WO2006006757A1

WO2006006757A1 - Composition for lowering blood glucose comprising hexose monophosphate

Info

Publication number: WO2006006757A1
Application number: PCT/KR2004/003254
Authority: WO
Inventors: Dae-Sil Lee; Hwan-Mook Kim; Joong-Su Kim; Chang-Woo Lee; Song-Kyu Park; Jung-Don Bae; Doo-Il Kim; Bo-Hyun Park; Jung-Eun Park; Myong-Lyoll Lee; Suk-Hoon Koh; Hyun-Jae Shin
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2004-07-15
Filing date: 2004-12-10
Publication date: 2006-01-19
Also published as: KR100720055B1; KR20060006353A

Abstract

Disclosed is a composition comprising a hexose monophosphate for lowering blood glucose by the action of orally administered hexose monophosphate of stimulating insulin production by the pancreatic beta cells and increasing serum insulin levels.

Description

COMPOSITION FOR LOWERING BLOOD GLUCOSE COMPRISING HEXOSE

MONOPHOSPHATE

Technical Field

The present invention relates to a composition for lowering blood glucose level comprising a hexose monophosphate, a derivative or a salt thereof.

Background Art

Diabetes mellitus (DM) is a complex chronic disorder of carbohydrate, fat and protein metabolism, which occurs due to relative or absolute deficiency of insulin secretion by pancreatic beta cells or deficient insulin receptors on cells. Normal people synthesize complementary hormones, insulin and glucagon, in the body to reduce or increase blood glucose, thus keeping the blood sugar within its normal limits. Insulin is produced in beta cells and stored therein, and is secreted into the blood when blood glucose levels increase. The secreted insulin is introduced into cells through binding to its receptors on muscle cells or hepatic cells and stimulates blood D-glucose uptake into the cells to trigger a glucose metabolism process.

In Korea, before the 1970' s, diabetes mellitus affected only about 0.5% of the total population, and there was relatively little interest therein. However, diabetic patients amount to 2-3% of the total population in the 1980' s and 4-6% (about 1.5-2 million people) in the 1990' s, and many diabetic patients do not recognize the onset of diabetes mellitus.

The symptoms of diabetes mellitus vary among patients. The common diabetes symptoms include excessive urination (polyuria) , excessive drinking (polydipsia) and excessive eating (polyphagia) . In spite of excessive eating, loss of sugar and water through urine also results in weight loss, which is caused because diabetic patients do not use glucose as an energy source and instead consume stored proteins and fats as an energy source.

Diabetes mellitus commonly occurs in two forms: insulin-dependent diabetes (type 1 diabetes) and insulin independent diabetes (type 2 diabetes) . Type 1 develops due to damage to the beta cells in the pancreas usually from hereditary factors and viral infections. This damage reduces or completely stops the production of insulin. Type 1 diabetes suddenly occurs mainly from age ten to twenty, and is also known as juvenile-onset, brittle or ketosis- prone diabetes. Type 2 diabetes is generally caused after the forties by factors including family history, obesity and stress. Individuals with type 2 diabetes have insulin resistance and glucose availability different from normal people although insulin secretion is sufficient in the pancreas, and, thus, blood glucose levels are not normalized despite high insulin levels. Type II diabetes is also known as maturity-onset, adult-onset, ketosis-resistant, or stable diabetes. Diabetes mellitus, not a lethal disease itself, becomes problematic due to its chronic complications resulting from late treatment, including diabetic retinopathy (causing optical defects, blindness, retinal hemorrhage and cataracts), diabetic nephropathy, diabetic neuropathy, diseases associated with the heart and the circulatory system (causing vasculopathy) , periodontitis, osteopenia, and skin diseases. These pathological complications of diabetes are known to vary in proportion to hyperglycemia (Porte, Jr. et al., 1996). Currently available therapeutic agents for diabetes mellitus are generally classified into oral hypoglycemic agents and insulin injectable preparations. Typically, insulin injections are applied to insulin-dependent diabetic patients or pregnant diabetic patients who are defective in insulin secretion, or insulin-independent diabetic patients whose blood glucose is not controlled within normal levels by oral hypoglycemic agents. Oral hypoglycemic agents are administered to insulin-independent diabetic patients whose blood glucose is not properly controlled by a regime of controlled diet and regular exercise. Insulin injections are carried out mainly via intravenous or intramuscular routes, and, if long-term administration is needed, mostly subcutaneousIy. Subcutaneous injections have the following disadvantages: they do not sharply increase insulin levels; they reduce insulin secretion upon calorie intake; and they release insulin into the peripheral circulation rather than the hepatic circulation, thus reducing the effect of the injected insulin (Goodman et al., The Pharmacological basis of Therapeutics, pl692) . Also, when genetically engineered insulin is injected, antibodies specific to the insulin are generated in the body due to the insulin' s structural defects. Thus, when a diabetic patient is administered with such insulin for a long period of time, antibodies continuously produced neutralize the administered insulin, thus causing the patient to require an increased dose of insulin. In this regard, many attempts have been made to develop insulin for oral administration. However, since insulin is structurally unstable, easily degraded in the gastrointestinal tract, and poorly absorbed due to its large molecular weight, such attempts are still in experimental stages.

Generally used oral hypoglycemic agents are classified into sulfonylureas, biguanides, alpha- glucosidase inhibitors, and the like (Deruiter, Endocrine Pharmacology Module, Spring, 2003) . The sulfonylureas include glipizide, gliclazide, gliquidone, glibenclamide and chlorpropamide, and stimulate pancreatic insulin secretion. Thus, the sulfonylureas cannot be applied to insulin- dependent diabetic patients who never secrete insulin in the pancreas, and their use is thus limited to insulin- independent diabetic patients who have relatively decreased insulin secretion ability. However, the sulfonylureas cannot be used to treat women of child-bearing age because they may cause deformed child birth, miscarriage, stillbirth, and the like. Also, the sulfonylureas cause hypoglycemia when administered in excessive amounts or upon fasting along with side effects including skin irritation, jaundice, poor appetite, nausea and diarrhea. The biguanide drugs, such as metformin, have a weak lowering effect on blood glucose compared to the sulfonylureas but rarely cause low blood sugar (hypoglycemia) . However, the biguanides have some gastrointestinal side effects including nausea, vomiting, diarrhea and irritation, and these side effects occur with high frequency at early stages of therapy. The biguanides also cause lactic acidosis and thus may have life-threatening side effects. Thus, in America, the biguanides are currently used only for experimental purpose.

