KR20050103259A - Anti-cancer agent containing interleukin-2 and monoacethyldiglyceride-3 as an effective ingredient - Google Patents

Abstract

The present invention relates to an anticancer agent containing monoacetyldiglyceride-3 (MADG-3) and interleukin-2 (IL-2) represented by Chemical Formula 1 as an active ingredient. When IL-2 is administered to a cancer-induced animal or MADG-3 is added to the cancer-induced animal, it is excellent in delaying the occurrence of cancer. Therefore, an anticancer agent containing the mixture as an active ingredient is useful for cancer treatment Can be.

<Formula 1>

Description

Anti-cancer agent containing interleukin-2 and monoacethyldiglyceride-3 as an effective ingredient}

The present invention relates to an anticancer agent containing a compound isolated from antler as an active ingredient and a cancer treatment method using the same.

Antler deer and is one of the herbal medicines most widely used in oriental medicine as well as dry as ginseng collected the deer is not abandonment yugak belonging to (Cornu cervi), as the origin of the antler has been used in our country animals belonging to the deer Cervus nippon Temminick var. Mantchuricus Swinhoe and Cervus elaphus L. Cornu Ceriv Pavum.

Deer antler has a variety of effects such as tonic, growth, growth promotion, hematopoietic, neurodebilitating, heart failure, and hyperfunction of the five intestines. And effects such as myocardial exercise improvement, fatigue recovery, physical resistance enhancement, pneumonia fiduciary effect, etc. are recorded in ancient literature.

Various studies have been conducted to investigate the mysterious effects of deer antler, and as a result, free amino acid and trace elements, hexasaccharide and octane sugar, hexamine, uronic acid, sialic acid, acid mucopolysaccharides, hyaluronic acid and sulfuric acid have been studied. The presence of chondroitin A, various fatty acids, prostaglandins and the like have been confirmed, and glycolipids and phospholipids, cholesterol, hypoxanthine, cholesterol, 5-en-3β, 7α-diol, cholesterol esters, polyamines, and the like have been reported. In addition, it is known that the antler contains estrone, estradiol receptor, and the like.

Currently, cancer disease, the leading cause of death in Korea, is known to increase every year. However, chemotherapy and radiation therapy used for chemotherapy damage not only cancer cells but also normal normal bone marrow cells, especially hematopoietic cells that regulate immunity and hematopoietic function. Paralysis has been pointed out as a serious problem.

Therefore, the present inventors completed the present invention by confirming that the mixture consisting of the active ingredient and interleukin-2 separated from the antler known to have an excellent medicinal effect has excellent anticancer action.

It is an object of the present invention to provide an anticancer agent containing monoacetyldiglyceride-3 and interleukin-2 as active ingredients.

In order to achieve the above object, the present invention provides a monoacetyldiglyceride-3 (hereinafter, abbreviated as 'MADG-3') and interleukin-2 (hereinafter, referred to as 'IL-2') described in Chemical Formula 1 below. It provides an anticancer agent containing an abbreviation) as an active ingredient.

<Formula 1>

Monoacetyldiglyceride-3 (Molecular Weight: 634)

Hereinafter, the present invention will be described in detail.

The anticancer agent of the present invention preferably contains 20 to 35% by weight, and more preferably 25 to 30% by weight of MADG-3. In addition, when administering the anticancer agent of the present invention, it is preferable to administer at a concentration of 25 to 50 mg / kg once to three times a day.

In addition, IL-2 is preferably generated in the cell by the expression of the gene encoding it. Expression of the IL-2 gene can be accomplished by conventional methods known in the art. Specifically, a group consisting of adenovirus, adenovirus, baculovirus, alphavirus, retrovirus, lentivirus, adeno-associated virus, and the like. Ii) Gene alone, consisting of the gene and calcium-phosphate, DEAE-dextran, polybrene, polyethylenimine and dendrimer When the compound is made through a complex of compounds selected from the group, or a cationic lipid (lipid) or liposome containing a gene, and (iii) electroporation, electropermeabilization, microinjection Selected from the group consisting of bead transfection and biolistic particles And the like when made by a physical method.

Adenovirus is more preferably used when the gene is to express the IL-2 gene through a virus, and the adenovirus is more preferably made of Ad / hIL-2, and the Ad / hIL-2 is 20 to 200. Most preferably, it is injected with a mupiplicity of infection (MOI).

The anticancer agent of the present invention may be prepared as a medicament by adding at least one pharmaceutically acceptable carrier together with an active ingredient having an anticancer effect. The carrier may include, but is not limited to, saline, buffered saline, water, glycerol and ethanol, and any suitable agent known in the art (Remingtons's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA) may be used. .

