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    • 专利摘要:
    PURPOSE: Pregnane glycoside of formula (1) and/or formula (2) which is isolated from Cynanchum wilfordii and its use as an anticancer agent are provided. CONSTITUTION: A pregnane glycoside represented by formula (1), isolated from Cynanchum wilfordii, is prepared by carrying out the following steps of: extracting the roots of Cynanchum wilfordii ground and drying in a shade with methanol and concentrating the extract under vacuum to obtain the methanol extract; fractionating to dichloromethane and water; performing a silica gel column chromatography and then obtaining an active fraction using n-hexane ethylacetate step gradient system; performing chromatography on a RPMPLC column to the active fraction; and recrystallizing with methanol. The compound increases the activities of the anticancer agent by preventing the anticancer agent from being released from a cell line having a multi-drug resistance, inhibits the replication of an endothelial cell necessary to the angiogenesis and inhibits cancer cellular infiltration by decreasing the activities of matrix metalloprotease and the expression of the gene encoding the enzyme.
    公开号:KR20000056561A
    申请号:KR1019990005977
    申请日:1999-02-23
    公开日:2000-09-15
    发明作者:이정준；김항섭；이정형；홍영수；김규원
    申请人:박호군；한국과학기술연구원；
    IPC主号:
    专利说明:

    Preganane glycosides, their preparation, and their use as anticancer agents
    The present invention relates to natural pregnan glycoside compounds isolated from Cynanchum wilfordii and their use as anticancer agents.
    More specifically, the present invention relates to the use of the pregnán glycoside compound of formula (I) isolated from Baek Sewage as a multi-drug resistance inhibitor, an angiogenesis inhibitor, and a cancer cell invasion inhibitor. The present invention relates to an angiogenesis inhibitor and a cancer cell invasion inhibitor.
    Until recently, with the development of various anticancer drugs, rapid progress has been made in the treatment of cancer by chemotherapy. However, the treatment of cancer by chemotherapy has failed in many cases because cancer cells gradually become resistant to drugs from the beginning or during the treatment.
    Recently, a phenomenon in which cancer cells show resistance to various anticancer drugs having different structures and mechanisms of action at the same time has been reported, which is called multi-drug resistance. This multidrug resistance is considered a major cause of chemotherapy failure in the treatment of cancer (Gottesman, M. M. et al., Annu. Rev. Biochem., 62, 3851, 1993).
    Multidrug resistance is mainly found in natural-derived fat-soluble anticancer drugs, such as adriamycin, vinblastine, taxol, etoposide and actinomycin D. Drugs, but bleomycin, methotrexate and alkylating agents do not exhibit multiple resistance (Lum, BL et al., Pharmacotherapy, 113, 88, 1993).
    The main mechanism by which cells express multi-drug resistance can be explained as follows: The cell membrane of multi-drug resistant cells has a p-glycoprotein (p-gp), a gene product that causes multi-drug resistance. This large amount is expressed, and this p-glycoprotein is energy-dependent to release anticancer drugs out of the cell membrane. Therefore, the cells expressing p-glycoprotein have a multi-drug resistance that is resistant to anticancer drugs because the concentration of anticancer drugs is reduced in the cells (Endicott, J. A., Annu. Rev. Biochem., 58, 137, 1989). It has also been reported that p-glycoprotein not only enhances drug release but also inhibits drug uptake. The p-glycoprotein is expressed in normal cells depending on the tissue, but it seems to have functions of transporting nutrients and exhaling toxic substances in vitro. Therefore, in the organs that carry nutrients and excrete toxic substances in vitro, many p-glycoproteins are expressed. Therefore, in the case of colon cancer, liver cancer and lung cancer, which are cancers derived from tissues expressing p-glycoproteins, There are many cases of multi-drug resistance from the beginning even without exposure to anticancer drugs (Lum, BL et al., Pharmacotherapy, 13, 88, 1993).
    To suppress or overcome multi-drug resistance, to date, calcium antagonists verapamil, nifedipine, calmodulin inhibitor trifluoroperazine, antihypertensive drugs lesperpine, quinoline Previous drugs, such as (quinoline) derivatives and cyclosporin, an immunosuppressant, have been studied (Ford, JM et al., Pharmacol. Rev., 42, 155, 1990). These drugs are structurally correlated with anticancer drugs that exhibit multi-drug resistance, thereby competing with the p-glycoprotein competitively with the anticancer drugs, thereby inhibiting the release of anticancer drugs present in the cells out of the cells, thereby maintaining a high concentration of intracellular anticancer drugs. It is known to partially modulate or overcome multi-drug resistance.
    However, there are many limitations to the clinical use of these drugs as multi-drug resistance modulators. For example, verapamil is a multi-drug resistant substance that has been studied for its effects and mechanisms, and its toxicity to cardiovascular system such as arrhythmia and hypotension has been reported at concentrations that can overcome its multi-drug resistance (0zols, RF et al. , j. Clin. Oncol., 5, 641, 1987). Therefore, it is very necessary to develop a new substance that regulates multi-drug resistance, which is less toxic and effective.
    In addition, among the development methods of new anticancer drugs, drugs that inhibit cancer cell angiogenesis (angiogenesis) to inhibit the metastasis and proliferation of cancer cells are attracting attention. Angiogenesis necessarily occurs during the initial growth of cancer cells, and the resulting blood vessels provide the necessary oxygen and nutrients for the cancer cells, enabling the cancer cells to continue to grow. These blood vessels are also used as a path for malignant cancer cells to spread to other organs, making cancer treatment impossible.