As described above, today's approaches for controlling diabetes mellitus are limited to several methods, and negatively affect the body because they control glucose metabolism in a fashion different from the control system of the body. Thus, there is a need for the development of drugs capable of replacing insulin or oral hypoglycemic agents.

Disclosure of the Invention

The present invention relates to a composition for lowering blood glucose comprising a hexose monophosphate, a derivative thereof or a salt thereof.

Brief Description of the Drawings

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

FIG. 1 is a graph showing the changes in blood glucose when D-glucose-alpha-1-phosphate is administered to normal mice in an oral glucose tolerance test (V.C: vehicle control, •: 30 mg/kg, A: 100 mg/kg, T: 300 mg/kg);

FIG. 2 shows the hypoglycemic effect of D-glucose- alpha-1-phosphate in a streptozotocin-induced diabetic mouse model (a: normal group, b: streptozotocin alone, c: streptozotocin + sterile DW(distilled water), d: 30 mg/kg, e:

100 mg/kg, f: 300 mg/kg, numbers 1 to 8: mouse number);

FIG. 3 is a graph showing the effects of D-glucose- alpha-1-phosphate and D-fructose-6-phosphate on serum insulin levels in a streptozotocin-induced diabetic mouse model;

FIG. 4 is a graph showing the effects of D-glucose- alpha-1-phosphate of inhibiting insulitis and stimulating insulin secretion in a streptozotocin-induced diabetic mouse model (control: normal group, STZ: streptozotocin alone, vehicle control: streptozotocin + sterile DW (distilled water) ) ; FIG. 5 is a graph showing the final blood glucose levels of a mouse model (db/db) of type 2 diabetes repeatedly administered with D-glucose-alpha-1-phosphate;

FIG. 6 is a graph showing the hypoglycemic effect of D-glucose-alpha-1-phosphate in an oral glucose tolerance test using a mouse model (db/db) of type 2 diabetes (■: vehicle control, •: 30 mg/kg, ▲: 100 mg/kg, T: 300 mg/kg) ;

FIG. 7 is a graph showing the changes in blood glucose when D-fructose-6-phosphate is administered to normal mice in an oral glucose tolerance test (V.C: vehicle control, •: 30 mg/kg, A: 100 mg/kg, T: 300 mg/kg);

FIG. 8 shows the hypoglycemic effect of D-fructose-6- phosphate in a streptozotocin-induced diabetic mouse model

(a: normal group, b: streptozotocin alone, c: streptozotocin

+ sterile DW(distilled water), d: 30 mg/kg, e: 100 mg/kg, f: 300 mg/kg, numbers 1 to 8: mouse number);

FIG. 9 is a graph showing the effects of D-fructose- 6-phosphate of inhibiting insulitis and stimulating insulin secretion in a streptozotocin-induced diabetic mouse model (control: normal group, STZ: streptozotocin alone, vehicle control: streptozotocin + sterile DW(distilled water)); FIG. 10 is a graph showing the final blood glucose levels of a mouse model (db/db) of type 2 diabetes repeatedly administered with D-fructose-6-phosphate; and

FIG. 11 is a graph showing the hypoglycemic effect of D-fructose-6-phosphate in an oral glucose tolerance test using a mouse model (db/db) of type 2 diabetes (■: vehicle control, •: 30 mg/kg, A: 100 mg/kg, T: 300 mg/kg) .

Best Mode for Carrying Out the Invention

The present invention provides a composition for lowering blood glucose level comprising a hexose monophosphate, a derivative or a salt thereof.

The term "hexose monophosphate", as used herein, means a six-carbon sugar that has one phosphate group at the first carbon position of the sugar, and includes a naturally occurring sugar or a synthesized sugar. The hexose comprising the hexose monophosphate of the present invention includes a six carbon sugar itself and derivatives thereof, for example, glucose, fructose, galactose, gulose, rhamnose, mannose, sorbose, allose, altrose, idose, tagatose, talose, fucose and derivatives thereof. Glucose or fructose may be preferred.

Sugars having one or more asymmetric carbons are present in a D or L from according to the absolute configuration of an asymmetric carbon the farthest away from an aldehyde or ketone group. The hexose of the present invention may be in a D or L form, and preferably a D form.

The monophosphate comprising the hexose monophosphate of the present invention refers to the phosphate attached to the C-I position of the hexose. The phosphate may be attached to any one of carbons 1 to 6, and preferably to carbon 1, 2 or 6.

Examples of the hexose monophosphate of the present invention may include D-glucose-alpha-1-phosphate, D- glucose-beta-1-phosphate, D-glucose-6-phosphate, D- galactose-1-phosphate, D-galactose-6-phosphate, D-mannose- alpha-1-phosphate, D-mannose-6-phosphate, D-allose-1- phosphate, D-allose-6-phosphate, D-altrose-1-phosphate, D- altrose-6-phosphate, D-rhamnose-1-phosphate, D-idose-1- phosphate, D-idose-6-phosphate, D-fucose-6-phosphate, D- gulose-1-phosphate, D-gulose-6-phosphate, L-sorbose-2- phosphate, D-tagatose-2-phosphate, D-talose-1-phosphate, D- talose-6-phosphate, and salts thereof.

Hexose derivatives of the present invention include non-substituted or substituted aminohexoses and deoxyhexoses. Aminohexoses include glucosamine, galactosamine and mannosamine, which are exemplified by D- glucosamine-1-phosphate, D-glucosamine-6-phosphate, N- acetyl-D-glucosamine-6-phosphate, D-galactosamine-1- phosphate, D-galactosamine-6-phosphate, N-acetyl-D- galactosamine-1-phosphate, N-acetyl-D-galactosamine-6- phosphate, D-mannosamine-1-phosphate, D-mannosamine-6- phosphate, N-acetyl-D-mannosamine-1-phosphate, N-acetyl-D- mannosamine-6-phosphate, and salts thereof.

