KR0173362B1 - Carbohydrate complex extracted from mycobacterium tuberculosis and process for the preparation thereof - Google Patents

Abstract

The present invention relates to a complex polysaccharide which is an anticancer component extracted from Mycobacterium tuberculosis and a method for producing the same, to obtain a cell as an aqueous fluid by culturing M. tuberculosis (M. tuberculosis), and pressurizing it. Under heating, followed by filtration, sulfosalicylic acid was added to the filtrate, and the precipitate was removed, and then dialyzed, phosphotungstic acid was added to the dialysate, the precipitate was removed, then dialyzed, and ethanol was added to the dialysate to obtain a precipitate, and the precipitate was obtained by ethanol and ether. Mannose, aribinose, glucose and galactose, which are prepared by removing lipids or protein components, are extracted as essential components of monosaccharides, and these monosaccharides are glycoside-linked linear or branched polysaccharides having a molecular weight of up to about 7000. Compound Toversin-5 can be used as an effective anticancer agent with little toxicity or side effects .

Description

Complex polysaccharide extracted from Mycobacterium tuberculosis and preparation method thereof

FIG. 1 shows the results of acid hydrolysis of the Toversin-5 of the present invention and analysis by Bio-LC equipped with a pulse current detector.

2 is a result of ¹ H-NMR analysis of the arabinomannan fraction 1, 2, 3 and 4 contained in the tubercin-5 of the present invention,

3 is a result of ¹³ C-NMR analysis of the arabinomannan fractions 1 and 3 contained in the tubercin-5 of the present invention,

4 shows the result of cationic FAB-MS analysis of oligosaccharide aldehyde in the tubercin-5 of the present invention.

The present invention relates to a complex polysaccharide, which is an anticancer component extracted from Mycobacterium tuberculosis, and a method for preparing the same. Specifically, a method for extracting and purifying a complex polysaccharide component of M. tuberculosis cell components and its It relates to the use as an anticancer agent.

In 1891, US surgeon William B. Coley was known to treat many cancer patients with bacterial extracts of the Coli vaccine, but scientific follow-up was not followed. Sixty years later, in the 1950s, Polly, Fren and Maine announced that experimentally induced animal cancers could be treated immunologically, and at the same time several cancer research institutes in France and the United States. Foley, EJ, Antigenic properties of methylcholanthrene-induced tumors in mice of the strain of origin, Cancer Res. 13, 885-837 (1953): Prehn, R. T. Main, J. M., Immunity to methylcholanthrene-induced sarcomas, J. of Nat. Cancer Inst. 18, 769-778 (1957). In 1959, reports from France's Biocci et al. Reported that Ehrlich ascites cancer in rats improved with BCG vaccination and BCG was used for cancer immunotherapy [Biozzi, G., Stiffel, C. , Halpern, BN Mouten, D., Effect del'inoculation du Bacille de Calmette-Guerin sur le development de la tumeur ascitique at Ehrlich Chez la souris, Compt. Rend. Soc. Biol., 153, 987-989 (1959)], and then the US, Old, and Vice have attempted to treat carcinoma in experimental animals and humans by inoculating BCG or tuberculosis bacteria or by injecting some component fractions. Old, LJ, Benacerraf. B., Clark, D. A., Carswell, E. A. Stochert, E., The role of the reticuloendothelial system in the host reaction to neoplasia, Cancer Res., 21, 1281-1300 (1961); Weiss, D.W., Donhag, R.S. Parks, J.A., Studies on the heterologous immunogenicity of a methanol-insoluble fraction of attenuated, tubercle bacilli (BCG), J. Exp. Med., 119, 53-70 (1964).

Since 1974, it has been found that the narcotic components of M. tuberculosis cell extracts are mainly present in the cell membrane and that the main components are polysaccharides and lipid derivatives. Recently, Azuma et al. Have isolated N-acetylmuramil-L-alanyl-D-isoglutamine (MDP) as an active ingredient [Azuma, L., Yamamura, Y. Fukushi, K., Fractionation of mycobacterial cell wall. Isolation of arabinose mycolate and arabinogalactan from cell wall fraction of mycobacterium tuberculosis strain Aoyama B., J. Bact., 96: 1885-1887 (1968)], 1992sus Barnes et al. Reported that lipoarabinomannnan promotes cytokine production. Barnes, P. F, Chatterjee, D., Abrams, JS, Lu, S., Wang, EW, Yamamura, M., Brennan, PJ Modlin, LR, Cytokin Production induced by Mycobacterium tuberculosis lipoarabinomannan, The J. of Immunol , 149, 541-547 (1992).

In Korea, in 1974, Chung et al. Isolated tubercin-3 as an anticancer substance without delayed hypersensitivity from M. tuberculosis. It has been used in tumors of animals [Chung, TH, Anti-tumor effect of a tuberculo-protein comple, Tubercin-3, and its isoelectrofocusing analysis, J. of Korean Med. Ass., 17, 427-431 (1974); Chung, T.H., Song, J.Y. Hong, S.S., Inhibitory effect of tuberculo-protein complex, Tubercin-3, on three cases of lepromatous leprosy, Yonsei Med. J., 17, 131-135 (1976); Kim, S.W., Lee, K.S. Chung, T.H., Effect of Tubercin-3 on a case of chronic dermatitis with selective hereditary complete IgA deficiency and partial T-lympocyte inactivation, The Korean Journal of Dermatology, 14, 289-295 (1978 /); Chung, T. H., Kim, S. M. Lee, S. H., Effect of Tubercin-3 on Rifampicin induced T-lymphocytopenia in tuberculosis patients, The Korean Journal of Thoracic and Cardiovascular Surgery, 11, 12-17 (1978); kim, Y.H. Chung, T.H., Anti-tumor effect of 1,3-bis (2-chloroethyl) -1-Nitrosourea with Tubercin-3 on Ehrlich ascites carcinoma in mice, Kyungpook Univ. Med. J., 19, 65-70 (1978); Chung, T.H., song, J.Y. Seo, J.K., Trial of Immunotherapeutic approach with tubercin-3 on lepromatous leprosy, Kyungpook Univ. Med. J., 22, 1-6 (1981); And Chung, T.H., Trial of Immunotherapeutic approach with tuberculo-protein carbohydrate complex, Tubercin-3, on 500 cases of various cancer in men, The Korean Journal of Biochemistry, 15, 65-70 (1983). Tubercin-3 is a mixture of nucleic acid proteins, lipoproteins, and polysaccharides obtained by extracting M. tuberculosis cells in aqueous solution, and then protein-polished with sulfosalicylic acid, phosphotungstic acid and the like, followed by precipitation with ethanol. Similarly, it is believed to have the effect of inhibiting several types of carcinoma.

