WO2005082349A1

WO2005082349A1 - Composition for the treatment of obesity comprising fumagillol derivative

Info

Publication number: WO2005082349A1
Application number: PCT/KR2005/000523
Authority: WO
Inventors: Sang-Joon Lee; Soon-Kil Ahn; Hong-Woo Lee; Joon-Kyum Kim; Jae-Soo Shin
Original assignee: Chong Kun Dang Pharmaceutical Corp.
Priority date: 2004-02-28
Filing date: 2005-02-25
Publication date: 2005-09-09
Also published as: KR20050087982A; KR100552043B1

Abstract

Disclosed herein is a composition for the treatment of obesity which comprises a compound selected from the group consisting of O-(4-methoxycinnamoyl)fumagillol, O-(3,4,5-trimethoxycinnamoyl)fumagillol, O-(4-dimethylaminoethoxycinnamoyl)fumagillol, and salts thereof; and a pharmaceutically acceptable carrier. Since the composition potently inhibits the proliferation and increases the apoptosis of vein endothelial cells in adipose tissues to reduce the adipose tissues, and decreases food intake to cause weight loss, it shows superior therapeutic effects on obesity with few or no side effects.

Description

COMPOSITION FOR THE TREATMENT OF OBESITY COMPRISING FUMAGILLOL DERTVATIVE

Technical Field The present invention relates to a composition for the treatment of obesity comprising a compound selected from the group consisting of O-(4- methoxycinnamoyl)fumagillol, O-(3,4,5-trimethoxycinnamo3^1)fumagillol and O-(4- dimethylaminoethoxycinnamoyl)fumagillol represented by -Formulae 1 to 3 below, respectively: Formula 1

Formula 2

Formula 3

and salts thereof; and a pharmaceutically acceptable c-r-rrier. Background Art The reason why obesity is a serious social problem is due to the related diseases caused by or closely associated with obesity, rather than due to overweight itself. The major medical problems associated with obesity include hypertension, coronary cardiovascular diseases, diabetes, endocrine disorders, cholelithiasis, and malignant tumors. According to statistical data published in 1994, it is estimated that about 55% of the population of the United States is overweight and 22.5% of this figure is classified as obese. More shockingly, the prevalence of obesity is sharply rising in many countries every year. In addition, obesity among adults and children has strikingly increased in almost all countries, including developed and underdeveloped countries, over the past decade. Without exception, the prevalence of obesity is also increasing in Korea. Korea's recent rapid economic growth has brought a steep change in diet. The prevalence of obesity in Korea is attributed to an increasingly westernized diet. Particularly, childhood obesity is issued as a social problem because of its severe symptoms. Overweight, the meaning of which is often confused with obesity, in a mild condition can be treated to some extent by an appropriately combined therapy of diet and exercise, contrast, obesity is a chronic disease that can be treated by a combination of diet therapy and appropriate long-term drug therapy. In addition, obese patients' inability to control their compulsive desire for food intake is a kind of morbidity. Despite the increased demand and recognition for obesity, development of therapeutic drugs to fight obesity is insufficient and the results are still unsatisfactory. It has recently been reported that sibutramine promotes energy consumption through the energy metabolism, leading to weight loss. As another drug with a pharmacological mechanism different from sibutramine, orlistat known as a lipase inhibitor was developed that interferes with the absorption and digestion of lipids in the small intestinal tract. The advantages of the drug are that lipid absorption is lowered and absorbed lipids are rapidly digested and discharged from the body, contributing to weight loss to some extent. However, lipase inhibitors cause serious adverse side effects, for example, abdominal pain, flatulence, fecal incontinence, increased frequency and urgency of defecation, etc. In particular, these side effects may be exacerbated depending on food intake between races and individuals (M. Malone, Orlistat. Drugs,

Nol (1), 232-235, 1998). In addition to these drug therapies, surgical therapies are employed to treat severe obesity by incision of the gastrointestine, intestines, vagus nerves, etc. However, since surgical therapies may cause lethal side effects, they cannot be recommended as general therapies. This surgical intervention is so tentative and aggressive that it can be applied only to patients suffering from severe obesity and is thus not an alternative for most overweight patients. It has been recently known that fumagillin-based drugs as angiogenesis inhibitors have inhibitory effects on the proliferation of human umbilical vein endothelial cells (S. Marui et al, Chem. Pharm. Bull., 40(1), 96-101, 1992, ibid., 40(3), 575-579, 1992). Angiogenesis refers to the excessive creation of new capillary vessels without being appropriately controlled and regulated, and is reported to be closely involved in growth of solid tumors, diabetic retinopathy, rheumatoid arthritis, psoriasis, and the like (D. C. Billington, Drug Design and Discovery, 8(3), 1991). In the case of obesity, increased vein endothelial cells in adipose tissues are implicated in excessive proliferation of adipose cells to a large degree. Accordingly, inhibition of the proliferation of vein endothelial cells and increased apoptosis of endothelial cells will be effective for the treatment of obesity. Judah Folkman recently reported that the administration of TΝP-470 as an angiogenesis inhibitor to obese animals (ob/ob mice) genetically deficient in the appetite-suppressing gene (leptin gene) inhibits weight gain, contributing to the treatment of obesity (J. Folkman et al, PΝAS., 99(16), 10730-10735, 2002; and USP 6,306,813). However, TNP-470 is known to cause various side effects, particularly, spleen toxicity (e.g., spleen size shrinkage), excessive vasodilation in the subcutis, and hematological changes (e.g., decrease in the number of white blood cells (leukopenia)), which cause a severe problem that TNP-470 is largely limited in its application to a therapeutic agent for obesity (R. Yarchoan et al, Clinical Cancer Research, Vol 17, 91-

