KR0145942B1 - New anticancer composition - Google Patents

Abstract

The present invention relates to a novel anticancer composition comprising as an active substance the anticancer substance GERI-BP002A of the formula (I) produced by the fungus Aspergillus fumigatus FM-F-93 isolated from soil. .

Description

New anticancer composition

Figure 1 shows the absorbance analysis spectrum of GERI-BP002A by ultraviolet-visible spectroscopy,

Figure 2 shows the molecular weight spectrum of GERI-BP002A by VG70-VSEQ mass spectrometer,

3 shows the ¹ H-NMR spectrum at 300 MHz of GERI-BP002A by Brooker AM-300,

4 shows the ¹³ C-NMR spectroscopy spectrum at 75 MHz of GERI-BP002A by Brooker AM-300.

The present invention relates to a novel anticancer agent composition. More specifically, the present invention relates to a novel anticancer composition comprising the anticancer substance GERI-BP002A produced by the fungus Aspergillus fumigatus FM-F-93 isolated from soil as an active substance.

Although the incidence of cancer is increasing as civilization develops, the cause of cancer is not known enough to point out the root cause of cancer development.

The treatment of cancer patients includes surgical surgery, radiation therapy, chemotherapy by the administration of chemotherapy, but most of them are limited to early cancer patients or certain cancers, and cancer deaths are steadily increasing.

Therefore, the present inventors found that the substance GERI-BP002A produced by the fungus Aspergillus pumigatus FM-F-93 isolated from soil showed anticancer activity in an effort to search for a highly active anticancer substance from a microbial metabolite. The present invention was completed.

It is an object of the present invention to provide a novel anticancer agent composition.

In accordance with the above object, the present invention provides a novel anticancer composition comprising a new anticancer substance GERI-BP002A produced by the genus Aspergillus pumigatus isolated from the soil.

Hereinafter, the present invention will be described in detail.

The novel anticancer composition of the present invention comprises as an active compound a compound of formula (I) exhibiting anticancer activity:

The compound is named GERI-BP002A and summarizes its physicochemical properties as shown in Table 1 below.

GERI-BP002A can be produced by culturing Aspergillus microorganisms, for example Aspergillus pumigatus FM-F-93 (KCTC 0111BP), on the other hand, can also be produced by chemical synthesis. The medium which can be used to produce GERI-BP002A by culturing Aspergillus fumigatus FM-F-93 is a medium composed of 1% glucose, 0.3% yeast extract and 0.3% malt extract and having a pH of 6.5 and cultured. As for conditions, a stirring speed of 100 rpm, aeration amount of 0.3 VVM, dissolved oxygen of 60% or more, a temperature of 28 ° C., and an initial pH of 6.5 are preferable. After the incubation was completed, the culture solution was filtered through cotton wool to collect the cells, and the crude extract obtained by sequentially extraction with acetone and ethyl acetate was purified using silica gel column chromatography and HPLC.

When intraperitoneal injection of GERI-BP002A into ICR mice to test the biotoxicity of GERI-BP002A, the LD was shown to be 500 mg / kg body weight.

Meanwhile, GERI-BP002A shows anticancer activity, and its anticancer activity is human tumor cell lines A549 (human non-small cell lung), SKOV-3 (human ovarian), HCT-15 (human colon), XF-498 (human CNS), SKMEL-2 (human melanoma), and confirmed the strong anti-cancer effect, it is expected to be effective in other tumor cell lines.

Accordingly, the present invention provides an anticancer composition comprising GERI-BP002A or a derivative thereof as an active ingredient.

Such pharmaceutical compositions may include pharmaceutically acceptable excipients, carriers, diluents, etc. together with GERI-BP002A or derivatives thereof, and the formulations of the compositions may be prepared according to known methods using readily available ingredients. can do. In preparation of the formulation, the active ingredient can be mixed with the carrier, diluted with the carrier, or contained in a carrier in the form of a capsule, sachet, paper or other container. If the carrier serves as a diluent, it may be a solid, semisolid or liquid substance which acts as a vehicle, excipient or medium for the active ingredient. Thus, the formulations may be tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (solid or in liquid media), soft and hard gelatin capsules, sterile injectable solutions, sterile packaged powders And the like.

Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, Cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition may further comprise lubricants, humectants, sweeteners, flavors, emulsifiers, suspending agents, preservatives and the like in addition to the above components. Compositions of the present invention can be formulated using techniques known in the art to release the active ingredient rapidly, continuously or delayed after administration to a patient.

