JP4825413B2 - Antitumor agent - Google Patents

Description

  The present invention relates to an antitumor agent comprising 1,5-D-anhydrofructose and / or ascopyrone as an active ingredient.

  1,5-D-anhydrofructose (hereinafter, sometimes abbreviated as 1,5-AF) is a starch or an enzyme α-1,4-glucan lyase possessed by certain ascomycetes and red algae. A starch degradation product can be produced as a substrate. 1,5-D-anhydrofructose is a sugar having a specific structure in which one water molecule is removed from glucose. It has already been reported that it has antioxidant activity (see Patent Document 1) and antibacterial activity (see Patent Document 2). Furthermore, recent research reports that hyperglycemia is suppressed (see Patent Document 3), and has attracted attention as a novel sugar having physiological activity.

  It has been reported that ascopyrone can be prepared from 1,5-D-anhydrofructose by an enzymatic reaction (see Patent Document 4, Patent Document 5 and Patent Document 6). Ascopirone is originally known to be biosynthesized by certain ascomycetes (see Non-Patent Document 1). Pesizales, for example, Picaria leiocarpa and Anthracobiala melanoma, and Tuberales, for example, Tubular melanosporum cell extract 1 , 5-D-anhydrofructose has also been reported to be prepared.

Ascopyrone P (2-Hydroxymethyl-5-hydroxy-2,3-dihydro-4H-pyran-4-one) was obtained in 1978 and 1981 by a group of US scientists as a starting material for organic synthesis. For the purpose of use as a preparation, it was prepared by thermally decomposing amylopectin, amylose, and cellulose (see Non-Patent Document 2).
Ascopyrone P (hereinafter sometimes abbreviated as APP) has been reported to have antioxidant activity and antibacterial activity in the same manner as 1,5-D-anhydrofructose (Patent Document 7, Patent Document 8 and Patent Document) 9).

Many of the antitumor agents currently used in clinical settings have a pro-oxidation effect due to their chemical properties, and there is a risk of side effects such as liver damage, kidney damage, bone marrow suppression, and lung damage. Is expensive. In contrast, 1,5-D-anhydrofructose and ascopyrone have antioxidative activity, and also inhibit inflammatory cell activation (reactive oxygen production). It is thought to attenuate inflammatory tissue damage. Therefore, in combination with other antitumor agents, not only can the antitumor effect be enhanced, but it can also be expected to be applied to chemotherapeutic adjuvants that reduce side effects.
Japanese National Patent Publication No. 9-505988 JP 2001-89377 A Special table 2003-519660 gazette International Publication No. WO03 / 38084 Pamphlet International Publication No. WO03 / 38085 Pamphlet International Publication No. WO03 / 38107 Pamphlet International Publication No. WO02 / 26060 Pamphlet International Publication No. WO02 / 26061 Pamphlet International Publication No. WO00 / 56838 Pamphlet M.M. A. Baute. , Phytochemistry, 33, (1991) 41-45. Shafizadeh, F.A. , Et al. , Carbohydr. Res. 67, (1978) 433-447 and Stevenson, F .; , Et al. , Carbohydr. Res. , 90, (1981) 319-325.

An object of the present invention is to provide an antitumor agent excellent in prognosis, which is expected to suppress inflammation, using 1,5-D-anhydrofructose tumor metastasis inhibitor and ascopyrone P as active ingredients. It is in.
Still other objects and advantages of the present invention will become apparent from the following description.

According to the present invention, the above objects and advantages of the present invention are achieved by an antitumor agent characterized by containing ascopyrone P.
Further, according to the present invention, the above objects and advantages of the present invention, Ru is achieved by tumor metastasis inhibitor comprising a 1, 5-D-anhydrofructose.

  By using 1,5-D-anhydrofructose and / or ascopyrone, tumor growth or metastasis can be suppressed with few side effects.

In the present invention, the term “anti-tumor” is a concept including an action of suppressing tumor growth or metastasis.
That is, tumor growth or metastasis can be significantly suppressed by administering an appropriate amount of the agent of the present invention to an individual having tumor cells in vivo.