97, 1997; S. Parkash et al, Journal of Clinical Oncology, 16 (4), 1444-1449, 1998; M.

J. Hawkins et al, Clinical Cancer Research, Vol. 5, 1989-1995, 1999; and R. Yarchoan et al, Clinical Cancer Research, Vol. 3, 1501-1505, 1997).

Disclosure of the Invention The present inventors have intensively conducted a series of studies, based on the finding that since obese patients have an excessively large volume of adipose tissues and vein endothelial cells are implicated in the proliferation of adipose cells to a considerable extent, fumagiUol derivatives will be considerably effective to reduce the adipose tissues. As a result, the present inventors have found that since new types of fumagiUol derivatives markedly improve the above-mentioned side effects of the prior art, potently inhibit the proliferation and increase the apoptosis of vein endothelial cells in adipose tissues to reduce the adipose tissues, and decrease food intake to cause weight loss, even at low doses, they are effective for the treatment of obesity. The present invention has been achieved based on these findings. Therefore, it is an object of the present invention to provide a composition for the treatment of obesity showing superior therapeutic effects on obesity with few or no side effects. According to the present invention, there is provided a composition for the treatment of obesity comprising, a compound selected from the group consisting of O-(4- methoxycinnamoyl)fumagillol, O-(3,4,5-1rimethoxycinnamoyl)fumagillol and O-(4- dimethylaminoethoxycinnamoyl)fumagillol represented by Formulae 1 to 3 below, respectively:

(3), and salts thereof; and a pharmaceutically acceptable carrier. The fumagiUol derivatives can be used in the form of free bases or common pharmaceutically acceptable salts. Particularly, the compound O-(4- dimethylaminoethoxycinnamoyl)fumagillol can be used in the form of a salt, e.g., acetate, lactate, benzoate, salicylate, mandelate, oxalate, methanesulfonate, or p- toluenesulfonate. Korean Patent No. 0357542 and its corresponding patents (U.S. Patent No. 6,063,812, Japanese Patent No. 3370985, and European Patent No. 1077964), filed by the present applicant, disclose fumagiUol derivatives, including the compounds used in the present invention. The composition of the present invention can be prepared in combination with pharmaceutically acceptable carriers commonly used in pharmaceutical formulations.

The composition of the present invention can be formulated into dosage forms, such as solid formulations (tablets and capsules) and liquid formulations (for oral and parenteral administrations). Examples of pharmaceutically acceptable carriers that can be included in the composition of the present invention include excipients, binders, lubricants, disintegrants, emulsifiers, suspending agents, solvents, stabilizers, absorption aids, water for injection, isotonic agents, etc., which are commonly used in the art. The dose of the composition according to the present invention may be varied depending on the age, sex and body weight of patients to be treated, but the daily dose is commonly in the range of 0.1 to 2,000 mg of the fumagiUol derivatives.

In another aspect, the present invention relates to a method for treating obesity in a mammal subject, including human, in need thereof which comprises administering a therapeutically effective amount of the composition according to the present invention. In another aspect, the present invention relates to use of the composition according to the present invention as a therapeutic agent for obesity. hi another aspect, the present invention relates to use of the composition according to the present invention in the manufacture of a medicine for treating obesity.

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 comparing the weight loss effects in experimental mice administered with compositions of the present invention and TNP-470; Fig. 2 is a graph comparing the decrease of feed intake in experimental mice administered with compositions of the present invention and TNP-470; Figs. 3 a and 3b are graphs comparing the inhibition of the proliferation and the increased apoptosis of vein endothelial cells in adipose tissues in experimental mice administered with compositions of the present invention and TNP-470, respectively; Fig. 4 shows photographs comparing the spleen toxicity (spleen size shrinkage) in experimental mice administered with TNP-470 as a positive reference material (A), those administered with a fumagiUol derivative used in Experimental Example 3 of the present invention (B) at the same dose as the positive reference material, and those administered with no drug (C); Fig. 5 shows photographs comparing the vasodilation in the subcutis of experimental mice administered with no drug (A), those administered with a fumagiUol derivative used in Experimental Example 3 of the present invention (B), and those administered with TNP-470 as a positive reference material (C) at the same dose as the fumagiUol derivative; and Fig. 6 is a graph comparing the hematological change (decrease of WBC) in experimental mice administered with no drug (A), those administered with a fumagiUol derivative used in Experimental Example 3 of the present invention at doses of 120 mg/kg (B) and 240 mg/kg (C), and those administered with TNP-470 as a positive reference material at doses of 100 mg/kg (D) and 200 mg/kg (E).