    Angiogenesis is indispensable for the growth and metastasis of cancer, especially solid cancer. Solid tumors up to about 1-2 mm 3 can be supplied with diffusion factors such as growth factor, oxygen, and nutrients necessary for growth, but in order to grow further, cancer cells grow through new angiogenesis. You must continue to receive growth factors, oxygen, and nutrients. Therefore, by blocking the angiogenesis by cancer cells can effectively prevent the growth of cancer.
    The research on the development of angiogenesis inhibitors was in 1990 when Dr. Judah Folkman of the United States proposed the hypothesis that angiogenesis is essential for cancer growth (Folkman, J. New England J. Medicine, 285, 1182, 1971). Fumagillin and TNP-470 (Ingber, D., Nature 348, 555, 1990), the first natural-derived angiogenesis inhibitors, developed by Dr. Folkman of Harvard Medical School in the United States, have since been released. In addition, a number of inhibitors of angiogenesis have been developed and are currently known to be in clinical trials. Angiostatin (0'Reilly, MS ,. et al, Cell 79, 315, 1994), subsequently reported in 1994, was the first endogenous angiogenesis inhibitor found in plasminogen isolated from the urine of C57BL / J mice implanted with Lewis Lung Carcinoma. 37 kDa fragment is known to strongly inhibit the proliferation of vascular endothelial cells. Later, endostatin (O'Reilly, MS. Et al, Cell 88, 277, 1997), a 20 kDa fragment of collagen 18, was detected from EOMA, a mouse hemangioendothelioma cell line. Both endostatin and angiostatin were potent angiogenesis in vivo and in vivo. In addition to its activity, it is known to exhibit excellent anticancer effects without showing resistance in animal models.
    Invasion of cancer cells is a three-step continuous reaction of cell adhesion, basement membrane degradation, and cell migration, which is essential for angiogenesis as well as metastasis of cancer cells. For example, in cancer metastasis, cancer cell infiltration is an essential step for cancer cells to migrate into the bloodstream or from other blood tissues, where they bind to adhesion molecules expressed on the basement membrane and secrete various types of protease. Decompose the basement membrane to move through the basement membrane. Marimastat, an inhibitor of matrix matalloproteinases involved in proteolysis essential for cell invasion, is known to inhibit cancer metastasis and inhibit angiogenesis by inhibiting cancer cell invasion.
    Therefore, the present inventors inhibit multi-drug resistance from natural products to enhance cytotoxicity against resistant cell lines, thereby increasing the therapeutic effect, inhibiting angiogenesis of cancer cells, and inhibiting cancer cell infiltration to inhibit cancer proliferation and metastasis. The study was carried out to obtain an anticancer agent that enables the treatment of cancer with the activity. The present inventors isolated and identified the pregnan glycoside of Formula 1 from the root extract of Cynanchum wilfordii, and the multidrug resistant cell line MCF-7 / ADR and vinblastine-induced multidrug resistant cell line KB derived from adriamycin, an anticancer agent. -V1 cells were found to have the activity of enhancing the effect of the anticancer agent by inhibiting the resistance to the anticancer drugs adriamycin, colchicine, vinblastine of the multi-drug resistant cell lines described above. Through the mechanism of action study, it was found that the pregnan glycoside of formula 1 has an effect of enhancing anticancer activity by inhibiting the outflow of intracellular anticancer drugs and suppressing MDR phenomenon. In addition to the inhibitory effect of multi-drug resistance, tuberculosis inhibitory effect of HUVEC cells, CMA analysis and in vivo matrigel plug analysis were found to be effective in inhibiting angiogenesis essential for cancer cell proliferation and metastasis. Infiltration through HT1080 cells It was found to have an inhibitory effect.
    In addition, it was found that the fregnan glycoside compound of Formula 2 (wilfoside KIN) registered in Korean Patent No. 143719 has an angiogenesis inhibitory effect and a cancer cell invasion inhibitory effect.
    It is an object of the present invention to provide a pregnan glycoside of formula (I) isolated from white sewage.
    Also provided is the use of a Pregnane glycoside of Formula 1 as a multidrug resistance inhibitor.
    Also provided is the use of a fregnan glycoside of formula 1 as an angiogenesis inhibitor.
    Also provided is the use of a pregnan glycoside of formula 1 as an inhibitor of cancer cell infiltration.
    Also provided is the use of the Fregnan glycoside of Formula 1 as an anticancer agent of various functions.
    Another object of the present invention is to provide the use of the frignan glycoside compound of formula (wilfoside KIN) as an angiogenesis inhibitor.
    Also provided is the use of the pregnane glycoside compound of formula (wilfoside KIN) as an inhibitor of cancer cell infiltration.
    Provided is the use of the pregnane glycoside compound of formula 2 (wilfoside KlN) as an anticancer agent of various functions.
    1 is an ultraviolet absorption spectrum of the Fregnan glycoside of formula (1).
    2 is a hydrogen nuclear magnetic resonance spectrum of the Fregnan glycoside of formula (1).
    3 is a carbon nuclear magnetic resonance spectrum of the Fregnan glycoside of the formula (1).
    FIG. 4 is a graph comparing the effects of pregnane glycoside of Formula 1 and verapamil on accumulation of vinblastine in resistant and sensitive cell lines.
