake can be used as an indirect measure of adipocyte differentiation.

Figure 3. PGG binds IR with Kd~ ^10-5M .

3T3-L1 adipocytes in 24-well plates were incubated overnight at 4°C with increasing concentrations of cold insulin (in the presence of 8 pM ¹²⁵ I-insulin) or increasing concentrations of cold PGG (in the presence of 20 μM ¹⁴ C-PGG), respectively, Cell- ^bound125I -insulin ^or14C -PGG was then measured after removal of unbound isotope.

Figure 4. PGG did not displace insulin binding to its receptor IR in 3T3-L1 adipocytes.

3T3-L1 adipocytes were incubated overnight at 4°C with increasing concentrations ^of14C -PGG in the presence of ^8pM125I -labeled insulin, and cellular binding ^of125I -insulin ^or14C -PGG was calculated. The quasi-constant insulin counts for PGG were between 0.1 and 20 [mu]M, indicating that PGG cannot displace insulin from the IR in this concentration range. Insulin binding increases at higher PGG concentrations.

Figure 5. Protein binding selectivity of PGG.

A fixed amount of ¹²⁵ I BSA was incubated with PGG in the presence of variable amounts of cold gel or BSA or ovalbumin. Bound was separated from free by precipitation and expressed as % bound in the absence of competitor. Three competition curves show that PGG selectively binds different proteins with significantly different affinities.

Figure 6. Three insulin signaling pathway-specific inhibitors also disrupt PGG-induced glucose transport in 3T3-L1 adipocytes.

Adipocytes were induced with insulin or PGG in the presence or absence of different inhibitors. The glucose transport activity of treated cells was measured by the uptake ^of3H glucose by the cells. HNMPA-(AM)3 inhibits Tyr kinase activity, Cytochalasin B inhibits GLUT4, and Wortmannin inhibits PI-3K.

Figure 7. PGG induces phosphorylation of Akt in 3T3-L1 adipocytes.

Differentially treated adipocytes were lysed, and cellular proteins were analyzed by SDS-PAGE. 1 = non-treated; 2 = insulin; 3 = PGG, 15 μM; 4 = PGG, 30 μM; M = protein size marker.

Figure 8. 3T3-L1 cells stained with Oil Red O induced by MDI or insulin plus 30 μM PGG.

After 10 days of induction, the cells were stained with Oil Red O and photographed at a magnification of ×200. Only those cells containing fat vesicles (triglycerides) can be stained.

Figure 9. Northern blot analysis of PPARγ and C/EBPa gene expression in 3T3-L1 preadipocytes. Differentially treated preadipocytes were analyzed for mRNA expression at different times after treatment. Lane 1: No treatment; Lane 2 = MDI; Lane 3 = 30 μM PGG + MDI; Lane 4 = 30 μM PGG only. Time = post-treatment time when mRNA was isolated.

Figure 10. Clonal expression in α-PGG and β-PGG treated 3T3-L1 preadipocytes.

MDI, or induction of clonal expansion of preadipocytes by α-PGG or β-PGG in the presence of MDI. 24 or 48 hours after induction, the medium was removed and the cells were lysed. Lactose dehydrogenase (LDH) activity of cell lysates was measured with an LDH kit. At the same time the cell growth medium was collected and the LDH activity of dead cells was measured (not shown in this graph). In a given cell type, LDH activity is constant and the measured LDH activity is proportional to the number of cells in the sample.

Figure 11. Effect of PGG on blood glucose levels in db/db and ob/ob mice. Oral administration of different doses of α-PGG without glucose to db/db mice (A) or oral administration of different doses of α-PGG with glucose to ob/ob mice (B). Glucose was measured in blood samples from the tail at various times after dosing.

Figure 12. PGG protects hyperglycemic ob/ob mice immediately after the glucose test and becomes hypoglycemic several days after the test. ob/ob mice were subjected to a glucose tolerance test, as shown in Figure 11B. Blood glucose levels were measured in tail blood at various time points after the glucose test.

Figure 13. Chemical structures of selected gallotannin variants. G represents trihydroxybenzoic acid.

Figure 14. Effect of PGG on plasma insulin levels in ob/ob mice.

Figure 15. Effect of PGG on blood glucose levels in mice with normal blood glucose levels.

Detailed description of the invention

definition

The term "diabetes mellitus" refers to a disease or condition, usually characterized by metabolic defects in the production and utilization of glucose that results in the inability of the body to maintain proper blood sugar levels. The result of these deficiencies is elevated blood glucose, known as "hyperglycemia." The two main forms of diabetes are type 1 diabetes and type 2 diabetes. As mentioned earlier, type 1 diabetes is usually caused by an absolute deficiency of insulin, the hormone that regulates glucose utilization. Type 2 diabetes often occurs in the presence of normal or even elevated levels of insulin, caused by tissues not responding normally to insulin. Most patients with type 2 diabetes are insulin resistant, with relative insulin deficiency because insulin secretion cannot compensate for the resistance of peripheral tissues to insulin response. Also, many type 2 diabetics are obese. Other types of glucose homeostasis disorders include impaired glucose tolerance, a metabolic stage intermediate between normal glucose homeostasis and diabetes; and gestational diabetes, which occurs in the absence of prior type 1 and type 2 diabetes. History of glucose intolerance in pregnancy in women.

Guidelines for diagnosing type 2 diabetes, impaired glucose tolerance, and gestational diabetes have been indicated by the American Diabetes Association (see eg, Expert Committee on Diagnosis and Classification of Diabetes, Diabetes Care, (1999), Vol. 2 (Suppl 1): S5-19).

The term "symptoms" of diabetes includes, but is not limited to, polyuria, polydipsia and bulimia, hyperinsulinemia and hyperglycemia as used herein, in combination with their usual usage. For example, "polyuria" refers to the production of large volumes of urine within a given period of time; "polydipsia" refers to chronic, excessive thirst; Syndrome refers to elevated blood insulin levels. Other symptoms of diabetes include, for example, increased susceptibility to certain infections (especially fungal and staphylococcal infections), nausea and ketoacidosis (increased production of ketone bodies in the blood).

The term "complications" of glucose includes, but is not limited to, microvascular complications and macrovascular complications. Microvascular complications are those that usually result in damage to small blood vessels. These complications include, for example, retinopathy (impairment or loss of vision due to damage to blood vessels in the eye); neuropathy (destruction of nerves and foot problems due to damage to blood vessels in the nervous system); and nephropathy (kidney disease due to damage to blood vessels in the kidneys). Macrovascular complications are those that usually result from damage to large blood vessels. These complications include, for example, heart disease and peripheral vascular disease. Cardiovascular disease refers to diseases of the blood vessels of the heart. See, eg, Kaplan, R.M. et al., "Cardiovascular diseases" in HEALTH AND HUMAN BEHAVIOR, pp. 206-242 (McGraw-Hill, New York 1993). Cardiovascular disease is usually one of several forms including, for example, hypertension (also known as high blood vessel pressure), coronary heart disease, stroke, and rheumatic heart disease. Peripheral vascular disease refers to any disease of the blood vessels outside the heart, which is often narrowly defined as the blood vessels that carry blood to the muscles of the legs and arms.

