AMELIORATING AGENT FOR OBESITY-RELATED METABOLIC DISEASE

Abstract

An object of the present invention is to provide a drug containing, as an active ingredient, a low-molecular-weight compound that ameliorates obesity-related metabolic diseases, for example, exhibits anti-obesity action without reduction of food intake through binding to Helz2 to lower its action, on the basis of the fact that Helz2 is intensely expressed in the liver both in humans and mice, and expression levels are significantly increased in the human liver with NAFLD, as an obesity-related metabolic disease, and in fatty liver in obese mice. The present invention provides an ameliorating agent for an obesity-related metabolic disease, in particular, an ameliorating agent for fatty liver, the ameliorating agent characterized by containing guanabenz or a salt of guanabenz as an active ingredient.

Claims

1.-14. (canceled)

15. A method for ameliorating a fatty liver comprising administrating guanabenz or a salt of guanabenz to a patient in need thereof.

16. The method for ameliorating a fatty liver according to claim 15, wherein guanabenz acetate is administered to a patient in need thereof.

17. The method for ameliorating a fatty liver according to claim 15, wherein the fatty liver is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

18. The method for ameliorating a fatty liver according to claim 15, wherein the fatty liver is caused by insulin resistance.

19. The method for ameliorating a fatty liver according to claim 15, wherein the fatty liver is accompanied by obesity disease.

20. The method for ameliorating a fatty liver according to claim 15, wherein the fatty liver is accompanied by hypertension.

21. The method for ameliorating a fatty liver according to claim 15, wherein the fatty liver is accompanied by metabolic syndrome.

22. The method for ameliorating a fatty liver according to claim 15, wherein guanabenz or the salt of guanabenz ameliorates the fatty liver through binding to Helz2 to lower its action.

23. The method for ameliorating a fatty liver according to claim 15, wherein guanabenz or the salt of guanabenz exhibits anti-obesity action without reduction of appetite through binding to Helz2 to lower its action.

24. The method for ameliorating a fatty liver according to claim 15, wherein the fatty liver is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH), and wherein guanabenz or the salt of guanabenz ameliorates non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) through binding to Helz2 to lower its action.

25. A method for treating a fatty liver comprising administrating guanabenz or a salt of guanabenz to a patient in need thereof.

26. The method for ameliorating a fatty liver according to claim 25, wherein guanabenz acetate is administered to a patient in need thereof.

27. The method for ameliorating a fatty liver according to claim 25, wherein the fatty liver is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

28. The method for treating a fatty liver according to claim 25, wherein the fatty liver is caused by insulin resistance.

29. The method for treating a fatty liver according to claim 25, wherein the fatty liver is accompanied by obesity disease.

30. The method for treating a fatty liver according to claim 25, wherein the fatty liver is accompanied by hypertension.

31. The method for treating a fatty liver according to claim 25, wherein the fatty liver is accompanied by metabolic syndrome.

32. The method for treating a fatty liver according to claim 25, wherein guanabenz or the salt of guanabenz ameliorates the fatty liver through binding to Helz2 to lower its action.

33. The method for treating a fatty liver according to claim 25, wherein guanabenz or the salt of guanabenz exhibits anti-obesity action without reduction of appetite through binding to Helz2 to lower its action.

34. The method for treating a fatty liver according to claim 25, wherein the fatty liver is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH), and wherein guanabenz or the salt of guanabenz ameliorates non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) through binding to Helz2 to lower its action.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0032] FIG. 1 shows graphs representing results of measurement of intracellular Leprb mRNA through qRT-PCR after addition of guanabenz acetate (FIG. 1-A) or salbutamol (FIG. 1-B) to the cultured hepatocyte HepG2.

[0033] FIG. 2 shows a graph representing temporal variation of body weight in male mouse fed either high-fat diet or low-fat diet.

[0034] FIG. 3 shows a graph representing expression of hepatic Leprb mRNA after 14-day oral administration of guanabenz acetate to obese mice fed high-fat diet. In all cases hereinafter, black columns each represent a group with administration of guanabenz acetate.

[0035] FIG. 4 shows graphs representing temporal variation of body weight gain (FIG. 4-A) and body weight (FIG. 4-B) with oral administration of guanabenz acetate to obese mice, where the data begin from pre-administration.

[0036] FIG. 5 shows graphs representing blood leptin concentrations (FIG. 5-A) after 14-day oral administration of guanabenz acetate to obese mice, and temporal variation of daily intake (FIG. 5-B) and total food intake accumulation (FIG. 5-C).