    (a) shows the outflow after accumulation of [ 3 H] VLB in KB-3-1 and KB-V1 cells.
    ●: without 0.3 μM tolyrin
    □: 0.3 μM tolyrin
    △: 30 μM VRP
    (b) VLB shows accumulation in KB-3-1 and KB-V1 cells
    ▧: 0 μM Fregnan glycoside of formula 1
    ▨: 0.1 μM Fregnan glycoside of formula 1
    ▥: 0.3 μM Fregnan glycoside of formula 1
    ▒: Pregnan Glycoside 1 μM
    ▦: 3 μM Fregnan glycoside of formula 1
    FIG. 5 is a graph comparing the effects of pregnane glycosides of Formula 1 and verapamil on the outflow of vinblastine in resistant and sensitive cell lines.
    (a) shows the outflow after accumulation of [ 3 H] VLB in KB-3-1 cells.
    (b) shows the outflow after accumulation of [ 3 H] VLB in KB-V1 cells.
    ●: without 0.3 μM tolyrin
    □: 0.3 μM tolyrin
    △: 30 μM VRP
    Figure 6 is a graph showing the effect of HUVECs (Human umbilical vein endothelial cel1) cell proliferation of the Fregnan glycoside of formula (1).
    Figure 7 is a photograph showing the tube forming inhibitory effect in the HUVEC of the formula 1 and Formula 2 glycogen glycosides.
    FIG. 8 is a photograph analyzing the angiogenesis inhibitory effect in the chick choriollantoic membrane (CAM) of the pragnan glycoside of Formula 1. FIG.
    9 is a photograph showing an angiogenesis inhibitory effect in the Matrigel analysis (invivo matrigel analysis method) of the living body of the Fregnan glycosides of Formula 1 and Formula 2.
    10 is a photograph showing the effect on the activity of MMP-9 and MMP-2 of the HT1080 cells of the pregnang glycosides of the formula (1) and formula (2) by gelatin-based zymography.
    Figure 11 is a photograph showing the effect of MMP-9 mRNA expression and MMP-9 protein amount of HTl080 cells of the Fregnan glycosides of Formula 1 and Formula 2.
    12 is a graph showing the infiltration inhibitory effect of HTl080 cells of the Pregnan glycosides of Formula 1 and Formula 2.
    Hereinafter, the present invention will be described in detail.
    The object of the present invention is to isolate and identify the Fregnan glycoside compound of Formula 1 from Baek Sewage, and to treat anti-cancer agent resistant cell line and susceptible cell line with anticancer agent and Formula 1 glycogen glycoside, respectively. And the cytotoxicity of the pregnan glycoside of Formula 1 itself, and then treated the resistant cell line and susceptible cell line with the anti-cancer agent with the Pregnan glycoside of Formula 1 to investigate the relationship between the accumulation of intracellular anticancer agent and the Fregnan glycoside of Formula 1 It was. In addition, the present invention is to investigate whether the endothelial cell proliferation inhibitory effect by treating the vascular endothelial cells with the pregnan glycoside compound of Formula 1 and Formula 2 and inhibit the angiogenesis in vivo through Chorioallantoic membrane (CAM) analysis method Activity was examined and angiogenesis inhibitory activity was confirmed in mice with matrigel plug. In addition, gelatin-based zymography and in vitro invasion assays were performed on HT1080 (human fibrosarcoma) cells, which are known to have excellent metastasis and invasiveness, to investigate the effects of cancer cell metastasis and invasion. It was confirmed that there is an inhibitory effect.
    Pregnan glycoside compound of formula (1) of the present invention is obtained by extracting methane from sewage and then separating by silica gel chromatography.
    The resistant and sensitive cell lines were treated with various anticancer agents and the Fregnan glycoside compound of Formula 1, respectively, and then cytotoxicity was measured by the sulforhodamine B (SRB) method. Investigate activity that enhances susceptibility to blastine. After the resistant cell line and the sensitive cell line are treated with a radioactive anticancer agent and a pregnan glycoside of Formula 1, the anticancer agent with radioactive activity is measured to investigate the accumulation and excretion of the anticancer agent. Human umbilical vein endothelial cell (HUVEC) cells were treated with the pregnan glycoside compounds of Formula 1 and Formula 2, followed by MTT (3- (4,5-dimethylthiazol-2-y1) 2,5-diphenyl tetrazolium bromlide) To investigate the effect of cell proliferation.
    The differentiation inhibitory activity of HUVEC cells into the tube was measured, and the angiogenesis inhibitory effect of pregnan glycosides was measured by CAM assay and matrigel plug assay. After treating pregnan glycoside compounds in HT1080 human fibrosarcoma cells, we investigated the inhibitory effect on the penetration of cancer cells by the pregnan glycoside compounds of formulas 1 and 2, and the coke involved in metastasis and invasion. The effect of the pregnan glycoside compounds of Formula 1 and Formula 2 on the activity and mRNA expression and protein expression of MMP-9, a collagenase, was identified.
    Pregnan derivatives of the present invention is highly useful as a multifunctional anticancer agent because it shows a variety of anticancer activity, such as the inhibitory effect of drug resistance of cancer cells, the activity of inhibiting angiogenesis and the inhibition of invasion, which are essential for cancer growth and metastasis.
    Pregnan glycoside compound of formula (1) can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical preparations.