As used herein, "hydrolyzable gallotannin" refers to a galloyl-glucose compound which is an ester of glucose with one or more trihydroxyphenyl carboxylic acids. A substantially pure hydrolyzable gallotannin composition of the invention comprising one or more hydrolyzable alpha or beta transomers of gallotannin having 5, 6, 7, 8, 9 or 10 galloyl groups, or Its pharmaceutically acceptable salt. The six, seven, eight, nine, and ten forms of hydrolyzable gallotannins all have an initial set of galloyl groups attached to carbons 1, 2, 3, 4, and 6 of the glucose core; and a secondary set of galloyl groups , from 1-5 additional galloyl groups, respectively. The galloyl group of the secondary setup attaches to the galloyl group separated in the primary setup. The substantially pure gallotannin compositions of the present invention may also comprise 1,2,3,4-tetra-O-galloyl-α-D-glucose.

The β-reverse form of hydrolyzable gallotannins is found in many plant-based foods, including common fruits (berries, bananas, grapes, apples); grains (barley, sorghum); and beverages of plant origin such as tea and wine. Typically, the beta isomer of hydrolyzable gallotannins is also found in tannin mixtures, which are commercially available.

Commercial tannin mixtures also contain varying amounts of methylgalloyl and galloylglucose compounds, containing 1, 2, 3, 11 and 12 galloyl groups. The substantially pure hydrolyzable gallotannin composition of the present invention comprises less than 5% by dry weight, preferably less than 3% by dry weight, more preferably less than 1% by dry weight of one or a mixture of the following compounds: Mono-O -galloyl-β-D-glucose, di-O-galloyl-β-D-glucose, tri-O-galloyl-β-D-glucose, or a mixture thereof. Thus, substantially pure hydrolyzable gallotannin compositions for use in the present invention are distinct from commercially available gallotannin mixtures.

As used herein, "gallotannin variant" refers to a compound, similar to but different from the following structure: 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, IX -O-galloyl-β-D-glucose, Deca-O-galloyl-β-D-glucose, Undeca-O-galloyl-β-D-glucose, Dodeca-O-galloyl-β-D -glucose.

A variant has the following structure:

RXA( _n )-XA( _q )-XA( _z ),

Wherein R is selected from: D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose , L-altrose, 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, α-D-arabinose, β-D-arabinose, α-D-ribose, β-D-ribose, D-trehalose, D-maltose, D-cellobiose, inositol, D-glucitol,

X is an ester or ether linkage,

A is trihydroxybenzoic acid selected from: 3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid; or dihydroxybenzoic acid, selected From: 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid; or monohydroxybenzoic acid selected from 3-hydroxybenzoic acid and 4-hydroxybenzoic acid,

where n is 5, q is 0, 1, 2, 3, 4 or 5, and, when 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-gulose, D-idose, L-idose , D-talose, L-talose, D-fructose, L-fructose, z is 0;

where n is 4, q is 0, 1, 2, 3 or 4, and, when R is: α-D-xylose, α-D-lyxose, β-D-lyxose, α-D -arabinose, β-D-arabinose, α-D-ribose, β-D-ribose, z is 0, 1 or 2.

wherein n is 6, q is 0, 1, 2, 3, 4, 5 or 6, and, when R is: D-glucitol or inositol, z is 0, and

wherein n is 8, q is 0, 1, 2, 3, 4, 5, 6, 7 or 8, and, when R is D-trehalose, D-maltose or D-cellobiose, z is 0 .

As used herein, "adipocyte" refers to fat cells. Morphologically, adipocytes are round, cells containing triglyceride (fat) vesicles. Biochemically, adipocytes express high levels of insulin receptors on the cell surface and exhibit a highly activated insulin-mediated glucose transport signaling pathway involving glucose transporter 4 (GLUT4). In the body, adipocytes are involved in the synthesis and storage of fat (triglycerides), as well as glucose metabolism (uptake of glucose from blood and conversion of glucose to fat).

As used herein, "preadipocyte" refers to adipocyte precursor cells that divide and differentiate into adipocytes under the action of hormones such as insulin and glucocorticoids. Morphologically, preadipocytes are fibroblast-like (thin and spindle-shaped) and lack triglyceride (fat) vesicles in their cytoplasm. Compared with adipocytes, preadipocytes contain low levels of insulin receptors and relatively high levels of insulin-like growth factor 1 (IGF-1) receptors for receiving mitogenic and differentiation signals. Without induction or full differentiation, preadipocytes do not express GLUT4 or other differentiation-associated genes such as PPAR-γ, C/EBP-α or C/EBP-γ. Preadipocytes have lower intracellular glucose transport activity than adipocytes.

As used herein, "lipogenesis" 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 (such as dogs or cats), pastoral animals (cows, horses or pigs), monkeys, rabbits, mice and laboratory animals.

In one aspect, the invention provides a method of stimulating glucose uptake in cells of a mammal, particularly a mammal suffering from diabetes, impaired glucose intolerance, insulin resistance or gestational diabetes. In another aspect, the present invention provides a method of inhibiting the differentiation of preadipocytes into adipocytes in a mammal, particularly a mammal that is obese, overweight or exhibiting symptoms of diabetes, glucose intolerance or gestational diabetes. This method is based in part on the inventors' discovery that certain hydrolyzable gallotannins and certain gallotannin variants are able to stimulate glucose transport into adipocytes and inhibit differentiation of preadipocytes into adipocytes. The method is also based in part on the inventors' discovery that certain hydrolyzable gallotannins lower blood glucose levels and blood insulin levels in mammals. The present method is also based at least in part on the inventors' discovery that certain hydrolyzable gallotannins do not cause hypoglycemia. Accordingly, the method is useful for treating and preventing diabetes, impaired glucose tolerance, insulin resistance and gestational diabetes in mammals.

In one embodiment, the method of the present invention for treating or preventing diabetes, impaired glucose tolerance, insulin resistance and gestational diabetes in a mammal comprises administering to the mammal a therapeutically effective amount of substantially pure hydrolyzable Gallotannin compositions. The substantially pure gallotannin composition comprises one or more hydrolyzable gallotannins selected from the group consisting of: 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-galloyl-β-D-glucose, 1,2,3,4,6-nona-O-galloyl-α-D-glucose, 1,2,3, 4,6-Nine-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 a pharmaceutically acceptable salt of a hydrolyzable gallotannin. A substantially pure hydrolyzable gallotannin composition comprising less than 5% by dry weight of one or more of the following compounds: mono-O-galloyl-β-D-glucose, di-O-galloyl-β-D - Glucose, Tris-O-galloyl-β-D-glucose, Tetra-O-galloyl-β-D-glucose, Undecyl-O-galloyl-β-D-glucose, Dodecanoyl-O-galloyl - beta-D-glucose or mixtures thereof.

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

Optionally, other agents used to treat or prevent diabetes, including insulin, sulfonylureas, Meglitinides, Biguanides (Glucophage or Metformin), Thiazolidinediones (TZDs), and alpha-glucosidase inhibitors, are administered to mammals in combination with the hydrolyzable gallotannin or gallotannin variant compositions of the present invention. For those mammals suffering from type 1 diabetes, it is preferred to administer insulin in combination with a gallotannin composition and/or a gallotannin variant composition.

object

The method is useful for treating mammals who have been diagnosed with diabetes, gestational diabetes, insulin resistance or impaired glucose tolerance. The method is also useful for treating mammals exhibiting symptoms of diabetes, gestational diabetes, insulin resistance, or impaired glucose tolerance. The methods are also useful for preventing or treating weight gain in individuals, particularly obese or overweight individuals.

