Inclusion Compounds of Fumagillol Derivative or its Salt, and Pharmaceutical Compositions Comprising the Same

Abstract

The present invention relates to an inclusion compound of fumagillol derivative or its salt with hydroxypropyl--cyclodextrin or sulfobutylether-7--cyclodextrin, and pharmaceutical compositions comprising the same. The inclusion compound according to the present invention has superior water solubility and stability while exhibiting low toxicity, rendering it valuable as an anticancer agent or inhibitor of tumor metastasis.

Claims

1. An inclusion compound of a fumagillol derivative or a salt thereof with hydroxypropyl--cyclodextrin, wherein the fumagillol derivative is O-(4-dimethylaminoethoxycinnamoyl)fumagillol.

2.-4. (canceled)

5. The inclusion compound of claim 1 characterized in that said fumagillol derivative salt is selected from a group consisting of salts of fumagillol derivative with hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, trifluoroacetic acid, oxalic acid, fumaric acid, tartaric acid, maleic acid, methanesulfonic acid, benzenesulfonic acid or para-toluenesulfonic acid.

6. The inclusion compound in claim 1 characterized in preparing by dissolving hydroxypropyl--cyclodextrin in distilled water, and adding fumagillol derivative or its salt under stirring.

7. The inclusion compound in claim 1 characterized in that the inclusion compound is prepared by dissolving hydroxypropyl--cyclodextrin in distilled water, and adding fumagillol derivative or its salt under stirring after adjusting the solution pH in a range of 6-8 with dilute hydrochloric acid or sodium hydroxide solution.

8. The inclusion compound in claim 1 characterized in that the inclusion compound is prepared by dissolving hydroxypropyl--cyclodextrin in buffer solution which pH was pre-adjusted in a range of 6-8 with phosphate, and adding fumagillol derivative or its salt under stirring.

9. The inclusion compound of claim 6 characterized in that the inclusion compound is obtained by further lyophilization step for the final solution obtained after stirring.

10. The inclusion compound of claim 6 characterized in that molar ratio of fumagillol derivative or its salt to hydroxypropyl--cyclodextrin is 1:1 to 1:10.

11. A pharmaceutical composition comprising the inclusion compound in claim 1 and pharmaceutically acceptable additives.

12. The pharmaceutical composition in claim 11 characterized in that the pharmaceutically acceptable additive is at least one selected from a group consisting of pharmaceutically acceptable diluents, buffers, flavors, binders, thickening agent, lubricants and preservatives.

13. The pharmaceutical composition in claim 12 characterized in that said buffer is phosphate buffer.

14. The pharmaceutical composition in claim 11 characterized in that it is formulated in oral or parenteral preparation.

15. The pharmaceutical composition in claim 14 characterized in that said parenteral preparation is injection.

16. The pharmaceutical composition in claim 11 characterized in that it is formulated in sustained-release dosage form.

17.-18. (canceled)

19. A lyophilized composition comprising the inclusion compound of claim 1.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0073] FIG. 1 shows two-dimensional nuclear magnetic resonance spectrum (NOSEY) for the inclusion compound of fumagillol derivative in Example 12.

[0074] FIG. 2 represents respective plasma concentration-time curve after intravenous administration of fumagillol derivative alone (.circle-solid.) and the inclusion compound of fumagillol derivative () in Example 17.

BEST MODE FOR CARRYING OUT THE INVENTION

[0075] The following Examples represent preferred embodiments of the present invention. However, the present invention is not limited to the following Examples.

Example 1

[0076] O-(4-dimethylaminoethoxycinnamoyl)fumagillol (30 mg) was added to 1:5, 3.5, 7.0, 14.0, 28.0 w/v % hydroxypropyl--cyclodextrin solution, respectively, and stirred at 4 C. After 72 hr, the mixture was filtered with 0.2 m membrane filter and O-(4-dimethylaminoethoxycinnamoyl)fumagillol in the filtrate was determined by high pressure liquid chromatography (HPLC). The solubility of O-(4-dimethylamino ethoxycinnamoyl)fumagillol as a function of the hydroxypropyl--cyclodextrin concentration was represented in Table 1.