    That is, the pregnan glycoside compound of formula 1 of the present invention can be administered in various oral and parenteral formulations during actual clinical administration, and when formulated, the commonly used fillers, extenders, binders, wetting agents, disintegrants, interfaces It is prepared using diluents or excipients such as active agents. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid form preparations include at least one excipient such as starch, calcium carbonate, or the like. It is prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations 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.
    The effective dose of the Pregnan glycoside compound of Formula 1 is 10-300 mg / kg, preferably 20-200 mg / kg, and may be administered 1-3 times a day.
    In addition, LD 50 of the pregnan glycoside compound of Formula 1 was 1 g / kg or more.
    In the present invention, the UV spectrum was obtained by Milton Roy 3000 spectrum analysis, the IR spectrum was advanced to KBr disk on RFX-65 FT-IR analytical to Raser Precision. 1H-NMR, 13 C-NMR, 1 H- 1 H COSY and 1 H- 13 C COSY spectra were obtained at various Unity 300 MHz (1 H-NMR) and 75 MHz ( 13 C-NMR), and the solvent was CDCl 3 It was. EIMS was measured with Hewlett Packard 5989A. MP was measured by the model Electrothermal 9100. Optical rotation was determined with a JASCO DIP-181 polarimeter. Si gel 60 (Merck) and C-18 (EM) were used for CC. Bovine serum media and additional materials and media for cell culture were purchased from GIBCO. Chemotherapy and imaging Verapamil hydrochloride was purchased from Sigma Chemical Co. [ 3 H] vinblastine sulfate (11.2 Ci / mmol) was purchased from Amersham Co. Calf pulmonary artery endothelial (CPAE) cells and human fibrosarcoma (HT1080) cells were purchased from the American Type Culture Collection (ATCC) (Rockvill, USA), while fetal bovine serum (FBS) and Dulbecco's modifide medium (DMEM) were used by Gibco BRL (Gaithesberg). , MD, USA) and [α- 32 P] dCTP was purchased from Amersham. The fertilized eggs used in the CAM analysis were purchased from Haegeumgang fertilized eggs from Hanshin Farm, Geojedo, and 10% fat emulsion (10% fat emulsion) was used for Green Cross. Transwells used in invasion assays were purchased from Corning and other reagents were purchased from Sigma Chemical Co. (St, Louis, MO).
    Hereinafter, the present invention will be described in more detail with reference to the following examples, which are not intended to limit the scope of the present invention.
    Example 1 Isolation and Purification of Pregnan Glycoside Compounds of Formula 1 from White Sea
    3 kg of dried white sewage roots were extracted with methanol three times at room temperature and concentrated under reduced pressure to obtain a methanol extract (480 g). The methanol extract was partitioned between dichloromethane and water to obtain a chloromethane layer (87 g). Was purified by silica gel column chromatography with dichloromethane-methanol (20: 1, 10: 1, 5: 1, 2: 1, methanol, 2 L each) to obtain five fractions. Fraction 2 (37.4 g) was again chromatographed on a silica gel column with hexane-acetone (3: 1, 2: 1, acetone, 2 L each) to obtain fraction 7-12, followed by active fraction 8 (1.8 g). 40 mg of Pregnan glycoside of Formula 1 was purified by RP MPLC (solvent 80% MeOH) and preparative HPLC (solvent 75% MeOH). Physicochemical properties of the Fregnan glycoside of Formula 1 are as follows.
    Appearance of substance: White amorphous powder
    Molecular Weight: 1193
    Molecular Formula: C 64 H 91 NO 20
    Melting Point: 185-187 ℃
    UV (MeOH) λ max nm (log ε): as shown in FIG. 1.
    FAB-MS m / z: 1194 [M + H] + , 1216 [M + Na] + ,
    1 H-NMR: as shown in FIG. 2 (Warashina, T. and Noro, T. Chem. Pharm Bull 44. 917, 1996).
    13 C-NMR (CDCl 3 ): as shown in FIG. 3 (Warashina, T. and Noro, T. Chem. Pharm Bull 44. 917, 1996).
    Solubility: Soluble in methanol, ethanol, ethyl acetate, acetone and chloroform, insoluble in water.
    Analysis on High Performance Liquid Chromatography (HPLC): instrument uses waters model (501 pump), column uses μ-Bondapak C18 (ψ3.9 x 300mm), mobile solvent water and methanol (15:85) It was detected at UV 280nm and the retention time (Rt) was 11.87 minutes.
    Example 2 Screening of the Multidrug Resistance Modulation of Pregnan Glycoside of Formula 1
    [Step 1] Cell Lines and Cell Culture
    KB-3-1, a human oral epidermoid cell line, and KB-V1, which induced resistance to vinblastine, MCF-7 and adriamycin, a human breast cancer cell line. Drug-resistant cell line MCF-7 / ADR was used. The KB-3-1 and KB-V1 cells were cultured in DMEM medium (Dulbecco's modifide Eagle's Medium, Gibco 430-2800) containing 10% fetal calf serum and 2 mM glutamine, MCF-7 and MCF- 7 / ADR cells were cultured in RPMI 1640 medium (Gibco 430-1800) containing 10% bovine serum 2 mM glutamine. All cultures were performed in 37 ° C., 5% CO 2 incubator.