In addition, the deoxyhexose refers to a sugar that has a substitution, hydrogen for a hydroxyl group at one or more carbons of a hexose. Examples of the deoxyhexose include 2-deoxy-D-glucose-l-phosphate, 3-deoxy-D-glucose-l- phosphate, 4-deoxy-D-glucose-l-phosphate, 6-deoxy-D- glucose-1-phosphate, 2, 3-dideoxy-D-glucose-l-phosphate, 2, 4-dideoxy-D-glucose-l-phosphate, 2, 6-dideoxy-D-glucose-l- phosphate, 3, 4-dideoxy-D-glucose-l-phosphate, 3, 6-dideoxy- D-glucose-1-phosphate, 4, β-dideoxy-D-glucose-1-phosphate, l-deoxy-D-fructose-6-phosphate, 3-deoxy-D-fructose-6- phosphate, 4-deoxy-D-fructose-6-phosphate, 1,3-dideoxy-D- fructose-6-phosphate, 1, 4-dideoxy-D-fructose-6-phosphate, 3, 4-dideoxy-D-fructose-6-phosphate, and salts thereof.

A carbon of the hexose may be replaced with a straight-linear or branched alkyl group of Ci to C^. For example, one or more carbons at carbon positions 1 to 6 may be replaced with methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, hexyl, isohexyl, heptyl, isoheptyl, and the like. The present composition comprising . hexose monophosphate may include one type of hexose monophosphate or a combination of several types of hexose monophosphate.

The hexose monophosphate of the present invention may be prepared in a salt form of being linked to a cation. The cation includes sodium salts, potassium salts, magnesium salts, calcium salts, lithium salts, rubidium salts, cesium salts, beryllium salts, strontium salts, barium salts, aluminum salts, boron salts, gallium salts, copper salts, silver salts, zinc salts, cadmium salts, mercury salts, scandium salts, nickel salts, manganese salts, chromium salts, valium salts, and titanium salts.

The hexose monophosphate of the present invention could be preserved as it' s own form or in an aqueous solution, and is advantageous in terms of being rarely degraded or deteriorated even upon long-term preservation because it is a chemically-stable oxidized product of carbohydrates.

In a preferred aspect, the present invention provides a composition for lowering blood glucose level comprising a glucose monophosphate or a fructose monophosphate, a derivative thereof, or a salt thereof. The present composition may comprise glucose monophosphate or fructose monophosphate alone, or a combination thereof. In a more preferred aspect, the present invention provides a composition for lowering blood glucose level comprising D-glucose-alpha-1-phosphate or D-fructose-β- phosphate, a derivative thereof, or a salt thereof.

The D-glucose-alpha-1-phosphate or D-fructose-6- phosphate used in the present invention may be synthesized by an enzymatic reaction using starch or glucose, or fructose as a raw material, or may be commercially available.

D-glucose-alpha-1-phosphate may be typically synthesized by an enzymatic reaction of α-glucan phospholyase in the presence of inorganic phosphate

(orthophosphate) using alpha-glucan (e.g., linear amylose, dextrin, starch, amylopectin, glycogen, etc.). The enzymatic reaction is preferably carried out at high temperature to reduce microbial infection in samples and increase the solubility of starch as a substrate. To achieve this purpose, an alpha-glucan phospholyase derived from a thermophilic bacterium (e.g.,

caldophilus GK24), which is able to exhibit its activity at 70⁰C, is preferred. In addition, D-fructose-6-phosphate may be prepared as follows. An enzymatic reaction of alpha-glucan phospholyase using alpha-glucan (starch, glycogen, amylopectic, etc.) is carried out to generate D-glucose-6- phosphate, and another enzymatic reaction of glucose-6- phosphate isomerase is- then allowed to generate D-fructose- β-phosphate. An alternative enzymatic method is to employ fructose kinase using fructose in the presence of ATP. Upon large-scale production, in order to prevent a reactor from being contaminated with a microorganism and increase the solubility of a polymeric substrate such as starch, an enzyme reaction should be carried out at high temperature.

Thus, a thermostable enzyme exhibiting its activity at 70⁰C is also preferable in the preparation of D-fructose-6- phosphate.

The reaction products may be isolated from reaction solutions by chromatography or other methods known in the art.

The present invention relates to a method of enhancing the production and secretion of insulin and lowering blood glucose level using a composition comprising a hexose monophosphate, a derivative thereof or a salt thereof.

Drugs should be absorbed by the body so as to display their functions in vivo. Gastrointestinal absorption of drugs depends on absorption surface area, blood flow at absorption sites, pharmaceutical formulations of drugs, physical properties such as solubility, and the like. In the gastrointestinal tract, since drugs are passively- absorbed, nonionic and liphophilic properties increase drug absorption. However, the hexose monophosphate of the present invention, despite its property of being negatively charged due to its phosphate group, is absorbed in the body upon oral administration and exerts its biological functions, such as lowering blood sugar level and stimulating insulin secretion. Sugar metabolites having two phosphate groups, such as D-fructose-1, 6-diphosphate, appear to be poorly absorbed because they do not affect blood sugar control. In contrast, D-glucose-alpha-1- phosphate and D-fructose-6-phosphate exhibit significant biological control functions, including stimulating insulin production and secretion in the pancreas, increasing blood insulin levels and lowering blood glucose, indicating that hexose monophosphate, having one phosphate group, can be effectively introduced into the body through oral administration.