Best Mode for Carrying Out the Invention The present invention will now be described in more detail with reference to the following preparative examples and experimental examples. However, these experimental examples are not to be construed as limiting the scope of the invention. Preparative Example of formulation 1 : Tablet O-(3,4,5- methoxycinnamoyl)fumagillol 500.0 mg Lactose BP 300.0 mg Croscamelose sodium 30.0 mg Povidone K-30 50.0 mg Magnesium stearate 1.0 mg O-(3,4,5-trimethoxycinnamoyl)fumagillol was dissolved along with Povidone

K-30 in a mixed solvent of ethanol and methylene chloride (1 : 1), and was then adsorbed and dispersed in a mixture of lactose and croscamelose sodium to obtain granules. The granules were dried to remove the solvents, and then magnesium stearate was added thereto. The mixture was compressed to produce a tablet.

Preparative Example of formulation 2: Capsule O-(4-dimethylaminoethoxycinnamoyl)fumagillol oxalate 100.0 mg Hydroxypropyl-β-cyclodextrin 200.0 mg Starch 1500 100.0 mg Magnesium stearate BP 1.0 mg O-(4-dimethylaminoethoxycinnamoyl)fumagillol oxalate was mixed with hydroxypropyl-β-cyclodextrin and starch 1500, and then water was added thereto until the resulting mixture was in a soupy state. The aqueous mixture was kneaded, granulated, and dried, followed by the addition of magnesium stearate. The resulting mixture was filled into a gelatin capsule to produce a capsule. Preparative Example of formulation 3: Injection O-(4-methoxycinnamoyl)fumagillol 100.0 mg Lactose 150.0 mg Diluted hydrochloric acid BP To pH of 3.5 Distilled water for inj ection 10 ml O-(4-methoxycinnamoyl)fumagillol and lactose were dissolved in distilled water for injection, and then the pH of the solution was adjusted to 3.5 using diluted hydrochloric acid BP to obtain a solution for injection. The solution was filtered, placed in a vial, rapidly frozen, and lyophilized to produce an injection.

Test for therapeutic effects of the compositions of the present invention on obesity To evaluate the therapeutic effects of the compositions according to the present invention on obesity, the weight loss effects, appetite-suppressing effects, inhibitory effects of the proliferation of vein endothelial cells in adipose tissues, increased apoptosis of endothelial cells, spleen toxicity, toxicity on the vasodilation in the subcutis, and hematological toxicity were determined in obese animals (ob/ob mice). As a positive reference material for the evaluation of pharmacological effects of the compositions according to the present invention, TNP-470, belonging to the same family as the fumagiUol derivatives used in the compositions of the present invention, was used.

<Test drugs and animals> FumagiUol derivatives used in the present experiments, i.e. O-(4- methoxycinnamoyl)fumagillol, O-(3,4,5-trimethoxycinnamoyl)fumagillol, and O-(4- dimethylaminoethoxycinnamoyl)fumagillol, were prepared by the process developed by the present inventors (USP 6,063,812). The positive reference material TNP-470 were prepared by the known process (S. Marui et al, Chem. Pharm. Bull, 40(1), 96-101,

1992). Animals (C57BL/6 ob/ob mice, male, aged 7 weeks) genetically deficient in the obesity regulatory gene (leptin gene) were purchased from Jackson Lab., U. S. A. The animals were housed in a polycarbonate cage, and acclimated to the cage for 7 days. Healthy animals were selected for use in the experiments. The selected experimental animals (ob/ob mice) had an average body weight of 49.6 ± 1.1 (n = 100), and were maintained at 22 ± 2°C with a humidity of 55 ± 5% on a 12-hour light/12-hour dark cycle. The animals were fed with solid mouse chow (Samyook Feed, Ltd.) and water ad libitum.