    [Step 2] Cytotoxicity and Multidrug Resistant Activity of Pregnan Glycoside of Formula 1
    Resistant cell lines KB-V1 and MCF-7 / ADR and susceptible cell lines KB-3-1 and MCF-7, which were cultured in step 1, were respectively treated with adriamycin (ADR), vinblastine (VLB), and colchicine (COL). Cytotoxicity was measured. Each cell in the logarithmic phase was trypsinized to make a single cell suspension and dispensed in an amount of 100 μl into a 96 well microplate. The divided cell numbers were 0.5 x 10 4 MCF-7 cell lines, 1.0 x 10 4 MCF-7 / ADR cell lines, 2.5 x 10 3 KB-3-1 cell lines and 5.0 per well, depending on the results of preliminary experiments on the cell growth of each cell line. x 10 3 KB-V1 cell line. After incubating each microplate for 24 hours, 100 μl of each concentration of anticancer agent was added, followed by further incubation for 48 hours. At this time, the anticancer agent was dissolved in a small amount of DMSO or methanol before dilution in the medium (final solvent concentration <0.5%). The control was incubated in a medium containing only solvent. The cells were fixed by adding 50 µl of cold 50% trichloroacetate (final concentration 10%) solution onto the growth medium of the wells, treated at 4 ° C for 1 hour, washed 5 times with tap water, and dried in air. Subsequently, staining was performed for 15 to 30 minutes with 0.4% (w / v) sulforhodamine B (SRB) solution containing 1% acetate and the excess dye solution was washed four times with acetic acid solution. After the plate was dried, the stained cells were lysed by treatment with 10 mM Tris base (pH10.5) for 5 minutes and the absorbance was measured at 570 nm. Inhibition of cell growth was expressed as a percentage of the absorbance of the control group to the absorbance of the sample treatment group, and the 50% inhibitory concentration (IC 50 ) for each anticancer agent of each cell line was determined by the Probit method. The relative resistance (RR) for each anticancer agent of the resistant cell line was calculated as in Equation 1 below.
    In order to determine the MDR inhibitory activity of the Fregnan glycosides of Formula 1, the activity of various anticancer agents for each cell was determined by 0, 0.1, 0.3. It was determined according to the presence of 1μM each and the case of Verapamil (VRP) 3, 10, 30μM each as a reference drug and the experimental results are shown in Table 1.
    Pregnan glycosides of formula 2 did not show particular cytotoxicity at IC 50 15.2-17.1 μM for all cell lines. As shown in Table 1, in KB-3-1 and MCF-7 to which 0.1-1 μM of the Fregnan glycoside of Formula 1 was added, the Pregnan glycoside of Formula 1 did not show the effect of increasing the sensitivity of the cell line to the anticancer agent. . However, in multi-drug resistant cells, the pregnan glycoside of Formula 1 increased the cytotoxicity of the resistant anti-cancer drug in a concentration-dependent manner, thereby reducing the relative resistance of the multi-drug resistant cells. In KB-V1 cells, 1 μM Fregnan glycosides reduced the resistance of KB-V1 cells to adriamycin from 644.2 fold to 0.64 fold, 1466.2 fold to 1.49 fold for VLB, and 428.1 fold to 1.37 fold for colchicine. Toxicity of the anticancer agent was fully restored to the level of sensitive cells KB-3-1. This effect was observed to the same extent in MCF-7 / ADR. Verapamil, a multidrug resistant active substance used as a control drug, partially recovers anticancer drug resistance even at a concentration of 30 μM, whereas the pregnan glycoside of Formula 1 is resistant to KB-V1 cells and MCF-7 / ADR cells at 1 μM. Showed excellent activity to completely inhibit
    Example 3 Effect Test of Pregnan Glycoside of Formula 1 on Intracellular Anticancer Drug Accumulation
    KB-3-1 and KB-V1 cell lines were seeded at 2.5 x 10 4 cells and 4 x 10 4 cells per well into 24-well tissue culture plates and grown to 90% of the culture area. After 48 hours, the medium was added to the serum-free DMEM medium containing [ 3 H] VLB 0.25 μCi / ml and the medium to which fregnan glycoside of Formula 1 and verapamil (VRP) were added and not added to the medium. Exchange and incubate for a further period of time. Cells were harvested at predetermined times, washed three times with cold phosphate buffer (PBS), dissolved in 200 μl of 0.2N NaOH, treated overnight at 37 ° C., followed by the addition of scintillation cocktails, and then radioactivity was measured and standardized. Vinblastine (VLB) concentration associated with cells was determined from the dose curve (FIG. 4).
    As shown in FIG. 4 (a), VLB accumulation increased with culture time in each cell and reached a stable state at 2 hours, and reached a stable state at 4 hours in KB-V1 cells. In the case of KB-V1 cells, the VLB accumulation at steady state was about 1/10 of that of KB-3-1 cells. When 0.3 μM Fregnan glycoside of Formula 1 or 30 μM VRP was added to the medium containing [ 3 H] VLB, the accumulation of VLB in KB-V1 cells was significantly increased to 1/2 level of KB-3-1 cells. There was no effect on KB-3-1 cells. As shown in FIG. 4 (b), the effect of increasing VLB accumulation in KB-V1 cells was increased in proportion to the concentration of pregnan glycoside of formula 1 and lμM of pregnan glycoside of formula 1 decreased VLB accumulation of KB-V1 cells. Increased to the same level as KB-3-1 cells. In part, VRP, which inhibits multidrug resistance, increased VLB accumulation in KB-V1 cells by 30 μM to 1/2 of the amount of VLB intracellular accumulation in KB-3-1 cells. Therefore, the multidrug-resistant inhibitory activity of the pregnan glycoside of Formula 1 may be directly related to increasing intracellular accumulation of anticancer drugs.
