Composition comprising oleanolic acid acetate as active ingredient for preventing, alleviating, or treating renal toxicity induced by medicine

Abstract

The present invention relates to a composition for preventing, improving or treating drug-induced nephrotoxicity, the composition comprising oleanolic acid acetate as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating drug-induced nephrotoxicity comprising oleanolic acid acetate or a pharmaceutically acceptable salt thereof as an active ingredient, a health functional food for preventing or improving drug-induced nephrotoxicity comprising the active ingredients, and an anticancer adjuvant comprising the active ingredients. The oleanolic acid acetate of the present invention is derived from a natural product and has no side effects and no cytotoxicity and has excellent effects of preventing, improving and treating nephrotoxicity caused by a drug, particularly a platinum-based anticancer drug, so that it can be utilized as a pharmaceutical composition, a health functional food, and a cancer adjuvant thereof.

Claims

1. A method for treating drug-induced nephrotoxicity, the method comprising: administering, to a patient, 0.001 mg/kg to 100 mg/kg per day of a compound represented by chemical formula I or a pharmaceutically acceptable salt thereof: ##STR00004## wherein the drug induced nephrotoxicity is induced by at least one platinum-based anticancer drug selected from the group consisting of cisplatin, carboplatin, oxaliplatin, and nedaplatin or antibiotic, wherein the compound or pharmaceutically acceptable salt thereof inhibits an inflammatory factor expression in kidney tissue wherein the inflammatory factor is a least one selected from the group consisting of TNF-α, IL-6, COX-2 and MCP-1.

2. The method of claim 1 for treating cisplatin-induced nephrotoxicity.

3. The method of claim 2 wherein the compound represented by the chemical of formula I or the pharmaceutically acceptable salt thereof is administered by parenteral administration of an aqueous solution.

4. The method of claim 2 wherein the compound represented by the chemical of formula I or the pharmaceutically acceptable salt thereof is administered through oral administration.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows results of MTT assay to show the cytotoxicity comparative evaluation between oleanolic acid and oleanolic acid acetate in HEK-293T cells, human embryonic kidney cells, (FIG. 1A), abdominal macrophages (FIG. 1B), and spleen cells (FIG. 1C) (*: p<0.05 significance with the control group).

(2) FIG. 2 shows changes in body weight (FIG. 2A), kidney tissue weight (FIG. 2B), death rate (FIG. 2C), and blood BUN value (FIG. 2d) after administration of oleanolic acid acetate at a concentration of 50 mg/Kg in cisplatin-induced nephrotoxicity animal experiments (*: P<0.05 significance with the control group, #: p<0.05 significance with only cisplatin-treated group).

(3) FIG. 3 shows changes in blood inflammatory cytokines TNF-α (FIG. 3A) and IL-6 (FIG. 3B) levels after administration of oleanolic acid acetate at a concentration of 50 mg/Kg in cisplatin-induced nephrotoxicity animal experiments (*: P<0.05 significance with the control group, #: p<0.05 significance with only cisplatin-treated group).

(4) FIG. 4 shows changes in inflammatory cytokines in the kidney tissues TNF-α (FIG. 4A), IL-6 (FIG. 4B), COX-2 (FIG. 4C), and MCP-1 (FIG. 4D) expression levels after administration of oleanolic acid acetate at a concentration of 50 mg/Kg in cisplatin-induced nephrotoxicity animal experiments (*: P<0.05 significance with the control group, #: p<0.05 significance with only cisplatin-treated group).

(5) Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of Adzuki Bean Ethanol Extract and Separation and Purification of Compound of the Chemical Formula I

Example 1-1: Preparation of Adzuki Bean Extract

(6) Adzuki beans (Phaseoli angularis Wight or Phaseolus calcaratus Roxburgh) were washed with water, dried in the shade, and then pulverized in a waring blender. 20 kg of the pulverized adzuki bean was put in 100 L of methanol, and cold-immersion extraction was performed at room temperature for 3 days. A filter paper (Whatman, USA) was used to perform filtration under reduced pressure, and the methanol solvent was removed from the filtered extract using a rotary vacuum evaporator at room temperature, so as to prepare 450 g of adzuki bean extract as an extraction residue.

Example 1-2: Preparation of Fraction

(7) In order to isolate an active fraction from the prepared adzuki bean extract, adzuki bean extract was suspended in 1 L of water, and an equal volume of n-hexane was added thereto, and mixed, followed by fractionation. This procedure was repeated four times to obtain 1 L of a water-soluble fraction and 4 L of n-hexane soluble fraction. Subsequently, the n-hexane soluble fraction was concentrated under reduced pressure to obtain 50 g of n-hexane soluble extract.