<Test dose and procedure> Each of the fumagiUol derivatives used in the compositions of the present invention was administered at five doses, i.e. 0.05 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg and 3 mg/kg. The positive reference material TNP-470 was administered in four high doses, i.e., 1 mg/kg, 2.5 mg/kg, 5 mg/kg and 7.5 mg/kg, in order to distinctly compare the pharmacological effects between TNP-470 and the fumagiUol derivatives. The drugs O-(4-methoxycinnamoyl)fumagillol, O-(3,4,5- trimethoxycinnamoyl)fumagillol, O-(4-dimethylaminoethoxycinnamoyl)fumagillol and TNP-470 were used in the form of a micro-emulsion. The micro-emulsion was prepared by feeding the corresponding drug into a self micro-emulsifying drug delivery system (SMEDDS, a solubilizer containing a mixture of triglyceride oil and a Cremophor surfactant) to dissolve the drug, and adding a proper amount of distilled water to the solution. On the other hand, O-(4- dimethylaminoethoxycinnamoyl)fumagillol oxalate as a fumagiUol derivative salt was diluted with phosphate buffer saline (PBS) for use. Each of the drugs was subcutaneously injected (s/c) into the animals once daily using the given daily dose for 21 days. The animals were grouped (5 mice/group) based on the used doses. The measured values were expressed as mean value (n = 5) ± standard deviation (S.D) with a significance level (p) less than 0.01. Experimental Example 1. Experiment for evaluating weight loss effect by administration of 0-(4-methoxycinnamoyι)fumagiIlol After O-(4-methoxycinnamoyl)fumagillol was subcutaneously injected (s/c) into experimental animals (ob/ob mice) once daily at five doses of 0.05 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg and 3 mg/kg for 21 days, the weight of the animals was measured daily at a given time point. 21 days after administration, the animals (ob/ob mice, n = 5) administered with the title compound at the highest dose (3 mg/kg) had an average body weight of 34.1 ± 1.5g, the mice admimstered with the title compound at the middle dose (0.3 mg/kg) had an average body weight of 38.0 ± 1.8g, and the mice administered with the title compound at the lowest dose (0.05 mg/kg) had an average body weight of 45.1 ± 1.5g. That is, the weight loss effect was dose-dependent. For comparison, after TNP-470 as a positive reference material was administered to experimental animals at four doses (1, 2.5, 5, and 7.5 mg/kg), the body weight of the animals was measured daily at a given time point. As a result, the mice administered with TNP-470 at the highest dose (7.5 mg/kg) had an average body weight of 41.8 ± 1.5g, and the mice administered with TNP-470 at the lowest dose (1 mg/kg) had an average body weight of 48.1 ± 1.5g. The weight loss effect of TNP-470 was dose- dependent to some extent, but was smaller than that of the title compound. On the other hand, a control group administered with physiological saline alone had an average body weight of 56.3 ± l.lg. The mice administered with the title compound at the highest dose showed a 39.4% decrease in body weight, compared to the control group. These experimental results are shown in Fig. 1 and Table 1. Experimental Example 2. Experiment for evaluating weight loss effect by administration of 0-(3,4,5-trimethoxycinnamoyl)fumagiIIol The administration of O-(3,4,5-trimethoxycinnamoyl)fumagillol at five doses (i.e. 0.05 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, and 3 mg/kg) and the measurement of the weight of animals were performed in the same manner as in Experimental Example 1. 21 days after administration, the animals (ob/ob mice, n = 5) administered with the title compound at the highest dose (3 mg/kg) had an average body weight of 32.1 ± 1.5g, the mice administered with the title compound at the middle dose (0.3 mg/kg) had an average body weight of 34.2 ± 1.5g, and the mice administered with the title compound at the lowest dose (0.05 mg/kg) had an average body weight of 43.1 ± 1.5g. That is, the weight loss effect was dose-dependent. For comparison, TNP-470 as a positive reference material was admimstered to experimental animals at different doses. As a result, the mice (n = 5) administered with TNP-470 at the highest dose (7.5 mg/kg) had an average body weight of 41.8 ± 1.5g, and the mice administered with TNP-470 at the lowest dose (1 mg/kg) had an average body weight of 48.1 ± 1.5g. However, the weight loss effect of TNP-470 was smaller than that of the title compound. On the other hand, a control group administered with physiological saline alone had an average body weight of 53.4 ± 1.8g. The mice administered with the title compound at the highest dose showed a 41.6% decrease in body weight, compared to the control group. These experimental results are shown in Fig. 1 and Table 1.

Experimental Example 3. Experiment for evaluating weight loss effect by administration of 0-(4-dimethylaminoethoxycinnamoyl)fumagillol oxalate The administration of O-(4-dimethylaminoethoxycinn-ιmoyl)fumagillol oxalate at five doses (i.e. 0.05 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, and 3 mg/kg) and the measurement of the weight of animals were performed in the same manner as in

Experimental Example 1. 21 days after administration, the animals (ob/ob mice) administered with the title compound at the highest dose (3 mg/kg) had an average body weight of 35.4 ± 1.3g, the mice administered with the title compound at the middle dose (0.3 mg/kg) had an average body weight of 38.6 ± 1.5g, and the mice administered with the title compound at the lowest dose (0.05 mg/kg) had an average body weight of 44.5 ± 1.4g. That is, the weight loss effect was dose-dependent. For comparison, TNP- 470 was administered to experimental animals at different doses. As a result, the mice (n = 5) administered with TNP-470 at the highest dose (7.5 mg/kg) had an average body weight of 41.8 ± 1.5g, and the mice administered with TNP-470 at the lowest dose (1 mg/kg) had an average body weight of 48.1 ± 1.5g. However, the weight loss effect of TNP-470 was smaller than that of the title compound. On the other hand, a control group administered with physiological saline alone had an average body weight of 56.3 ± l.lg. The mice administered with the title compound at the highest dose showed a 37.1% decrease in body weight, compared to the control group. These experimental results are shown in Fig. 1 and Table 1.