[0037] FIG. 6 shows graphs representing the HOMA-IR index (FIG. 6-A), an insulin resistance index, blood insulin levels (FIG. 6-B), and blood glucose levels (FIG. 6-C) after 14-day oral administration of guanabenz acetate to obese mice.

[0038] FIG. 7 shows graphs representing pancreatic weight (FIG. 7-A) and the HOMA-β index (FIG. 7-B), an index of pancreatic β-cell function, after 14-day oral administration of guanabenz acetate to obese mice.

[0039] FIG. 8-1 shows graphs representing hepatic triglyceride content (Tg, FIG. 8-A) and cholesterol content (Chol, FIG. 8-B) after 14-day oral administration of guanabenz acetate to obese mice.

[0040] FIG. 8-2 shows amounts of fat accumulated in hepatocyte per cell and change in shape of fat. As shown in representative photographs, fat globules in hepatocytes positive in BODIPY staining are exhibited (FIG. 8-C). A graph representing area of staining-positive cell (FIG. 8-D) and a graph representing size of staining-positive cell (FIG. 8-E) for different groups are shown. “Administration of G agent” refers to administration of guanabenz acetate.

[0041] FIG. 9 shows graphs representing weight change of liver (FIG. 9-A) and heart (FIG. 9-B) after 14-day oral administration of guanabenz acetate to obese mice.

[0042] FIG. 10 shows graphs representing blood chylomicron-triglyceride (CM-Tg, FIG. 10-A), very-low-density lipoprotein-triglyceride (VLD-Tg, FIG. 10-B), low-density lipoprotein-cholesterol (LDLc, FIG. 10-C), and high-density lipoprotein-cholesterol (HDLc, FIG. 10-D) levels after 14-day oral administration of guanabenz acetate to obese mice.

[0043] FIG. 11 shows graphs representing weight changes in spleen (FIG. 11-A) and kidney (FIG. 11-B) after 14-day oral administration of guanabenz acetate to obese mice.

[0044] FIG. 12 shows graphs representing weights of intestinal membrane fat (FIG. 12-A), peritesticular fat (FIG. 12-B), total visceral fat (FIG. 12-C), soleus muscle (FIG. 12-D), plantaris muscle (FIG. 12-E), and gastrocnemius muscle (FIG. 12-F) after 14-day oral administration of guanabenz acetate to obese mice.

DESCRIPTION OF EMBODIMENTS

[0045] Guanabenz, which is an active ingredient of an ameliorating agent of the present invention for metabolic control disorder, is a known compound that is represented by a formula:

##STR00001##

[0046] acts as a central α2-adrenaline receptor agonist, attenuates a sympathetic nervous action for heart to reduce blood pressure, and is used in clinical site as a therapeutic drug for hypertension to ameliorate peripheral vascular resistance. The Ki value of guanabenz to α2-adrenaline receptor is 7×10.sup.−9M.

[0047] The molecular formula of guanabenz is C.sub.8H.sub.8Cl.sub.2N.sub.4 and the molecular weight is 291.1, and the name defined by International Union of Pure and Applied Chemistry is 2-{[(2,6-dichlorophenyl)methylidene]amino}guanidine. The half-life of guanabenz in blood is 4.3 to 6.4 hours, and an absorptivity from a gastrointestinal tract is approximately 75%, and guanabenz is mostly metabolized in the liver.

[0048] The ameliorating agent of the present invention for an obesity-related metabolic disease contains guanabenz or a salt thereof such as acetate salt thereof as an active ingredient, and is formulated into an appropriate dosage form, singly or together with a pharmaceutically acceptable additional component.

[0049] The ameliorating agent of the present invention for an obesity-related metabolic disease may be in a dosage form to be used as a pharmaceutical composition, and can be formulated, for example, as any of tablet (including orally disintegrating tablet), capsule, controlled-release formulation, enteric-coated formulation, granule, powder, syrup, troche, rectal formulation, injection, or transdermal formulation, by using a known method. In manufacturing a formulation, formulation may be performed in accordance with regulation and rule in any country, and can be performed in accordance with the latest Japanese pharmacopoeia, for example, The Japanese Pharmacopoeia 17th Edition.

[0050] Excipient to be contained in such pharmaceutical formulation is not limited, and in an example the following can be appropriately combined for use: diluents such as starch, mannitol, and lactose; binders such as carboxymethyl cellulose sodium and hydroxypropyl cellulose; disintegrators such as microcrystalline cellulose and carboxymethyl cellulose; lubricants such as talc and magnesium stearate; and flow improvers such as light anhydrous silicic acid.