(8) Further, an equal volume of ethyl acetate was added to 1 L of the water-soluble fraction, and mixed with each other, followed by fractionation. This procedure was repeated three times to obtain 1 L of a water-soluble fraction and 3 L of ethyl acetate soluble fraction.

(9) The ethyl acetate soluble fraction thus obtained was concentrated under reduced pressure so as to obtain 35 g of an ethyl acetate soluble extract, and the remaining water-soluble fraction was concentrated under reduced pressure to obtain 35 g of concentrate for a water fraction.

Example 1-3: HPLC Analysis of Adzuki Bean Extract and Fraction

(10) Each of the adzuki bean extracts and fractions obtained in Examples 1-1 and 1-1-2 was subjected to HPLC analysis.

(11) In this regard, Agilent Technologies 1200 series was used for HPLC, and YMC J′sphere ODS-H80 (YMC, 4 μm, 4.6 mm I.D.×150 mm) column was used as an analysis column. At this time, 5% to 90% CH.sub.3CN was applied as an analysis solvent at a flow rate of 1 ml/min, and analysis was performed at 210 nm and 10 μl of the sample was injected (Table 1).

(12) TABLE-US-00001 TABLE 1 Time Solvent (%) (minutes) CH.sub.3CN H.sub.2O 0 5 95 15 5 95 25 30 70 30 30 70 50 90 10 70 90 10

(13) The peaks of catechin-7-glucopyranoside (catechin-7-glu), rutin, oleanolic acid acetate (OAA) and stigmasterol were observed at 5.5, 24.5, 35.5, and 35.5 minutes, respectively, and HPLC chromatograms of Phaseoli angularis Wight and Phaseolus calcaratus Roxburgh showed a similar pattern.

Example 1-4: Purification of Active Ingredient

(14) 80 g of the n-hexane fraction obtained in Example 1-2 was applied to silica gel column chromatography using a step gradient solvent system consisting of hexane:ethyl acetate (100:1->1:1) to obtain 5 active fractions (Fr.1-5). Recrystallization was performed by adding methanol to Fraction 3 and Fraction 4 among the active fractions, thereby purifying 2 types of compounds as a white powder.

(15) The 2 types of compounds thus purified were applied to instrumental analysis (1H-, 13C-NMR, MS) and reference values (Voutquenne L. et al. Phytochemistry 2003, 64, 781-789; Kongduang D. et al. Tetrahedron letters 2008, 49, 4067-4072) to identify oleanolic acid acetate (chemical formula I), respectively.

(16) ##STR00003##

(17) 4aS,6aR,6aS,6bR,8aR,10S,12aR,14bS-10-hydroxy-2,2,6a,6b,9,9,12a-heptamethyl-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylic acid acetate

Example 2: Comparison of Cytotoxicity Between Oleanolic Acid and Oleanolic Acid Acetate

(18) In order to compare the cytotoxicity of oleanolic acid and oleanolic acid acetate, human kidney cell lines, mouse peritoneal macrophages, and spleen primary cells were used.

Example 2-1: Culture of Cell Lines and Separation of Primary Cells

(19) HEK-293T cells, human embryonic kidney cells, were cultured in Dulbecco's modified eagle medium (Cat No. 12800-017, Gibco, USA) supplemented with heat-inactivated 10% FBS.

(20) For the isolation of peritoneal macrophage of mice, 3 ml of 3% thioglycollate medium was administered intraperitoneally to 5-6 week old ICR male mice (Oriental Bio, Seoul, Korea). After 3 days, 6 ml to 7 ml of PBS was administered intraperitoneally, followed by a massage of peritoneal cavity to collect the peritoneal fluid. The collected peritoneal fluid was centrifuged and suspended in RPMI (10% FBS, 1% antibiotics), transferred to a plate, and stabilized at 37° C. for 2 hours.

(21) For the isolation of mouse spleen cells, 5-6 week old ICR male mice (Oriental Bio, Seoul, Korea) were sacrificed with CO.sub.2 and then their spleen was aseptically extracted. The spleen was ground with a filter to separate single spleen cells, centrifuged using centrifugation, and erythrocytes were dissolved using ficoll. Cells from which erythrocytes had been removed were stabilized with RPMI (10% FBS, 1% antibiotics) at 37° C. for 2 hours.