Among the anticancer agents of the present invention, the formulation for pharmacological MADG-3 can be administered orally during clinical administration and can be used in the form of general pharmaceutical formulations, and when formulated, the commonly used fillers, extenders, binders, humectants, It is prepared using diluents or excipients such as disintegrants and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and liquid preparations for oral use include suspensions, solvents, emulsions, and syrups. In addition to liquids and paraffins, various excipients may be included, such as wetting sweeteners, fragrances, preservatives and the like.

The IL-2 gene in the anticancer agent of the present invention can be administered parenterally during clinical administration and can be used in the form of a general pharmaceutical preparation. That is, it can be administered in various parenteral dosage forms during actual clinical administration, and solid preparations include tablets, pills, powders, granules, capsules, and the like, and liquid preparations include suspensions, contents, emulsions, syrups, and the like. This may include a variety of excipients, such as wetting agent sweeteners, fragrances, preservatives, etc., in addition to commonly used simple diluents such as water, liquid, paraffin. Specifically, preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used. In addition, calcium or vitamin D ₃ may be added to enhance the efficacy of anticancer drugs.

The formulation of the present invention may be applied differently depending on the age, sex, condition, absorption of the active ingredient in the body, inactivation rate and excretion rate, the drug used in combination, for example, 1 to 1.5 g may be administered per day. It is not limited to this. The invention also includes formulations of dosage units. The formulations are present in individual dosage forms, such as tablets, coated tablets, capsules, pills, suppositories, and ampoules, wherein the amount of active compound in the drug corresponds to the fraction or multiple of the individual dosage. Dosage units may contain, for example, one, two, three or four times the individual dosage, or 1/2, 1/3 or 1/4 times. The individual dosages preferably contain an amount in which the active compound is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dosage.

The effective dose of the anticancer agent is 25-50 mg / kg, preferably 35-45 mg / kg, and may be administered 2-3 times a day. The composition as described above is not necessarily limited to this, and may vary depending on the condition of the patient and the degree of development of neurological diseases.

MADG-3 is known to promote hematopoietic stem cell proliferation and immune boosting activity by promoting lymphocyte activation. However, it has not been found to have an anticancer effect. In the present invention, it was confirmed that MADG- 3 has an immune enhancing effect because it promotes proliferation of T cells, monocytes, and dendritic cells, which are immune cells (see FIGS . 1 to 4 ). In addition, after injecting lymphoma into mice to induce cancer, adenoviruses containing human interleukin-2 gene (hereinafter abbreviated as " hIL-2 gene") are injected. It was confirmed to increase (see FIGS . 5 to 7 ). In addition, cancer cells were injected into hamsters to induce the development of cancer, and then MADG-3 was confirmed to be delayed in the development of cancer, and when MADG-3 and hIL-2 genes were administered in combination, cancer development was much more effective. The delay was confirmed (see FIGS . 8 to 11 ). Therefore, MADG-3 and IL-2 also had anticancer effects, respectively, and when mixed, it was found that the anticancer effect was even better. Therefore, in the present invention, MADG- 3 and hIL-2 genes are contained as an active ingredient, wherein MADG- 3 is administered as soft capsules and tablets, and the hIL-2 gene is intravenously injected with Ad / hIL-2 together with BMSC. The present invention was completed by administration (see Preparation Example 1 ).

In addition, the kind of cancer which can be treated using the anticancer agent of the present invention is not limited, but is preferably selected from biliary tract cancer or kidney cancer.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Effect of MADG-3 on T Cell Proliferation and Monocyte Cell Proliferation

<1-1> Effect of MADG-3 on T Cell Proliferation

Splenocytes were isolated from the spleen of C57BL / 6 mice (provided by Asan Life Science Research Institute Animal Lab). Splenocytes were made into single cells through aspiration and flushing. Red blood cells were removed using ammonium chloride, and cell debris and clumps were removed while passing through nylon wool. Next, magnetic beads containing anti-goat IgG (Miltenyi Biotec, bergich gladbach, Germany), anti-mouse CD4 (Miltenyi Biotec, bergich gladbach, Germany) or anti-mouse CD8 antibodies (Miltenyi Biotec, bergich gladbach, Germany) T cells were isolated using MACS bead, Miltenvi Biotec, Bergich gladbach, Germany (Turner and Dockrell (1996) Immunology , 87 : 339-342).

The isolated T cells were subjected to IMDM medium (Isocove's modified dulbecco's medium) (Gibco, Grand Island, NY) and 10% fetal bovine serum (hereinafter abbreviated as 'FBS') (Gibco, Grand Island, NY). Suspended in a medium containing and then inoculated in each well of a 94-well plate with 5 × 10 ⁴ cell number, MADG-3 (Ewha Womans University Department of Chemistry, Ewha Womans University) concentration of 0.01, 0.1 or 1 ㎍ / ㎖ in the inoculated cells Or IL-2 at a concentration of 20 ng / ml. On day 6 of the culture, ³ H-thymidine was treated at 1 μCi concentration per each cell culture well and reacted for 24 hours. Cells were obtained on the 7th day of culture, and then the degree of ³ H-thymidine incorporation was measured to calculate a stimulation value (hereinafter, abbreviated as 'SI') by the following formula (1).