The present composition functions to control blood glucose level by activating sugar metabolism-controlling functions of the body. When D-glucose-alpha-1-phosphate and D-fructose-β-phosphate were administered to streptozotocin- induced diabetic mice, insulin production was sustained for several days. These results indicate that D-glucose-alpha- 1-phosphate and D-fructose-β-phosphate serve as signal molecules for increasing the expression of the insulin gene. Therefore, the hexose monophosphate of the present invention, such as D-glucose-alpha-1-phosphate or D- fructose-6-phosphate, stimulates the pancreatic beta cells to increase the expression of the insulin gene and to suitably secrete the produced insulin to the blood stream, resulting in an increase in serum insulin levels and a decrease in blood glucose levels. Thus, the hexose monophosphate controls blood glucose in a manner similar to the body's sugar metabolism control functions. In an embodiment, when a composition comprising D- glucose-alpha-1-phosphate or D-fructose-6-phosphate was orally administered to normal mice, a db/db mouse model of type 1 diabetes and a db/db mouse model of type 2 diabetes, in all of the tested mice, insulin production increased in the pancreatic beta cells, and blood sugar subsequently decreased with elevated serum insulin levels.

Type 1 diabetes may be induced in mice by administration of streptozotocin capable of selectively disrupting the pancreatic beta cells. In embodiments of the present invention, when D-glucose-alpha-1-phosphate and D- fructose-6-phosphate were individually administered to streptozotocin-induced diabetic mice, both of them exhibited inhibitory effects against an increase in blood glucose levels. When administered in low doses, D-fructose- 6-phosρhate has stronger inhibitory effects against blood glucose increase. When administered in high doses, D- glucose-alpha-1-phosphate was found to have an inhibitory effect against blood glucose increase similar to or slightly better than D-fructose-β-phosphate (Examples 2 and 5) .

In addition, when insulin levels were measured in blood samples collected from streptozotocin-induced diabetic mice administered with the hexose monophosphate of the present invention, D-glucose-alpha-1-phosphate and D- fructose-6-phosphate all increased serum insulin levels in a dose-dependent manner, and reduced blood glucose at levels similar to the increased insulin levels (Examples 2 and 5) . D-fructose-β-phosphate exhibited stronger effects both on increasing serum insulin and lowering blood glucose than D-glucose-alpha-1-phosphate. Thus, hexose monophosphate, such as D-glucose-alpha-1-phosphate or D- fructose-β-phosphate, is a potential insulin-substituting drug applicable to diabetic patients.

When pancreatic tissue samples from normal mice and streptozotocin-induced diabetic mice, which were administered with the present composition, were subjected to selective insulin staining and microscopically observed, insulin levels present in the pancreatic beta cells were consistent with the decreased levels of blood glucose and the increased levels of blood insulin, and the distribution of beta cells containing insulin increased in the pancreas. These results indicate that the present composition comprising hexose monophosphate stimulates the pancreatic beta cells to produce and secrete insulin.

The present composition also has the effect of lowering a blood glucose level on type 2 diabetes. In an embodiment, when D-glucose-alpha-1-phosphate and D- fructose-6-phosphate were administered to a db/db mouse model of type 2 diabetes ^" for two weeks or in an oral glucose tolerance test, both of them were found to reduce blood glucose levels (Examples 3 and 6) . As described above, the present composition comprising hexose monophosphate is applicable to patients with type 1 diabetes as well as type 2 diabetes with an aim of lowering blood glucose levels.

Thus, the present invention provides a method of treating diabetes mellitus and associated symptoms using the present composition. Diabetes mellitus and associated symptoms include insulin-dependent diabetes (type 1 diabetes), insulin-independent diabetes (type 2 diabetes), insulin resistance, hyperinsulinemia and hypertension induced by diabetes mellitus. Other symptoms associated with diabetes mellitus include obesity and damage to blood vessels, eyes, the kidney, nerves, the autonomic nervous system, the skin, the connective tissue, the genital system and the immune system. The term "treatment", as used herein, refers to the prevention, inhibition and reduction of diabetes mellitus or associated symptoms. The present composition may be administered alone in a therapeutically effective amount or may be administered along with a therapeutically effective amount of insulin with an aim of lowering blood glucose. Also, the present composition may be administered along with other hypoglycemic agents in order to treat type 2 diabetes, insulin resistance, hyperinsulinemia, hypertension induced by diabetes mellitus, obesity, and damage to blood vessels, eyes, the kidney, nerves, the autonomic nervous system, the skin, the connective tissue, the genital system and the immune system.

The term "therapeutically effective amount", as used herein, means an amount needed to prevent diabetes mellitus or associated symptoms, or an amount needed to treat or alleviate the disease. Typically, when the present composition is administered along with insulin and/or a hypoglycemic agent, the insulin and/or hypoglycemic agent may be administered in smaller amounts than when administered alone. The use of the insulin and/or hypoglycemic agent in smaller amounts may lead to a decrease in their adverse effects and delay the incidence of complications of diabetes mellitus or associated diseases.

Examples of hypoglycemic agents capable of being used along with the present composition include metformin, acarbose, acetohexamide, glimepiride, tolazamide, glipizide, glyburide, tolbutamide, chlorpropamide, thiazolidinediones, alpha-glucosidase inhibitors, biguanide derivatives, troglitazone, and mixtures thereof. The present composition may be preferably administered orally, or subcutaneously, intramuscularly, intravenously in an injectable formulation, or in the form of a spray or an ointment. For these types of administration, the present composition may be formulated into tablets, capsules, powders, granules, microgranules, microspheres, suspensions, emulsions, or single-dose or multidose preparations such as syrups or injectable preparations. The present composition is capable of being absorbed in the body even when orally administered, and may be preferably administered orally for convenient administration, thereby overcoming the inconvenience and side effects due to frequent insulin injections.

To be formulated into a pharmaceutical preparation, the present composition may be mixed with one or more inert, non-toxic, solid or liquid pharmaceutically acceptable carriers. Available pharmaceutical carriers include physiological saline, Ringer's solution, phosphate- buffered saline and others known in the art. Also, a pharmaceutical composition may further include stabilizers, antioxidants, excipients, dyes, binders, precipitating agents, dispersing agents, diluting agents, surfactants, and preservatives. For example, available are inorganic substances, such as calcium carbonate, magnesium carbonate, calcium phosphate tribasic, magnesium phosphate, kaolin, talc, magnesium stearate, silicon dioxide, titanium dioxide, zirconium dioxide and colloidal silica, cellulose and derivatives thereof, alginate, carraginate, chitosan derivatives, vegetable rubber, such as tragacanth rubber, guar gum and derivatives thereof, xanthan gum, starch, maltodextrin and organic substances of vegetable oils.

