CN1093763C - Anticancer substance suppressing cancerous metastasis - Google Patents

Abstract

It is an object of the present invention to provide an anticancer agent that alone can inhibit cancer cell metastasis, has few side effects and has very little toxicity even when administered for a long period of time. The anticancer agent for suppressing cancer metastasis of the present invention is characterized by containing sialic acid, sialic acid salt, polymer of sialic acid and/or salt of polymer of sialic acid as an active ingredient.

Description

Anticancer agent that inhibits cancer metastasis

technical field

The present invention relates to an anticancer agent that significantly inhibits the metastasis of cancer cells, and provides an agent that can cure cancer more perfectly by preventing the metastasis of cancer cells.

Background technique

In recent years, chemotherapy to administer various anticancer agents, immunotherapy to promote the production of antibodies against cancer cells, surgery to remove cancer cells, and radiation therapy to kill cancer cells by irradiation have been used to treat cancer. However, due to the development of these treatment methods, surgery and radiation therapy are limited in means and cannot effectively prevent the metastasis of cancer. In addition, chemotherapy using anticancer agents directly acts on cancer cells, but in this case, it often produces harmful side effects on normal cells of the host, and is not necessarily effective in preventing cancer metastasis. Furthermore, immunotherapy, which treats cancer by enhancing the host's immune ability, has not found a good effect on preventing cancer metastasis. Now the effect of treating primary cancer has been greatly improved, but even if the primary cancer is completely cured, there are still many cases of death due to cancer cell metastasis. For the suppression of cancer metastasis, the development of agents capable of preventing cancer cell metastasis is strongly desired. It can be considered that cancer cells metastasize extensively by adhering to the cell membranes of tissues such as the mesentery.

As a medicine that inhibits the metastasis of cancer cells, it will be When mycocin is used in combination with a chemotherapeutic agent, an effect of inhibiting cancer cell metastasis can be seen, but there is no commercially available drug that independently inhibits cancer cell metastasis. In addition, JP-A-60-190791, JP-A-61-83125, and JP-A-62-223124 disclosed the effect of sialic acid derivatives on cancer metastasis inhibition, but did not describe the sialic acid used as an active ingredient in the present invention. or their derivatives and their inhibitory effect on cancer metastasis.

Issues to be solved

In view of the above problems, the present invention aims to provide an anticancer agent capable of independently inhibiting metastasis of cancer cells. Another object of the present invention is to provide an anticancer agent that suppresses cancer cell metastasis and has few side effects and very little toxicity even when administered for a long period of time.

means of solving problems

The present invention is accomplished by screening agents for inhibiting cancer cell metastasis from nature, and relates to an anticancer agent that uses sialic acid and/or its salt as an active ingredient and has the effect of inhibiting cancer metastasis.

In addition, the present invention relates to an anticancer agent having an effect of inhibiting cancer metastasis, comprising a polymer of sialic acid and/or a salt of the polymer as an active ingredient.

The above polymer of sialic acid is preferably a polymer composed of 2 to 13 molecules of sialic acid. Since the 13-mer fraction can be efficiently separated, the upper limit is limited to the 13-mer. Salivary polymers of 2- to 13-mers and salts thereof are expected to have the same pharmacological effects as sialic acid and salts thereof. In addition, various pharmaceutically acceptable salts can be used as salts of sialic acid or its polymers, and sodium salts, potassium salts, calcium salts, and magnesium salts can be used as salts of sialic acid monomers. As the salt of the substance, the sodium salt can be used. In addition, the sialic acid mentioned in this specification means [N-acetylneuraminic acid].

The preparations of the present invention include oral preparations such as tablets, capsules, and powders, percutaneous absorption preparations such as suppositories and pessaries, and injections for subcutaneous administration, intraperitoneal administration, and intravenous administration. In the prevention of diseases, oral preparations are preferred, but in emergencies, injections are preferred.

Oral preparations, percutaneous absorption preparations and injection preparations can be prepared by common preparation methods, and the preparation examples of oral preparations and injection preparations are given below.

1) Preparation example of injection:

Dissolve 50 g of sialic acid or its polymer in 1000 ml of distilled water (pyrogen-free), adjust the pH to 7.0 using caustic soda solution, filter and sterilize by the usual method, and then, under aseptic conditions Enclosed in 20ml ampoules to prepare injections.

2) The preparation example of oral agent:

Fill 280 mg of sialic acid or its polymer through a 60-mesh sieve into No. 3 gelatin capsules to make capsules.

The dose varies depending on the patient's age, sex, disease degree, etc., so it cannot be generalized, but as the sodium salt of sialic acid or its polymer contained in the injection, it is 1 to 2000 mg/kg per day for adults, preferably It is 10-500mg/kg, and the administration frequency is preferably 1-6 times a day, and intravenous drip injection is also an effective method.

Since sialic acid is mostly distributed at the ends of sugar chains on the surface of cells constituting the human body and at the ends of sugar chains of glycoproteins present in blood and body fluids, it is a drug that has very little adverse effect on the human body.

The sialic acid and its polymers used in the present invention can be chemically synthesized, using sialic acid aldolase or cytosine monophosphate-N-acetylneuraminic acid (CMP-NANA) synthetase and CMP-NANA transfer Enzyme-synthesized products obtained by enzymes, and hydrolyzed products of choridinic acid obtained by acid-decomposing choridinic acid (corosin), but the present invention is not limited thereto.

Brief description of the drawings

Fig. 1 shows the adhesion inhibitory effect of sialic acid on cancer cell M cells in rats.

Fig. 2 shows the influence of the interaction of sialic acid on the adhesion inhibitory effect of LFA-1 antibody to cancer cell-rat M cells.