    Example 4 Effect Test of Pregnan Glycoside of Formula 1 on Extracellular Excretion of Anticancer Agents
    After incubating KB-3-1 and KB-V1 cells for 48 hours, the medium was replaced with serum free DMEM. KB-3-1 cells and KB-V1 were [H 3 ] VLB in two cell lines in serum-free DMEM medium containing O.25 μCi / mL [H 3 ] VLB and 2.5 μCi / mL [H 3 ] VLB, respectively. Incubated for 4 hours so that the accumulation of. Each cell was washed with cold PBS to remove residual VLB, and then the cells were recovered every 30 minutes while the Pregnan glycoside of Formula 1 was cultured in a medium containing VRP and a medium without washing, and then washed with PBS. Was measured. Cellular radioactivity is expressed as% of the radioactivity of the cell when no Fregnan glycoside of formula (1) was added.
    As shown in FIG. 5, VLB was slowly discharged into the medium in KB-3-1 cells, and VLB was maintained at 60% or more of the initial concentration even after 2 hours. In contrast, VLB was rapidly excreted in KB-V1 cells, resulting in 38% of the initial concentration after 15 minutes and 17% after 2 hours. The release of VLB from KB-V1 cells was markedly inhibited by the Fregnan glycoside of formula (1). At least 90% of the VLB was maintained in KB-V1 cells with 2 μM Fregnan glycoside of Formula 1 until 2 hours later. A 30 μM VRP maintained more than 70% of the amount of VLB after 2 hours. Thus, the Fregnan glycosides of Formula 1 have been shown to increase anticancer agent concentration in cells by preventing the outflow of the anticancer agent extracellularly and consequently inhibit multidrug resistance (FIG. 5).
    Example 5 Effect of Pregnan Glycosides of Formula 1 and Formula 2 on HUVEC Cell Proliferation
    HUVEC cells were passaged for 2-3 days in a 5% CO 2 incubator maintained at 37 ° C. in a medium containing 1% penicillin-streptomycin in DMEM with 10% FBS. After seeding the cultured HUVEC cells to 1 x 10 5 cells in a 24-well culture plate, 10 μM of Pregnan glycoside of Formula 1 and 2 μM of Pregnan glycoside of Formula 2 were added and cultured for 24, 48, and 72 hours. MTT (3- (4,5-dimethylthia zol-2-yl) 2,5-diphenyl tetrazolium bromide) was incubated for 4 hours after the final concentration was 0.25 mg / ml. Subsequently, the medium was removed, and 1 ml of dimethyl sulfoxide (DMSO) was added to dissolve the produced formazan crystal, and the absorbance was measured at 540 nm. The experimental results showed that the control showed a slow growth from 24 hours when the control group was treated with the pregranular glycoside of Formula 1 compared to the normal cell growth curve, but there was no toxicity to vascular endothelial cells and significantly increased the growth. Was observed to decrease. Pregnan glycosides of formula 2 showed significant cytotoxicity over 24 hours (FIG. 6).
    Example 6 In Vitro Tube Formation Assay in HUVEC Cells
    In vitro angiogenesis assays were performed on HUVEC cells, vascular endothelial cells, using the Fregnan glycosides of Formula 1 and Formula 2, respectively, at 10 μM and 2 μM concentrations that did not affect vascular endothelial cell growth. 24, 48) significantly reduced the tube formation compared to the control. Pregnan glycosides of formula (1) markedly inhibited tube formation with little or no toxicity to vascular endothelial cells within 48 hours, and pregnan glycosides of formula (2) also showed up to 24 hours before cytotoxicity was observed. The formation of tubes was significantly inhibited. However, the pregnan glycoside of formula (2) caused cytotoxicity to HUVEC cells after 36 hours, and it is thought that the inhibition of tube formation of the pregnan glycoside of formula (2) observed after 36 hours may be due to cytotoxicity ( 7).
    Example 7 Chorioallantonic Membrane (CAM) Analysis
    The embryos were grown for 3 days in a 37 ° C. incubator maintained at 90% humidity, then punctured and 2 ml of albumin was extracted using an 18-gauge subcutaneous needle. When the cells were doubled for 4 days, the outer membrane of the CAM was peeled off with a circular window, and then the Fregnan glycoside of Formula 1 was applied to thermanox coverslip at 1, 2, 10, 30, 50, and 100 µg / CAM at 4.5 days. It was applied and the solvent, ethanol, was blown off. The coverslip was placed on the developing embryo CAM with a diameter of 3-5 mm and incubated for another 2 days, followed by injection of 10% fat emulsion, and the inhibition of angiogenesis was observed under a dissecting microscope. At this time, the anti-anglogenic response was examined by observing the lower avascular zone of the coverslip. In the control group, only the coverslip was attached to the CAM, and retinoic acid (1㎍ / CAM), which is known to have an angiogenesis inhibitory effect, was treated with a positive control and compared three days later. All showed more than 55% of angiogenesis inhibitory effect. In particular, when treated with 30 μg / egg as shown in Table 2 showed an angiogenesis inhibitory effect of about 78% (Fig. 8).