<Equation 1>

SI = ³ H-thymidine absorbed from experimental wells (experimental group CPM) / ³ H-thymidine absorbed from wells of control group (control CPM)

As a result, the SI of T cells was 2.05 in the group treated with MADG-3, which was similar to the group treated with IL-2 ( Table 1 ).

Treatment group SI IL-2 (20 ng / ml) * 2.05 ± 0.24 MADG-3 (0.01 μg / mL) ** 1.83 ± 0.32 MADG-3 (0.1 μg / ml) * 1.96 ± 0.18 MADG-3 (1 μg) * 2.05 ± 0.64

In the above table, * P <0.05, ** P <0.005, and each value is the result of three experiments.

<1-2> Effect of MADG-3 on Monocyte Cell Proliferation

Histopark 1077 was used to separate monocytes from human whole blood. The isolated monocytes were inoculated into a tissue culture flask at a concentration of 5 × 10 ⁶ cells / ml and then incubated in a 5% CO ₂ incubator for 3 hours. After incubation for 3 hours, the adsorbed cells were obtained and suspended in RPMI 1640 medium (GIBCO, Grand Island, NY) containing 10% FBS. 5 × 10 ⁴ viable cells per well were incubated in 96 well plates with MADG-3 at a concentration of 1 μg / ml. On day 6 of culture, 1 μCi ³ H-thymidine was added to each culture well and incubated for 24 hours. Finally, cells were obtained on day 7 of culture, and then the incorporated ³ H-thymidine was quantified. SI was calculated according to Equation 2 using the quantified values.

<Equation 2>

SI = number of monocytes in sample wells / number of monocytes in control wells

As a result, it was found that the treatment of MADG-3 stimulated the monocytes by 10.86-fold increase in the SI value of the monocytes compared to the control group ( Table 2 ).

Treatment group SI (± S.E) Untreated control One MADG-3 (1 μg / ml) * 10.68 ± 0.13

In the above * P <0.001, each value is the result of two experiments.

Example 2 Effect of MADG-3 on T Cell Activity

The ELispot assay is a highly sensitive assay capable of quantifying cytokines, since the bottom of each well of an ELispot (ESAT-6 enzyme-linked immunospot assay) plate is coated with a cytokine-specific capture antibody. Thus, ELispot assay was performed to quantify cytokine with higher sensitivity to measure T cell activity. T cells were seeded in 24 well sterile tissue culture plates (Nunc, Denmark) at a concentration of 2 × 10 ⁶ cells / ml and treated with MADG-3 at 0.01, 0.1, 1 μg / ml or IL-2 at 20 The cells were incubated at a concentration of ng / ml. After 7 days of culture, the cells were harvested and mouse IL-2 was inoculated at 5 × 10 ⁵ cell count / ml in a multi-test plate coated with primary antibody (Elispot system kit, AID, Straberg, Germany). The plates were incubated in a 5% CO ₂ incubator for 24 hours and then the amount of IL-2 secreted by T cells was analyzed using the IL-2 ELispot kit according to the manufacturer's instructions. Each sample was tested twice and the amount of IL-2 produced by the cells was determined by ELispot analysis using an Elispot reader (AID ELispot Reader System).

As a result, in the group treated with MADG-3, the activity of T cells increased 1.5 times compared to the untreated control ( Fig. 1 ).

Example 3 Effect of MADG-3 on the Expression of Adhesion Molecules of Dendritic Cells

<3-1> dendritic cell culture

Bone marrow cells were isolated from the femur and tibia of C57BL / 6 mice by known methods (Park, J. et al . (2003) J. Korean Med. Sci ., 18 : 372-380). After washing the separated cells three times with RPMI medium, only monocytes (mononuclear cells) were isolated, and the isolated monocytes were inoculated into a tissue culture flask using RPMI medium and a medium containing 10% FBS for 3 hours. The culture was allowed to adhere to the culture. After incubation, the adherent cultured monocytes were removed and the unattached cells were inoculated into 100 mm tissue culture dishes. At the inoculation, the cell concentration was 1 × 10 ⁵ cell number / ml, and 10% FBS was added to RPMI medium, and additionally 20 g / ml of mouse rGM-CSF (R & D systems, minneapolis, MN, USA) Medium with 10 ng / ml IL-4 (R & D systems) and 2.5 ng / ml mouse TNF-α (R & D systems) was used. The medium of the culture dish was changed every 3 days. TNF-α was added on the 6th day of culture, and every 3 days until 11 days thereafter. Mature dendritic cells were used for RT-PCR analysis for adhesion molecule studies.

