JP2006273751A - Antitumor agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a safe and excellent antitumor agent having reduced side effects with the use of 1,5-D-anhydrofructose or ascopyrone. <P>SOLUTION: The antitumor agent or a kit contains (1) 1,5-D-anydrofructose and/or ascopyrone and (2) at least one selected from the group consisting of vitamin A and vitamin A derivatives as active ingredients. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an agent for treating a tumor using 1,5-D-anhydrofructose or ascopyrone. More specifically, the present invention relates to an antitumor agent comprising 1,5-D-anhydrofructose and / or ascopyrone and vitamin A and / or vitamin A derivatives as active ingredients.

  1,5-D-anhydrofructose can be produced by allowing α-1,4-glucan lyase, an enzyme derived from certain ascomycetes and red algae, to act on starch or starch degradation products. 1,5-D-anhydrofructose is a sugar having a specific structure in which one water molecule is removed from glucose. Already, it has been reported so far that it exhibits antioxidant activity (see Patent Document 1) and antibacterial activity (see Patent Document 2). Furthermore, in recent studies, a hypoglycemic action (see Patent Document 3) has also been reported. It is also attracting attention for its physiological activity.

  It has been reported that ascopyrone can be prepared by enzymatic reaction using 1,5-D-anhydrofructose as a starting material (see Patent Document 4, Patent Document 5 and Patent Document 6). Originally, it is known that ascopyrone is biosynthesized by certain ascomycetes (see Non-Patent Document 1), and the order of Pezzales (for example, Picaria leiocarpa and Anthracobiala melanoma) and Tuberales (for example, Tuber melanosporum) It has also been reported that the bacterial cell extract can be prepared by acting on 1,5-D-anhydrofructose. In addition, the structure of ascopyrone that is biosynthesized depends on the type of fungus.

Ascopyrone P (2-Hydroxymethyl-5-hydroxy-2,3-dihydro-4H-pyran-4-one), which is one of the ascopyrones, was introduced by a group of American scientists in 1978 and 1981 by the group of amylopectin, It was reported to be synthesized by pyrolysis of amylose and cellulose and used as a starting material for organic synthesis. (See Non-Patent Document 2 and Non-Patent Document 3).
Ascopyrone P has been reported to have an antioxidant activity (see Patent Document 7) and an antibacterial activity in the same manner as 1,5-D-anhydrofructose (see Patent Document 8 and Patent Document 9).

Many of the antitumor agents currently used in the clinical setting have cytotoxicity due to free radical production due to chemical properties, and due to the inflammation-inducing action, side effects (liver damage, kidney damage, bone marrow suppression) , Lung disorder, etc.). On the other hand, since 1,5-D-anhydrofructose and ascopilone have antioxidant activity, it is considered that there are few side effects as seen in conventional antitumor agents. Moreover, in combination with other antitumor agents, not only can the antitumor effect be enhanced, but it can also be expected to be applied to chemotherapy adjuvants that reduce side effects.
Japanese National Patent Publication No. 9-505988 Special table 2001-89377 Special table 2003-519660 gazette WO03 / 38084 WO03 / 38085 WO03 / 38107 WO00 / 56838 WO02 / 26060 WO02 / 26061 M.M. A. Baute. , Phytochemistry, 33, (1991) 41-45. Shafizadeh, F.A. , Et al. , Carbohydr. Res. , 67, (1978) 433-447. Stevenson, F.M. , Et al. , Carbohydr. Res. , 90, (1981) 319-325.

  An object of the present invention is to provide a safe and excellent antitumor agent with reduced side effects by using a combination of 1,5-D-anhydrofructose and / or ascopyrone and vitamin A and / or a vitamin A derivative. Is to provide.

  Still other objects and advantages of the present invention will become apparent from the following description.

  In general, in the clinical setting, there is a limit to the effect of an antitumor agent alone in tumor chemotherapy, and the current situation is that combined chemotherapy, that is, a treatment method used in combination with other agents is frequently used. As a result of intensive studies on the antitumor ability of 1,5-D-anhydrofructose and ascopyrone, the present inventors have found that vitamin A and / or vitamin A derivatives, which are nutrients that can be taken from meals, and 1,5 The present inventors have found that by using -D-anhydrofructose and / or ascopilone in combination, the antitumor effect is even better than when each compound is used alone, and the present invention has been achieved.

According to the present invention, the above objects and advantages of the present invention are as follows.
(1) 1,5-D-anhydrofructose and / or ascopyrone
(2) It is achieved by an antitumor agent comprising at least one selected from the group consisting of vitamin A and vitamin A derivatives.