In addition, the present composition may be formulated into edible solid or liquid food, or a cosmetic.

Daily dosage of the hexose monophosphate of the present invention may be 30-300 mg per kg mouse body weight and 10-100 mg per kg human body weight, and may be divided into several smaller doses. The dosage for a particular patient may vary according to the patient's weight, age and gender, state of health and diet, administration duration, administration methods, excretion rates, and severity of the illness. The present composition comprising hexose monophosphate never exhibited acute toxicity when orally administered to mice in high doses. The present composition did not induce antibody production upon administration to the body, thereby overcoming problems resulting from insulin resistance due to antibody production, which may emerge upon administration of genetically engineered insulin or biochemically synthesized insulin.

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

EXAMPLE 1: Measurement of the hypoglycemic effect of D- glucose-alpha-1-phosphate by oral glucose tolerance test

An oral glucose tolerance test is used for measuring the body' s ability to secrete insulin and insulin action upon an increase of blood sugar levels in order to collectively predict the function of the beta cells of the pancreas and D-glucose uptake of target cells.

Six week-old normal mice (ICR, Daehan Biolink Co. Ltd., Korea) weighing about 22 g were grouped (6 mice per test group) and starved for 16 hrs before being used in this test. D-glucose-alpha-1-phosphate (Sigma Co.) was dissolved in sterile distilled water in a concentration of 30 mg/ml, diluted, and orally administered to mice in doses of 30, 100 and 300 mg/kg body weight. After 90 min, D- glucose dissolved in physiological saline was orally administered to the mice in a dose of 2 g/kg and a volume of 10 ml/kg. Blood glucose levels were measured before

(time zero) and 15, 60 and 120 min after D-glucose administration. For measurement of blood glucose levels, blood samples were collected from the orbital sinus of the mice using capillary tubes. Blood glucose levels were measured using an Accutrend alpha blood glucose meter

(Boehringer Mannheim Co.) . A control was administered with an equal volume of sterile distilled water instead of D- glucose-alpha-1-phosphate. When D-glucose was administered, in all of the tested mice, blood glucose levels were elevated, with the highest levels at 15 min, and thereafter decreased to normal levels after 120 min. At 15 min, at the highest blood glucose levels, the blood glucose of the control group increased by 261.5 mg/dl compared to before D-glucose administration. In contrast, when D-glucose-alpha-1-phosphate was administered to mice in doses of 30, 100 and 300 mg/kg, it displayed antihyperglycemic effects of 38.6% (p<0.01), 37.2% and 29.3% (p<0.05), respectively, compared to the control group. These results indicate that D-glucose-alpha-1- phosphate has an effect of lowering blood glucose (hypoglycemic effect) upon oral administration (Table 1 and FIG. 1) .

TABLE 1

The hypoglycemic effect of D-glucose-alpha-1-phosphate upon oral administration in oral glucose tolerance test

^*p<0.05, ^**p<0.01 ^a Antihyperglycemic rate (%)= [(sample treatment group/control group) increase in blood glucose x 100] - 100

GlP: D-glucose-alpha-1-phosphate EXAMPLE 2: Evaluation of the hypoglycemic effect and blood insulin-increasing effect of D-glucose-alpha-1-phosphate in streptozotocin-induced diabetic mice

Streptozotocin is a substance capable of inducing insulin-dependent diabetes mellitus, which is known to selectively disrupt the beta cells of the pancreas. Streptozotocin (Sigma Co.) was dissolved in citrate buffer (pH 4.2) immediately before being used. Streptozotocin was intraperitoneally administered to mice (ICR) in a dose of 50 mg/kg three times to induce diabetes mellitus. Each test group was composed of eight mice, and mean blood glucose was calculated.

On Day 11 of the test period, normal mice not administered with streptozotocin showed a mean blood glucose of 175.3 mg/dl, and mice (ICR) administered with streptozotocin and sterile distilled water displayed a blood glucose increase by 219.0 mg/dl compared to that measured on Day 0, indicating that diabetes mellitus was induced (Table 2) . When streptozotocin (50 mg/kg, intraperitoneally administered three times) was administered along with D- glucose-alpha-1-phosphate of 30, 100 and 300 mg/kg, blood glucose increased by 237.8, 174.2 and 82.7 mg/dl, respectively. D-glucose-alpha-1-phosphate did not have an inhibitory effect against an increase of blood glucose in a dose of 30 mg/kg. In contrast, the administration of 100 and 300 mg/kg of D-glucose-alpha-1-phosphate resulted in antihyperglycemic rates of 20.5% and 62.2% in comparison with the control group administered with streptozotocin and sterile distilled water (Table 2 and FIG. 2) .

TABLE 2

The hypoglycemic effect of D-glucose-alpha-1-phosphate in streptozotocin-induced diabetic mice

^*p<0.05 ^a Antihyperglycemic rate (%) = [ (sample treatment group/control group) increase in blood glucose x 100] - 100

STZ : Streptozotocin

GlP: D-glucose-alpha-1-phosphate

In addition, after all of the tested mice were sacrificed on Day 11, serum samples were collected, and serum insulin levels were measured using a mouse insulin ELISA kit (Schibayagi, Japan) . That is, an anti-insulin coated plate was washed with washing buffer (Schibayagi buffer) four times, and 100 μl of a biotin-conjugated anti- insulin and 10 μl of a plasma sample were added to each well of the anti-insulin coated plate and incubated at room temperature for 2 hrs. After the plate was washed with the washing buffer (I) four times, 100 μl of an HRP-conjugated streptavidin solution was added to each well and incubated at room temperature for 30 min. After the plate was again washed with the washing buffer four times, 100 μl of a substrate chromogen reagent was added to each well and allowed to react at room temperature for 30 min. Then, 100 μl of a Reagent stopper was added to each well to terminate the reaction. Absorbance was measured at 450 nm using a Precision Microplaste Reader (Molecular Devices, USA) . As a result, when 300 mg/kg of D-glucose-alpha-1-phosphate was administered along with streptozotocin, the maximum serum insulin levels were six times in comparison with when streptozotocin and sterile distilled water were administered (Table 3 and FIG. 3) .