    Example 8 Matrigel plug assay
    Matrigel containing bFGF, which is known to promote angiogenesis between the peritoneum and skin of C57BL / 6 mice, 0.5 mg of Fregnan glycoside of Formula 1, and 0,5 mg of Fragnan glycoside of Formula 2, was added to the mouse. 7 days after the injection, the matrigel was extracted and paraffin-embedded sections were made into paraffins, and after Masson-Trichrome staining, image proPlus analysis was performed to observe the degree of angiogenesis, and on the other hand, the amount of hemoglobin in the formed blood vessels was quantified. The degree of inhibition of the samples used for bFGF-induced angiogenesis was compared. When bFGF with angiogenesis-promoting effect was used as a positive control and only matrigel was injected as a negative control, angiogenesis occurred remarkably in the matrigel plug treated with bFGF. When bFGF was treated with bFGF, Induced angiogenesis was clearly inhibited. As a result, the hemoglobin content was measured and quantified, and the formula 1 showed an angiogenesis inhibitory effect more than the pregran glycoside of formula 2, and the pregnan glycoside of formula 1 showed less Hb elution than the pregran glycoside of formula 2.
    Example 9 Cytotoxicity of Pregnan Glycosides against HT1080 Human Fibrosarcoma Cells
    HT1080 cells were passaged for 2-3 days with 0.05% trypsin-EDTA in a 5% CO 2 incubator maintaining 37 ° C saturation humidity in medium containing 1% penicillin-streptomycin in DMEM with 10% FBS. After seeding the cultured HT1080 cells to 1 x 10 5 on a 24-well culture plate, and treated with 10 μM of Fregnan glycoside of Formula 1 and 2 μM of Pregnan glycoside of Formula 2 for 1, 3, 6 days and MTT ( 3- (4,5-dimethylthia zo1-2-yl) 2,5-diphenyl tetrazolitlum bromide) was incubated for 4 hours after the final concentration was 0.25 mg / ml. Subsequently, the medium was removed, and 1 ml of dimethyl sulfoxide (DMSO) was added to dissolve the produced formazan crystal, and the absorbance was measured at 540 nm. As a result, it was confirmed that 10 μM of Fregnan glycosides of Formula 1 and 2 μM of Pregnan glycosides of Formula 2 were concentrations that did not affect viability of HT 1080 human fibrosarcoma cells.
    Example 10 Effect of Pregnan Glycosides of Formulas 1 and 2 on the Activity of Matrix Metalloprotease (MMP)
    HT1080 cells were planted in a 6-well dish at 2 x 10 4 and treated with 10 μM of Fregnan glycosides of Formula 1 and 2 μM of Pregnan glycosides of Formula 2 in the culture medium, and then maintained at 5% CO 2. After incubation in the incubator for 1 day and 3 days, the medium was collected and subjected to gelatin-based zymography. The gel for electrophoresis was 10% SDS-polyacrylamaide gel containing 1 mg / ml gelatin, and the amount of the medium applied to electrophoresis was based on a medium corresponding to 1-2 x 10 4 cells. After loading the medium onto the gel, electrophoresis was performed at 130 volts for about 3 hours, and after separation of the protein, the gel was purified by 50 mM Tris-Cl (pH 7.5), 10 mM CaCl 3 , 2.5% Triton X-100, 1 μM ZnCl 2 . Washed twice for 30 minutes at room temperature with the configured washing buffer. To investigate the activity of Matrix metalloprotease (MMP), immerse the gel in a culture medium containing 50 mM Tris-Cl (pH 7.5), 0.15 M NaCl, 10 mM CaCl 2 , O.O2% NaN 3 and incubate overnight at 37 ° C. It was. Thereafter, after staining with 0.1% coomassie brilliant blue R-250, it was decolorized. The part with MMP is gelatin decomposed and appears transparent without being stained with coomasie blue. Experimental results showed that the Fregnan glycosides of Formula 1 and Formula 2 inhibited the activity of matrix metalloproteinase 92 Kd type IV collagenase (MMP-9), which plays an important role in cancer cell metastasis and invasion. There was no effect on activity (FIG. 10).
    Example 11 Effect of Pregnan Glycosides on MMP-9 Gene Expression and Protein Amount
    To investigate the effect of the Fregnan glycosides of Formula 1 and Formula 2 on the mRNA expression of collagenase or collagenase inhibitors involved in metastasis and infiltration, 10 μM of the Fregnan glycosides of Formula 1 and 2 μM of the Fregnan glycosides of Formula 2 were applied to HT1080 cells. After 24 hours and 72 hours of treatment, total RNA was isolated and nothern blotting was performed using MMP-9, MMP-2, TIMP-1, TIMP-2, uPA, and PAI as probes. As a result, it was confirmed that both compounds reduced the expression of the MMP-9 gene, but there was no significant effect on the expression of other genes (FIG. 11A). To investigate the amount of collagenase protein affecting invasion and metastasis, HT1080 cells were treated with 10 μM of Fregnan glycoside of Formula 1 for 24 and 72 hours and 2 μM of Pregnan glycoside of Formula 2 for 24 hours, followed by conditioned medium. Western blotting was performed by treating primary Ab and secondary Ab for MMP-9. Both compounds had an effect of reducing the amount of MMP-9 protein (FIG. 1 lb).