Example 2-2: Evaluation of Cytotoxicity

(22) The survival rate of cultured cells was measured by MTT reduction method. As an MTT solution, formazan was formed by mitochondrial dehydrogenases in living cells to confirm cell viability. In order to confirm the cytotoxicity, cells were cultured in a 96-well plate at 5×10.sup.4 cells/well at 37° C., followed by treatment with oleanolic acid and oleanolic acid acetate per concentration thereof, and the cells were cultured for 24 hours. After 24 hours, 20 μl of MTT solution was added to each well, followed by further culturing for 2 hours. Then, the culture solution was removed, and 100 μl of dimethylsulfoxide was added thereto. The absorbance thereof was measured at 570 nm.

Example 2-3: Comparison of Cytotoxicity

(23) In order to evaluate the cytotoxicitiy of oleanolic acid and oleanolic acid acetate, MTT assay was performed using human kidney cell lines, peritoneal macrophage, and spleen cells. As a result, the cytotoxicity of oleanolic acid acetate was concentration-dependently less than that of oleanolic acid in human kidney cell lines. Oleanolic acid and oleanolic acid acetate, respectively, were found to have a cell survival rate of 27% and 43% at the highest concentration of 1000 μg/ml, indicating that oleanolic acid acetate induced less cytotoxicity (FIG. 1A).

(24) In peritoneal macrophages collected from mouse peritoneal cavity, oleanolic acid showed cytotoxicity in a concentration-dependent manner, while oleanolic acid acetate showed a cell survival rate of 81% at the maximum concentration of 1000 μg/ml, indicating almost no cytotoxicity. In addition, the cell survival rate of 81% at 1000 μg/ml of oleanolic acid acetate was found to be about twice that of 41% of the oleanolic acid (FIG. 1B).

(25) In the spleen cells isolated from the mouse spleen, oleanolic acid acetate had higher cell survival rate than that of oleanolic acid at all concentrations (100, 300, and 600 μg/ml), resulting in less cytotoxicity (FIG. 1C).

Example 3: Inhibitory Effect of Oleanolic Acid Acetate on Nephrotoxicity in Cisplatin-Induced Nephrotoxicity Animal Model

(26) In order to examine the inhibitory effect of oleanolic acid acetate on cisplatin-induced nephrotoxicity, animal experiments were conducted.

Example 3-1: Cisplatin-Induced Nephrotoxicity

(27) 8 week old C57BL/6 male mice having a weight of 20 g to 25 g (Oriental Bio, Seoul, Korea) were purchased and randomly divided into saline-treated group (Control, CON), oleanolic acid acetate-treated group (OAA), only cisplatin-treated group (CP) and cisplatin and oleanolic acid acetate-treated group (CP+OAA). For the experiment, the mice were kept under constant temperature (23±3° C.), humidity (55±15%) and light irradiation (7:00 to 19:00). After the purchase, the mice were stabilized in the SPF animal breeding room for 1 week and used in the experiment. Cisplatin (Sigma, St. Louis, Mo., USA) was dissolved in physiological saline at a concentration of 2 mg/ml and then administered intraperitoneally at a dose of 20 mg/kg. Oleanolic acid acetate was dissolved in distilled water, and the mixture was orally administered at a dose of 50 mg/ml 1 hour before the cisplatin administration and 1, 3, and 5 days after the cisplatin administration. Mice in all groups were sacrificed on day 5 after the cisplatin administration.

Example 3-2: Monitoring of Body Weight, Kidney Weight, Death Rate and Analysis of BUN

(28) After the mouse sacrifice, the body weight was measured, the kidney was isolated, the blood was collected from the heart, and the blood urea nitrogen (BUN), a biochemical indicator related to nephrotoxicity, was measured. The collected mouse blood was centrifuged at 3000 rpm and 4° C. for 15 minutes using a centrifuge to separate the only serum, and the BUN was measured using an automatic analyzer (Fuji Dry-Chem NX500i, Tokyo, Japan).

(29) As shown in FIGS. 2A and 2B, in the case of mice exposed to cisplatin, the body weight and the kidney weight were decreased. The results indicate that the nephrotoxicity was induced. As shown in FIG. 2C, the death rate was 100% in the cisplatin-treated group, but the death rate was reduced to 44% in the cisplatin and oleanolic acid acetate-treated group. The death rate was expressed as a percentage by dividing the number of mice killed in each group by the total number of mice.

(30) As shown in FIG. 2D, the BUN value is a blood biochemical indicator indicating the blood content of urea nitrogen, which is a metabolic product of proteolysis. Further, BUN elevation in blood generally indicates the presence of kidney disease. Accordingly, it was confirmed that the BUN value was significantly increased due to renal toxicity in the cisplatin-treated group, but the BUN value was decreased in the cisplatin and oleanolic acid acetate-treated group.