Method of administration

The compositions are administered to a subject by injection, including subcutaneous, parenteral and intravenous injection, or by oral administration. Due to the ease of administration, the preferred route of administration is oral administration.

formula

The gallotannin and gallotannin variant compositions used in the present invention are formulated into pharmaceutical compositions using conventional methods. These pharmaceutical formulations comprise one or more hydrolyzable gallotannins of the invention and/or one or more gallotannin variant compositions of the invention. Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or diluent. The term "pharmaceutically acceptable" refers to a non-toxic material that does not interfere with the effectiveness of the gallotannin or gallotannin variant composition. Many such carriers are routinely used and can be identified by reference to pharmaceutical texts. The nature of the carrier will depend upon 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 skill in the art. The formulation may further comprise other agents that enhance or supplement the activity of the gallotannin or variant of the gallotannin. The formulation may further comprise fillers, salts, buffers, stabilizers, solubilizers and other materials known in the art.

The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods known in the art of pharmacy. The term "unit dose" refers to a desired defined amount of gallotannin or gallotannin variant composition, sufficient to be effective in treating the target disease or disorder. All methods involve contacting the gallotannin composition, the gallotannin variant composition, or both with a carrier or diluent and any other optional additional ingredients.

For oral administration, the pharmaceutical composition may take the form, for example, of tablets or capsules prepared by conventional means plus pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinized cornstarch, polymer vinylpyrrolidone or hydroxypropylmethylcellulose), fillers (such as lactose, microcrystalline cellulose or calcium hydrogen phosphate), lubricants (such as magnesium stearate or talc), disintegrants (such as potato starch or carboxylate sodium starch glycolate), or a wetting agent (such as sodium lauryl sulfate). Tablets may be coated by techniques well known in the art. Liquid preparations for oral administration may take the form of, for example, aqueous solutions, syrups or suspensions, or they may be presented as a dry product to be dissolved in water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with the addition of pharmaceutically acceptable additives such as suspending agents (such as sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifiers (such as lecithin or gum arabic) ), non-aqueous vehicles (such as almond oil, oily esters, ethanol, or fractionated vegetable oils), and preservatives (such as methyl or propyl-p-hydrobenzoate or sorbic acid). The preparation may contain suitable buffer salts, flavoring, coloring and sweetening agents. The formulation may also take the form of a nutritional supplement. Formulations for oral administration may be formulated as controlled release gallotannin compositions. Due to the high levels of proline-containing proteins present in saliva, the oral formulation is preferably in the form of a capsule comprising an outer layer to protect the gallotannin composition from reaction with saliva.

The gallotannin and gallotannin variant compositions may be prepared for parenteral administration by injection, such as by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, eg, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, tablet or capsule form for mixing with a suitable vehicle, eg sterile pyrogen-free water, before use.

The gallotannin and gallotannin variant compositions may also be formulated in rectal compositions such as suppositories or enemas, eg, containing conventional suppository bases such as cocoa butter, other glycerides, or Carbowax.

In addition to the formulations described above, gallotannin and gallotannin variant compositions may also be formulated as depot formulations. Such long-acting formulations may be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (such as emulsions in acceptable oils) or ion exchange resins, or as sparingly soluble derivatives, eg, as sparingly soluble salts.

dose

The gallotannin or gallotannin variant composition is administered to the individual in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" refers to a total amount sufficient to show meaningful benefits, such as alleviation of hyperglycemia (lower blood sugar levels), alleviation of hyperinsulinemia (lower blood insulin levels), improvement in glucose tolerance, weight gain Prevention and weight loss. The dosage of the gallotannin composition or the gallotannin variant composition required to obtain meaningful results can be determined by routine experimentation with appropriate controls by one of ordinary skill in the art in light of this disclosure. Comparison of appropriate treatment groups with controls will show whether a particular dose is effective in reducing blood glucose levels or inhibiting lipogenesis in an individual.

The amount of gallotannin composition required will depend on the nature and severity of the condition being treated, and on the nature of the individual's previous treatment. Finally, the dosage will be obtained using clinical studies. Initially, clinicians can administer doses from animal studies. An effective amount can be achieved by one administration of the composition. Alternatively, an effective amount can be achieved by administering the composition to an individual multiple times. In vitro, biologically effective amounts, ie, amounts sufficient to induce glucose uptake, were administered in two-fold increments to determine the full range of activity. The efficacy of oral, subcutaneous and intravenous administration is determined in clinical studies. Although a single administration of the gallotannin composition may be beneficial, administration of multiple doses is preferred.

Method for determining the dose to stimulate glucose uptake in cells

Glucose uptake activity in cells can be assayed by standard assays measuring uptake of 2-deoxy-D-[ ^3H ]glucose. Confluent 3T3-L1 adipocytes grown on 12-well plates were washed twice with serum-depleted DMEM and incubated with 1 mL of the same medium for 2 hours at 37°C. Cells were washed 3 times with Krebs-Ringer-Hepes (KRP) buffer and incubated with 0.9 ml of KRP buffer at 37°C for 30 minutes. Then, insulin (positive control) or gallotannin or gallotannin variant (experimental) was added at a predetermined concentration and the adipocytes were incubated at 37°C for 15 minutes. Glucose uptake was initiated by adding 0.1 ml KRP buffer and 37 MBq/L 2-deoxy-D-[ ³ H]glucose with a final concentration of 1 mmol/L glucose. After 10 minutes, glucose uptake was terminated by washing the cells 3 times with cold PBS. Cells were lysed with 0.7 mL of 1% Triton X-100 at 37°C for 20 minutes. Radioactivity of cell lysates was determined by scintillation counter. A dose can be selected that induces maximal glucose uptake in the test sample.

Method for determining doses to stimulate glucose uptake in animals

Eight week old male db/db (leptin receptor deficient) mice can be used to determine the dose to stimulate glucose uptake in vivo. Mice were divided into groups of 3 to 4 depending on how many doses were to be analyzed. 10 µl of the test solution with a predetermined concentration of the test gallotannin composition was orally administered to the test mice. Negative control mice received the same amount of water. After dosing, blood was collected from the tails of mice at various times after oral dosing. Blood glucose levels in mice at specific post-dose times were measured by applying 6 μl of blood to a One Touch Basic Complete Diabetes Monitoring System (obtained from Lifescan). Effective dosage ranges and optimal dosages can be determined by comparing the reduction in blood glucose levels of different dosages relative to the blood glucose levels of a negative (water) control group.

Inhibition of lipogenesis and weight gain

In another aspect, the invention provides methods of inhibiting the differentiation of preadipocytes into adipocytes. Adipocytes can be in culture or in a mammalian subject. In one embodiment, the method comprises contacting a preadipocyte with a biologically effective amount of a hydrolyzable gallotannin composition. The gallotannin composition is substantially pure, comprising a gallotannin composition comprising one or more of the following hydrolyzable gallotannins: 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-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.

In another embodiment, the method comprises contacting a preadipocyte with a biologically effective amount of a gallotannin variant composition comprising one or more gallotannin variants. In another embodiment, the method comprises simultaneously contacting a preadipocyte with a hydrolyzable gallotannin composition and a gallotannin variant composition of the present invention.

In vitro dosing procedure

To determine the effective concentration of gallotannin or gallotannin variants for preventing adipogenesis in vitro, undifferentiated preadipocytes were treated with a differentiation-inducing cocktail (comprising 3-isobutyl-1-methylxanthine, Dexamethasone, and insulin (MDI)) or MDI plus gallotannin or gallotannin variants were incubated. MDI induced differentiation after about 10 days, which was clearly visible as a change from fibroblast-like preadipocytes to round, fat vesicle-containing adipocytes. The degree of cell differentiation was assessed by microscopic observation of lipid accumulation and Oil Red O staining (only vesicles containing triglycerides were stained red), and by glucose uptake activity exhibited by treated cells at the end of the incubation period. A glucose uptake assay was selected and performed to determine the degree of differentiation of adipocytes based on the observation that differentiated adipocytes can be induced by insulin to uptake glucose, whereas preadipocytes cannot.