[0051] A liquid such as an injection can be employed as a pharmaceutical formulation, and, for example, a formulation obtained by dispersing or solubilizing in physiological saline, distilled water, or the like containing a surfactant, a dispersant, or the like may be employed, and a lyophilized formulation that is dispersed or solubilized at time of use, as necessary, may be employed. A pH adjustor or stabilizer may be added to a liquid.

[0052] A method of administration of an ameliorating agent of the present invention for an obesity-related metabolic disease can be appropriately set in a manner compatible with symptom of an obesity-related metabolic disease of interest or a patient therewith.

[0053] A dose within the range of 0.01 to 1000 mg may be typically administered per day, and the dose may be 0.1 mg or more, 1 mg or more, or 500 mg or less, 200 mg or less, 100 mg or less, 50 mg or less, or 20 mg or less within that range.

[0054] The frequency of administration is not limited, and may be one administration per day or multiple administrations per day. Administration may be performed at intervals of 1 day or longer. The timing of administration is appropriately set, for example, before meal, after meal, or during meal.

[0055] Even the route of administration is not limited, and administration can be performed with a dosage form of any of oral agent, nasal drop, oro-mucosal agent, vaginal agent, suppository, injection, and so on, and oral administration or parenteral administration is performed, and examples of parenteral administration include intravenous, intraarterial, intramuscular, subcutaneous, intratissue, intranasal, intradermal, intracerebral, intrarectal, intravaginal, intraperitoneal administrations, and so on. In being administered as an injection, the injection may be administered in one shot or through infusion.

[0056] There is no limitation to patient for administration, but administration to human is preferred.

[0057] The present invention may include a method for ameliorating an obesity-related metabolic disease by administrating a therapeutically effective amount of guanabenz or a salt of guanabenz to a patient in need thereof.

[0058] In the present invention, an ameliorating agent containing guanabenz or a salt of guanabenz for an obesity-related metabolic disease can be administered to a patient in need thereof.

[0059] The ameliorating agent for an obesity-related metabolic disease can be a prophylactic or therapeutic agent for an obesity-related metabolic disease, and the method for ameliorating an obesity-related metabolic disease can be a prophylactic or therapeutic method for an obesity-related metabolic disease.

[0060] The present invention also provides the following.

[0061] Guanabenz or a salt of guanabenz for ameliorating an obesity-related metabolic disease.

[0062] Guanabenz or a salt of guanabenz for preventing or treating an obesity-related metabolic disease.

[0063] Use of guanabenz or a salt of guanabenz for ameliorating an obesity-related metabolic disease.

[0064] Use of guanabenz or a salt of guanabenz for preventing or treating an obesity-related metabolic disease.

[0065] Use of guanabenz or a salt of guanabenz for adjustment/manufacture of a pharmaceutical composition/pharmaceutical formulation for ameliorating an obesity-related metabolic disease.

[0066] Use of guanabenz or a salt of guanabenz for adjustment/manufacture of a pharmaceutical composition/pharmaceutical formulation for preventing or treating an obesity-related metabolic disease.

[0067] The obesity-related metabolic disease in each embodiment described herein may be an obesity-related metabolic disease caused by insulin resistance and/or an obesity-related metabolic disease caused by obesity. Examples of the obesity-related metabolic disease in the present invention include, but are not limited to, impaired glucose tolerance or diabetes mellitus, dyslipidemia, hypertension, hyperuricemia or gout, coronary artery disease, cerebral infarct, fatty liver, and obesity-related kidney disease. Especially, fatty liver is preferred as the obesity-related metabolic disease, and the obesity-related metabolic disease may be non-alcoholic fatty liver disease or non-alcoholic steatohepatitis. In the present invention, the obesity-related metabolic disease caused by insulin resistance and/or caused by obesity may be fatty liver or fatty liver caused by insulin resistance and/or caused by obesity, and such fatty liver may be non-alcoholic fatty liver disease or non-alcoholic steatohepatitis.

[0068] The ameliorating agent for various obesity-related metabolic diseases may exhibit anti-obesity action without reduction of appetite, and may be an ameliorating agent for obesity-related metabolic disease including metabolic syndrome.

[0069] In the present invention, the salt of guanabenz to be used for ameliorating or preventing or treating an obesity-related metabolic disease is not limited, but preferably guanabenz acetate.