Table 1: Weight loss effect of fumagiUol derivatives in experimental animals

^a) The drugs were administered once daily by subcutaneous injection ⁾ Weight values measured 21 days after administration: Mean value (n = 5) ± standard deviation (S.D), with a significance level (p) less than 0.01.

Experimental Example 4. Experiment for measuring decrease in feed intake by administration of O-(4-methoxycinnamoyι)fumagiIloI O-(4-methoxycinnamoyl)fumagillol was subcutaneously injected (s/c) into experimental animals (ob/ob mice) once daily at five doses of 0.05 mg/kg, 0.1 mg/kg,

0.3 mg/kg, 1 mg/kg and 3 mg/kg for 21 days. lOg of solid mouse chow (Samyook

Feed, Ltd.) was placed daily in a feed tub attached to a polycarbonate cage to measure the feed intake in the mice. The daily feed intake was measured by subtracting the weight of the remaining feed at a specified time point on the next day from the given amount (lOg) on the previous day. The measurement of feed intake was continued daily for 21 days. The animals had ad libitum access to water. 21 days after administration, the mice administered with the title compound at the highest dose (3 mg/kg) showed an average daily feed intake of 1.4g, those admimstered with the title compound at the middle dose (0.3 mg/kg) showed an average daily feed intake of 2.3 g, and those administered with the title compound at the lowest dose (0.05 mg/kg) showed an average daily feed intake of 3.2g. That is, the feed intake was significantly decreased in a dose-dependent manner. For comparison, after TNP-470 as a positive reference material was administered to experimental animals at four doses (1, 2.5, 5, and 7.5 mg/kg), feed intake was measured in the same way as in the title compound. As a result, the mice administered with TNP-470 at the highest dose (7.5 mg/kg) showed an average daily feed intake of 1.1 g, and those administered with TNP-470 at the lowest dose (1 mg/kg) showed an average daily feed intake of 3.0g. TNP-470 showed a smaller decrease in feed intake than the title compound. On the other hand, a control group administered with no drug showed an average daily feed intake of 6.3 g. The mice administered with the title compound at the highest dose (3 mg/kg) showed a 77.8% decrease in feed intake, compared to the control group. These experimental results are shown in Fig. 2 and Table 2. Experimental Example 5. Experiment for measuring decrease in feed intake by administration of O~(3,4,5-trimethoxycinnamoyι)fumagillol The administration of O-(3,4,5-trimethoxycinnamoyl)fumagillol and the measurement of feed intake in animals (ob/ob mice) were performed in the same manner as in Experimental Example 4. 21 days after administration, the mice administered with the title compound at the highest dose (3 mg/kg) showed an average daily feed intake of 1.2g, those administered with the title compound at the middle dose (0.3 mg/kg) showed an average daily feed intake of 2.2g, and those administered with the title compound at the lowest dose (0.05 mg/kg) showed an average daily feed intake of 3.2g. That is, the feed intake was effectively decreased in a dose-dependent manner. For comparison, after TNP-470 as a positive reference material was administered to experimental animals at different doses, feed intake was measured. As a result, the mice administered with TNP-470 at the highest dose (7.5 mg/kg) showed an average daily feed intake of 1.1 g, and those admimstered with TNP-470 at the lowest dose (1 mg/kg) showed an average daily feed intake of 3.0g. TNP-470 showed a smaller decrease in feed intake than the title compound. On the other hand, a control group administered with no drug showed an average daily feed intake of 6.3g. The mice administered with the title compound at the highest dose (3 mg/kg) showed an 81.0% decrease in feed intake, compared to the control group. These experimental results are shown in Fig. 2 and Table 2.