Methods for Determining Dosage in Vivo

In order to determine the in vivo anti-adipogenic effect and effective dosage of the gallotannin composition or the variant gallotannin composition of the present invention, 5-week-old genetically diabetic female mice (type 2, KK-A ^y ) were used. Gallotannin or gallotannin variants were administered orally or intraperitoneally into mice at different concentrations daily for 6 to 10 weeks. Monitor the food intake and body weight of the mice. At the end of the experiment, the parauterine fat tissues of the treated and control mice were removed, weighed and compared. The livers of treated and control mice were also removed, and the lipid content of the livers was measured. The dose that causes the greatest decrease in the lipid content of the parauterine adipose tissue as well as the liver without significant changes in food intake is considered to be the optimal dose for the anti-adipogenic activity of the gallotannin or gallotannin variant composition of the present invention.

Typical Process for Preparation of Hydrolyzable Gallotannins

A. Separation method

The beta form of hydrolyzable gallotannins can be reisolated from commercial tannin preparations using HPLC. The HPLC system was a Beckman System Gold consisting of a 125 solvent module, a 168 PDA detector and a 508 autosampler. For separation, a Beckman Ultrasphere C-18 reverse semi-preparative column (10.0 mm x 250 mm inner diameter, 5 μm) was used. The detection wavelength is set at 320nm or 330nm. Eluent A was water with 0.1% trifluoroacetic acid added and eluent B was acetonitrile with 0.1% trifluoroacetic acid. A Foxy Jr. fraction collector from ISCO was used to collect peaks within a time window. Separation was achieved with a constant solvent gradient A:B of 82:18 over 40 minutes at a flow rate of 3 mL/min. In these cases, the approximate retention times of the β-form gallotannins 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-beta-D-glucose 30.0 minutes.

Dec-O-galloyl-β-D-glucose 36.3 minutes.

Undecyl-O-galloyl-β-D-glucose 39.8 minutes.

B. Synthesis method

The chemical synthesis of the alpha and beta forms of PGG consists of four steps:

(i) Starting from the methyl ester of gallic acid, reaction with chlorotoluene in the presence of potassium carbonate and potassium iodide in acetone produces the protected gallic acid derivative methyl 3,4,5-tri-O-benzyl gallate .

(ii) Hydroxidation of the nacetoester in aqueous ethanol to form 3,4,5-tris-O-benzyl gallic acid.

(iii) 3,4,5-tri-O-benzyl gallic acid is used in the presence of 4-(dimethylamino)pyrimidine in 1,2-dichloroethane to mediate dicyclohexylcarbodiimide Guided D-glucose esterification. Penta-O-(3,4,5-tri-O-benzylgalloyl)-D-glucopyranose is obtained as a mixture of α- and β-anti-isomers.

(iv) Hydrogenation of penta-O-(3,4,5-tri-O-benzylgalloyl)-D-glucopyranose in the presence of a palladium catalyst in tetrahydrofuran, deprotection. Chromatographic (Chromatotron) separation of alpha/beta mixed PGG yielded individual pure anti-isomers.

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

Using a mixture of α-penta-O-galloylglucose, β-penta-O-galloylglucose as starting material, α-hexa-O-galloylglucose, β-hexa-O-galloylglucose, α - Hepta-O-galloyl glucose, β-Septa-O-galloyl glucose, α-Octa-O-galloyl glucose, β-Octa-O-galloyl glucose, α-Nine-O-galloyl glucose, β -nona-O-galloylglucose, alpha-deca-O-galloylglucose, beta-deca-O-galloylglucose. The addition of one or more gallic acids to α/β-penta-O-galloylglucose is the same or very similar to the reaction steps described previously for the synthesis of α/β-penta-O-galloylglucose. This reaction will synthesize a mixture of galloylglucose with 6, 7, 8, 9 or 10 gallic acids. These distinct compounds can be separated into single species by HPLC, and their individual structural identities can be verified by mass spectrometry and NMR analyses.

α and β-tetra-O-galloylglucose can be prepared by protecting one hydroxyl group on the glucose prior to the addition of gallic acid and deprotecting the hydroxyl group after the addition reaction.

Exemplary manufacturing method for preparing gallotannin variants

1) Synthesis of five-O-(3,4-dihydroxybenzoyl)-β-D-glucopyranose-

Step 1: Ethyl 3,4-dibenzyloxybenzoate

method:

A mixture of ethyl 3,4-dibenzyloxybenzoate (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 20 minutes. Chlorotoluene (14.85 g, 117 mmol) dissolved in 100 mL of acetone was added. The suspension was refluxed for 18 hours. The solid was filtered and the filtrate was evaporated. The residue was redissolved in 300 mL of dichloromethane and filtered again. The solvent was evaporated. A dark yellow oil is obtained. It was used in the next step without any further purification.

Step 2: 3,4-Dibenzyloxybenzoic acid

method:

The crude product from the previous step was suspended in 95% ethanol (300 mL) and precipitated by adding sodium hydroxide (3.54 g, 88.5 mmol). The mixture was heated at reflux for 3 hours. The hot solution was poured into 500mL of water and 25mL of concentrated hydrochloric acid. After swirling the flask for 10 minutes, the product was filtered 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 placed under oil pump vacuum (-0.1 bar) and dried overnight at room temperature.

Yield over two steps: 18.6 g (93%)

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

method:

D-glucose (0.2g, 1.11mmol), 3,4-dibenzyloxybenzoic acid (2.78g, 8.31mmol), dicyclohexylcarbodiimide (DCC, 1.84, 8.92mmol) and N, N- Dimethylaminopyridine (DMAP, 1.08 g, 8.84 mmol) was added to dry dichloromethane (130 mL). The suspension was refluxed for 2.5 days. After cooling to room temperature, the urea by-product was filtered off. 8 g of silica gel were 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). Pure fractions were combined and evaporated.

Yield: 1.72 g (88%) of a highly viscous clear oil.

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

method:

The benzyl protected starting material (392 mg, 0.222 mmol) was dissolved in anhydrous THF (50 mL). The solution was degassed by a water pump for about 30 seconds while magnetically stirring. The flask was then flushed with argon. Degassing and flushing were performed 2 more times. Add 10% palladium on carbon (287mg, 0.27mmol). The mixture was degassed, then flushed with hydrogen. Degassing and rinsing were repeated 2 more times. The suspension was then stirred at maximum speed under atmospheric hydrogen at 40°C for 5 hours. The mixture was cooled, filtered through celite, and the filtrate was evaporated.

Yield: 190 mg (99%) of foamed amorphous solid.

Other gallotannin variants containing dihydroxybenzyloxybenzoyl groups were prepared as previously described, except that different ethyl dihydroxybenzoates were used as starting material for step 1 .

2) Synthesis of tetra-O-(3,4-dibenzyloxybenzoyl)-β-D-glucopyranose

Step 1: Methyl 3,4,5-tribenzyloxybenzoate

method:

A mixture of 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) was stirred at room temperature for 20 minute. Chlorotoluene (22 g, 174 mmol) dissolved in 100 mL of acetone was added. The suspension was refluxed for 18h. The solid was filtered and the filtrate was evaporated. The residue was taken up in 400 mL of dichloromethane. The suspension was filtered through celite and the filtrate was evaporated. The residue was placed in an oil pump vacuum and dried at room temperature for 45 minutes.