TABLE-US-00001 TABLE 1 Solubility Concentration of hydroxypropyl--cyclodextrin (w/v %) (mg/ml) 0 0.05 1.5 1.03 3.5 2.93 7.0 6.95 14.0 11.54 28.0 22.64

[0077] As in apparent from Table 1, the solubility of O-(4-dimethylaminoethoxycinnamoyl)fumagillol was improved when hydroxypropyl--cyclodextrin was added to form a complex and when the concentration of hydroxypropyl--cyclodextrin was increased.

Example 2

[0078] O-(4-dimethylaminoethoxycinnamoyl)fumagillol (30 mg) was added to 1.5, 3.5, 7.0, 14.0, 28.0 w/v % sulfobutylether-7--cyclodextrin solution, respectively, and stirred at 4 C. After 72 hr, the mixture was filtered with 0.2 m membrane filter and O-(4-dimethylaminoethoxycinnamoyl)fumagillol in the filtrate was determined by high pressure liquid chromatography (HPLC). The solubility of O-(4-dimethylaminoethoxycinnamoyl)fumagillol as a function of the sulfobutylether-7--cyclodextrin concentration was represented in Table 2.

TABLE-US-00002 TABLE 2 Solubility Concentration of sulfobutylether-7--cyclodextrin (w/v %) (mg/ml) 0 0.05 1.5 0.87 3.5 2.24 7.0 5.42 14.0 10.43 28.0 21.30

[0079] As in apparent from Table 2, the solubility of O-(4-dimethylaminoethoxycinnamoyl)fumagillol was improved when sulfobutylether-7--cyclodextrin was added to form a complex and when the concentration of sulfobutylether-7--cyclodextrin was increased.

Example 3

[0080] O-(3,4,5-trimethoxycinnamoyl)fumagillol (20 g) was added to 1.5, 3.5, 7.0, 14.0, 28.0 w/v % hydroxypropyl--cyclodextrin solution, respectively, and stirred at 4 C. After 72 hr, the mixture was filtered with 0.2 m membrane filter and O-(3,4,5-trimethoxycinnamoyl)fumagillol in the filtrate was determined by high pressure liquid chromatography (HPLC). The solubility of O-(3,4,5-trimethoxycinnamoyl)fumagillol as a function of the hydroxypropyl--cyclodextrin concentration was shown in Table 3.

TABLE-US-00003 TABLE 3 Solubility Concentration of hydroxypropyl--cyclodextrin (w/v %) (mg/ml) 0 0.002 1.5 0.43 3.5 1.52 7.0 3.54 14.0 6.72 28.0 12.02

[0081] As in apparent from Table 3, the solubility of O-(3,4,5-trimethoxycinnamoyl)fumagillol was improved when hydroxypropyl--cyclodextrin was added to form a complex and when the concentration of hydroxypropyl--cyclodextrin was increased.

Example 4

[0082] O-(3,4,5-trimethoxycinnamoyl)fumagillol (20 mg) was added to 1.5, 3.5, 7.0, 14.0, 28.0 w/v % sulfobutylether-7--cyclodextrin solution, respectively, and stirred at 4 C. After 72 hr, the mixture was filtered with 0.2 m membrane filter and O-(3,4,5-trimethoxycinnamoyl)fumagillol in the filtrate was determined by high pressure liquid chromatography (HPLC). The solubility of O-(3,4,5-trimethoxycinnamoyl) fumagillol as a function of the sulfobutylether-7--cyclodextrin concentration was shown in Table 4.

TABLE-US-00004 TABLE 4 Solubility Concentration of sulfobutylether-7--cyclodextrin (w/v %) (mg/ml) 0 0.002 1.5 0.58 3.5 1.38 7.0 2.75 14.0 5.69 28.0 11.67

[0083] As in apparent from Table 4, the solubility of O-(3,4,5-trimethoxycinnamoyl) fumagillol was improved when sulfobutylether--cyclodextrin was added to form a complex and when the concentration of sulfobutylether--cyclodextrin was increased.