As a result, when the round granule cells were cultured for 3 days, the cells formed clusters, adhered to the bottom of the cell culture well, and showed the cultured form, and mature dendritic cells were clustered at 6 to 7 days of culture. It showed that the growing form, the dendritic cells on the ninth day of culture was confirmed to exhibit the form of specifically long and small protruding ( Fig. 2 ).

<3-2> Phenotypic Determination of Dendritic Cells

Cells that were negative for trypan blue staining and large in size were counted as living cells and their respective morphology was identified. 1 × 10 ⁶ cells were incubated, washed and fixed with 1% paraformaldehyde solution. Fixed cells were phenotyped with antibodies to the following markers by flow cytometric analysis using FACScan (Beckton Dickinson, Mountain View, Calif., USA); Isotype control for hamster IgG, murine IgG 2 _a , DC markers: DEC 205 (NLDC-145) and CD 11C, co-stimutatory / adhesion molecule: CD 80 (B7-1 ) And CD 86 (B7-2), macrophage markers: CD 14 and F4 / 80, granulocyte markers: Gr-1 (Pharmingen, Hamburg, Germany).

As a result, the costimulatory specific molecular markers CD80 and CD86 were high, and the dendritic cell specific markers CD11c and DEC-205 were high. However, the dendritic cells isolated in the present invention exhibited low values for CD14 and F4 / 80, which are monocyte-specific markers, and Gr-1, which is a granulocyte-specific marker, and exhibited the phenotype of dendritic cells accurately. It can be seen that the purity ( Fig. 3 ).

<3-2> Expression Analysis of Treatment and Adhesion Molecules of MADG-3

The relationship between cells is known to stimulate various hematopoietic and immune cells. Thus, we tried to determine whether MADG-3 affects various adhesion molecules of the cells. Specifically, the dendritic cells cultured in the above Example were treated with MADG-3 at a concentration of 1 μg / ml, and the cells were obtained to perform RT-PCR.

Specifically, primers used for RT-PCR include Icam-1 (SEQ ID NO: 1 and SEQ ID NO: 2), Icam-2 (SEQ ID NO: 3 and SEQ ID NO: 4), Vcam-1 (SEQ ID NO: 5 and SEQ ID NO: 6), VLA-4 (SEQ ID NO: 7 and SEQ ID NO: 8), VLA-5 (SEQ ID NO: 9 and SEQ ID NO: 10), LFA-1 (SEQ ID NO: 11 and SEQ ID NO: 12), and GAPDH (SEQ ID NO: 13 and SEQ ID NO: 14) Primer set was used with 2 μl DNA, 10 × buffer, 1.5 μl mgCl ₂ , 2 μl dNTP, 0.5 μl forward primer, 0.5 μl reverse primer, 0.2 μl polymerase (polymerase) and a mixed solution consisting of 15.8 μl of distilled water were used.

Total RNA isolated from MS-5 and dendritic cells cultured using oligo (dt) -primer were reverse transcribed and reacted for 30 seconds at 94 ° C, 30 seconds at 65 ° C and 50 seconds at 72 ° C. PCR was performed. The number of PCR runs was performed a total of 34 times, confirming the production of the product increased by 2 times per PCR run. When performing RT-PCR, expression of Vcam-1, Icam-1, Icam-2, VLA-4, VLA-5, and LFA-1 molecules was confirmed, and PCR reaction was performed on GAPDH for comparative quantification. The presence of the corresponding cDNA was confirmed.

As a result, dendritic cells treated with MADG-3 increased the expression of adhesion molecules Icam-2, VLA-5, and LFA-1 compared to the untreated control ( FIG. 4 ).

Example 4 Analysis of the Effect of IL-2 on the Development and Metastasis of Cancer in vivo  test)

<4-1> Preparation of stromal cells transformed with hIL-2 expressing adenovirus

First, human adenovirus serotype 5 by homologous recombination, adenovirus containing adenovirus Ad / LacZ containing a β-galactosidase gene, human interleukin-2 gene ( hIL-2 gene) Adenovirus Ad / ΔE1, in which the Ad / hIL-2 and E1 genes were deleted, was prepared ( Leukemia & Lymphoma , vol. 44, No. 11 (November 2003), pp1973-1978).

Next, X-gal analysis was performed to measure the infection efficiency of adenovirus. Specifically, adenovirus Ad / LacZ was transduced into bone marrow stromal cells (BMSC) with 0, 10, 20, and 50 multiplicity of infection (MOI). 24 hours after transduction, fixation solution was added and reacted at room temperature for 5 minutes, then fixation solution was removed, and 5-bromo-4-chloro-3-indolyl-β-D-galactopy was obtained. Lanoside (X-gal) was added and stained overnight at 37 ° C. Infection efficiency was determined with the degree of blue to the stained cells. The distribution of infected cells to the number of infected adenoviruses was 5-15% at 10 MOI and 20-40% at 20 MOI, 60-70% at 50 MOI. It was judged that it was proper to infect adenovirus with 25 MOI, and the number of subsequent adenovirus infections was 25 MOI. Interstitial cells transduced with Ad / ΔE1 were termed 'BMSC + Ad / ΔE1' and interstitial cells transduced with Ad / hIL-2 were named 'BMSC + Ad / hIL-2'.