[0070] An ameliorating agent of the present invention for an obesity-related metabolic disease may be used in combination with an additional drug known to be used for obesity-related metabolic disease, and may be formulated into a drug combination. In use in combination, any of a kit formulation, independent pharmaceutical formulation, and a drug combination is acceptable.

[0071] The additional drug known to be used for obesity-related metabolic disease is not limited, and any drug known to be applicable for preventing or treating impaired glucose tolerance or diabetes mellitus, dyslipidemia, hypertension, hyperuricemia or gout, coronary artery disease, cerebral infarct, fatty liver (including non-alcoholic fatty liver disease and non-alcoholic steatohepatitis), or obesity-related kidney disease may be used.

[0072] Examples of mode of administration include, but are not limited to:

[0073] oral administration of a single agent of guanabenz or an acetate salt thereof as any of tablet (including orally disintegrating tablet), capsule, controlled-release formulation, enteric-coated formulation, granule, powder, syrup, or troche;

[0074] parenteral administration of guanabenz or an acetate salt thereof as any of rectal formulation, injection, or transdermal formulation;

[0075] administration of a combination drug of guanabenz or an acetate salt thereof and a metformin agent in any of the above mode of administration;

[0076] administration of a combination drug of guanabenz or an acetate salt thereof and a pioglitazone agent in any of the above mode of administration;

[0077] administration of a combination drug of guanabenz or an acetate salt thereof and a dipeptidyl peptidase-4 inhibitor in any of the above mode of administration;

[0078] administration of a combination drug of guanabenz or an acetate salt thereof and an α-glucosidase inhibitor in any of the above mode of administration;

[0079] administration of a combination drug of guanabenz or an acetate salt thereof and a sodium/glucose cotransporter-2 inhibitor in any of the above mode of administration;

[0080] administration of a combination drug of guanabenz or an acetate salt thereof and a sulfonylurea-based drug in any of the above mode of administration;

[0081] administration of a combination drug of guanabenz or an acetate salt thereof and a glinide-based drug in any of the above mode of administration; and

[0082] administration of a combination drug of guanabenz or an acetate salt thereof and a glucagon-like peptide-1 analog or a receptor agonist therefor in any of the above mode of administration.

EXAMPLES

Procedures

[0083] 1. High-Throughput Screening for Low-Molecular-Weight Compounds that Bind to Helz2

[0084] HEK293 cells were allowed to express Helz2 by using a Helz2 expression vector (p3×FLAG-CMV10-pDIP2), and Helz2 protein was purified. Subsequently, high-throughput screening assay through surface plasmon resonance (Flexera Japan KK, Tokyo) was performed for low-molecular-weight compounds that bind to Helz2 using an array panel holding 1,200 low-molecular-weight compounds, all approved by the U.S. Food and Drug Administration, to find low-molecular-weight compounds that showed binding and dissociation against Helz2.

2. Identification of Low-Molecular-Weight Compound that Promotes Leprb Expression in Cultured Hepatocytes

[0085] Because Leprb gene expression is promoted on lowering of the action of Helz2 through binding of a low-molecular-weight compound, the low-molecular-weight compounds that bound to Helz2 were screened with use of increase of Leprb gene expression in cultured hepatocyte as an index. Each of the low-molecular-weight compounds that bound to and dissociated from Helz2 with high to middle affinity (Sigma-Aldrich Co. LLC) was diluted to a final concentration of 10.sup.−9 to 10.sup.−7M, and added to HepG2 cells, a cultured hepatocyte strain (10.sup.5 cells/well in a 6-well plate). The hepatocytes were cultured at 37° C. for 24 to 48 hours, and Leprb gene expression therein was measured through a quantitative real time polymerase chain reaction (qRT-PCR, TaqMan Gene expression assays using LEPRB probes) method.

3. Hepatic Leprb Expression with Oral Administration of Low-Molecular-Weight Compound

[0086] Six-week-old male mice (C57BL6J) were grown with high-fat diet (f.sub.at content: 60 kcal %, Research diets, Inc.) or low-fat diet (fat content: 10.5 kcal %, CLEA Japan, Inc.) for 18 weeks. Thereafter, obese mice fed high-fat diet were separated into two groups, and 0.3 mL of physiological saline was orally administered to one group through a gastric tube for mouse, and 0.3 mL of a solution of a low-molecular-weight compound dissolved in physiological saline was orally administered to the other group through the gastric tube for mouse. Oral administration was performed every day over 14 days during 30 minutes before turning lights off at 18:00 (turning lights on at 6:00, 12-hour light-dark cycle, room temperature: 22° C.). On day 15, fasting was initiated at 9:00, each mouse was anesthetized with isoflurane at 14:00, blood was collected from the abdominal inferior vena cava (blood glucose was measured for tail vein blood), and the weight of each organ was measured. Thereafter, the organs were stored at −80° C. Hepatic Leprb expression was measured by means of the qRT-PCR.