Example 5

[0084] O-(4-dimethylaminoethoxycinnamoyl)fumagillol (50 mg) was added to phosphate buffer (pH 6.7) containing 1.5, 3.5, 7.0 and 14.0 w/v % hydroxypropyl--cyclodextrin, respectively, and stirred at 4 C. After 72 hr, the mixture was filtered with 0.2 m membrane filter and O-(4-dimethylaminoethoxycinnamoyl)fumagillol in the filtrate was determined by high pressure liquid chromatography (HPLC). The solubility of O-(4-dimethylaminoethoxycinnamoyl)fumagillol as a function of the hydroxypropyl--cyclodextrin concentration was shown in Table 5.

TABLE-US-00005 TABLE 5 Solubility Concentration of hydroxypropyl--cyclodextrin(w/v %) (mg/ml) 0 2.38 1.5 4.24 3.5 9.86 7.0 17.59 14.0 32.25

[0085] As in apparent from Table 5, the solubility of O-(4-dimethylaminoethoxycinnamoyl)fumagillol was improved when hydroxypropyl--cyclodextrin was added to form a complex and when the concentration of hydroxypropyl--cyclodextrin was increased

Example 6

[0086] Hydroxypropyl--cyclodextrin (13 g) was put to mass flask and 60 ml of distilled water was added and stirred or subjected to sonication at 4 C. until clear solution was obtained. The pH of the solution was adjusted in a range of 6-8 with dilute hydrochloric acid or sodium hydroxide. O-(4-dimethylaminoethoxycinnamoyl)fumagillol (1 g) was added and completely dissolved by stirring at 4 C. If necessary, the pH of the final solution was adjusted between 6-8 with dilute hydrochloric acid or sodium hydroxide and filtered through 0.2 m membrane filter and the filtrate was lyophilized.

Example 7

[0087] Hydroxypropyl--cyclodextrin (13 g) was put to mass flask and 60 ml of distilled water was added and stirred or subjected to sonication at 4 C. until clear solution was obtained. The pH of the solution was adjusted in the range of 6-8 with dilute hydrochloric acid or sodium hydroxide. O-(3,4,5-trimethoxycimamoyl) fumagillol (1 g) was added and completely dissolved by stirring at 4 C. If necessary, the pH of the final solution was adjusted in a range of 6-8 with dilute hydrochloric acid or sodium hydroxide and filtered through 0.2 m membrane filter and the filtrate was lyophilized.

Example 8

[0088] Potassium phosphate (6.8 g) and hydroxypropyl--cyclodextrin (21.67 g) were put to mass flask and 100 ml of distilled water was added and stirred or subjected to sonication at 4 C. until clear solution was obtained. O-(4-dimethylaminoethoxycinnamoyl)fumagillol (1.67 g) was added and completely dissolved by stirring at 4 C. The final solution was filtered through 0.2 m membrane filter and the filtrate was lyophilized.

Example 9

[0089] According to the same method as in Example 8 except using sulfobutylether-7--cyclodextrin instead of hydroxypropyl--cyclodextrin, inclusion compound of sulfobutylether-7--cyclodextrin with O-(4-dimethylaminoethoxy cinnamoyl)fumagillol was prepared.

Example 10

[0090] Potassium phosphate (6.8 g) and hydroxypropyl--cyclodextrin (21.67 g) were put to mass flask and 100 ml of distilled water was added and stirred or subjected to sonication at 4 C. until clear solution was obtained. O-(4-dimethylaminoethoxycinnamoyl)fumagilloloxalate (1.84 g) was added and completely dissolved by string at 4 C. The final solution was filtered through 0.2 m membrane filter and the filtrate was lyophilized.

Example 11

[0091] According to the same method as in Example 10 except using sulfobutylether-7--cyclodextrin instead of hydroxypropyl-f-cyclodextrin in Example 10, inclusion compound of O-(4-dimethylamino ethoxycinnamoyl)fumagilloloxalate with sulfobutylether-7--cyclodextrin was prepared.