TABLE 3

The effect of D-glucose-alpha-1-phosphate on serum insulin levels in streptozotocin-induced diabetic mice

STZ : Streptozotocin On the other hand, a histopathological test was carried out to determine whether D-glucose-alpha-1- phosphate has an inhibitory effect on insulitis induced by the destruction of the beta cells by streptozotocin and a stimulatory effect on pancreatic insulin production and/or secretion. In insulin-dependent diabetes mellitus, immune cells such as macrophages or T cells invade the islet of the pancreas and destroy the beta cells, thus causing insulitis. To investigate the inhibition of insulitis and insulin production by the pancreatic beta cells upon hexose monophosphate administration, after mice administered with D-glucose-alpha-1-phosphate were sacrificed, their chests were opened to excise the pancreas, present between the duodenum and the spleen, using surgical scissors. The excised pancreas was fixed with 10% neutral formalin, embedded in paraffin according to a general method, and sectioned into a thickness of 5 μm using a microtome. Then, the tissue sections were subjected to insulin immunostaining using an ABC kit (Vector Co.) containing an anti-human guinea pig insulin antibody.

As a result of the histopathological test, when mice were administered with streptozotocin alone, the number of insulin-positive beta cells markedly decreased in comparison with a normal control group. In contrast, administration with D-glucose-alpha-1-phosphate resulted in an increase in the number of insulin-positive cells and an enhancement in staining degree in a dose-dependent manner, indicating that D-glucose-alpha-1-phosphate has a strong inhibitory effect against insulitis and a strong stimulatory effect on insulin secretion (FIG. 4, three photographs per test group) . These results indicate that D- glucose-alpha-1-phosphate effectively reduces blood glucose levels and strongly stimulates insulin production/secretion in diabetes mellitus induced by streptozotocin administration.

EXAMPLE 3: Evaluation of the therapeutic effect of D- glucose-alpha-1-phosphate upon oral administration in db/db mouse model of type 2 diabetes (insulin-independent diabetes)

D-glucose-alpha-1-phosphate was evaluated for a lowering effect on blood glucose when orally administered to a db/db mouse model of type 2 diabetes (insulin- independent diabetes) . In type 2 diabetes, insulin resistance increases in peripheral tissues due to obesity and other factors, thereby bringing about hyperinsulinemia. The beta cells of patients with type 2 diabetes exhibit reduced response to insulin secretion due to elevated blood sugar, and thus, do not recognize that sugars are secreted into urine and continuously secrete insulin. This mouse model (db/db) exhibiting these symptoms is the most- characterized genetic animal model of diabetes mellitus for studies of insulin-independent diabetes mellitus in humans. Mice (db/db, 21-31g, SLC Co.) were orally administered with D-glucose-alpha-1-phosphate in doses of 30, 100 and 300 mg/kg once per day for two weeks from 5.5 weeks to 7.5 weeks, and blood glucose levels were measured on the last day of administration. Model mice (db/db) of a control group administered with an equal volume of sterile distilled water instead of D-glucose-alpha-1-phosphate exhibited a mean blood glucose level of 700.8 mg/dl on the last day of administration. In contrast, model mice (db/db) of a test group administered with D-glucose-alpha-1-phosphate in doses of 30, 100 and 300 mg/kg displayed blood glucose levels of 711.1, 662.4 and 543.1 mg/dl, respectively, indicating that D-glucose- alpha-1-phosphate has a lowering effect on blood glucose in a mouse model of type 2 diabetes (Table 4 and FIG. 5) .

TABLE 4

Final blood glucose levels in db/db mouse model of type 2 diabetes repeatedly administered with D-glucose-alpha-1- phosphate

GlP: D-glucose-alpha-1-phosphate

In addition, an oral glucose tolerance test was carried out in a db/db mouse model of type 2 diabetes

(insulin-independent diabetes) according to the same method as in Example 1 in order to evaluate the hypoglycemic effect of D-glucose-alpha-1-phosphate.

D-glucose-alpha-1-phosphate (Sigma Co.) was orally administered to model mice (db/db) starved for 16 hrs in doses of 30, 100 and 300 mg/kg body weight. After 90 min, D-glucose was orally administered to the mice in a dose of 2 g/kg and a volume of 10 ml/kg. Blood glucose levels were measured before (time zero) and 15, 30, 60, 120 and 180 min after D-glucose administration. A control was orally administered with an equal volume of sterile distilled water instead of D-glucose-alpha-1-phosphate.

When D-glucose was administered, in all of the tested mice, blood glucose levels were elevated to the highest levels after 15-30 min. In the control group, the time required for the elevated blood glucose level to come back down to the initial blood glucose level after D-glucose administration was about 180 min, and in the treatment groups administered with D-glucose-alpha-1-phosphate of 30, 100 and 300 mg/kg, the time was 180 min, 120 min and 30 min, respectively (Table 5 and FIG. 6) . These results indicate that D-glucose-alpha-1-phosphate has a strong hypoglycemic effect in a mouse model (db/db) of type 2 diabetes.

TABLE 5

The hypoglycemic effect of D-glucose-1-phosphate in oral glucose tolerance test using db/db mouse model of type 2 diabetes

GlP: D-glucose-alpha-1-phosphate

EXAMPLE 4: Measurement of the hypoglycemic effect of D- fructose-6-phosphate by oral glucose tolerance test

Six week-old normal mice (ICR, Daehan Biolink Co. Ltd., Korea) weighing about 22 g were grouped (6 mice per test group) and starved for 16 hrs before being used in this test. D-fructose-6-phosphate (Sigma Co.) was dissolved in sterile distilled water in a concentration of 30 mg/ml, diluted, and orally administered to mice in doses of 30, 100 and 300 mg/kg body weight. After 90 min, D-glucose dissolved in physiological saline was orally administered to the mice in a dose of 2 g/kg and a volume of 10 ml/kg.