<4-2> Anticancer Effects of IL-2 in Tumor-induced Mice with Lymphoma Injection

The tails of 6-8 week old Balb / c AnN mice were injected with A-20 lymphoma cells (ATCC: Rockville, MD, USA) at 5 × 10 ⁴ cells per horse. 7 days after lymphoma cell injection, PBS, BMSC (1 × 10 ⁶ cells / horse), BMSC (1 × 10 ⁶ cells / horse) + Ad / ΔE1 (25 MOI), BMSC (1 × 10 ⁶ cells / Mari) + Ad / hIL-2 (25 MOI) were injected respectively. Two to four weeks after injection, mice were sacrificed and observed for tumor formation.

As a result, in the control group injected with only BMSC alone and in the BMSC + Ad / ΔE1 injection group, large tumors were formed in the liver, but the BMSC + Ad / hIL-2 injection group showed normal liver morphology ( FIG. 5 ).

In addition, as a result of extraction and histologic staining of livers with a difference in tumor formation, lymphoma cells showed diffuse or nodular infiltration in the BMSC-injected group, and the BMSC + Ad / ΔE1 infusion group. Lymphoma cells showed interstitial infiltrates and no lymphoma cells were found in the BMSC + Ad / hIL-2 injection group ( FIG. 6 ). Therefore, it could be seen that the tumor formation was delayed and suppressed when IL-2 was injected into mice inducing tumorigenesis by lymphoma cell injection.

In addition, the survival rate was measured while observing the mice of each injection group. As a result, mice in which BMSC or BMSC + Ad / ΔE1 was injected after tumor induction into lymphoma cells after 8 weeks of breeding had a 20% survival rate, but BMSC + Ad / hIL-2 was injected after tumor induction into lymphoma cells. The survival rate of one mouse was 100% ( FIG. 7 ). Therefore, it could be seen that administration of IL-2 to tumorigenic cells induced by lymphoma cell injection suppressed tumorigenesis and increased survival rate.

Example 5 Analysis of Anticancer Effect and Anticancer Effect of MADG-3 by Subcutaneous Tumor Transplantation (Local Model) and Intravenous Tumor Injection (Full Body Model)

Six-week-old female Syrian golden hamsters (Harlan, Indianapolis, India, USA) were bred in specific pathogen-free kennels. 5 x 10 ⁵ biliary cancer cells KIBG-5 ( Molcular therapy , Vol. 3, No4, pp431-437) were suspended in 100 µl of serum-free RPMI 1640 medium and immediately injected intravenously into the femoral vessels (IV). . In addition, 1 × 10 ⁵ KIBG-5 cells were suspended in 100 μl of serum-free RPMI 1640 medium and subcutaneously injected (SC) in the flank. At this time, hamsters injected with KIBG-5 cells were divided into the following seven groups; 1) control group treated with RPMI medium, 2) unmodified BMSC cells (2.5 × 10 ⁶ cells) ( Leukemia & Lymphoma , Vol. 44, No11, pp1973-1978) treated group, 3) modified with Ad / ΔE1 100 MOI One BMSC cell population ( Leukemia & Lymphoma , Vol. 44, No11, pp1973-1978), 4) DC + tumor lysate (5 × 10 ⁶ ) treatment group, 5) BMSC cell population modified with Ad / hIL-2 100 MOI Group ( Leukemia & Lymphoma , Vol. 44, No11, pp1973-1978), 6) BMSC cell group modified with Ad / hIL-2 100 MOI + MADG-3 treatment group and 7) MADG-3 10 mg / day treatment group. In the case of BMSC cell injection group, only one injection of 2.5 × 10 ⁶ BMSCs per hamster was injected after a week of tumor cell injection or subcutaneous transplantation. In the DC + tumor lysate treatment group, 12 x weekly injection of 5 × 10 ⁶ DC + tumor lysates per hamster every 1, 2, 3, 4, 6, or 8 weeks in the local or intravenous infusion group. Was observed. MADG-3 treated groups were implanted with 10 mg / kg hamsters and treated for 8 weeks twice a week from -7 to 7 days.

As a result, tumors were formed in the control group RPMI injection group, BMSC cell injection group and BMSC + Ad / ΔE1 injection group at 4 weeks after tumor injection ( FIG. 5 ), but DC + tumor lysate injection group, MADG No tumors were observed in the -3 and BMSC + Ad / hIL-2 injection groups.