Experimental Example 6. Experiment for measuring decrease in feed intake by administration of 0-(4-dimethylaminoethoxycinnamoyl)fumagillol oxalate The administration of O-(4-dimethylaminoethoxycinnamoyl)fumagillol oxalate and the measurement of feed intake in animals (ob/ob mice) were performed in the same manner as in Experimental Example 4. 21 days after administration, the mice administered with the title compound at the highest dose (3 mg/kg) showed an average daily feed intake of 1.3g, those administered with the title compound at the middle dose (0.3 mg/kg) showed an average daily feed intake of 2.3g, and those administered with the title compound at the lowest dose (0.05 mg/kg) showed an average daily feed intake of 3.1 g. That is. the feed intake was effectively decreased. For comparison, after

TNP-470 was administered to experimental animals at different doses, feed intake was measured. As a result, the mice administered with TNP-470 at the highest dose showed an average daily feed intake of 1.1 g, and those administered with TNP-470 at the lowest dose showed an average daily feed intake of 3.0g. TNP-470 showed a smaller decrease in feed intake than the title compound. On the other hand, a control group administered with no drug showed an average daily feed intake of 6.3g. The mice admimstered with the title compound at the highest dose showed a 79.4% decrease in feed intake, compared to the control group. These experimental results are shown in Fig. 2 and Table 2.

Table 2: Decreasing effect of fumagiUol derivatives on feed intake in experimental animals

^a) The drugs were administered once daily by subcutaneous injection ^b) Feed intake measured 21 days after administration: Mean value (n = 5) ± standard deviation (S.D), with a significance level (p) less than 0.01.

Experimental Example 7. Experiment for evaluating inhibitory effect on proliferation and increased apoptosis of vein endothelial cells in adipose tissues by administration of 0-(4-methoxycinnamoyl)fumagilIol O-(4-methoxycinnamoyl)fumagillol was administered at the same doses by the same procedure as in Experimental Example 4. The testes were isolated from the experimental animals (ob/ob mice) administered with the title compound at the respective doses, and then adipose tissues were obtained therefrom. To evaluate inhibitory effect on the proliferation and increased apoptosis of vein endothelial cells in the adipose tissues, the adipose tissues were treated by a specific fluorescence staining technique and observed under a microscope. As a result, the inhibition rates on the proliferation of endothelial cells in the adipose tissues of the animals (ob/ob mice) administered with the compound at the highest dose (3 mg/kg), the middle dose (0.3 mg/kg) and the lowest dose (0.05 mg/kg) were 1.13%, 4.51% and 6.11%, respectively, indicating about 85.0%, 40.1% and 18.9% increases, respectively, in the inhibitory effect when compared to that in the control group (7.54%). That is, the proliferation of endothelial cells in the adipose tissues was inhibited in a dose-dependent manner by the administration of the title compound. For comparison, TNP-470 as a positive reference material was administered to experimental animals at different doses. As a result, the inhibition rates on the proliferation of endothelial cells in the adipose tissues of the mice administered with TNP-470 at the highest dose (7.5 mg/kg) and the lowest dose (1 mg/kg) were 1.31% and 6.50%, respectively, indicating about 82.6% and 13.8% increases in the inhibitory effect when compared to the control group. The inhibitory effect of the title compound on the proliferation of endothelial cells in adipose tissues was superior to that of TNP-470. These experimental results are shown in Fig. 3a and Table 3. Further, increased apoptosis of endothelial cells in the adipose tissues in mice administered with the title compound at different doses was measured. As a result, the apoptosis rate was 9.45% at the highest dose (3 mg/kg), 6.50% at the middle dose (0.3 mg/kg), and 1.93% at the lowest dose (0.05 mg/kg), which indicates that the apoptosis of endothelial cells was increased by 91.3%, 87.3%, and 57.5%, respectively, compared to that in the control group (0.82%), in a dose-dependent manner by the administration of the title compound. For comparison, increased apoptosis of endothelial cells in the adipose tissues in mice administered with TNP-470 at different doses was measured. As a result, the apoptosis rate was 5.81% at the highest dose (7.5 mg/kg) and 1.2% at the lowest dose (1 mg/kg), which indicates that the apoptosis of endothelial cells was increased by 85.8% and 31.6%, respectively, compared to that in the control group.

The increased apoptosis rate of endothelial cells in adipose tissues by administration of the title compound was superior to that of TNP-470. These experimental results show good agreement with the experimental results (weight loss effects) obtained in

Experimental Example 1. These experimental results are shown in Fig. 3b and Table

3.

Experimental Example 8. Experiment for evaluating inhibitory effect on proliferation and increased apoptosis of vein endothelial cells in adipose tissues by administration of 0-(3,4,5-trimethoxycinnamoyl)fumagiIlol After O-(3,4,5-trimethoxycinnamoyl) fumagiUol was administered at the same doses as in Experimental Example 7 and adipose tissues were obtained in accordance with the same procedure as in Experimental Example 7, inhibitory effect on the proliferation and increased apoptosis of vein endothelial cells in the adipose tissues were evaluated. As a result, the inhibition rates on the proliferation of endothelial cells in the adipose tissues of the animals (ob/ob mice) administered with the compound at the highest dose (3 mg/kg), the middle dose (0.3 mg/kg) and the lowest dose (0.05 mg/kg) were 1.11%, 4.48% and 6.01%, respectively, indicating about 85.2%, 40.5%, and 20.2% increases, respectively, in the inhibitory effect when compared to the control group. That is, the proliferation of endothelial cells in the adipose tissues was inhibited in a dose-dependent manner by the administration of the title compound. For comparison, TNP-470 as a positive reference material was administered to experimental animals at different doses. As a result, the inhibition rates on the proliferation of endothelial cells in the adipose tissues of the mice administered with TNP-470 at the highest dose (7.5 mg/kg) and the lowest dose (1 mg kg) were 1.31% and 6.50%, respectively, indicating about 82.6% and 13.8% increases in the inhibitory effect when compared to the control group. The inhibitory effect of the title compound on the proliferation of endothelial cells in adipose tissues was superior to that of TNP-470.