    Example 12 effect on cancer cell invasion by in vitro assay method
    HT1080 cells were planted in a T-25 culture plate in a number of 5 × 10 4 and treated with 10 μM of Fregnan glycoside of Formula 1 and 2 μM of Pregnan glycoside of Formula 2 in the culture medium for 1 day and 3 days, Subcultures were carried out after treatment with pregran glycosides adjusted to concentration in transwells containing the matrix matrix of cells in the upper layer of the filter and type I collagen in the lower layer. After 16 hours of incubation in a 37 ° C. incubator, the cells were fixed and stained, the transwell filter was cut out and spread on a slide glass, and the infiltration effect was determined by counting the number of cells that passed through the matrigel through a phase contrast microscope to the bottom of the filter. As a result of the experiment at a magnification of 200 times under a microscope, the pregnan glycoside of Formula 1 showed about 60% inhibition of the control group. On the other hand, the Fregnan glycoside of Formula 2 showed an inhibitory activity of about 80% or more, but is believed to be due to cytotoxicity. In the case of the Fregnan glycoside of Formula 2, the inhibitory effect persisted after 72 hours (FIG. 12).
    On the other hand, the following experiment was performed to determine the acute toxicity of the compound of Formula 1.
    Example 13 Anticancer Effect Test of Pregnan Glycoside in P388 Celiac Cancer Model
    Adriamycin 0.5 mg of Pregnan glycosides of Formulas 1 and 2 were injected into mice intraperitoneally implanted with adriamycin resistant cell line P388 / ADR cells. After co-administration with 1, 2mg / kg, the life-extension effect of the mice was measured. By 9 days, there was no significant difference in the body weight of the Ga-administrated group. Compared with the average survival rate, the group treated with adriamycin or the formula 1 and 2 pregnan glycosides alone did not differ from the control group, but the group administered with 2 mg / kg of adriamycin and 30 mg / kg of the formula 1 or formula 2 Survival rates increased by 78% and 58%, compared to the control group.
    Example 14 Acute Toxicity in Oral Administration in Rats
    Acute toxicity test was performed using 6 week-old SPF SD rats. Five animals per group were suspended orally at a dose of 0.5 g / kg / 15 ml of the Fregnan glycoside compounds of Formulas 1 and 2 in 0.5% methylcellulose solution, respectively. After administration of the test substance, the mortality, clinical symptoms, and weight changes of the animals were observed and necropsy, and the abdominal and thoracic organ abnormalities were visually observed. As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no change in toxicity was observed in weight change or autopsy findings. As a result, all of the tested compounds did not show toxicity change up to 0.5 g / kg in rats, and the minimum lethal dose for oral administration was determined to be more than 0.5 g / kg.
    As described above, the present invention enhances the activity of the anticancer agent because the novel pregnan glycoside of formula (I) isolated from Baek HaoSoo prevents the release of anticancer agent in multi-drug resistant sapons and accumulates anticancer agent in the cells. Pregnan glycosides of Formula 1 and Formula 2 inhibit the proliferation of endothelial cells essential for angiogenesis and inhibit angiogenesis, as well as inhibit the invasion of cancer cells by reducing the activity of matrix metalloprotease and its gene expression. It is effective as a new multifunctional anticancer agent.
    权利要求:
    Claims (9)
    [1" claim-type="Currently amended] Pregnan glycoside compound represented by the formula (1).
    Formula 1

    [2" claim-type="Currently amended] A multi-drug resistance inhibitor comprising the compound of claim 1 as an active ingredient.
    [3" claim-type="Currently amended] Angiogenesis inhibitor comprising the compound of claim 1 as an active ingredient.
    [4" claim-type="Currently amended] A cancer cell infiltration inhibitor comprising the compound of claim 1 as an active ingredient.
    [5" claim-type="Currently amended] An anticancer agent comprising the compound of claim 1 as an active ingredient.
    [6" claim-type="Currently amended] After drying the dried white sewage roots with methanol and concentrating under reduced pressure to obtain a methanol extract, and fractionated with dichloromethane and water, the dichloromethane layer was partitioned with n-hexane and methanol, and the methanol fraction was subjected to silica gel chromatography to obtain a fraction. This fraction was repeatedly chromatographed on a silica gel column with dichloromethanol, and then an active fraction was obtained using an n-hexane-ethyl acetate step gradient system, and the active fraction was chromatographed on an RPMPLC column and recrystallized with methanol to purify. Method for producing a pregnan glycoside of claim 1
    [7" claim-type="Currently amended] Angiogenesis inhibitor comprising the pregnane glycoside compound represented by the formula (2) as an active ingredient.
    Formula 2

    [8" claim-type="Currently amended] A cancer cell infiltrating inhibitor using the pregnan glycoside compound represented by the formula (2) as an active ingredient.
    [9" claim-type="Currently amended] An anticancer agent comprising a pregnan glycoside compound represented by the formula (2) as an active ingredient.
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    同族专利:
    公开号 | 公开日
    KR100293258B1|2001-06-15|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1999-02-23|Application filed by 박호군, 한국과학기술연구원
    1999-02-23|Priority to KR1019990005977A
    2000-09-15|Publication of KR20000056561A
    2001-06-15|Application granted
    2001-06-15|Publication of KR100293258B1
    优先权:
    申请号 | 申请日 | 专利标题
    KR1019990005977A|KR100293258B1|1999-02-23|1999-02-23|Preganane glycosides, their preparation, and their use as anticancer agents|
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