The A Sukopiron, alkali Tatoebako ascomycete (Ascomycetes) derived from 1, 5-D-anhydrofructose anhydrase by 1, 5-D-anhydrofructose de seafood or 1, 5-D-anhydrofructose, Mention may be made of dehydrated products of 1,5-D-anhydrofructose by chemical or physical manipulation, such as by treatment under conditions or by heat treatment. As an example of ascopyrone, several structural formulas are shown in FIG. In FIG. 1, the structural formula shown on the left is Ascopilone P.

The agent of the present invention can be administered by various methods known per se, and the dosage, administration site, administration interval, period, etc., can be determined based on the patient's age, weight, medical condition or other drugs or treatment methods. It can be determined in consideration of the case of using together.
The administration method can be, for example, intravenous, subcutaneous, intraperitoneal, or oral administration by injection or infusion, and is not particularly limited.
It is also possible to add the agent of the present invention to food, take the food and take it into the body.

The dose varies depending on the administration method, the administration interval, the type of tumor, and the severity of the patient. For example, the dose per dose is 0.000001 μg / kg to 1,000 mg / kg, preferably 0 0.001 μg / kg to 500 mg / kg, and for 1,5-anhydrofructose, 0.001 μg / kg to 10,000 mg / kg, preferably 0.01 mg / kg to 1,000 mg / kg. can do.
Moreover, this single dose can be divided into several doses.

Examples of the form of the agent of the present invention include, but are not particularly limited to, tablets, capsules, powders, granules, suppositories, injections, and transdermal absorption agents. The agent of the present invention can also contain components necessary for preparing a preparation, such as a preparation carrier, excipient, and stabilizer. Furthermore, as long as the effects of the present invention are exhibited, other antitumor agents, other pharmacological components, or nutritional components such as glucose can also be included.
The results of studying the present invention will be described in detail below. In the following tests, 1,5-D-anhydrofructose and ascopyrone were prepared based on known methods.

Test Example Cell line and culture method Adhesive cancer cell line C57 BL / 6 mouse melanoma cell line (B16 melanoma), human lung adenocarcinoma cell line (A549), human keratinocyte-derived tumor-like cell line (HaCaT), human cervix The cancer cell line (HeLa) was cultured in a DMEM medium supplemented with 10% FCS (fetal bovine serum) and 2% penicillin / streptomycin at 37 ° C. and a CO ₂ concentration of 5%. The suspension tumor cell THP-1 (promyelocytic leukemia cell line) was cultured in RPMI 1640 medium containing 10% FCS and 2% penicillin / streptomycin under conditions of 37 ° C. and 5% CO ₂ concentration.
Method for measuring the number of viable cells B16 melanoma cells grown on a 6-well plate were fixed with 2% glutaraldehyde, and then stained with 4% crystal violet, and the staining cells were determined to be viable cells.

Method for measuring the rate of cell death and the proportion of cells in each cell cycle Stimulate the cell culture solution with various concentrations of ascopyrone P solution (dissolved in dimethylsulfoxide (DMSO)) The cells were collected in a suspended state using trypsin. Then, after fixing with 70% ethanol for 30 minutes at 4 ° C., staining was performed with a 50 μg / ml propidium iodide (PI) solution. Thirty minutes later, FACS was used to generate a DNA histogram to examine the rate of cell death and the cell cycle.

Method for measuring number of adherent cells 1,5-D-anhydrofructose solution (1,5-D-anhydrofructose dissolved in DMSO) was added to THP-1 cell culture solution, and then phorbol ester (PMA ; Stimulated with phosphoryl acetate). After culturing at 37 ° C. and 5% CO ₂ for 1 hour, the adhered cells were stained by the Giemsa staining method.

Results and Discussion
1) Cell growth inhibitory effect by Ascopilone P 5 × 10 ³ cells / ml B16 melanoma cells (C57 BL / 6 mouse melanoma cells) are seeded on a 6-well plate, and after the cells adhere to the bottom surface (24 hr), the culture solution Ascopirone P solution (using DMSO as a solvent) was added in equal amounts so that the final concentration in the solution was 0 to 0.70 mM. After culturing at 37 ° C. and 5% CO ₂ for one week, the number of viable cells was measured.
The results are shown in FIG. When the number of viable cells in the control (adding only DMSO) is 100%, the number of viable cells is remarkably reduced in a concentration-dependent manner by adding Ascopyrone P, and the proportion is the final concentration (0.70 mM). Decreased to 50% or less. From these results, it was considered that ascopyrone P has the ability to suppress the growth of tumor cells.