These experimental results are shown in Fig. 3a and Table 3. Further, increased apoptosis of endothelial cells in the adipose tissues in mice administered with the title compound at different doses was measured. As a result, the apoptosis rate was 9.27% at the highest dose (3 mg/kg), 6.30% at the middle dose (0.3 mg/kg), and 1.89% at the lowest dose (0.05 mg/kg), which indicates that the apoptosis of endothelial cells was increased by 91.1%, 86.9%, and 56.6%, respectively, compared to that in the control group (0.82%), in a dose-dependent manner by the administration of the title compound. For comparison, increased apoptosis of endothelial cells in the adipose tissues in mice administered with TNP-470 at different doses was measured. As a result, the apoptosis rate was 5.81% at the highest dose (7.5 mg/kg) and 1.20% at the lowest dose (1 mg/kg), which indicates that the apoptosis of endothelial cells was increased by 85.8% and 31.6%, respectively, compared to that in the control group. The increased apoptosis rate of endothelial cells in adipose tissues by administration of the title compound was superior to that of TNP-470. These experimental results show good agreement with the experimental results (weight loss effects) obtained in Experimental Example 2. These experimental results are shown in Fig. 3b and Table 3.

Experimental Example 9. Experiment for evaluating inhibitory effect on proliferation and increased apoptosis of vein endothelial cells in adipose tissues by administration of 0-(4-dimethylaminoethoxycinnamoyl)fumagillol oxalate After O-(4-dimethylaminoethoxycinnarnoyl)fumagillol oxalate was administered at the same doses as in Experimental Example 7 and adipose tissues were obtained in accordance with the same procedure as in Experimental Example 7, inhibitory effect on the proliferation and increased apoptosis of vein endothelial cells in the adipose tissues were evaluated. As a result, the inhibition rates on the proliferation of endothelial cells in the adipose tissues of the animals (ob/ob mice) admimstered with the compound at the highest dose (3 mg/kg), the middle dose (0.3 mg/kg) and the lowest dose (0.05 mg/kg) were 1.10%, 4.50% and 5.92%, respectively, indicating about 85.4%, 40.5%, and 21.4% increases, respectively, in the inhibitory effect when compared to the control group (7.54%). That is, the proliferation of endothelial cells in the adipose tissues was inhibited in a dose-dependent manner by the administration of the title compound. These experimental results are shown in Fig. 3 a and Table 3. Further, increased apoptosis of endothelial cells in the adipose tissues in mice admimstered with the title compound at different doses was measured. As a result, the apoptosis rate was 9.09% at the highest dose (3 mg/kg), 6.47% at the middle dose (0.3 mg/kg), and 1.90% at the lowest dose (0.05 mg/kg), which indicates that the apoptosis of endothelial cells was increased by 90.9%, 87.3%, and 56.4%, respectively, compared to that in the control group (0.82%), in a dose-dependent manner by the administration of the title compound. For comparison, increased apoptosis of endothelial cells in the adipose tissues in mice administered with TNP-470 at different doses was measured. As a result, the apoptosis rate was 5.81% at the highest dose (7.5 mg/kg) and 1.20% at the lowest dose (1 mg/kg), which indicates that the apoptosis of endothelial cells was increased by 85.8% and 31.6%, respectively, compared to that in the control group. The increased apoptosis rate of endothelial cells in adipose tissues by administration of the title compound was superior to that of TNP-470. These experimental results show good agreement with the experimental results (weight loss effects) obtained in Experimental Example 3. These experimental results are shown in Fig. 3b and Table

3.

Table 3: Inhibitory effect on proliferation and increased apoptosis of vein endothelial cells in adipose tissues by administration of fumagiUol derivatives

^a) The drugs were administered once daily by subcutaneous injection ^b) Inhibition rates on the proliferation and increased apoptosis rates of endothelial cells in adipose tissues were measured by treating the tissues using a specific fluorescence staining technique, followed by microscopic observation, 21 days after administration. Measured values: Mean value (n = 5) ± standard deviation (S.D), with a significance level (p) less than 0.01.