Example 3-3: Measurement of Inflammatory Cytokines (TNF-α, IL-6) in Blood

(31) In order to measure inflammatory cytokines in the blood (TNF-α and IL-6), serum was separated and used from blood, and TNF-α and IL-6 were measured using a mouse ELISA kit (R&D system, Minneapolis, Minn., USA). ELISA color development was measured using a Varioskan™ LUX multimode microplate reader (Thermofisher, Sunnyvale, Calif., USA) at a wavelength of 450 nm.

(32) As shown in FIGS. 3A and 3B, TNF-α and IL-6, which are inflammatory cytokines in the blood, were significantly increased in the cisplatin-treated group, but TNF-α and IL-6 were decreased in the cisplatin and oleanolic acid acetate-treated group, indicating an effect of inhibiting the inflammatory response in the blood by nephrotoxicity.

Example 3-4: Analysis of Inflammatory Factor (TNF-α, IL-6, COX-2 and MCP-1) Expression in Kidney Tissue

(33) After the mouse sacrifice, kidneys were separated and homogenized by adding Trizol reagent (Invitrogen, Carlsbad, Calif., USA). To this, chloroform was added to extract RNA, and isopropanol was added to precipitate. After the RNA precipitate was washed with 75% ethanol, the RNA concentration and purity were measured with a 2100 Bioanalyzer System (Agilent Technologies, Santa Clara, Calif., USA), and Taqman reverse transcription reagents kit (Applied Biosystems, Foster City) was used to synthesize cDNA. The expression level of inflammatory factors was measured by real-time PCR using SYBR Green PCR master mix kit (Applied Biosystem, Foster City, Calif., USA).

(34) In order to examine the expression levels of TNF-α and IL-6, inflammatory cytokines, COX-2, a prostaglandin E.sub.2 (PGE.sub.2) synthase involved in increasing the vascular permeability of the inflammatory response, and MCP-1, an inflammatory chemokine, Real-time PCR was performed on each gene. The results are shown in FIGS. 4A to 4D. As shown in FIGS. 4A to 4D, the amount of mRNA expression of TNF-α, IL-6, COX-2 and MCP-1 was significantly increased in the cisplatin-treated group, but the amount of mRNA expression of TNF-α, IL-6, COX-2 and MCP-1 was decreased in the cisplatin and oleanolic acid acetate-treated group, indicating an effect of inhibiting the inflammatory response in the blood by nephrotoxicity.

(35) The sum of results suggests that oleanolic acid acetate inhibits the cisplatin-induced nephrotoxicity and effectively inhibits the expression and secretion of inflammatory factors. Furthermore, oleanolic acid acetate shows less cytotoxicity with excellent nephrotoxicity-inhibiting effect than oleanolic acid. Therefore, oleanolic acid acetate can be utilized as a pharmaceutical composition, health functional food, anticancer drug, etc. having an excellent effect of preventing, improving, treating nephrotoxicity without adverse effect in animals or human body.

Preparation Example 1: Preparation of Powder

(36) 0.1 g of oleanolic acid acetate, the adzuki bean extract including the same, or the fraction thereof, 1.5 g of lactose and 0.5 g of talc were mixed and filled in the air-tight bag to prepare a powder.

Preparation Example 2: Preparation of Tablet

(37) 0.1 g of oleanolic acid acetate, the adzuki bean extract including the same, or the fraction thereof, 7.9 g of lactose, 1.5 g of crystalline cellulose, and 0.5 g of magnesium stearate were mixed, and a 500 mg-tablet containing 50 mg of the active ingredient was prepared by a direct tableting method.

Preparation Example 3: Preparation of Powder

(38) 0.1 g of oleanolic acid acetate, the adzuki bean extract including the same, or the fraction thereof, 5 g of corn starch, and 4.9 g of carboxy cellulose were mixed well to prepare a powder, and 500 mg of the powder was put in a hard capsule to prepare a capsule.

Preparation Example 4: Preparation of Injectable Formulation

(39) According to the conventional preparation method of injectable formulations, 2 ml-volume ampule for injection containing 0.1 g of oleanolic acid acetate, the adzuki bean extract including the same, or the fraction thereof, a proper amount of sterile distilled water for injection and a pH adjuster was prepared.

Preparation Example 5: Preparation of Liquid Formulation

(40) According to the conventional preparation method of liquid formulations, 0.1 g of oleanolic acid acetate, the adzuki bean extract including the same, or the fraction thereof, 10 g of isomerized glucose syrup and 5 g of mannitol were added to and dissolved in purified water, and a proper amount of lemon flavor was added thereto. The ingredients were mixed, and then the total volume was adjusted to 100 ml by adding purified water. The mixture was filled in a brown bottle and sterilized to prepare a liquid formulation.