Example 12

[0092] The filtrate obtained in Example 6 was lyophilized. The resulting lyophilized product was dissolved in heavy water (D.sub.2O) and analyzed by using two-dimensional .sup.1H-NMR (NOESY). The results are shown in FIG. 1. The cross peaks indicate that there are interactions between four protons of O-(4-dimethylaminoethoxycinnamoyl)fumagillol and the protons of the glucose skeleton of hydroxypropyl-4-cyclodextrin. These cross peaks were not observed for the spectrum of O-(4-dimethylaminoethoxycinnamoyl)fumagillol alone. These results indicate that O-(4-dimethylaminoethoxycinnamoyl)fumagillol forms an inclusion compound with hydroxypropyl--cyclodextrin.

Example 13

[0093] The stability 6f the lyophilized powders obtained in Example 6 was compared with that of O-(4-dimethylaminoethoxycinnamoyl)fumagillol alone under storage at 25 C. The residual amount of O-(4-dimethylaminoethoxycinnamoyl)fumagillol was determined by HPLC. The results are shown in Table 6.

TABLE-US-00006 TABLE 6 Residual amount of O-(4-dimethylaminoethoxy- cinnamoyl)fumagillol (%) after after after after 1 month 3 month 6 month 12 month O-(4-dimethylaminoethoxy- 72.8 54.3 cinnamoyl)fumagillol Inclusion compound of 99.3 98.7 97.5 93.6 O-(4-dimethylaminoethoxy- cinnamoyl)fumagillol

[0094] As in apparent from Table 6, the stability of complex of O-(4-dimethylaminoethoxycinnamoyl)fumagillol with hydroxypropyl--cyclodextrin was improved as compared to that of O-(4-dimethylaminoethoxycinnamoyl)fumagillol alone.

Example 14

[0095] The stability of the lyophilized powders obtained in Example 7 was compared with that of O-(3,4,5-trimethoxycinnamoyl)fumagillol alone at 25 C. The residual amount of O-(3,4,5-trimethoxycinnamoyl)fumagillol was determined by HPLC. The results are shown in Table 7.

TABLE-US-00007 TABLE 7 Residual amount of O-(3,4,5-trimethoxy- cinnamoyl)fumagillol (%) after after after after 1 month 3 month 6 month 12 month O-(3,4,5-trimethoxycinna- 65.3 moyl)fumagillol Inclusion compound of 99.5 97.7 94.4 93.2 O-(3,4,5-trimethoxycinna- moyl)fumagillol

[0096] As in apparent from Table 7, the stability of complex of O-(3,4,5-trimethoxycinnamoyl)fumagillol with hydroxypropyl--cyclodextrin was improved as compared to that of O-(3,4,5-trimethoxycinnamoyl)fumagillol alone.

Example 15

[0097] The stability of O-(4-dimethylaminoethoxycinnamoyl)fumagillol was investigated in the presence of various concentrations of hydroxypropyl--cyclodextrin in acidic, neutral and basic solutions at 50 C. The residual amount of O-(4-dimethylaminoethoxycinnamoyl)fumagillol in each solution was determined by HPLC. The results are shown in Table 8.

TABLE-US-00008 TABLE 8 Concentration of hydroxypropyl-- Acidic (pH 3.2) Neutral (pH 7.2) Basic (pH 11.5) cyclodextrin Inhibition Inhibition Inhibition (w/v %) k.sub.obs (h.sup.1).sup.a ratio (%) k.sub.obs (h.sup.1) ratio (%) k.sub.obs (h.sup.1), ratio (%) 0 0.2042 0.0162 0.0918 2 0.0976 52.20 0.0141 12.96 0.0816 5.00 5 0.0848 58.47 0.0137 15.43 0.0580 16.55 10 0.0754 63.08 0.0122 24.96 0.0453 22.77 20 0.0665 67.43 0.0115 29.01 0.0294 30.56 .sup.ak.sub.obs: Hydrolysis Rate Constant

[0098] As in apparent from Table 8, hydroxypropyl-f-cyclodextrin suppressed the hydrolysis rate of O-(4-dimethylaminoethoxy cinnamoyl)fumagillol significantly.

