KR19990013196A - New anticancer composition - Google Patents

Abstract

The present invention relates to a novel anticancer composition, and more particularly, to a cell wall component of Gram-negative bacteria, which is a substance capable of enhancing the anticancer action of macrophage (Macrophage), which plays a pivotal role in biological defense mechanisms corresponding to the proliferation of tumor cells. Lipopolysaccharide (LPS), capsular polysaccharides isolated from pneumococci, tumor necrosis factor (TNF) and gamma interferon (γ-Interferon, IFN-), which are anticancer substances secreted by macrophages and cells of various immune systems By containing γ) as an active ingredient, it induces a higher defense mechanism that each active substance cannot induce alone, and at the same time reduces the toxicity that each active substance can exhibit at concentrations having anticancer activity. The present invention relates to a novel anticancer agent composition for the prevention and treatment of tumors capable of killing cancer cells.

Description

New anticancer composition

The present invention is an active ingredient of gram-negative cell wall components of lipopolysaccharide (LPS), capsular polysaccharide isolated from pneumococcal, tumor necrosis factor (TNF) and gamma interferon (γ-Interferon, IFN-γ) It relates to a novel anticancer composition for the prevention and treatment of various malignancies.

More specifically, by the substances (liposaccharides and capsular polysaccharides) and macrophages and cells of various immune systems that can enhance the anticancer action of macrophage (Macrophage), which plays a pivotal role in the biological defense mechanism in response to the proliferation of tumor cells It contains a large amount of anticancer substances (tumor necrosis factor and gamma interferon), which makes it less toxic to living organisms through higher defense mechanisms and immunomodulatory activities that each active substance cannot induce alone, and effectively kills cancer cells. It relates to a composition of an anticancer agent.

As a biological defense mechanism corresponding to the occurrence of cancer and the proliferation of tumor cells, the immune recognition function by the immune function of the living body plays an important role, and the anti-cancer effect is T lymphocytes, B lymphocytes, macrophages and natural killer (NK) cells. And the like. In particular, macrophages play a pivotal role in the immune response to antigens, which directly target cancer cells in relation to nonspecific immunity such as antigen presenting and the secretion of cytokines necessary for the proliferation and activation of lymphocytes. Can be killed. Macrophages are activated upon exposure to various stimulants, which show functional changes such as phagocytosis, surface adhesion, increased prostaglandin secretion, and increased protein synthesis. In particular, macrophages activated by immunomodulators such as interferon and lipopolysaccharide have a cytotoxic effect on cancer cells. The mechanisms are tumor necrosis factor (TNF) and interleukin (IL) secreted by activated macrophages. -1), hydrogen peroxide (H ₂ O ₂ ), nitric oxide (NO), cytolytic protease (cytolytic protease) and the like to induce cytotoxicity to cancer cells.

Activated macrophages have the ability of antimicrobial and anticancer activity, but in cancer patients, anticancer activity in vivo is not effective alone, and generally sufficient immune capacity is induced to remove tumor cells. The multi-pharmaceutical concept of the Biological Response Modifier (BRM) has been introduced as an attempt to increase immunity because concentrations cause systemic side effects such as extreme high fever, sweating, headache and vomiting. It has been applied to therapy.

Biological response modifiers are immune modulators that can affect the biological interaction of immune cells with cancer cells and activate immune cells that specifically attack critical steps in cancer growth and metastasis. Currently, the composition of multi-drugs that induce biological response control by various anticancer substances includes 'interferon + tumor necrosis factor + chemotherapeutic agent', 'interferon + tumor necrosis factor + anti-inflammatory agent' and 'tumor necrosis factor + interleukin'. Is being used. These are mixed examples to reduce the toxicity since each anticancer substance is toxic at a concentration having anticancer activity.

Considering the fact that conventional chemotherapy and radiotherapy result in widespread biotoxicity and the composition of various biological response modifiers shows significant anti-cancer activity, the multi-drug body's defense mechanism and immunomodulatory effect Development of anticancer drugs is very important.

In order to improve the activity of existing biologically regulated multi-drugs, the inventors of the present invention have evaluated the anti-cancer activity in combination with lipopolysaccharide, capsular polysaccharide, tumor necrosis factor and gamma interferon. At the same time, the present invention was completed by finding that there is an additive or synergistic effect at a concentration that is not expected to be toxic.

Accordingly, an object of the present invention is to provide a biological multi-pharmaceutical with low toxicity to a living body with excellent anticancer effect in various malignant tumor patients.