Yield: 26.528 g (107%) (product contains some chlorotoluene as an impurity)

Step 2: 3,4,5-tribenzyloxybenzoic acid

method:

The crude product from the previous step was suspended in 95% ethanol (300 mL) and precipitated by adding sodium hydroxide (3.54 g, 88.5 mmol). The mixture was heated at reflux for 3 hours. The hot solution was poured into a mixture of 500 mL of water and 25 mL of concentrated hydrochloric acid. After swirling the flask for 10 minutes, the product was filtered 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 placed under oil pump vacuum (-0.1 bar) and dried overnight at room temperature.

Yield over two steps: 22.42 g (94%)

Step 3: Tetra-O-(3,4,5-tribenzyloxybenzoyl)-D-xylopyranose

method:

D-xylose (0.2g, 1.33mmol), 3,4,5-tribenzyloxybenzoic acid (3.515g, 7.98mmol), dicyclohexylcarbodiimide (DCC, 1.77, 8.57mmol) and N, N-Dimethylaminopyridine (DMAP, 1.04 g, 8.49 mmol) was added to anhydrous dichloromethane (130 mL). The suspension was refluxed for 2.5 days. After cooling to room temperature, the urea by-product was filtered off. 9 g of silica gel were 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). Pure fractions were combined and evaporated.

Yield: 208 mg of beta isomer

220mg of the alpha isomer

770mg of mixed distillate (α+β)

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

method:

The benzyl protected starting material (150 mg, 0.0815 mmol) was dissolved in anhydrous THF (20 mL). The solution was degassed by a water pump for about 30 seconds while magnetically stirring. The flask was then flushed with argon. Degassing and flushing were repeated 2 more times. Add 10% palladium on carbon (287mg, 0.27mmol). The mixture was degassed, then flushed with hydrogen. Degassing and rinsing were repeated 2 more times. The suspension was then stirred at maximum speed for 5 h at 40° C. under atmospheric pressure of hydrogen. The mixture was cooled, filtered through celite, and the filtrate was evaporated.

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

Other gallotannin variants contain a sugar core other than glucose, such as galactose, mannose, trehalose, maltose, cellobiose, cellulose, and glucitol, prepared as previously described, except that in step 3 the of xylose is replaced by another sugar.

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

The first three steps of the synthesis are methods reported in the literature (E.Eich, H.Pertz, M.Kaloga, J.Schulz, M.R.Fesen, A.Mazumder, Y.Pommier, (-)-Arctigenin as a Lead Structure for Inhibitor of Human Immunodeficiency Virus Type-1 Integrase, J.Med.Chem.1996, 39, 86-95).

Subsequent steps are similar to standard carbohydrate benzyl protection/deprotection chemistry. Finally hydrogenolysis will bind trishydroxybenzyl groups faster for benzylphenols than for carbohydrates. It is expected that the reduced susceptibility of the ether linkages to acid hydrolysis will increase the stability (half-life) of the molecule, thereby increasing the duration of in vivo action and overall overt biological activity.

Example

The following examples are for illustration only and do not limit the scope of the appended claims. All documents cited herein are hereby incorporated in their entirety.

Example 1: Stimulation of glucose uptake in cells with penta-O-galloyl-D-glucose (PGG)

A 50:50 mixture of α-PGG and β-PGG was synthesized as before. The α and β anti-isomers were isolated as previously described. The glucose transport activity of the two transisomers was compared with that of authentic plant-derived β-PGG. Chemically synthesized PGG is spectrally identical to genuine plant-derived PGG.

3T3-L1 adipocytes were purchased from ATCC and maintained in DMEM supplemented with 10% calf serum in a 37 °C incubator containing 10% _CO2 required for the cells as preadipocytes. By adding MDI inducing cocktail, cells were induced to differentiate into adipocytes, such as Liu, F., Kim, J., Li, Y., Liu, X., Li, J. & Chen, X. (2001), in 3T3- Lagerstroemia speciosa L. extracts in L1 cells have insulin-like glucose uptake stimulating and adipocyte differentiation-inhibiting activities as described in J. Nutrition 131:2242-224, which is incorporated herein by reference. Different amounts of α-PGG and β-PGG were then added to the medium, and glucose uptake by control and treated cells was performed using a standard glucose uptake assay as described by Liu et al. As shown in Figure 2A, chemically synthesized and plant-derived β-PGG have similar glucose transport stimulating properties (GTS). The GTS activity of α-PGG is generally 10-20% higher than that of β-PGG (Figure 2).

To investigate the mechanism by which PGG induces GTS activity, ¹⁴ C-labeled PGG was synthesized and used for cell studies. In order to determine whether PGG acts on the cell membrane or intracellularly, radioactive PGG was administered to 3T3-L1 adipocytes at 37°C or 4°C for different times. After incubation, excess radioactive PGG was removed by washing, cells were harvested, and cells were separated into membrane and intracellular components using published protocols. More than 90-95% of the radioactivity was bound to membrane components, proving that PGG acts on 3T3-L1 cells by binding to the cell membrane.

Ligand displacement studies were performed using 3T3-L1 cells incubated overnight at 4°C ^with14C -PGG and increasing amounts of unlabeled PGG. Radioactive insulin was used as a receptor binding control. PGG functions as a ligand, binding the target with an apparent receptor binding constant (Kd) of ~10 ^-5 M in the concentration range tested (Fig. 3). For GTS and AD activity, the estimated Kd is very similar to what the EC50 of PGG does not represent. Since PGG has insulin-like GTS activity and was shown to induce GTS activity in a similar way to insulin, it can be speculated that PGG induces GTS activity by binding to insulin receptors in 3T3-L1 cells.

To determine whether PGG and insulin share a common target, 3T3-L1 cells were incubated with a constant concentration of radioactive insulin plus increasing amounts of cold PGG, or with radioactive PGG plus increasing amounts of cold insulin. Binding assay results showed that PGG did not compete with insulin for the insulin binding site on IR (Figure 4). Conversely, it was also found that insulin does not compete with PGG for PGG binding sites located on the cell membrane. Thus, PGG appears to bind to IR sites other than the insulin binding site, or to membrane receptors other than IR.

Using radiolabeled bovine serum albumin (BSA) as a tracer, a competition binding assay was used to determine whether PGG could selectively bind to the protein. The relative affinities of PGG for the three standard proteins varied by at least 10-fold (FIG. 5), and the binding affinities of PGG for gel and ovalbumin varied by more than 100-fold. This suggests that the PGG-protein response is specific and that PGG can act selectively on a single biochemical target, such as IR.

Chemical inhibitors that specifically inhibit proteins/enzymes involved in insulin-mediated signaling pathways were used to further identify molecular targets of PGG. Three inhibitors were tested: hydroxy-2-naphthylmethylphosphonic acid triacetoxymethyl ester (HNMPA-(AM) _{3 )} , a chemical agent capable of inhibiting IR tyrosine kinase (Qiu, Z., etc. (2001) J.Biol.Chem.276:11988-11995); Wortmannin (wortmannin), a compound (Saperstein, R., etc. (1989) Biochemistry 28:5694-5701 specificity inhibits PI-3K ); and cytochalasin B, a compound that specifically inhibits glucose transport mediated by GLUT4 (Tomiyama, K. et al. (1995) Biochem.Biophy.Res.Commu.212:263-269; Kletzien, RF et al. ( 1972) J.Biol.Chem.247:2964-2966).All three inhibitors completely inhibit GTS activity mediated by insulin or PGG (Figure 6), demonstrating that all GTS activity mediated by PGG is through, and Only through the insulin-mediated signaling pathway. In fact, the inhibitor HNMPA-(AM) ₃ of the first enzyme (IR tyrosine kinase) in the insulin signaling pathway can also completely inhibit the GTS activity of PGG, proving that the molecular The target is IR. The idea that PGG employs the insulin-mediated GTS pathway is further supported by Western blot showing that Akt, a key protein kinase involved in this pathway (zz), is phosphorylated by PGG (Figure 7).