Example 16

[0099] Pre-prepared tumor mass (Lewis lung carcinoma) of 8 mm.sup.3 was subcutaneously implanted into the right axillary region of BDF1 mice (4 weeks). When the tumor size was 100-200 mm.sup.3, mice were divided randomly into treatment group and control group. The treatment group was administered subcutaneously with O-(4-ethylaminoethoxycinnamoyl)fumagillol or the complex of O-(4-ethylaminoethoxycinnamoyl)fumagillol with hydroxypropyl--cyclodextrin at a dose of 30 mg/kg or 120 mg/kg as O-(4-ethylaminoethoxycinnamoyl)fumagillol every other day for 5 injections, while the control group was given injections of 0.2 ml of phosphate buffered saline. The tumors were weighed on the final day, and tumor volume was calculated using the following equation:

Tumor volume (mm.sup.3)=ab.sup.20.5

(a: the longest diameter, b: the shortest diameter)
Inhibition ratio (IR %) of the treatment group relative to the untreated control group was calculated using the following equation:

Inhibition Ratio %=(1Tumor volume of Treatment group/Tumor volume of Control group)100 Inhibition Ratio %

(1Tumor weight of Treatment group/Tumor weight of Control group)100

[0100] The results are shown in Table 9.

TABLE-US-00009 TABLE 9 Inhibition Ratio (%), Total Dose Tumor Tissue Tumor Tissue (mg/kg) Volume Weight Control Group 150 0 0 600 0 0 Group administered 150 37.7 33.6 with O-(4-dimethyl- 600 63.3 71.4 aminoethoxycinna- moyl)fumagillol Group administered 150 32.2 34.7 with inclusion 600 60.4 70.8 compound of O-(4- dimethylaminoethoxy- cinnamoyl)fumagillol

[0101] As in apparent from Table 9, the inclusion compound of O-(4-dimethylaminoethoxycinnamoyl)fumagillol exhibits comparable antitumor activity with O-(4-dimethylaminoehoxycinnamoyl)fumagillol alone.

Example 17

[0102] O-(4-dimethylaminoethoxycinnamoyl)fumagillol alone and the complex of O-(4-dimethylaminoethoxycinnamoyl)fumagillol with hydroxypropyl--cyclodextrin containing the same amount of O-(4-dimethylaminoethoxycinnamoyl)fumagillol were injected via intravenous route, and blood level of the drug was determined. As test animal, 5 male rats were used per 1 group.

[0103] Under light ether anesthesia, the femoral arteries and veins of rats were cannulated with PB-50 polyethylene tubing. After complete recovery from anesthesia, O-(4-dimethylaminoethoxycinnamoyl)fumagillol alone or the complex of O-(4-dimethylaminoethoxycinnamoyl)fumagillol with hydroxypropyl--cyclodextrin dissolved in phosphate buffered saline (pH 6.2) was administered intravenously to the femoral vein through the catheter at a dose of 20 mg/kg as O-(4-dimethylaminoethoxycinnamoyl)fumagillol, respectively. Blood samples (0.15 ml) were collected via the femoral artery immediately after the dose and at designated time intervals (15, 30, 45, 60, 120, 180 and 240 min). The blood samples were centrifuged immediately and the concentrations of O-(4-dimethylaminoethoxycinnamoyl)fumagillol in the plasma were determined by HPLC.

[0104] As apparent in FIG. 2, there were no significant differences between the plasma concentrations of O-(4-dimethylaminoethoxycinnamoyl)fumagillol alone and those of the complex of O-(4-dimethylaminoethoxycinnamoyl)fumagillol with hydroxypropyl--cyclodextrin. However, it was advantageous that the administration of the complex of O-(4-dimethylaminoethoxycinnamoyl)fumagillol with hydroxypropyl--cyclodextrin causes little pain on the injection site in contrast to that of O-(4-dimethylaminoethoxycinnamoyl)fumagillol alone.

INDUSTRIAL APPLICABILITY

[0105] The inclusion compound of fumagillol derivative or its salt with hydroxypropyl--cyclodextrin or sulfobutylether-7--cyclodextrin according to the present invention shows improved water-solubility, superior long-term stability at room temperature and reduced irritancy effect to injection site with unaltered tumor growth inhibitory activity when compared to fumagillol derivative alone, and thus may be useful for the treatment of tumors as an angiogenesis inhibitor.