4. Daily Variation of Body Weight with Oral Administration of Low-Molecular-Weight Compound

[0087] The body weight of each mouse with administration of physiological saline or administration of a low-molecular-weight compound was measured every day before each administration at 18:00.

5. Change in Blood Leptin Concentration and Food Intake after Oral Administration of Low-Molecular-Weight Compound

[0088] Blood leptin concentration after administration of physiological saline or administration of a low-molecular-weight compound were measured by using a mouse leptin ELISA kit (Morinaga Institute of Biological Science, Inc.). The daily food intake of each mouse was measured every day before each administration at 18:00.

6. Change in Insulin Resistance and Blood Glucose Level after Oral Administration of Low-Molecular-Weight Compound

[0089] Fasting blood glucose and blood insulin concentration after administration of physiological saline or administration of a low-molecular-weight compound were measured respectively by using an enzyme-electrode method with glucose oxidase and a mouse insulin ELISA kit (Morinaga Institute of Biological Science, Inc.). The insulin resistance of each mouse was represented as Homeostatic model assessment of insulin resistance (HOMA-IR) [insulin (mU/L)×blood glucose (mM)÷22.5] index (×100) (Brit J Nutr 101: 701-708, 2009).

7. Change in Pancreatic Weight and Pancreatic β-Cell Function after Oral Administration of Low-Molecular-Weight Compound

[0090] Pancreatic weight after administration of physiological saline or administration of a low-molecular-weight compound was measured. The pancreatic β-cell function of each mouse was represented by Homeostatic model assessment-β cell function (HOMA-β) {[insulin (mU/L)×20]÷[blood glucose (mM)−3.5]} index (×100) (Brit J Nutr 101:701-708, 2009).

8. Change in Hepatic Lipid Content after Oral Administration of Low-Molecular-Weight Compound

[0091] Hepatic Tg and Chol contents after administration of physiological saline or administration of a low-molecular-weight compound were measured by using a LipoSEARCH method (Skylight Biotech, Inc.).

[0092] To visualize and quantify the amount of fat accumulated in hepatocyte per cell and change in shape of fat, fat globule fluorescent staining with BODIPY (excitation light: 493 nm; fluorescence: 503 nm) was performed. Staining-positive area and size of staining-positive cell were measured in 10 views per mouse hepatocyte in each group. ANOVA was used for intergroup significance test in FIGS. 8-D and 8-E (****. P<0.001).

9. Change in Weights of Liver and Heart after Oral Administration of Low-Molecular-Weight Compound

[0093] The weights of the liver and heart after administration of physiological saline or administration of a low-molecular-weight compound were measured.

10. Change in Blood Lipid Concentration after Oral Administration of Low-Molecular-Weight Compound

[0094] Fasting blood CM-Tg, VLDL-Tg, LDLc, and HDLc concentrations after administration of physiological saline or administration of a low-molecular-weight compound were measured by using the LipoSEARCH method.

11. Change in Weights of Spleen and Kidney after Oral Administration of Low-Molecular-Weight Compound

[0095] The weights of the spleen and kidney after administration of physiological saline or administration of a low-molecular-weight compound were measured.

12. Change in Weights of Fat and Muscle after Oral Administration of Low-Molecular-Weight Compound

[0096] The amounts of visceral fat, which evokes metabolic disorder, and muscle (right side) after administration of physiological saline or administration of a low-molecular-weight compound were measured.

13. Significance Test

[0097] Data were represented as mean±standard deviation for each group. Paired Student's t test was used for significance test among two groups, and two-way ANOVA followed by Bonferroni's test was used for significance test for three or more, multiple groups.

Results

[0098] 1. High-Throughput Screening for Low-Molecular-Weight Compounds that Bind to Helz2

[0099] Analysis was performed for 1,200 low-molecular-weight compounds that bind to and dissociate from Helz2. Selected considering the result were 14 low-molecular-weight compounds that bound and dissociated with high to middle affinity (KD value: 10.sup.−9 to 4.9×10.sup.−7 M) (amifostine trihydrate, brompheniramine maleate, clemastine fumarate, fenbendazole, guanabenz acetate, loperamide hydrochloride, pirenperone, ritodrine hydrochloride, rivastigmine tartrate, salbutamol, sibutramine hydrochloride, sulbactam, voriconazole, zoxazolamine). Other low-molecular-weight compounds exhibited low affinity with KD values of 5×10.sup.−7 M or higher.