Example 2: Effect of PGG on lipogenesis

3T3-L1 adipocytes were purchased from ATCC, maintained and passed as preadipocytes in DMEM supplemented with 10% calf serum, placed in a 37 °C incubator with 10% _CO2 as needed for cells. To determine the effect of PGG on adipogenesis, preadipocytes were treated with a differentiation-inducing cocktail (MDI, a 3-isobutyl-1-methylxanthine, dexamethasone (MD) and PGG, a 3-isobutyl Butyl-1-methylxanthine, dexamethasone (MD) cocktail) co-incubation; or co-incubation with MDI plus PGG. When insulin was replaced by PGG in MDI, the new cocktail failed to induce differentiation of preadipocytes (see Figure 2B). These results suggest that PGG cannot replace insulin to induce differentiation of preadipocytes into adipocytes.

Cell proliferation assays were used to determine whether PGG inhibits adipocyte differentiation by arresting clonal expansion. This assay showed that the first round of clonal expansion was not inhibited by α- or β-PGG. The second round of clonal expansion was partially inhibited by α-PGG and completely inhibited by β-PGG ( FIG. 10 ). The basis for the difference between the two transforms in clonal expansion and differentiation is not understood. However, our results suggest that inhibition of clonal expansion or apoptosis (programmed cell death) in preadipocytes does not lead to the observed inhibition of differentiation, as α-PGG only inhibits differentiation with limited inhibition of clonal expansion, unlike MDI-induced Cell killing was not significantly improved compared to cells (data not shown). Therefore, it is likely that the critical step of repression is a different step than clonal expansion.

To investigate the mechanism by which PGG inhibits 3T3-L1 preadipocyte differentiation, we applied gene expression studies. This study allowed us to determine whether PGG acts specifically on genes involved in the differentiation process. Also, gene expression studies overcome difficulties in the study of the insulin receptor (which expresses 10-fold less IR on the cell surface) in preadipocytes (Modan-Moses et al. (1998) Biochem. J. 333:825-831). To study genes affected by PGG during AD, preadipocytes were incubated with a differentiation-inducing cocktail containing 3-isobutyl-1-methylxanthine, dexamethasone, and insulin; or with MDI plus PGG Incubation. After about 10 days, MDI induced differentiation, and the change was clearly visible due to the change from fibroblast-like preadipocytes to round adipocytes containing fat vesicles (Figure 8 middle). In contrast, preadipocytes treated with MDI plus PGG retained their fibroblast-like morphology and remained devoid of fat vesicles (Fig. 8 right).

At various times, treated cells were harvested, total RNA was isolated, and expression was analyzed by Northern blot with probes complementary to the different genes involved in the differentiation process. Northern blot analysis showed that the expression of genes PPAR-γ, c/EBP-α required for differentiation and induced by MDI was completely inhibited by PGG ( FIG. 9 ). The relatively consistent β-actin levels in differentiation-treated cells showed that other cellular processes such as β-actin expression were not significantly affected by PGG ( FIG. 9 ).

Example 3: Effect of PGG on Lowering Blood Glucose Levels in Diabetic Animals

To determine whether α-PGG is capable of exhibiting anti-diabetic activity in vivo, α-PGG in aqueous solution was orally administered to 8-week-old male fasted diabetic db/db mice. A single dose of α-PGG at a concentration of 25 mg/kg body weight was found to significantly reduce blood glucose levels in db/db mice compared to db/db mice receiving vehicle alone (the same aqueous solution without α-PGG) (Fig. 11A). The reduction in glucose levels was about 15-20%, depending on the time after α-PGG administration (P<0.01, FIG. 11A ).

To determine whether α-PGG is effective in improving glucose tolerance in diabetic and obese mice, a glucose tolerance test was performed using ob/ob mice. Glucose or glucose plus α-PGG was orally administered to male ob/ob mice, and blood glucose levels were measured at different time points after glucose/PGG administration. Ob/ob mice receiving glucose plus α-PGG had significantly lower blood glucose levels compared to those mice treated with glucose only (P<0.001, FIG. 11B ). Interestingly, after 24 hours of α-PGG treatment, elevated blood glucose levels were still observed, demonstrating that the effects of α-PGG are relatively long-lasting.

A single high dose of glucose can be fatal in ob/ob mice because of very high levels of blood glucose levels ~24 hours after the glucose test, or because of abnormally low glucose levels 2-3 days after the glucose test. We found that α-PGG not only protected ob/ob mice from very high glucose levels shortly after the glucose test (Fig. 11B), but also protected mice from very low glucose levels 2–5 days later ( Figure 12). Although the protective mechanism is currently unknown, it is likely that α-PGG protects ob/ob mice not only with its GTS activity, but also with some other activities, such as AD-related activities. No PGG-related toxic effects were found in these mice.

These animal studies clearly demonstrate that α-PGG is effective not only in 3T3-L1 cells but also in diabetic/obese animal models.

Example 4 Effects of gallotannin variants on glucose uptake by cells

We have tested several types of polymerized polyphenols for GTS in the 3T3-L1 cell system (Table 1). Lagerstroemia grandis extracts and gallotannin preparations (tannic acid (a mixture of galloylglucose); and purified PGG) had high activity. Proanthocyanidins have limited activity and tea catechin EGCG has no activity. This supports the notion that a specific protein-PGG reaction is responsible for GTS activity, since only PGG and closely related compositions are active. If GTS activity is simply the result of normal polymeric polyphenolic protein binding, flavonoid-based proanthocyanidins and tea catechol preparations would be active.

Reproducible differences in GTS and AD activity between α-PGG and β-PGG ( FIG. 2 ) and between PGG and Hexa-GG (Table 1 ) were routinely obtained. It is possible that the orientation of the gallic acid at carbon 1 of the sugar is responsible for the observed difference in activity between α-PGG and β-PGG. Pentagalloylgalactose (PGGal), a gallotannin variant of the present invention, and pentagalloyldeoxynojirimycin (PG-DJM) were synthesized ( FIG. 13 ). We found that PGGal had 60-70% of the activity of PGG, whereas PG-DJM had no measurable GTS activity.

Table. Glucose transport stimulating activity of PGG derivatives-β-D-pyran

The GTS activity of α-PGG was +++++, and the activity of β-PGG was ++++.

Example 5: Effect of PGG on Plasma Insulin Levels

Diabetic and obese ob/ob mice were injected intraperitoneally with water or α-PGG. Plasma from each mouse was isolated at various times after injection and insulin levels were measured. As shown in Figure 15, ob/ob diabetic and obese mice injected with a single injection of α-PGG had significantly lower plasma insulin levels compared to ob/ob diabetic mice treated with water alone (negative control). Based on these results and the effect of PGG on glucose uptake, it is believed that PGG enhances the glucose transport stimulating activity of insulin in vivo. Accordingly, PGG is expected to be therapeutically useful for increasing insulin resistance in mammalian subjects.