According to the present invention, the above objects and advantages of the present invention are secondly,
(1) 1,5-D-anhydrofructose and / or ascopyrone
(2) It is achieved by a kit for treating tumors characterized by containing at least one selected from the group consisting of vitamin A and vitamin A derivatives as separate preparations.
That is, by administering an appropriate amount of 1,5-D-anhydrofructose and / or ascopyrone and vitamin A and / or a vitamin A derivative to an individual who possesses tumor cells in a living body, the tumor is significantly increased. It is possible to suppress the growth or metastasis of

  By using 1,5-D-anhydrofructose and / or ascopilone and vitamin A and / or vitamin A derivatives for tumor growth inhibition or metastasis inhibition, compared to when each compound is used alone. Not only can the dosage be significantly reduced, but the antitumor effect can be significantly improved.

  Ascopilone in the present invention is, for example, 1,5-D-anhydrofructose dehydration product derived from 1,5-D-anhydrofructose dehydrase derived from Ascomycetes, and other 1,5-D-anhydrous. It may be a compound obtained by dehydrating 1,5-D-anhydrofructose by chemical or physical operation such as treatment of fructose under alkaline conditions or heat treatment. As an example of ascopirone, the following formula

で表わされる化合物を挙げることができる。

The compound represented by these can be mentioned.

  Vitamin A and vitamin A derivatives, which are one of the active ingredients of the present invention, are widely contained mainly in animal foods including dairy products, and are compounds called retinol, retinal, retinoic acid and the like. In addition, vegetable foods, especially green and yellow vegetables, contain a large amount of carotene as provitamin A, and when ingested, it changes in the body to become vitamin A. Examples of vitamin A and vitamin A derivatives include:

で表わされる化合物を挙げることができる。

The compound represented by these can be mentioned.

All-trans-retinoic acid (atRA), which is one of the vitamin A derivatives in the present invention, is an antitumor agent used in chemotherapy for acute promyelocytic leukemia in clinical settings.
Examples of the vitamin A derivative that can be used in the present invention include retinal and retinoic acid as described above. Among them, retinoic acid, more specifically 9-cis-retinoic acid and atRA are preferable.

  The preparation in the present invention can be administered by various methods known per se, and the dose, 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, a method of direct administration into the body such as intravenously, subcutaneously or intraperitoneally by a method such as injection or infusion, or oral administration, and is not particularly limited.

  In addition, the antitumor agent in the present invention includes not only a single composition containing vitamin A and / or vitamin A derivatives and 1,5-D-anhydrofructose and / or ascopyrone as active ingredients, but also each compound. Each may be in the form of a kit composed of a combination of preparations each independently contained as an active ingredient. In the case of a kit, each preparation may be mixed immediately before administration, or each preparation may be administered by different administration methods.

  The dose of the antitumor agent in the present invention varies depending on the administration method, the type of tumor, and the severity, but for example, 1,5-D-anhydrofructose and / or ascopyrone as a single dose per kg body weight. Is 0.000001 μg to 1,000 mg, vitamin A and / or vitamin A derivatives can be 0.001 mg to 10 mg. Also preferably, when 1,5-D-anhydrofructose and / or ascopyrone is 0.0001 μg to 100 mg, vitamin A and / or vitamin A derivatives can be 0.03 mg to 3 mg, and vitamin A excess Administration to the extent that no symptom occurs is possible.

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 other 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.
Also, 1,5-D-anhydrofructose and / or ascopyrone and vitamin A and / or vitamin A derivatives are added to the food together to take the form of a health food for cancer prevention. Is also possible.

  The antitumor agent of the present invention can also be administered to mammals other than humans. That is, in that case, treatment of a tumor is performed by administering to a mammal at least one selected from the group consisting of 1,5-anhydrofructose and / or ascopyrone and vitamin A and a vitamin A derivative in combination. It can be performed.

Hereinafter, the present invention will be described in more detail by way of examples. The present invention is not limited in any way by such examples.
In the following tests, ascopyrone was prepared based on a known method.

Example 1
[Synergistic effect of antitumor activity of ascopilone P and all-trans-retinoic acid]
all-trans-retinoic acid (atRA) is an antineoplastic agent used in the clinical setting of chemotherapy for acute promyelocytic leukemia. Therefore, the synergistic effect of the antitumor activity of ascopilone P and atRA was examined in a cell line derived from acute promyelocytic leukemia (U937).
The cell culture solution of U937 cells was stimulated with various concentrations of ascopyrone P solution, all-trans retinoic acid (atRA), and a mixed solution of ascopyrone P and atRA. The solutions used at this time were all dissolved in dimethyl sulfoxide (DMSO), and the one added only with DMSO was used as a control.