Experimental Example 10. Experiment for comparing spleen toxicity To compare the toxicity of O-(4-dimethylaminoethoxycinnamoyl)fumagillol oxalate used in Experimental Example 3 and TNP-470 as a positive reference material, five specific pathogen-free Sprague-Dawley (SD) rats (male, average weight: 210 ± lOg) as experimental animals were purchased from Samtako Bio Korea h e, Korea, and acclimated for a period of one week before use. After the drugs were injected subcutaneously into the abdominal cavity of the rats at a high dose of 200 mg/kg in nine consecutive administrations, the rats were excised and the spleen size shrinkage was observed. As a result, the size of the spleen ("A" designated in Fig. 4) was considerably reduced (i.e. spleen toxicity was caused) in the group admimstered with

TNP-470 as compared with the size ("C" designated in Fig. 4) in the group admimstered with vehicle alone. The size of the spleen ("B" designated in Fig. 4) was slightly reduced in the group administered with the title compound. This spleen size shrinkage was because the dose was sufficiently high to cause spleen toxicity. Little or no toxicity was observed within the actual dose range. These experimental results are shown in Fig. 4. Experimental Example 11. Experiment for comparing toxicity on vasodilation in the subcutis After the compound used in Experimental Example 3 and TNP-470 were administered at the same dose by the same procedure as in Experimental Example 10, vasodilation in the subcutis was measured. Little or no vasodilation in the subcutis was observed in the group administered with the compound used in Experimental

Example 3 ("B" designated in Fig. 5) when compared to in the group administered with vehicle alone ("A" designated in Fig. 5), and was very close to the normal state, hi the group administered with TNP-470 ("C" designated in Fig. 5), excessive vasodilation in the subcutis as a serious side effect was observed. These experimental results are shown in Fig. 5. Experimental Example 12. Experiment for comparing hematological toxicity Male SD rats (weight: 210 ± lOg, n = 5) were used as experimental animals. To measure hematological side effects, the compound used in Experimental Example 3 was administered at doses of 120 mg/kg and 240 mg/kg, and TNP-470 was administered at doses of 100 mg/kg and 200 mg/kg. Although the doses of the compound used in Experimental Example 3 were higher than those of TNP-470, the WBC values were slightly decreased in the group administered with the compound used in Experimental Example 3 and those were considerably decreased in the TNP-470 group, compared to a control group administered with vehicle alone. Particularly, the TNP-470 group showed a hematological side effect at the higher dose (200 mg/kg), i.e. the WBC value was considerably decreased. These experimental results are graphically shown in Fig. 6 (a significance level (p) less than 0.01). As is apparent from the above experimental results, the fumagiUol derivatives used in the composition of the present invention exhibit superior pharmacological effects even at low doses, potently inhibit the proliferation and increase the apoptosis of vein endothelial cells in adipose tissues to reduce the adipose tissues, and decrease food intake to cause weight loss. Accordingly, the fumagiUol derivatives exhibit superior therapeutic effects on obesity. In addition, since the fumagiUol derivatives used in the composition of the present invention can effectively decrease food intake in a dose- dependent manner, they can control food intake in obese patients to enhance the therapeutic effects on obesity. Despite the potent pharmacological effects of the fumagiUol derivatives, few or no side effects were observed. Furthermore, when compared to TNP-470, which belongs to the same family as the fumagiUol derivatives used in the compositions, the compositions of the present invention are superior in pharmacological effects and are greatly safe from spleen size shrinkage, excessive vasodilation in the subcutis and hematological side effects.

Industrial Applicability As apparent from the above description, since the composition of the present invention potently inhibits the proliferation and increases the apoptosis of vein endothelial cells in adipose tissues to reduce the adipose tissues, and decreases food intake to cause weight loss, it shows superior therapeutic effects on obesity with few or no side effects.

Claims

1. A composition for the treatment of obesity comprising, a compound selected from the group consisting of O-(4- methoxycinnamoyl)fumagillol, O-(3,4,5-trimethoxycinnamoyl)fumagillol and O-(4- dimethylaminoethoxycinnamoyl)fumagillol represented by Formulae 1 to 3 below, respectively:

(3), and salts thereof; and a pharmaceutically acceptable carrier.

2. The composition according to claim 1, wherein the composition comprises a salt of O-(4-dimethylaminoethoxycinnamoyl)fumagillol (Formula 3) and a pharmaceutically acceptable carrier.

3. The composition according to claim 2, wherein the salt of O-(4- dimethylaminoethoxycinnamoyl)fumagillol (Formula 3) is selected from the group consisting of acetate, lactate, benzoate, salicylate, mandelate, oxalate, methanesulfonate, and p-toluenesulfonate.

4. A method for treating obesity in a mammal subject, including human, in need thereof which comprises admimstering a therapeutically effective amount of the composition according to any one of claims 1 to 3 to said subject.

5. Use of the composition according to any one of claims 1 to 3 as a therapeutic agent for obesity.

6. Use of the composition according to any one of claims 1 to 3 in the manufacture of a medicine for treating obesity.