Blood glucose levels were measured before (time zero) and 15, 60 and 120 min after D-glucose administration. For measurement of blood glucose levels, blood samples were collected from the orbital sinus of the mice using capillary tubes. Blood glucose levels were measured using an Accutrend alpha blood glucose meter (Boehringer Mannheim Co.) - A control was administered with an equal volume of sterile distilled water instead of D-fructose-β- phosphate.

When D-glucose was administered, in all of the tested mice, blood glucose levels were elevated to the highest levels at 15 min, and thereafter, decreased to normal levels after 120 min. At 15 min, at the highest blood glucose levels, the blood glucose of the control group increased by 256.1 mg/dl compared to before D-glucose administration. In contrast, when D-fructose-6-phosphate was administered to mice in doses of 30, 100 and 300 mg/kg, it displayed antihyperglycemic effects of 25.0, 48.4% (p<0.001) and 66.5% (p<0.001), respectively, compared to the control group. These results indicate that D-fructose- 6-phosphate has an effect of lowering blood glucose upon oral administration (Table 6 and FIG. 7) .

TABLE 6

The hypoglycemic effect of D-fructose-6-phosphate upon oral administration in oral glucose tolerance test

"^*p<0.01 ^a Antihyperglycemic rate (%)= [(sample treatment group/control group) increase in blood glucose x 100] - 100

F6P: D-fructose-6-phosphate

EXAMPLE 5: Evaluation of the hypoglycemic effect and blood insulin-increasing effect of D-fructose-6-phosphate in streptozotocin-induced diabetic mice

D-fructose-6-phosphate was evaluated for its effects of lowering blood glucose and increasing blood insulin in streptozotocin-induced diabetic mice according to the same method as in Example 2.

Mice (ICR) not administered with streptozotocin exhibited a final blood glucose level of 175.3 mg/dl, and mice (ICR) administered with streptozotocin and sterile distilled water showed a blood glucose level increased by 219.0 mg/dl compared to the initial blood glucose level. In contrast, model mice (db/db) of a test group administered with streptozotocin and D-fructose-6-phosphate of 30, 100 and 300 mg/kg displayed blood glucose levels of 203.1, 176.0. and 89.2 mg/dl, respectively, and thus had antihyperdemic effects of 7.3%, 19.6% and 59.3% compared to the control group, indicating that D-fructose-6-phosphate has an antihyperdemic effect in a mouse model (db/db) of type 2 diabetes (Table 7 and FIG. 8) . Compared to when D- glucose-alpha-1-phosphate was administered in Example 2, in low doses, D-fructose-6-phosphate had stronger antihyperdemic effects, and in high doses, D-fructose-6- phosphate had similar or slightly weaker antihyperdemic effects, than D-glucose-alpha-1-phosphate.

TABLE 7

The hypoglycemic effect of D-fructose-6-phosphate in streptozotocin-induced diabetic mice

^**p<0.01 ^a Antihyperglycemic rate (% ) = [ (sample treatment group/control group) increase in blood glucose x 100] - 100

STZ : Streptozotocin

F6P: D-fructose-6-phosphate

In addition, according to the same method as in Example 2 , after all of the tested mice were sacrificed on Day 11 , serum samples were collected, and serum insulin levels were measured using a mouse insulin ELISA kit (Schibayagi, Japan) . As a result, when 300 mg/kg of D- fructose-6-phosphate was administered along with streptozotocin, serum insulin levels were elevated to maximal nine times in comparison with when streptozotocin and sterile distilled water were administered. Herein, serum insulin levels were higher than those of normal mice

(Table 8 and FIG. 3) . These results indicate that D- fructose-6-phosphate has stronger effects of increasing serum insulin and lowering blood glucose than D-glucose- alpha-1-phosphate of Example 2.

TABLE 8

The effect of D-fructose-6-phosphate on serum insulin levels in streptozotocin-induced diabetic mice

STZ: Streptozotocin

In addition, after oral administration with D- fructose-6-phosphate, a histopathological test was carried out to determine whether D-glucose-alpha-1-phosphate has an inhibitory effect on insulitis induced by the destruction of the beta cells by streptozotocin and a stimulatory effect on pancreatic insulin production and/or secretion. After mice were administered with D-fructose-6- phosphate, according to the same method in Example 2, the pancreas was excised from the mice, fixed with formalin, embedded in paraffin and sectioned. Then, the tissue sections were subjected to insulin immunostaining.

As a result of the histopathological test, when mice were administered with streptozotocin alone, the number of insulin-positive beta cells markedly decreased in comparison with a normal control group. In contrast, the administration with D-fructose-6-phosphate resulted in an increase in the number of insulin-positive cells and an enhancement in staining degree in a dose-dependent manner, indicating that D-fructose-6-phosphate has a strong inhibitory effect against insulitis and a strong stimulatory effect on insulin secretion (FIG. 9, three photographs per test group) . These results indicate that D- fructose-6-phosphate effectively reduces blood glucose levels and strongly stimulates insulin production/secretion in diabetes mellitus induced by streptozotocin administration.

EXAMPLE 6: Evaluation of the therapeutic effect of D- fructose-6-phosphate upon oral administration in db/db mouse model of type 2 diabetes (insulin-independent diabetes) D-Fructose-6-phosphate was evaluated for a lowering effect on blood glucose when orally administered to a db/db mouse model of type 2 diabetes (insulin-independent diabetes) . Model mice (db/db) of a control group administered with an equal volume of sterile distilled water instead of D-fructose-β-phosphate exhibited a mean blood glucose level of 700.8 mg/dl on the last day of administration. In contrast, model mice (db/db) of a test group administered with D-fructose-β-phosphate in doses of 30, 100 and 300 mg/kg displayed blood glucose levels of 663.1, 611.6 and 603.0 mg/dl, respectively, indicating that D-glucose-alpha- 1-phosphate has a lowering effect on blood glucose in a mouse model of type 2 diabetes (Table 9 and FIG. 10) .