  After culturing for a predetermined time, the cells were collected, fixed with 70% ethanol at 4 ° C. for 30 minutes, and then stained with a 50 μg / ml propidium iodide (PI) solution. After 30 minutes, a DNA histogram was prepared using FACS to examine the rate of cell death. The results are shown in FIG.

  The percentage of dead cells after stimulation with atRA (1 μM), ascopyrone P (0.3 mM) and ascopyrone P (0.7 mM) was 3.4%, 7.2% and 20.7%, respectively. Antitumor activity was confirmed. Furthermore, in the mixed solution of ascopilone P and atRA, the ratio of dead cells was 34.0% and 39.6%, which was higher than that of the single agent. From these results, it was suggested that the combined use of ascopilone P and atRA enhances the antitumor effect.

  Next, an experiment similar to that of U937 cells was attempted with HeLa cells (APP1.4 mM, the ratio of dead cells being 8.3 ± 2.0%) that had the lowest cell killing effect with ascopilone P alone (FIG. 2). Even when the concentration of atRA was lowered to 1/10 (0.1 μM), and when Ascopirone P was lowered to 3.5 nM and 0.35 nM, the cell killing effect was enhanced by using both in combination. From this, it was suggested that ascopirone P exhibits an antitumor effect at a very low dose when used in combination with atRA.

Example 2
[Synergistic effect of antitumor activity of ascopirone P and retinoic acid]
The synergistic effect of the antitumor activity of ascopilone P and 9-cis retinoic acid (RA) was evaluated in the same manner as in Example 1 using HeLa cells (FIG. 3). At both concentrations of ascopilone and retinoic acid at the concentrations used in this experiment, almost no cell killing effect was observed. However, when both are used in combination, the proportion of dead cells increases significantly, suggesting that the antitumor effect is enhanced.

Example 3
[In vivo anti-tumor test against B16 melanoma cells]
C57 BL / 6 mice were seeded with B16 melanoma cells (1 × 10 ⁵ cells) subcutaneously at the tail, and when a tumor of about 3 mm was formed, local subcutaneous injections were started (4 days, 5 times in total). The dosage was 2.5 mg / kg for Ascopirone P and 30 μg / kg for 9-cis retinoic acid (RA), and the antitumor effect was evaluated by the increase / decrease in the diameter of the tumor. At this time, ascopirone P and RA used were those dissolved in PBS (phosphate buffer), and those administered only with PBS were used as controls. The results are shown in FIG.

The group using both Ascopilone P and RA was clearly different from the other 3 groups, and tumor atrophy was observed from the first day (24 hours later), and the tumor growth rate was slow. That is, it shows the synergistic effect of the antitumor activity of ascopilone P and RA in vivo. In addition, in the anatomical findings after slaughter, tumor metastasis was observed in several buttocks in the PBS administration group and the retinoic acid single administration group, but almost no metastasis was observed in the group using ascopirone P and retinoic acid in combination.

Quantitative evaluation of dead cells by flow cytometry (FACS) (Effects of U937 cells / Ascopilone P and atRA) Quantitative evaluation of dead cells by flow cytometry (FACS) (Effects of HeLa cells / Ascopyrone P and atRA) Quantitative evaluation of dead cells by flow cytometry (FACS) (Effects of HeLa cells / Ascopyrone P and retinoic acid) Tumor volume in mice after administration of ascopirone P and retinoic acid

Claims (9)

An antitumor agent comprising (1) 1,5-D-anhydrofructose and / or ascopyrone and (2) at least one selected from the group consisting of vitamin A and a vitamin A derivative as an active ingredient.   The agent according to claim 1, wherein the ascopyrone is ascopyrone P.   The agent according to claim 1, wherein the vitamin A derivative is retinoic acid.   The agent according to claim 1, wherein the vitamin A derivative is 9-cis retinoic acid or all-trans-retinoic acid. (1) 1,5-D-anhydrofructose and / or ascopyrone, and (2) at least one selected from the group consisting of vitamin A and vitamin A derivatives, each as a separate preparation, Kit for the treatment of cancer.   The kit according to claim 5, wherein the ascopyrone is ascopyrone P.   The kit according to claim 5, wherein the vitamin A derivative is retinoic acid.   The kit according to claim 5, wherein the vitamin A derivative is 9-cis retinoic acid or all-trans-retinoic acid. Characterized in that (1) 1,5-D-anhydrofructose and / or ascopyrone and (2) at least one selected from the group consisting of vitamin A and vitamin A derivatives are administered to a mammal in combination. To treat tumor.

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