In addition, 8 weeks after tumor injection, multiple metastatic (metastatic) groups were used in the control group RPMI injection group, BMSC cell injection group, BMSC + Ad / ΔE1 injection group, DC + tumor lysate injection group, and MADG-3 injection group. ) Lung lesions were formed, but the BMSC + Ad / hIL-2 infusion group did not show any signs in the lungs ( FIGS. 6, 7A and 7B ).

In addition, 8 weeks after tumor injection, multiple metastatic lung lesions were formed in the control group of the RPMI injection group, the BMSC cell injection group, and the BMSC + Ad / ΔE1 injection group, but the BMSC + Ad group was formed. The / hIL-2 infusion group did not show any signs in the lungs ( FIGS. 9, 10A and 10B ).

In addition, 12 weeks after subcutaneous injection of tumor, control group RPMI injection group, BMSC cell injection group, BMSC + Ad / ΔE1 injection group, DC + tumor lysate injection group, and MADG-3 injection group had tumor formation at the injection site. However, no tumors were formed in the BMSC + Ad / hIL-2 injection group and the BMSC + Ad / hIL-2 + MADG-3 injection group ( FIG. 8 ).

Example 6 Toxicity Test of MADG-3

The organic chemically synthesized MADG sample was dissolved in 5% ethanol and used orally (p.o.) at a dose of 0.1 ml per 20 g of body weight. The control group was administered ethanol 5% solution. ICR mice that were fed and reared under SPF conditions were used. Samples were fasted one night before oral administration and water and feed were allowed to drink freely.

The experimental animals consisted of 8-10 males of ICR mice weighing 25-30 g and weighing 62.5 mg / kg, 125 mg / kg, 250 mg / kg, 500 mg / kg, 1 g / kg, From the day of oral administration at a dose of 2 g / kg, the number of mice to die and visual abnormalities were observed for 14 days. LD ₅₀ was calculated by the Lichfield-Wilcoxon method (白 須, 泰 彦, 吐 山, 豊 秋: 新 毒性 試驗 法-方法 と 評價-acute toxicity test, Realize Inc., Tokyo, 1988), weight gain rate Was calculated according to the following equation.

<Equation 3>

% Weight gain = {weight on day 14-weight on day 0} ÷ {weight on day 0} × 100

As a result, as shown in Table 3 , oral administration of MADG-3 at a dose of 62.5 mg / kg to 2 g / kg in mice showed no toxicity at all, indicating that both LD _{50 and} 2 g / kg or more. Could. In addition, no abnormality was observed when observed for 14 days after drug administration. The body weight of the animals that received the sample increased substantially steadily, as in the group that did not receive the sample, and as shown in Table 4 , no specific weight gain or weight loss effect was observed.

% Mortality by MADG-3 Dosage MADG-3 Dose (mg / kg) 0 62.5 125 250 500 1000 2000 Number of death mice / Oral administration mice 0/10 1/8 0/8 1/8 2/9 0/9 0/9 LD ₅₀ 0 12.5 30 12.5 22.2 0 0

Weight gain 14 days after oral MADG-3 MADG-3 Dose (mg / kg) 0 63 125 250 500 1000 2000 Body weight (g) after 14 days of administration 38.7 ± 1.1 34.3 ± 0.1 38.1 ± 1.3 35.6 ± 1.4 36.5 ± 1.9 35.5 ± 1.1 35.9 ± 2.7 % Weight gain 16.9 11.9 17.3 11.2 10.1 9.5 14.4

On the other hand, IL-2 is a natural substance produced in the cell during the immune response of the animal cells, and did not perform a separate toxicity test.

Preparation Example 1 Preparation of Anticancer Agents Containing MADG-3 and IL-2 as Active Ingredients

The present inventors confirmed that MADG-3, IL-2 and mixtures thereof have excellent anticancer activity through the above examples, thereby preparing an anticancer agent containing MADG-3 and IL-2 as an active ingredient as follows. In addition, the preparation of the following therapeutic agent can be used for the application of the health food as well as the therapeutic agent. IL-2 included in the anticancer agent may use the adenovirus Ad / hIL-2 prepared in the present invention, and any medium other than IL-2 may be used.