2. Identification of Low-Molecular-Weight Compound Guanabenz that Promotes Leprb Expression in Cultured Hepatocyte

[0100] Concentration-dependent increase in Leprb expression by each of the 14 low-molecular-weight compounds added to cultured hepatocyte was used as an index of lowering of Helz2 action, and guanabenz acetate, a candidate low-molecular-weight compound, was identified (FIG. 1-A). The KD value for binding to and dissociating from Helz2 was 2.3×10.sup.−9M, indicating high affinity. In contrast, addition of salbutamol, which exhibited middle affinity with Helz2 as 2.2×10.sup.−7M, caused no significant influence on Leprb expression (FIG. 1-B). The KD values of clonidine hydrochloride and methyldopa, each of which acts as a central α2-adrenaline receptor agonist to exhibit hypotensive action similarly to guanabenz, for Helz2 were 10.sup.−6M or higher, indicating low affinity.

3. Hepatic Leprb Expression after Oral Administration of Guanabenz

[0101] The body weight of male mice reached a plateau of approximately 50 g at 14 weeks after the beginning of intake of high-fat diet (N=16), indicating significant obesity as compared with male mice with intake of low-fat diet (N=4) (FIG. 2). The dose of guanabenz acetate orally administered was set to a 0.32 mg/kg body weight of mouse/day, a usual dose for humans considering the half-life of guanabenz acetate in blood, the absorptivity from the gastrointestinal tract, and so on. Hepatic Leprb gene expression was significantly increased after 14-day continuous oral administration of guanabenz acetate to obese mice fed high-fat diet for 18 weeks (black column, N=8), as compared with that after administration of physiological saline (white column, N=8) (FIG. 3).

4. Daily Variation of Body Weight with Oral Administration of Guanabenz

[0102] No body weight change was found with administration of physiological saline to obese mice (white circles, N=8) (FIG. 4). In contrast, the body weight gain was significantly reduced with administration of guanabenz acetate (black circles, N=8) after day 4 from the beginning of administration (FIG. 4-A), and significant body weight reduction was found on day 8 from the beginning of administration (FIG. 4-B), and this body weight reduction was maintained during the period of administration. These results indicate that anti-obesity action is found with administration of guanabenz acetate even in a state with high-fat diet.

5. Change in Blood Leptin Concentration and Food Intake after Oral Administration of Guanabenz

[0103] The increased blood leptin concentration found in obese mice was rather slightly decreased after administration of guanabenz acetate (black column, N=8) as compared with that after administration of physiological saline (white column, N=8), and the concentration was 10-fold or more higher than the leptin concentration in non-obese mice (2.3±0.5 ng/mL, N=4) (FIG. 5-A). After administration of guanabenz acetate to obese mice (black circles, N=8), no change in daily food intake was found except on day 3 from the beginning of administration, as compared with that after administration of physiological saline (white circles, N=8) (FIG. 5-B), and no significant change was found in total food intake accumulation (FIG. 5-C), similarly. These results revealed that oral administration of guanabenz acetate in a usual dose does not reduce appetite even under growing with high-fat diet, and provides anti-obesity action through energy expenditure enhancing action.

6. Change in Insulin Resistance and Blood Glucose Level after Oral Administration of Guanabenz

[0104] The HOMA-IR index (FIG. 6-A), which is an insulin resistance index, blood insulin levels (FIG. 6-B), and blood glucose levels (FIG. 6-C) in obese mice fed high-fat diet were higher than the indexes in non-obese mice (HOMA-IR index: 0.65±0.12, insulin level: 0.48±0.08 ng/mL, blood glucose level: 142.8±9.8 mg/dL, N=4). Administration of guanabenz acetate to obese mice (black column, N=8) suppressed the HOMA-IR index, mitigated hyperinsulinemia, and lowered the level of hyperglycemia to that in non-obese mice, as compared with administration of physiological saline (white column, N=8). These results indicate that 2-week administration of guanabenz significantly reduces insulin resistance to ameliorate hyperglycemia as an obesity-related metabolic disease.