1 is a graph showing the results of in vitro anticancer activity of macrophages induced by capsular polysaccharide isolated from pneumococcal at different concentrations,

2 is a graph showing the results of in vitro anticancer activity of macrophages induced by lipopolysaccharide by concentration,

3 is a graph showing the results of in vitro anticancer activity by gamma interferon (γ-Interferon) by concentration,

4 is a graph showing the results of in vitro anticancer activity by alpha-tumor necrosis factor by concentration.

FIG. 5 shows the results of in vitro anticancer activity by a composite composition of capsular polysaccharide (10 μg / ml), lipopolysaccharide (100 μg / ml), gamma interferon (10 IU / ml) and tumor necrosis factor (10 ng / ml) of pneumococcal bacteria. Is a graph showing

Figure 6 shows in vivo anti-cancer by complex composition of capsular polysaccharide (100 mg / kg), lipopolysaccharide (100 mg / kg), gamma interferon (200 IU / kg) and tumor necrosis factor (3 µg / kg) of pneumococcal A graph showing the results of the action.

The present invention is characterized by an anticancer composition containing four biological response modifiers: lipopolysaccharide, capsular polysaccharide, tumor necrosis factor and gamma interferon.

Hereinafter, the present invention will be described in more detail.

In the present invention, by a substance (lipopolysaccharide and capsular polysaccharide), macrophages and cells of various immune systems that can enhance the anticancer action of macrophage (Macrophage) that plays a pivotal role in the biological defense mechanism corresponding to the proliferation of tumor cells By combining a large amount of anticancer substances (tumor necrosis factor and gamma interferon), each active substance is less toxic to the living body through higher defense mechanisms and immunomodulatory actions that each active substance cannot induce alone, and effectively prevents cancer cells. Provided are anticancer drug compositions that can be killed.

In general, all drugs have a dose-dependent propensity for their toxicity, so if a multi-drug composition exhibits an additive or synergistic effect on its efficacy, the composition exhibits high potency due to low concentrations of the drug and does not have the toxicity of each drug. It is expected to be reduced.

According to the composition of the present invention it is possible to fundamentally treat a variety of malignant tumors and diseases in need of bioimmunity and to minimize the generation of various toxicity during treatment.

In this example, each titer for each of the four immunomodulators was searched for concentration levels (Tables 1 to 4). According to the results, the cell membrane showed significant lysis of 10 µg / ml for pneumococcal polysaccharide, 100 µg / ml for lipopolysaccharide, 100 IU / ml for gamma interferon, and 10 ng / ml for tumor necrosis factor. On these basis, each immunomodulator concentration showing about 30% cytolysis was taken and mixed. In all cases, the increase in titer was shown (Table 5), especially when all four components were contained, the highest increase in potency.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

Example 1

Isolation of Macrophage

Macrophages were obtained by washing the abdominal cavity of 6-8 weeks old SPF, ICR mice received with the National Institutes of Health. The obtained cells were centrifuged to wash the RPMI cultures twice, and RPMI cultures containing 10% fetal bovine serum (FBS) and 2% penicillin / streptomycin were added to the concentration of 1 × 10 ⁶ cells / ml. Cells were placed in a 35 mm Teflon-coated petri dish and incubated for 2 hours, unattached cells were removed, and fresh culture was added. Cell viability was determined by trypan blue exclusin technique.

Example 2

Isolation and Purification of Capillary Polysaccharides from Pneumonia

Pneumococcal inoculation was inoculated in BHI broth and incubated at 37 ° C. until absorbance reached about 0.6 at 550 nm, followed by lysis by adding 1% phenol. 1/2 volume of ethanol cooled at −70 ° C. was added, left at 4 ° C. for 3 hours, then centrifuged at 10,000 rpm at 4 ° C. for 10 minutes, and the supernatant was discarded. The precipitate was completely dissolved in distilled water in 1/20 volume, extracted with 1/5 volume of chloroform: butanol (5: 1), followed by centrifugation at 10,000 rpm for 15 minutes to obtain a supernatant. 0.2% cetavilon (cetavlon) was added to the supernatant, and 2 volumes of cooled ethanol was added and left at 4 ° C for 1 hour. The supernatant was discarded by centrifugation, and the precipitate was dissolved in physiological saline to prepare a sample solution.

All samples were dissolved in physiological saline and applied, and the control group was treated with physiological saline without drugs.

Example 3

Evaluation of in vitro anticancer effects induced by capsular polysaccharides

Capsular polysaccharides isolated from pneumococci were added to the isolated macrophages at concentrations of 0, 1, 10, 100 and 200 µg / ml, respectively, and cultured for 20 hours. The supernatant was removed and washed twice with culture. B16 cancer cells (1 × 10 ⁶ cells / ml) were labeled with 100 μCi sodium chromat for 2 hours and then incubated with activated macrophages for 40 hours. 100 μl of the supernatant was harvested and radioactivity was measured on a gamma counter ( ⁵¹ Cr release microcytotoxicity assay). The results are shown in Table 1 and FIG.