<1-1> soft gelatin capsules containing MADG-3 and IL-2  Preparation of Intravenous Formulations Containing Genes

<1-1-1> soft capsule containing MADG-3

MADG-3 20%

Vitamin C 4.5%

Vitamin D ₃ 0.001%

Manganese Sulfate 0.1%

10% wax

Palm oil 25%

Safflower Seed Oil 30.399%

<1-1-2> IL-2  Intravenous preparations containing genes

BMSC + Ad / hIL-2 (25 MOI) 0.2%

Mannitol 0.3%

Saline 9.5%

<1-2> tablet containing MADG-3 and IL-2  Preparation of Intravenous Formulations Containing Genes

<1-2-1> Tablet containing MADG-3

MADG-3 35%

Vitamin C 10%

Vitamin D ₃ 0.001%

Manganese Sulfate 0.1%

Crystalline cellulose 25.0%

Lactose 17.999%

Magnesium Stearate 2%

<1-2-2> IL-2  Intravenous preparations containing genes

BMSC + Ad / hIL-2 (25 MOI) 0.2%

Mannitol 0.3%

Saline 9.5%

As described above, it was confirmed that the occurrence of cancer was delayed when the cancer cells were injected with MADG-3 and IL-2, which were isolated from antler, to hamsters that induced cancer development. Therefore, anticancer agents containing MADG-3 and IL-2 as active ingredients can be usefully used for cancer treatment.

Figure 1 is a photograph showing the T cell activity of the untreated control group, IL-2 treated group (20 ng / ml treatment), MADG-3 treated group (1 µg / ml treatment). Each number in the figure represents the number of spots that captured an IL-2 specific antibody.

Figure 2 is a dendritic cell (DC) is isolated from the mouse bone marrow cells, and the cell morphology photograph after each step of treatment with GM-CSF, IL-4 and TNF-α cultured.

A : Photograph immediately after inoculation of mouse bone marrow cells at a concentration of 2 × 10 ⁶ cells / 100 mm dish (100 × magnified micrograph),

B : When the rounded dendritic granule cells were incubated for 3 days, the cells formed clusters and adhered to the bottom of the cell culture well (400 × enlarged micrograph),

C : Photograph showing the growth and growth of mature dendritic cells in 6 to 7 days of culture (400 × magnified micrograph).

D : Photograph confirming that the dendritic cells showed the shape of a special long, small protrusion on the 9th day of culture (1000 × magnified micrograph). Picture,

FIG. 3 shows FACS analyzing the expression of dendritic cell specific markers, monocyte specific markers, and granulocyte specific markers on day 11 of culturing bone marrow dendritic cells isolated from Balb / c AnN mice. Analytical graph (where isomer control staining for hamster IgG and rat IgG2a was used as the setting marker line (straight line)).

CD80 and CD86: costimulatory specific molecular markers,

CD11c and DEC-205: dendritic cell specific markers,

CD14 and F4 / 80: monocyte / macrophage specific markers,

Gr-1: granulocyte specific marker,

Figure 4 is an RT-PCR electrophoresis picture confirming whether or not to affect the expression of adhesion molecules when MADG-3 is treated to dendritic cells.

Lane 1: Vcam-1, Lane 2: Icam-1, Lane 3: Icam-2,

Lane 4: VLA-4, lane 5: VLA-5, lane 6: LFA-1,

Lane 7: GAPDH, (+): MADG-3 treated group, (-): untreated control group,

FIG. 5 shows A-20 lymphoma cells injected intravenously to induce tumor formation, followed by 2-4 weeks after intravenous injection of BMSC, BMSC + Ad / ΔE1 or BMSC + Ad / hIL-2. Photograph (pictured above) and cancer showing lung cancer (pictured below).

FIG. 6 shows tumor formation by injecting A-20 lymphoma cells intravenously followed by infusion of BMSC, BMSC + Ad / ΔE1 or BMSC + Ad / hIL-2 via vein 2-4 weeks after lung tissue This is a tissue stained picture.

A : BMSC injection group, 200 × magnification,

B : BMSC + Ad / ΔE1 injection group, 400 × magnification,

C : BMSC + Ad / hIL-2 injection group, 400 × magnification.

7 is a graph showing the survival rate for 8 weeks after A-20 lymphoma cells were injected intravenously to induce tumor formation and after injection of BMSC, BMSC + Ad / ΔE1 or BMSC + Ad / hIL-2 via vein .

FIG. 8 is a photograph showing tumor formation at the site of tumor cell injection after 4 weeks of biliary cancer cell KIGB-5 was injected by intravenous injection and treated under different conditions.

A : RPMI injection group, B : BMSC injection group,

C : BMSC + Ad / ΔE1 injection group, D : DC + tumor lysate injection group,

E : MADG injection group, F : BMSC + Ad / hIL-2 injection group.

FIG. 9 is a photograph showing the tumor formation in the lung after 8 weeks of biliary cancer cell KIGB-5 injected by intravenous injection and treated under different conditions.

A : RPMI injection group, B : BMSC (2.5 × 10 ⁶ cells / day) injection group,

C : BMSC (2.5 × 10 ⁶ cell number / day) + Ad / ΔE1 (50 MOI) injection group,

D : DC (5 × 10 ⁶ cell count / day) + tumor lysate infusion group,

E : BMSC (2.5 x 10 ⁶ cell count / day) + Ad / hIL-2 (50 MOI) injection group,

F : MADG-3 (50 mg / kg / day) injection group,

10A and 10B are histologically stained tumor formations in the lung after 8 weeks of biliary cancer cell KIGB-5 injected intravenously and treated under different conditions.