Best way to implement the invention

According to the present invention, the sialic acid, sialic acid salt, sialic acid polymer, and sialic acid polymer salt contained as an active ingredient in the anticancer agent can inhibit the adhesion of cancer cells to cell membranes outside the mesentery, thereby preventing Cancer cells metastasize. Moreover, as an active ingredient, sialic acid, a sialic acid salt, a sialic acid polymer, and a sialic acid polymer salt can be used individually, and can also use it in combination of 2 or more types.

Cancer metastasis can be effectively suppressed by the anticancer agent of the present invention.

Example

The present inventors adopted a system in which extremely malignant cancer cells - rat ascites liver cancer adhered to the rat mesentery - as a model system for measuring the inhibitory effect on cancer cell metastasis, using a more convenient method of cell lines. The former used AH66F-cells for cancer cells, and the latter used M-cells for mesenteric cells.

When various substances existing in nature were screened using the above assay system, sialic acid and its polymers were seen to inhibit the adhesion of cancer cells to mesenteric cells, and further, animal experiments were carried out using rats with AH66F ascites liver cancer , a significant life-prolonging effect was also seen. Hereinafter, it demonstrates in detail using an Example.

Example 1

Effects of Sialic Acid and Sialic Acid Polymers on Inhibiting Adhesion of Cancer Cells

Use a cell culture plate to cultivate mesenteric cells (M-cells) from rats in Dalbeck's modified Eagle's medium (DMEM) containing 5% fetal calf serum (FCS). After all the wells are covered with cells, After washing with FC3-free DMEM, culture was carried out at 37°C with DMEM containing 10 μg/ml or 100 μg/ml of sialic acid. After 45 minutes, wash the wells with DMEM, add rat AH66F cells (4×10 ⁴ cells/well) and sialic acid (concentration shown in Figure 1), add DMEM, and incubate at 37 degrees for 1 hour. After incubation, stir for 30 seconds, and transfer the culture supernatant to a sampling tube. Add 200 μl of new DMEM to each well, stir for 15 seconds and wash in the same way. This washing operation was performed twice more, and the obtained washing solution was combined with the culture supernatant. It was centrifuged at 12,000 rpm for 5 minutes, and after the supernatant was removed, 100 μl of new DMEM was added to suspend the cells, and the number of cells (number of non-adherent cells) was counted under a microscope using a hemocytometer.

In addition, only cancer cells were cultured as a control, and the number of cells (number of control cells) was measured after performing the same operation.

The adhesion rate of cancer cells can be obtained by the following calculation formula. The results are shown in Fig. 1, but it is the average value of the concentration of each sialic acid in 4 wells.

Adhesion rate = [1-(non-adhesive cell number)/(control cell number)]×100

As described in Example 1, sialic acid concentrations of 10 μg/ml and 100 μg/ml prevented 27% and 31% of cancer cell adhesion to mesenteric cells, respectively.

Example 2

Effects of sialic acids on inhibiting cancer cell adhesion in the presence of anti-LFA-1 antibody

The contact culture of M-cells and AH66F cells was exactly the same as in Example 1, except that the concentration of sialic acid was 0 and 100 μg/ml, and the anti-LFA-1 antibody was coexisted at 10 μg/ml.

The result is shown in Figure 2.

In the presence of rat anti-LFA-1 antibody (anti-leukocyte function-related antigen β-chain antibody), which is known to have the ability to prevent cancer cell adhesion, 40% inhibition can be achieved without adding sialic acid, and when 100 μg/ml of sialic acid is added, Shown to prevent 70% of sticking. This means that the anti-LFA-1 antibody prevents the adhesion of cancer cells in a different mechanism from that of sialic acid, and also shows that the two substances have a synergistic effect.

Example 3

Experiments on model systems revealed that sialic acids have the effect of inhibiting cancer cell adhesion, so the effects of sialic acid and its polymers on rats with metastatic cancer (AH66F) were studied. Effect of sialic acid on life extension of cancer rats

After administering AH66F cancer cells (1×10 ⁶ cells) into the abdominal cavity of Donryu male rats (6 weeks old, 100-120 g, 6 rats in each group), immediately intraperitoneally administered 0.5 ml of sialic acid solution (dosage: 10 mg/kg and 100 mg/kg) or physiological saline (control group). Then, after intraperitoneal administration of sialic acid or saline for 2 days, the survival days of the rats were observed.

The prolongation rate was calculated with the following formula, and the results are shown in Table 1.

Life extension rate=[(survival days of treatment group)-(survival days of control group)/

(survival days of the control group)]×100 Table 1 The life extension effect of sialic acid in AH66F cancer cell carcinogenic rats Treatment group Survival days Life extension rate control 7.3±0.3 -sialic acid 10mg/kg×3 days 41.5±0%

100mg/kg×3 days 13.4±0.4 84%

The results confirmed that the survival days of the group not given sialic acid were 7.3±0.3 days, the group given 10 mg/kg of sialic acid had a 1.6-fold life-prolonging effect, and the group given 100 mg/kg had a 1.8-fold life-prolonging effect.

Acute Toxicity Test

The LD50 value obtained for intravenous administration using Wister rats (male) was 2,000 mg/kg or more for sialic acid and the sodium salt (pH 7.0) of the trimer of sialic acid.

Claims (3)

1. Use of sialic acid or a salt thereof in the preparation of an anticancer agent. 2. Use of a polymer of sialic acid or a salt thereof in the preparation of an anticancer agent. 3. The application according to claim 2, wherein the polymer of sialic acid is a polymer composed of 2-13 molecules of sialic acid.

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