The pharmaceutical compositions of the present invention may be administered orally or parenterally, and the active compounds are effective for a wide range of dosages. For example, daily dosages of active compounds typically range from about 10 to 50 mg / kg body weight. However, the amount of compound actually administered will be determined by the physician in light of the relevant circumstances, including the condition of the cancer to be treated, the route of administration chosen, the age, weight and response of the individual patient, and the severity of the disease, and thus the range of administration will be It is not intended to limit the scope of the invention in any way.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited thereto.

Example 1

Microbial Isolation and Identification

Soil samples collected from Jang-dong, Yuseong-gu, Daejeon, Korea were diluted in sterile water and smeared on a commonly used potato-dextrose agar plate medium.

The fungus is described in R.A. Samson and J. I. Pitt, Advances in Penicillium and Aspergillus systematics, 1985, Plenum Press, New York and London] and R. A. Samson and J. I. Pitt, Modern Conceptain Penicillium and Aspergillus Classification 1989, Plenum Press, New York and London, identified as Aspergillus pumigatus.

The isolated fungus was named Aspergillus pumigatus FM-F-93 and deposited on May 24, 1994 as the accession no. KCTC 0111BP to the Genetic Bank of Korea (KCTC).

Example 2

Fermentation Production of GERI-BP002A

Aspergillus pumigatus FM-F-93 strain was inoculated into a 1000 ml Erlenmeyer flask containing 200 ml of sterile spawn medium (pH 6.5) consisting of 1% glucose, 0.3% yeast extract and 0.3% malt extract. After shaking for 2 days at 28 ℃.

200 ml of the seed culture was inoculated into a 14 l bioreactor containing 8 l of sterile production medium (1.5% glucose, 0.3% yeast extract, 0.3% malt extract, pH 6.5) at 121 ° C. for 30 minutes, The fermentation was completed in 120 hours by stirring at 100 rpm, aeration rate of 0.3 VVM, maintaining 60% or more of dissolved oxygen, and pH adjustment at a temperature of 28 ° C.

Example 3

Isolation and Purification of GERI-BP002A

GERI-BP002A was isolated and purified from the fermentation broth of Aspergillus pumigatus FM-F-93 obtained in Example 2 in the following manner. After passing through 8 L of the fermentation broth to separate the cells, the same amount of acetone was added to the separated cells and extracted twice, and the extract was concentrated under reduced pressure. 500 ml of ethyl acetate and 500 ml of distilled water were added to the concentrated solution, which was transferred to a separatory filter, and the ethyl acetate layer was separated. The remaining water layer was extracted twice with ethyl acetate, and the extracted solution was mixed with ethyl acetate layer, dehydrated with anhydrous manganese, and dried under reduced pressure to obtain 6 g of a yellowish brown crude extract.

120 g of silica gel 60 (US Merck, 230-400 mesh) was charged with CHCl, and slurried to fill a 4.5 X 30 cm column. The crude extract was dissolved in chloroform, 6 g of silica gel was added to the column, and dried by adsorption. The adsorbed crude extract was eluted gradually from the chloroform: methanol (100: 0) solution in steps of gradually increasing the ratio of methanol and collected in fractions of 50 ml. Fractions containing the active material were collected and dried under reduced pressure to obtain 910 mg of the active material, which was finally purified using HPLC (Perkin-Elmer, S410). At this time, an HPLC column was used for Ultracarb (Phenomenex). Ultracarb) 10 ODS (250 x 22.5mm, 10㎛) was used and the active material was eluted with 80% acetonitrile at a flow rate of 5ml per minute to identify the peak at 225nm ultraviolet (UV) absorption wavelength. The material showing anticancer activity showed a peak at 32 minutes of retention time (Rt), and the pure material obtained through HPLC was 20 mg.

Example 4 Structure Determination of GERI-BP002A

Various instrumental analyzes were performed to determine the structure of the anticancer active material.

1) UV-visible absorbance analysis

0.1 mg of the active substance finally purified by HPLC was dissolved in 2 ml of 100% methanol, and the absorption wavelength was analyzed by using an ultraviolet-visible spectrometer (Shimazu, UV-265), and the absorption peak was observed at 212 nm and 281 nm. (Figure 1).