Example 6 Penta-O-(3,4-dihydroxybenzoyl)-B-D-glucopyranose and tetra-O-(3,4,5-trihydroxy Effects of Benzoyl-a-D)xylopyranose on Glucose Uptake in Adipocytes

Confluent 3T3-L1 adipocytes grown on 12-well plates were washed twice with serum-depleted DMEM and incubated with 1 mL of the same medium for 2 hours at 37°C. Cells were washed 3 times with Krebs-Ringer-Hepes (KRP) buffer and incubated with 0.9 ml of KRP buffer at 37°C for 30 minutes. Then, the compounds listed in the table below were added at 20-40 μM (final concentration?), and the adipocytes were incubated at 37° C. for 15 minutes. Glucose uptake was initiated by adding 0.1 mL of KRP buffer and 37 MBq/L 2-deoxy-D-[ ³ H]glucose and 1 mmol/L glucose as a final concentration. After 10 minutes, glucose uptake was terminated by washing the cells 3 times in cold PBS. Cells were lysed with 0.7 mL of 1% Triton X-100 for 20 minutes at 37°C. Radioactivity retained by cell lysates was determined by scintillation counter. As indicated in the table, the gallotannin variants penta-O-(3,4-dihydroxybenzoyl)-β-D-glucopyranose and tetra-O-(3,4,5-trihydroxybenzoyl) -α-D-Xylopyranose increases cellular glucose uptake.

Example 7: PGG does not cause hypoglycemia

Normal (healthy) mice were orally administered 40 mg of glucose at time 0. After 2 hours (120 minutes) but when blood glucose was at normal or basal levels, mice were given oral administration of different concentrations of PGG or water (negative control), or intraperitoneal injection of insulin. At various time intervals between PGG or insulin administration, blood was collected from control and treated animals to determine blood glucose levels. As shown in Figure 15, insulin injection induced hypoglycemia in mice, whereas PGG administration did not. Thus, PGG reduces blood glucose levels that are higher than normal. However, PGG does not further reduce blood sugar levels below normal or baseline levels.

Claims (24)