2) Tumor cell apoptosis-inducing effect by ascopirone P It was investigated whether the mechanism of the tumor cell proliferation inhibitory effect of ascopilone P on B16 melanoma cells is due to the cell killing effect as seen in other antitumor agents. In this study item, in addition to B16 melanoma cells, four types of human cancer cell lines (THP-1: promyelocytic leukemia, HeLa: cervical cancer, A549: alveolar epithelial cancer, HaCaT: skin cancer model cell) are added. The proportion of dead cells induced by ascopirone P stimulation was examined. Here, ascopirone P was added to 1.4 mM to each cancer cell culture solution, and the ratio of dead cells after 48 hours was measured. The results are shown in Table 1.

  From Table 1, it can be seen that Ascopyrone P exhibits a cell killing effect with a wide spectrum regardless of the type of cancer derived from each cancer cell. Furthermore, in an experimental system using HaCaT cells, apoptosis-specific DNA fragmentation was observed 48 hours after addition of Ascopilone P (1.4 mM) (FIG. 3). From this, it was confirmed that the cell death by ascopilone P was apoptosis, and the same result was obtained in the system using A549 cells. Furthermore, in the system using THP-1 cells, ascopirone P was 0.35 mM and was found to induce cell death within 48 hours (FIG. 4).

3) Specific cell death-inducing action of Ascopirone P HaCaT cells have characteristics as tumor cells in the presence of 10% FCS, but become normal cells in the presence of a low concentration of FCS (1% or less). It is known to have a close personality. Using this property, the effect of ascopilone P on normal cells was examined. The results are shown in FIG. Under the condition of 10% FCS growing like a tumor, about 30% of cell death was induced within 48 hours after addition of Ascopilone P (1.4 mM) (FIG. 5 (c)), whereas normal cell-like In the characteristic FCS 1%, almost no dead cells were observed even in the presence of 1.4 mM ascopilone P (FIG. 5B). From this, it was suggested that ascopyrone P specifically acts on highly proliferative cells (tumor cells) and hardly affects slow-growing cells (normal cells).
On the other hand, in HeLa cells proliferating like a tumor with FCS 10%, a marked increase in the S phase cell population and a decrease in the G2 / M phase cell population were observed 24 hours after the addition of Ascopirone P (1.4 mM). (FIG. 6) After that, about 48% cell death was induced as shown above at 48 hours. This suggests that the action point on the cell cycle of ascopilone P is the S phase, which is the DNA synthesis period, which inhibits the transition to the G2 / M phase and consequently induces cell death. Therefore, when Ascopyrone P is administered to a living body as an anti-tumor agent, the cell killing effect is specific to only tumor cells without damaging normal cells, most of which are considered to be in the G0 / 1 phase on the cell cycle. It is expected to demonstrate.

4) Integrin function inhibitory action by 1,5-D-anhydrofructose When a leukemia cell line is stimulated with phorbol ester (PMA), activation of the adhesion molecule integrin is observed, resulting in the generation of reactive oxygen as a cellular phenomenon. It is known that cell adhesion enhancement and cell infiltration are induced. Therefore, the THP-1 cell line was cultured in the presence of 1,5-D-anhydrofructose, and the integrin function was evaluated using the number of adherent cells after stimulation with phorbol ester as an index.
The results are shown in FIG. 12.3 mM 1,5-D-anhydrofructose suppressed cell adhesion by phorbol ester to about 25%. Therefore, it was speculated that 1,5-D-anhydrofructose can suppress the function of integrin molecules which are thought to play an important role in tumor cell metastasis.

  Hereinafter, the present invention will be described in more detail by way of examples. The present invention is not limited to the embodiment.