Cytotoxic capacity was calculated as follows.

The spontaneous release was determined by measuring only cancer cells, and the maximum release of ⁵¹ Cr was determined by adding 0.1 ml of triton X-100 to the cancer cells.

Example 4

Evaluation of in vitro anticancer effects induced by lipopolysaccharide

Cells were carried out in the same manner as in Example 3, except that lipopolysaccharide (E. coli of antigen 055: B5, Sigma Co., Ltd.) was used at concentrations of 0, 1, 10, 100, and 200 µg / ml instead of the capsular polysaccharide. Toxicity was measured. The results are shown in Table 2 and FIG.

Example 5

Evaluation of in vitro anticancer effects induced by gamma interferon

Cytotoxic activity was carried out in the same manner as in Example 3, except that gamma interferon (genome recombination gamma interferon in rats, Genzyme, Inc.) was used at concentrations of 0, 1, 10, 100, 200 IU / ml instead of capsular polysaccharides. Was measured. The results are shown in Table 3 and FIG.

Example 6

Evaluation of in vitro anticancer effects induced by tumor necrosis factor

Alpha tumor necrosis factor (Genome alpha tumor necrosis factor, Genzyme) in place of the capsular polysaccharide was carried out in the same manner as in Example 3, except that the concentration of 0, 1, 10, 100, 200ng / ml Cytotoxic activity was measured. The results are shown in Table 4 and FIG.

Example 7

Evaluation of in vitro anticancer effects induced by complex components

Capsular polysaccharide + lipopolysaccharide, capsular polysaccharide by combining pneumococcal capsular polysaccharide (10 µg / ml), lipopolysaccharide (100 µg / ml), gamma interferon (10 IU / ml) and tumor necrosis factor (10 ng / ml) instead of capsular polysaccharide + Interferon, capsular polysaccharide + tumor necrosis factor, liposaccharide + interferon, liposaccharide + tumor necrosis factor, interferon + tumor necrosis factor, capsular polysaccharide + lipopolysaccharide + interferon, capsular polysaccharide + lipopolysaccharide + tumor necrosis factor, capsular polysaccharide + interferon Cytotoxic activity was measured in the same manner as in Example 3 with respect to each of the composite compositions such as + tumor necrosis factor, capsular polysaccharide + lipopolysaccharide + interferon + tumor necrosis factor. The results are shown in Table 5 and FIG.

Example 8

In vivo anticancer effect evaluation

As experimental animals, athymic rats (athymic NC4-nu) weighing about 25 ± 5g were adapted to a large isolator maintained at constant temperature and humidity for at least one week. Used for. Animal breeding environment is artificial lighting (from 7 am to 7 pm) at 12 hour intervals, illuminance 200 ~ 300Lux, temperature 22 ± 1 ℃, humidity 55 ± 5%, exhaust 15 ~ 17 times / hr, noise below 60db, Four dogs were kept in a polycarbonate breeding box (W280 × L400 × H170mm) under a wind speed of 15-20 cm / sec and ammonia concentration of 20 ppm or less. Solid feed and tap water were freely consumed. All items used in the experiment including feed and water were sterilized with ethylene oxide gas (EO gas, ethylene oxide).

After pre-prepared MX-1 tumor sections (2 × 2 × 2 mm 2) were transferred to the injection site, the mice were fixed with the left hand and the right waist skin was incised with sterile scissors. Tumor sections were inserted into the skin incision site by implantation, and dice were implanted subcutaneously by pushing the dice over the right chest. The incision was sterilized with iodine solution. After a certain period of time, the tumor was grown and the tumor site was cut out with sterile scissors, the connective and adipose tissues were removed, and the tumor weight was measured. The drug was administered topically subcutaneously at 24 hour intervals with a mixture of pneumococcal capsular polysaccharide (100 mg / kg), lipopolysaccharide (100 mg / kg), gamma interferon (200 IU / kg) and tumor necrosis factor (3 µg / kg). The number of animals per experimental group was 10. The result is shown in FIG.

TABLE 1

TABLE 2

TABLE 3

TABLE 4

TABLE 5

Stenosis: Capsular polysaccharide

Ji: Polysaccharide

Phosphorus: Interferon

Species: Tumor necrosis factor

The present invention can reduce the toxicity that can be exhibited at high concentrations by using a multi-drug of immunomodulators, which induces a higher biological defense mechanism that each active substance cannot induce alone and also exhibits anticancer activity at low doses. .

Claims (1)

An anticancer composition comprising pneumococcal capsular polysaccharide, lipopolysaccharide, gamma interferon and tumor necrosis factor as active ingredients.