1. A method of treating or preventing diabetes in a mammalian individual, comprising: administering to an individual in need thereof a therapeutically effective amount of a gallotannin composition, Wherein, the gallotannin composition comprises one or more hydrolyzable gallotannins selected from: 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-galloyl-β-D-glucose, 1,2,3,4,6-nona-O-galloyl-α-D-glucose, 1,2,3 , 4,6-Nine-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 a salt thereof, and Wherein, the gallotannin composition comprises less than 5% dry weight of one or more of the following compounds: mono-O-galloyl-β-D-glucose, di-O-galloyl-β-D- Glucose, Tris-O-galloyl-β-D-glucose, Tetra-O-galloyl-β-D-glucose, Undecyl-O-galloyl-β-D-glucose, Dodecanoyl-O-galloyl-β-D-glucose, Beta-D-glucose or mixtures thereof. 2. The method of claim 1, wherein said 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 , Conjugates of 4,6-penta-O-galloyl-β-D-glucose. 3. The method of claim 1, wherein said gallotannin composition comprises at least 50% by dry weight of 1,2,3,4,6-penta-O-galloyl-[alpha]-D-glucose. 4. The method of claim 1, wherein said gallotannin composition comprises at least 50% by dry weight of 1,2,3,4,6-penta-O-galloyl-[beta]-D-glucose. 5. The method of claim 1, wherein said gallotannin composition is administered to the individual orally or by injection. 6. The method of claim 1, further comprising the step of administering insulin to the individual. 7. A method of treating or preventing diabetes in an individual comprising: administering a gallotannin variant composition to an individual, Wherein, the modified gallotannin composition comprises one or more modified gallotannin compounds or salts thereof, wherein each of the modified gallotannin compounds has the following structure: RXA( _n )-XA( _q )-XA( _z ), Wherein R is selected from: D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose , L-altrose, 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, α-D-arabinose, β-D-arabinose, α-D-ribose, β-D-ribose, D-trehalose, D-maltose, D-cellobiose, inositol, D-glucitol, X is an ester or ether linkage, A is trihydroxybenzoic acid selected from: 3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid; or dihydroxybenzoic acid, selected From: 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid; or monohydroxybenzoic acid selected from 3-hydroxybenzoic acid and 4-hydroxybenzoic acid, n is 5, q is 0, 1, 2, 3, 4 or 5, and, when 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-gulose, D-idose, L-idose, D - When talose, L-talose, D-fructose, or L-fructose, z is 0; n is 4, q is 0, 1, 2, 3 or 4, and, when R is: α-D-xylose, α-D-lyxose, β-D-lyxose, α-D-arabino In the case of sugar, β-D-arabinose, α-D-ribose, β-D-ribose, z is 0, 1 or 2. n is 6, q is 0, 1, 2, 3, 4, 5 or 6, and, when R is: D-glucitol or inositol, z is 0, and n is 8, q is 0, 1, 2, 3, 4, 5, 6, 7 or 8, and z is 0 when R is D-trehalose, D-maltose or D-cellobiose. 8. The method of claim 7, wherein X is an ether linkage. 9. The method of claim 7, wherein R is L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D- Altrose, L-altrose, 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, α-D-arabinose, β-D-arabinose, α-D-ribose, β-D - ribose, D-trehalose, D-maltose, D-cellobiose, inositol, or D-glucitol. 10. The method of claim 4, wherein A is a trihydroxybenzoic acid selected from the group consisting of: 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid; or a dihydroxybenzoic acid Formic acid selected from: 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid; or a monohydroxybenzoic acid selected from 3-hydroxybenzoic acid and 4- Hydroxybenzoic acid. 11. The method of claim 4, wherein each of said modified gallotannin compounds has a structure other than: 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-β-D-glucose. 12. A method of reducing elevated blood glucose levels without causing hypoglycemia in an individual in need thereof, comprising: administering to the individual one or both of the following compositions (a) and (b), (a) a gallotannin composition comprising one or more hydrolyzable gallotannins selected from the group consisting of 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-galloyl-β-D-glucose, 1,2,3,4,6-nona-O-galloyl-α-D-glucose, 1,2,3 , 4,6-Nine-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 a pharmaceutically acceptable salt thereof, and Wherein, the gallotannin composition comprises less than 5% dry weight of one or more of the following compounds: mono-O-galloyl-β-D-glucose, di-O-galloyl-β-D- Glucose, Tris-O-galloyl-β-D-glucose, Tetra-O-galloyl-β-D-glucose, Undecyl-O-galloyl-β-D-glucose, Dodecanoyl-O-galloyl-β-D-glucose, Beta-D-glucose or mixtures thereof. (b) a gallotannin composition, comprising one or more compounds having the following structure, or a pharmaceutically acceptable salt thereof: RXA( _n )-XA( _q )-XA( _z ), Wherein R is selected from: D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose , L-altrose, 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, α-D-arabinose, β-D-arabinose, α-D-ribose, β-D-ribose, D-trehalose, D-maltose, D-cellobiose, inositol, D-glucitol, X is an ester or ether linkage, A is trihydroxybenzoic acid selected from: 3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid; or dihydroxybenzoic acid, selected From: 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid; or monohydroxybenzoic acid selected from 3-hydroxybenzoic acid and 4-hydroxybenzoic acid, n is 5, q is 0, 1, 2, 3, 4 or 5, and, when 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-gulose, D-idose, L-idose, D - talose, L-talose, D-fructose, L-fructose, z is 0; n is 4, q is 0, 1, 2, 3 or 4, and, when R is: α-D-xylose, α-D-lyxose, β-D-lyxose, α-D-arabino z is 0, 1 or 2 for sugar, β-D-arabinose, α-D-ribose, β-D-ribose. n is 6, q is 0, 1, 2, 3, 4, 5 or 6, and, when R is: D-glucitol or inositol, z is 0, and n is 8, q is 0, 1, 2, 3, 4, 5, 6, 7 or 8, and z is 0 when R is D-trehalose, D-maltose or D-cellobiose. 13. A method of treating an individual exhibiting one or more of obesity, type 2 diabetes, glucose intolerance, insulin resistance, hyperglycemia, or hyperinsulinemia, comprising administering to an individual a gallotannin composition, The gallotannin composition described therein comprises one or more hydrolyzable gallotannins selected from 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-galloyl-β-D-glucose, 1,2,3,4,6-nona-O-galloyl-α-D-glucose, 1,2,3,4 , 6-Nine-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 its salts, and Wherein, the gallotannin composition comprises less than 5% dry weight of one or more of the following compounds: mono-O-galloyl-β-D-glucose, di-O-galloyl-β-D- Glucose, Tris-O-galloyl-β-D-glucose, Tetra-O-galloyl-β-D-glucose, Undecyl-O-galloyl-β-D-glucose, Dodecanoyl-O-galloyl-β-D-glucose, Beta-D-glucose or mixtures thereof. 14. The method of claim 13, wherein administering the gallotannin composition inhibits differentiation of preadipocytes to adipocytes in the individual. 15. The method of claim 13, wherein the gallotannin composition is administered to the individual orally or by injection. 16. A method of inhibiting differentiation of preadipocytes into adipocytes in vitro or in vivo, comprising: contacting the preadipocytes with the gallotannin composition, Wherein, the gallotannin composition comprises one or more hydrolyzable gallotannins selected from 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-galloyl-β-D-glucose, 1,2,3,4,6-nona-O-galloyl-α-D-glucose, 1,2,3, 4,6-Nine-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 a salt thereof. 17. The method of claim 16, wherein said gallotannin composition comprises less than 5% by dry weight of one or more of 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, undecyl-O-galloyl-β-D-glucose, Dodecanoyl-O-galloyl-β-D-glucose or mixtures thereof. 18. The method of claim 16, wherein said 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 galloyl-α-D-glucose and 1,2,3,4,6-penta-O-galloyl-β- Conjugates of D-glucose. 19. The method of claim 16, wherein said gallotannin composition comprises at least 50% by dry weight of 1,2,3,4,6-penta-O-galloyl-[beta]-D-glucose. 20. A method of inhibiting the differentiation of preadipocytes into adipocytes in vitro or in vivo, comprising The preadipocytes are contacted with a composition comprising one or more compounds having the following structures or salts thereof: RXA( _n )-XA( _q )-XA( _z ), Wherein R is selected from: D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose , L-altrose, 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, α-D-arabinose, β-D-arabinose, α-D-ribose, β-D-ribose, D-trehalose, D-maltose, D-cellobiose, inositol, D-glucitol, X is an ester or ether linkage, A is trihydroxybenzoic acid selected from: 3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid; or dihydroxybenzoic acid, selected From: 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid; or monohydroxybenzoic acid selected from 3-hydroxybenzoic acid and 4-hydroxybenzoic acid, n is 5, q is 0, 1, 2, 3, 4 or 5, and, when 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-gulose, D-idose, L-idose, D - talose, L-talose, D-fructose, L-fructose, z is 0; n is 4, q is 0, 1, 2, 3 or 4, and, when R is: α-D-xylose, α-D-lyxose, β-D-lyxose, α-D-arabino z is 0, 1 or 2 for sugar, β-D-arabinose, α-D-ribose, β-D-ribose. n is 6, q is 0, 1, 2, 3, 4, 5 or 6, and, when R is: D-glucitol or inositol, z is 0, and n is 8, q is 0, 1, 2, 3, 4, 5, 6, 7 or 8, and z is 0 when R is D-trehalose, D-maltose or D-cellobiose. 21. A modified gallotannin composition comprising one or more modified gallotannin compounds or pharmaceutically acceptable salts thereof, wherein each of said modified gallotannin compounds has the following structure: RXA( _n )-XA( _q )-XA( _z ), Wherein R is selected from: D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose , L-altrose, 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, α-D-arabinose, β-D-arabinose, α-D-ribose, β-D-ribose, D-trehalose, D-maltose, D-cellobiose, inositol, D-glucitol, X is an ester or ether linkage, A is trihydroxybenzoic acid selected from: 3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid; or dihydroxybenzoic acid, selected From: 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid; or monohydroxybenzoic acid selected from 3-hydroxybenzoic acid and 4-hydroxybenzoic acid, n is 5, q is 0, 1, 2, 3, 4 or 5, and, when 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-gulose, D-idose, L-idose, D - talose, L-talose, D-fructose, L-fructose, z is 0; n is 4, q is 0, 1, 2, 3 or 4, and, when R is: α-D-xylose, α-D-lyxose, β-D-lyxose, α-D-arabino z is 0, 1 or 2 for sugar, β-D-arabinose, α-D-ribose, β-D-ribose. n is 6, q is 0, 1, 2, 3, 4, 5 or 6, and, when R is: D-glucitol or inositol, z is 0, and n is 8, q is 0, 1, 2, 3, 4, 5, 6, 7 or 8, and, when R is D-trehalose, D-maltose or D-cellobiose, z is 0, as well as wherein each of said gallotannin variant compounds is not the beta isomeric form of: tetra-O-galloyl-D-glucose, penta-O-galloyl-D-glucose, hexa-O-galloyl-D-glucose, hexa-O-galloyl -alpha-D-glucose, hepta-O-galloyl-D-glucose, octa-O-galloyl-D-glucose, nona-O-galloyl-D-glucose, and deca-O-galloyl-D-glucose glucose. 22. The composition of claim 21, wherein the composition comprises penta-O-(3,4-dihydroxybenzoyl)-β-D-glucopyranose tetra-O-(3,4,5-tri hydroxybenzoyl)-α-D-xylopyranose. 23. A composition for the treatment of diabetes, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia or obesity in an individual, said composition having the following structural formula: RXA( _n )-XA( _q )-XA( _z ), Wherein R is selected from: D-glucose, L-glucose, D-mannose, L-mannose, D-galactose, L-galactose, D-allose, L-allose, D-altrose , L-altrose, 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, α-D-arabinose, β-D-arabinose, α-D-ribose, β-D-ribose, D-trehalose, D-maltose, D-cellobiose, inositol, D-glucitol, X is an ester or ether linkage, A is trihydroxybenzoic acid selected from: 3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid; or dihydroxybenzoic acid, selected From: 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid; or monohydroxybenzoic acid selected from 3-hydroxybenzoic acid and 4-hydroxybenzoic acid, n is 5, q is 0, 1, 2, 3, 4 or 5, and, when 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-gulose, D-idose, L-idose, D - talose, L-talose, D-fructose, L-fructose, z is 0; n is 4, q is 0, 1, 2, 3 or 4, and, when R is: α-D-xylose, α-D-lyxose, β-D-lyxose, α-D-arabino z is 0, 1 or 2 for sugar, β-D-arabinose, α-D-ribose, β-D-ribose. n is 6, q is 0, 1, 2, 3, 4, 5 or 6, and, when R is: D-glucitol or inositol, z is 0, and n is 8, q is 0, 1, 2, 3, 4, 5, 6, 7 or 8, and, when R is D-trehalose, D-maltose or D-cellobiose, z is 0, Or a pharmaceutically acceptable salt of said compound. 24. A pharmaceutical composition for treating insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia or obesity in an individual, said pharmaceutical composition comprising an effective amount of a compound of claim 23 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.

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