Example 1
C57 BL / 6 mice were seeded with B16 melanoma cells (5 × 10 ⁶ cells) intraperitoneally, and PBS (phosphate buffer) and Ascopirone P solution were intraperitoneally administered daily from the 15th day (dosage was 200 mg / day). kg), survival rate was examined (cancer peritonitis model). In this experimental system, when B16 melanoma cells were seeded in the abdominal cavity of the mouse in advance, it was confirmed that the individual (mouse) died from the 14th day onwards in the case of no administration and PBS administration. FIG. 8 shows the results of examining the life-prolonging effect of ascopirone P on the terminal cancer model using this experimental system. The average survival days were 8 days in the Ascopirone P administration group and 4 days in the PBS group, and it was proved to have an antitumor effect even in vivo. Furthermore, in 40% (n = 2) of the ascopirone P administration group, the survival period after administration of ascopilone P was more than 3 times that of the control (PBS group). This means the antitumor effect of ascopilone P in vivo.

Example 2
In the same manner as in Example 1, B16 melanoma cells (5 × 10 ⁶ cells) were seeded intraperitoneally into C57 BL / 6 mice, and then PBS and 1,5-D-anhydrofructose were intraperitoneally administered daily from the second day. (Dose was 200 mg / kg). The survival rate is shown in FIG. The average survival days after tumor cell seeding were 19 days for the 1,5-D-anhydrofructose group and 14 days for the control PBS group. From these results, it was confirmed that 1,5-D-anhydrofructose has a life-prolonging effect on cancer-bearing mice.

Example 3
Next, B57 melanoma cells (1 × 10 ⁵ cells) were subcutaneously seeded on the back of C57 BL / 6 mice, and PBS and Ascopilone P were subcutaneously injected every other day to the tumor site from day 3 to day 13 ( The antitumor effect was evaluated based on the tumor volume (major axis × minor axis ² × 0.52) using a solution dissolved in PBS (the dose was 25 mg / kg) (FIG. 10). Although no difference was observed until day 7 after tumor cell seeding, it was found that the volume of tumor in the Ascopirone P administration group was significantly smaller than that in the PBS administration group after Day 7. In general, the slower the rate of tumor growth in clinical settings, the better the prognosis. That is, it shows the antitumor effect of ascopilone P in vivo. In addition, almost no tumor metastasis was observed in the anatomical findings after sacrifice in the Ascopilone P administration group.

Example 4
A cell culture solution of promyelocytic leukemia cells (THP-1 cells) with various concentrations of ascopirone P solution (ascopirone P dissolved in DMSO), cisplatin solution (also dissolved in DMSO), and further, ascopirone P and cisplatin After stimulating with the mixed solution and culturing for 48 hours, it was thoroughly pipetted and collected in the state of a suspension consisting of uniform single cells. Here, in part, the number of cells was measured under a microscope using a hemocytometer. The remaining cell suspension was fixed with 70% ethanol at 4 ° C. for 30 minutes and then stained with a final 50 μg / ml propidium iodide (PI) solution. After 30 minutes, a DNA histogram was prepared using FACS, the rate of cell death was examined, and the number of each living cell was calculated (FIG. 11). When viable cells in the control (added with DMSO only) were taken as 100%, the ratio was reduced from about 30% to about 50% by adding Ascopyrone P, and the cell growth inhibitory effect was confirmed. On the other hand, the ratio of viable cells in the group in which cisplatin and ascopirone P were used in combination was around 10%, and a synergistic effect between ascopirone P and cisplatin was confirmed.

Structural formula of ascopirone Evaluation of cell growth inhibitory ability of Ascopilone P by crystal violet staining method DNA fragmentation analysis by agarose gel electrophoresis Quantitative evaluation of dead cells by flow cytometry (FACS) Quantitative evaluation of dead cells by flow cytometry (FACS) Cell cycle by flow cytometry (FACS) Functional evaluation of integrins in leukemia cell lines (Example 1) Survival rate of mice after administration of Ascopilone P (Example 2) Survival rate of mice after administration of 1,5-D-anhydrofructose (Example 3) Evaluation of tumor volume of mice after administration of Ascopilone P (Example 4) Evaluation of cell growth inhibitory ability of ascopilone P and cisplatin

Claims (2)

An antitumor agent for tumors comprising ascopyrone P as an antitumor component. A tumor metastasis inhibitor comprising 1,5-D-anhydrofructose as a tumor metastasis inhibitor component.