In FIG. 10A ,

A : RPMI injection group,

B : BMSC (2.5 × 10 ⁶ cell number / day) injection group,

C : BMSC (2.5 × 10 ⁶ cell number / day) + Ad / ΔE1 (50 MOI) injection group,

D : DC (2.5 × 10 ⁶ cell number / day) + Ad / hIL-2 (50 MOI) injection group.

In FIG. 10B ,

A : RPMI injection group,

B : DC (5 × 10 ⁶ cell number / day) + tumor lysate injection group,

C : MADG-3 (50 mg / kg / day) injection group,

FIG. 11 is a photograph of tumors formed after 12 weeks of biliary cancer cell KIGB-5 subcutaneously transplanted (1 × 10 ⁵ cells) and treated with different conditions, and their size.

A : RPMI injection group,

B : BMSC (2.5 × 10 ⁶ cell count / day) + Ad / ΔE1 (100 MOI) injection group,

C : BMSC (2.5 × 10 ⁶ cell number / day) injection group,

D : DC (5 × 10 ⁶ cell count / day) + tumor lysate infusion group,

E : BMSC (2.5 × 10 ⁶ cell number / day) + Ad / hIL-2 (100 MOI) injection group,

F : BMSC (2.5 x 10 ⁶ cell counts / day) + Ad / hIL-2 (100 MOI) + MADG-3 (10 mg / kg / day) injection group,

G : MADG-3 (10 mg / kg / day) injection group

<110> KIM, Sang-Hee B. <120> Anti-cancer agent containing interleukin-2 and monoacethyldiglyceride-3 as an effective ingredient <130> 4p-01-69 <160> 14 <170> KopatentIn 1.71 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ICAM-1 forward primer <400> 1 ccaactggaa gctgtttgag ct 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ICAM-1 reverse primer <400> 2 ttctgtcgaa ctccacagtc a 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ICAM-2 forward primer <400> 3 catatggtcc gagaagcaga t 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ICAM-2 reverse primer <400> 4 aagcatagca ggacagatgt c 21 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> VCAM-1 forward primer <400> 5 ggttgggaag ccggtcacag tcaa 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> VCAM-1 reverse primer <400> 6 gcacacgtca gaacaaccga atcc 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> VLA-4 forward primer <400> 7 ctcagatctc cttgttggag cagc 24 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> VLA-4 reverse primer <400> 8 tgaatgcctg gtgtgtccta c 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> VLA-5 forward primer <400> 9 cagtggtgat gacactgatg a 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> VLA-5 reverse primer <400> 10 agaagctaag gttgatgcag g 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LFA-1 forward primer <400> 11 tgacacttta cttgcgacca 20 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LFA-1 reverse primer <400> 12 gatgggtagt cgaactcatt g 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 13 accacagtcc atgccatcac 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 14 tccaccaccc tgttgctgta 20

Claims (10)

An anticancer agent containing monoacetyldiglyceride-3 (MADG-3) and interleukin-2 (IL-2) represented by the following Chemical Formula 1 as an active ingredient. <Formula 1> According to claim 1, wherein the MADG-3 is an anticancer agent, characterized in that contained 20 to 35% by weight relative to the total weight of the anticancer agent. The anticancer agent according to claim 1, wherein the IL-2 is produced in a cell by expression of a gene encoding the IL-2. The method of claim 3, wherein the expression of the IL-2 gene is adenovirus (adenovirus), baculovirus, alphavirus, retrovirus, lentivirus, adeno-binding virus (adeno). Anti-cancer agent, characterized in that made by a virus selected from the group consisting of -associated virus. The anticancer agent according to claim 4, wherein the adenovirus is Ad / hIL-2. The anticancer agent according to claim 5, wherein the Ad / hIL-2 is administered at 20 to 200 muiplicity of infection (MOI). According to claim 3, the expression of the IL-2 gene is the gene alone, the gene and calcium-phosphate (calcium-phosphate), DEAE-dextran (polybrene), polyethylenimine (polyethylenimine) and dendrimer (dendrimer) An anticancer agent comprising a complex of compounds selected from the group consisting of C) or a cationic lipid (lipid) or liposome comprising a gene. 4. The method of claim 3, wherein the expression of the IL-2 gene is characterized by electroporation, electropermeabilization, microinjection, bead transfection and biolistic particle injection. Anticancer agent, characterized in that made by a physical method selected from the group consisting of. The anticancer agent according to claim 1, wherein the anticancer agent is administered at 25 to 50 mg / kg of body weight. The anticancer agent according to claim 1, wherein the anticancer agent is for treating biliary tract cancer or kidney cancer.