2) Molecular weight analysis

Molecular weights were measured using the High-Resolution Electron Impaction (HERI) -MS method using the VG70-VSEQ Mass Spectrometer (Vacuum Generator, UK). Ion molecule peaks appeared (Figure 2). The molecular weight 340.2418 obtained by the above measurement and mathematical calculation method yielded CHO from the molecular formula of the substance.

3) Nuclear Magnetic Resonance (NMR) Analysis

The active material was completely dried, dissolved in 0.5 ml of CDCl, and placed in a 5 mm diameter tube, and subjected to NMR analysis with a Brooker AM-300. H-NMR is 300.13 MHz, C-NMR was measured at 75.47 MHz, H spectrum and As a result of analyzing the C spectrum (FIGS. 3 and 4), the molecular structure of the active material was found to be as follows.

Therefore, when the results of the various analytical methods described above are comprehensively interpreted, the structure of the anticancer active substance found by the present inventors is the same as that of the structural formula (I).

Example 5

Anticancer Activity Assay of GERI-BP002A

Assays for anticancer activity include human solid tumor cell lines A549 (non-small cell lung), SKMEL-2 (melanoma), HCT-15 (colon), SKOV-3 (ovarian), and XF- 498 (INS) and the like at 37 ° C using RPMI 1640 medium with 10% bovine serum albumin (FBS) (with L-glutime and no sodium bicarbonate, Gibco Cat. No. 31800-022) Cultures were incubated in a 5% CO ₂ incubator and passages were performed twice a week. Cells were isolated from adherent surfaces using a solution of 0.25% trypsin and 3 mM CDTA (1,2-cyclohexane diamine tetraacetic acid) in phosphate buffer (PBS). 5 x 10 ³ to 2 x 10 ⁴ cells were added to each well of a 96 well plate (Nunc) and incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator. GERI-BP002A was dissolved in a small amount of dimethylsulfoxide (DMSO) and diluted with experimental medium to the desired concentration for the test so that the final DMSO concentration was less than 0.5%. This was filtered through a 0.22㎛ ultrafiltration membrane and diluted with each concentration step.

After removing all of the medium from each cell culture medium incubated in a 96 well plate for 24 hours, GERI-BP002A prepared at each concentration was added to each well 200 μl and incubated for 48 hours. Tz (Time zero) plates were collected at the time GERI-BP002A was added.

Tz plates and each plate after incubation were subjected to the method of sulforhodamine B (SRB) analysis (Skehan, P., et al., J. Natl. Cancer Inst., 82, 1107-1112 (1990)). Cell fixation with trichloroacetic acid (TCA), staining with 0.4% SRB solution, 1% acetic acid washing, dye extraction with 10 mM Tris solution and OD value at 520 nm with a microplate reader (Dynatech Model MR 700). Measured.

ED ₅₀ for various cancer cells was calculated as follows. The cells were collected at the time of initiation of the drug and the amount of SRB 4 protein was collected to determine the number of living cells, which was determined as Tz. C). The OD values of wells treated and cultured with GERI-BP002A were called drug treated test values (T). The effects of GERI-BP002A such as growth stimulation, net growth inhibition and net killing from Tz, C and T could be determined. If T ≥ Tz, the cellular response function is 100 x (T-Tz) / (C-Tz), and if T ≤ Tz, it is calculated as 100 x (T-Tz) / Tz. .

As a control sample, cis-platinum was used to secure the objectivity of the experiment, and ED ₅₀ of GERI-BP002A could be calculated from the results in Table 2 below.

As a result of the experiment, the concentration showing anticancer activity in 50% of cells, that is, ED is about 5.45 μg / ml in A549 cells, about 8.83 μg / ml in SKOV-3 cells, about 5.79 μg / ml in HCT-15 cells, It was about 7.30 μg / ml in XF-498 cells and about 5.39 μg / ml in SKMEL-2 cells.

[Preparation Example]

Preparation of a pharmaceutical composition comprising GERI-BP002A

Hard gelatin capsules were prepared using a pharmaceutical composition comprising the following ingredients:

The ingredients were mixed thoroughly and filled into hard gelatin capsules in a total amount of 500 mg.

Claims (2)

An anticancer composition comprising, together with a suitable pharmaceutical excipient, an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient: The anticancer composition according to claim 1, wherein the compound of formula (I) is a substance produced by Aspergillus fumigatus FM-F-93 (KCTC 0111BP).