7. Change in Pancreatic Weight and Pancreatic β-Cell Function after Oral Administration of Guanabenz

[0105] The pancreatic weight of obese mice was more than the pancreatic weight of non-obese mice (0.34±0.01 g, N=4), and the pancreatic weight increased by obesity was not changed after administration of guanabenz acetate (black column, N=8) as compared with that after administration of physiological saline (white column, N=8) (FIG. 7-A). The HOMA-β index, an index of pancreatic β-cell function, was not changed after administration of guanabenz acetate, similarly (FIG. 7-B). These results indicate that administration of guanabenz in a usual dose causes no influence on pancreatic β-cell function.

8. Change in Hepatic Lipid Content after Oral Administration of Guanabenz

[0106] The hepatic Tg content and Chol content in fatty liver (NAFLD) found in obese mice fed high-fat diet were significantly higher than those in liver of non-obese mice (Tg content: 11.3±2.6 mg/g, Chol content: 2.5±0.1 mg/g, N=4). The increased hepatic Tg content (FIG. 8-A) and Chol content (FIG. 8-B) were significantly reduced after administration of guanabenz acetate (black column, N=8), as compared with those after administration of physiological saline (white column, N=8). These results indicate that even 2-week oral administration of guanabenz ameliorates fatty liver characterized by Tg and Chol accumulations, as an obesity-related metabolic disease.

[0107] In addition, the fat globule area and fat globule cell size, which reflect the amount of fat per hepatocyte, in fatty liver caused by intake of high-fat diet significantly decreased after administration of guanabenz acetate (FIGS. 8-C to 8-E), which indicated anti-fatty liver action of guanabenz acetate.

9. Change in Weights of Liver and Heart after Oral Administration of Guanabenz

[0108] The weights of liver (FIG. 9-A) and heart (FIG. 9-B) of obese mice increased over those of non-obese mice (liver: 1.17±0.02 g, heart: 0.14±0.09 g, N=4), and the weights were significantly decreased after administration of guanabenz acetate (black column, N=8) as compared with those after administration of physiological saline (white column, N=8). These results indicate that liver enlargement evoked by obesity is reduced in association with amelioration of fatty liver with administration of guanabenz acetate. Further, it is suggested that guanabenz acetate mitigates heart burden induced by obesity through central α2-adrenaline receptor action.

10. Change in Blood Lipid Concentration after Oral Administration of Guanabenz

[0109] The blood CM-Tg (FIG. 10-A), VLDL-Tg (FIG. 10-B), and HDLc (FIG. 10-D) concentrations in obese mice were not changed after administration of guanabenz acetate (black column, N=8) as compared with those after administration of physiological saline (white column, N=8), but the LDLc concentration (FIG. 10-C), which acts to cause promotion of arteriosclerosis, was significantly decreased after administration of guanabenz acetate.

11. Change in Weights of Spleen and Kidney after Oral Administration of Guanabenz

[0110] No change was found in the weights of spleen and kidney after administration of guanabenz acetate (black column, N=8) as compared with those after administration of physiological saline (white column, N=8) (FIGS. 11-A, 11-B).

12. Change in Weights of Fat and Muscle after Oral Administration of Guanabenz

[0111] The amounts of intestinal membrane fat (FIG. 12-A), peritesticular fat (FIG. 12-B), and total visceral fat (FIG. 12-C) increased with obesity were not changed after administration of guanabenz acetate (black column, N=8) as compared with those after administration of physiological saline (white column, N=8). The muscle weights of soleus muscle (FIG. 12-D), plantaris muscle (FIG. 12-E), and gastrocnemius muscle (FIG. 12-F) of obese mice were not affected by administration of guanabenz acetate. These results indicate that 2-week administration of guanabenz to obese mice does not cause reduction of the amounts of visceral fat or muscle.

[0112] Significant difference was as follows.