TABLE 9

Final blood glucose levels in db/db mouse model of type 2 diabetes repeatedly administered with D-fructose-6- phosphate

F6P: D-fructose-6-phosphate

In addition, an oral glucose tolerance test was carried out in a db/db mouse model of type 2 diabetes (insulin-independent diabetes) according to the same method as in Example 1 in order to evaluate the hypoglycemic effect of D-fructose-6-phosphate.

D-fructose-β-phosphate was orally administered to model mice (db/db) starved for 16 hrs in doses of 30, 100 and 300 mg/kg body weight. After 90 min, D-glucose was orally administered to the mice in a dose of 2 g/kg and a volume of 10 ml/kg. Blood glucose levels were measured before (time zero) and 15, 30, 60, 120 and 180 min after D- glucose administration. A control was orally administered with an equal volume of sterile distilled water instead of D-fructose-6-phosphate.

When D-glucose was administered, in all of the tested mice, blood glucose levels were elevated to the highest levels after 15-30 min. In the control group, administered with sterile distilled water, the time required for the elevated blood glucose level to come back down to the initial blood glucose level after D-glucose administration was about 180 min, and in the treatment groups administered with D-fructose-6-phosphate in doses of 30, 100 and 300 mg/kg, the time was 180 min, 180 min and 120 min, respectively (Table 10 and FIG. 11) . These results indicate that D-fructose-6-phosphate has a strong hypoglycemic effect in a mouse model (db/db) of type 2 diabetes.

TABLE 10 The hypoglycemic effect of D-fructose-6-phosphate in oral glucose tolerance test using db/db mouse model of type 2 diabetes

F6P: D-fructose-6-phosphate

Industrial Applicability

As described hereinbefore, the present composition comprising a hexose monophosphate, a derivative thereof or a salt thereof stimulates insulin production by the pancreatic beta cells and increases serum insulin levels, thereby lowering blood sugar.

Claims

1. A composition for lowering blood glucose level comprising a hexose monophosphate, a derivative thereof or a salt thereof.

2. The composition according to claim 1, wherein the hexose is selected from among glucose, fructose, galactose, gulose, rhamnose, mannose, sorbose, allose, altrose, idose, tagatose, talose, and fucose.

3. The composition according to claim 1, wherein the hexose monophosphate is selected from the group of hexose-

1-phosphate, hexose-2-phosphate, and hexose-β-phosphate.

4. The composition according to claim 1, wherein the hexose monophosphate is glucose monophosphate or fructose monophosphate.

5. The composition according to claim 1, wherein the hexose monophosphate has one or more carbons replaced with methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, hexyl, isohexyl, heptyl or isoheptyl group.

6. The composition according to claim 1, wherein the salt is selected from the group of sodium salts, potassium salts, magnesium salts, calcium salts, lithium salts, rubidium salts, cesium salts, beryllium salts, strontium salts, barium salts, aluminum salts, boron salts, gallium salts, copper salts, silver salts, zinc salts, cadmium salts, mercury salts, scandium salts, nickel salts, manganese salts, chromium salts, valium salts, and titanium salts.

7. The composition according to claim 1, wherein the hexose monophosphate is selected from the group of D- glucose-alpha-1-phosphate, D-glucose-beta-1-phosphate, D- glucose-6-phosphate, D-galactose-1-phosphate, D-galactose- 6-phosphate, D-mannose-alpha-1-phosphate, D-mannose-6- phosphate, D-allose-1-phosphate, D-allose-6-phosphate, D- altrose-1-phosphate, D-altrose-β-phosphate, D-rhamnose-1- phosphate, D-idose-1-phosphate, D-idose-β-phosphate, D- fucose-β-phosphate, D-gulose-1-phosphate, D-gulose-6- phosphate, L-sorbose-2-phosphate, D-tagatose-2-phosphate, D-talose-1-phosphate, and D-talose-6-phosphate.

8. The composition according to claim 7, wherein the hexose monophosphate is D-glucose-alpha-1-phosphate.

9. The composition according to claim 7, wherein the hexose monophosphate is D-fructose-6-phosphate.

10. The composition according to claim 1, wherein the derivative of the hexose monophosphate is aminohexose monophosphate or deoxyhexose monophosphate.

11. The composition according to claim 10, wherein the derivative of the hexose monophosphate is selected from the group of D-glucosamine-1-phosphate, D-glucosamine-6- phosphate, D-fructose-6-phosphate, D-fructose-2-phosphate, N-acetyl-D-glucosamine-1-phosphate, N-acetyl-D-glucosamine- 6-phosphate, D-galactosamine-1-phosphate, D-galactosamine- 6-phosphate, N-acetyl-D-galactosamine-l-phosphate, N- acetyl-D-galactosamine-6-phosphate, D-mannosamine-1- phosphate, D-mannosamine-6-phosphate, N-acetyl-D- mannosamine-1-phosphate, and N-acetyl-D-mannosamine-β- phosphate.

12. The composition according to claim 10, wherein the derivative of the hexose monophosphate is selected from the group of 2-deoxy-D-glucose-l-phosphate, 3-deoxy-D- glucose-1-phosphate, 4-deoxy-D-glucose-l-phosphate, 6- deoxy-D-glucose-1-phosphate, 2, 3-dideoxy-D-glucose-l- phosphate, 2, 4-dideoxy-D-glucose-l-phosphate, 2,6-dideoxy- D-glucose-1-phosphate, 3, 4-dideoxy-D-glucose-l-phosphate, 3, 6-dideoxy-D-glucose-l-phosphate, 4, 6-dideoxy-D-glucose-l- phosphate, 1-deoxy-D-fructose-^β-phosphate, 3-deoxy-D- fructose-6-phosphate, 4-deoxy-D-fructose-β-phosphate, 1,3- dideoxy-D-fructose-6-phosphate, 1, 4-dideoxy-D-fructose-6- phosphate, and 3,4-dideoxy-D-fructose-6-phosphate.

13. The composition according to any one of claims 1 to 12, which is formulated into an oral preparation, an aerosol spray, an ointment or an injectable preparation.

14. The composition according to any one of claims 1 to 12, which is formulated into a solid or liquid food or a cosmetic.