[0113] FIG. 1: **p<0.01

[0114] FIG. 3: *p<0.05

[0115] FIG. 4: *p<0.05, **p<0.01

[0116] FIG. 5: *p<0.05, **p<0.01

[0117] FIG. 6: **p<0.01

[0118] FIG. 8: **p<0.01

[0119] FIG. 9: *p<0.05, **p<0.01

[0120] FIG. 10: **p<0.01

Comparison of Action Among Ameliorating Agents for Insulin Resistance

[0121] Metformin and pioglitazone are oral drugs that ameliorate insulin resistance associated with obesity-related metabolic disease (insulin dependence). Metformin primarily activates AMPK phosphorylation in the liver to ameliorate insulin resistance, and (1) exhibits hyperglycemia-mitigating action, but (2) does not exhibit anti-fatty liver action and (3) does not exhibit anti-obesity action, either. Pioglitazone dose not bind to Helz2, but binds to the functional activation domain-2 in the C-terminal side of PPARγ protein, enhancing the PPARγ transcription activity to ameliorate insulin resistance, and (1) exhibits hyperglycemia-mitigating action and (2) exhibits anti-fatty liver action in clinical situations, but there are results that administration of the ligand specific to PPARγ rather induces fatty liver (Matsusue K et al; J Clin Invest 111: 737, 2003). The growing action for fat cell (3) causes body weight gain rather than anti-obesity action. In contrast, guanabenz or a salt thereof such as an acetate salt thereof, which is identified as a low-molecular-weight compound that binds to Helz2 to lower its action, ameliorates insulin resistance, and (1) exhibits hyperglycemia-mitigating action (2) with anti-fatty liver action, (3) exhibits anti-obesity action without reduction of appetite, and (4) exhibits reducing action for high LDL cholesterol. These obesity-related metabolic diseases and the characteristics of the drug actions are summarized in Table 1 in the following.

TABLE-US-00001 TABLE 1 Metformin Pioglitazone Guanabenz Insulin resistance ↓ ↓ ↓ Hyperglycemia ↓ ↓ ↓ Hypertension .fwdarw. ↓ ↓* Blood LDLc .fwdarw. .fwdarw. ↓ Blood HDLc .fwdarw. ↑ .fwdarw. Fatty liver .fwdarw. ↓ ↓ Body weight .fwdarw. ↑ ↓ ↓: lowering or mitigating action ↑: increasing action .fwdarw.: no change. *based on clinical data

[0122] The above results revealed that oral administration of guanabenz or a salt thereof such as an acetate salt thereof in a usual dose not only provides conventionally known hypotensive action through binding to central α2-adrenaline receptor, but also binds to Helz2 to lower its action and ameliorates obesity and/or insulin resistance to thereby exhibit anti-obesity action and so on without reduction of appetite, and exerts pharmacological effects for the above four different obesity-related metabolic diseases by using a single agent (drug repositioning).

[0123] Therefore, examples of disease indicated for administration of the ameliorating agent for an obesity-related metabolic disease according to the present invention, the ameliorating agent containing guanabenz or a salt of guanabenz as an active ingredient, include the following caused by obesity and/or insulin resistance.

[0124] Diabetes mellitus associated with insulin resistance

[0125] Diabetes mellitus associated with obesity

[0126] Hypertension associated with insulin resistance

[0127] Hypertension associated with obesity

[0128] Hyperlipidemia associated with insulin resistance

[0129] Hyperlipidemia associated with obesity

[0130] Fatty liver (NAFLD and NASH) associated with insulin resistance

[0131] Fatty liver (NAFLD and NASH) associated with obesity

[0132] Complication of diabetes mellitus associated with insulin resistance and all or any of hypertension, hyperlipidemia, fatty liver, coronary artery disease, and obesity disease

[0133] Complication of diabetes mellitus associated with obesity and all or any of hypertension, hyperlipidemia, fatty liver, coronary artery disease, and obesity disease

[0134] Obesity disease caused by insulin resistance

[0135] Obesity disease caused by obesity

[0136] Metabolic syndrome (consisting of increased visceral fat, abnormal glucose tolerance, mild hypertension, and mild hyperlipidemia) caused by insulin resistance

[0137] Metabolic syndrome caused by obesity

[0138] Metabolic syndrome caused by obesity

[0139] With use of formulas (content ratios) shown in Table 2, formulations are obtained in accordance with a known method described in General Rules for Preparation in The Japanese Pharmacopoeia 17th Edition.

TABLE-US-00002 TABLE 2 Formulation Formulation Formulation Formulation Formulation Example 1 Example 2 Example 3 Example 4 Example 5 (Dosage form) Component capsule capsule tablet tablet tablet Guanabenz acetate 2.525 2.525 2.525 2.525 2.525 Anhydrous lactose 76.475 — — 58.675 — D-Mannitol — — 63.375 — — Calcium hydrogen — 67.175 — — 81.875 phosphate Microcrystalline — 30 25 — cellulose Corn starch 20 30 — 10 10 Croscarmellose — — 3 — 5 sodium Crospovidone — — — 3 — Sodium stearyl — — 1 — — fumarate Magnesium stearate — 0.3 0.8 0.3 Talc 0.5 — — — 0.3 Light anhydrous 0.5 — 0.1 — — silicic acid Total 100.0 100.0 100.0 100.0 100.0