Infection of M. tuberculosis bacteria in humans results in granulomatous inflammatory changes, particularly in the lungs, when cavities form simultaneously with these inflammatory changes. It was discussed before 1912 that these inflammatory changes and cavities can be caused by proteins and complex lipids that are contained in and secreted from the Mycobacterium tuberculosis cell wall components, and a test for tuberculosis infection called tuberculin testing has emerged. . Therefore, polysaccharides that exclude proteins and lipids as M. tuberculosis cell extracts are not considered to be toxic when injected into animals within the toxicological range, which is low protein-treated in M. tuberculosis cell extracts. It can also be predicted by the fact that the tuberculin reaction of the substance was negative.

In other words, when immunotherapy is used to treat cancer, if the drug itself contains a protein component or a high molecular weight polysaccharide component, there is a high possibility of causing an immune response or a direct inflammatory response irrelevant to the anticancer effect. The present inventors pay attention to the fact that the anticancer component of M. tuberculosis cell extracts is mainly polysaccharides and lipid derivatives present in the cell membrane, and further isolates and analyzes the components of tubercin-3 comprising the complex components. The pure polysaccharide material obtained therefrom was found to be an anticancer component, which led to the completion of the present invention.

That is, an object of the present invention is to provide a complex polysaccharide compound which is an anticancer component obtained from Mycobacterium tuberculosis, and a method for extracting and preparing the same.

In order to achieve the above object, in the present invention, mannose, arabinose, glucose and galactose isolated from Mycobacterium tuberculosis are essential constituent monosaccharide components, and these monosaccharides are linear or branched glycosides. Provided are a complex polysaccharide compound having a combined molecular weight of up to about 7000 and an anticancer composition comprising the same.

In order to achieve the above object, the present invention is cultured Mycobacterium tuberculosis (Mycobacterium tuberculosis) to obtain the cells as an aqueous suspension, it is heated under pressure and then separated by filtration, sulfosalicylic acid is added to the filtrate and the precipitate is removed And then dialysis, phospho tungstic acid is added to the dialysate, the precipitate is removed, and then dialysed, ethanol is added to the dialysate to obtain a precipitate, and the precipitate is extracted with ethanol and ether to remove lipid or protein components. Provided are methods for preparing the compounds.

The complex polysaccharide compound of the present invention isolated from M. tuberculosis was named Tubercin-5, and is also referred to below.

Toversin-5 of the present invention is cultured M. tuberculosis (M. tuberculosis) to obtain the aqueous suspension of the cells, the cells are extracted by heating with water, and then the sulfo salicylic acid and phospho tung acid to precipitate the protein in the extract The acid used at this time can be removed by dialysis, followed by ethanol to obtain a precipitate, and the precipitate is repeatedly extracted with ethanol and ether to completely remove lipid or protein components.

At this time, polysaccharides and protein complexes can be extracted by heating the aqueous suspension of M. tuberculosis culture cells in a high pressure autoclave, and sulfosalicylic acid and phosphotungst used for protein precipitation. The acid is preferably added so as to have a concentration of 1 to 10% and 1 to 15%, respectively. The precipitate obtained by adding ethanol was washed with ethanol-water (1: 3) mixture, and then repeatedly extracted with phenol, ether and ethanol in Tris-hydrochloric acid buffer-EDTA to remove the remaining lipid or protein, and the ion exchange resin column was By passing through to remove the minerals incorporated during the extraction process it is possible to purely separate the complex polysaccharide compound of the present invention.

In order to analyze the constituent monosaccharides of purely isolated tubuscin-5 and to confirm the binding method thereof, per-O-methylation, followed by acid hydrolysis, reduction and acetylation, and analysis through GLC-MS system, It showed arabinomannan structure and was found to be present in high amounts of glucose, aribinose and mannose.

Subsequently, acid hydrolysis and neutralization of tubosecin-5 were carried out by high-speed anion exchange chromatography (HPAEC) to confirm that monosaccharides and polysaccharide constructs having various lengths were mixed and then divided into four arabino-mannan fractions. Aliquoted.

As a result of ¹ H NMR analysis of the four types of usable arabinomannans, α- or β-furanosyl and α-pyranosyl residues were confirmed. In addition, ¹³ C NMR analysis showed that ① 5-linked α-Araf residues ② branched (3 → 5) -linked α-Araf units substituted with 5-bonded α-Araf residues at the branched positions and ③ disaccharides It was confirmed that it has a β-Araf- (1 → 2) -α-Araf and two (3 → 5) -bonded α-Araf units substituted at the same time.

For more accurate structural analysis, O-alkylation of tubercin-5, followed by partial acid hydrolysis and reduction with sodium borohydride (NaB [ ² H] ₄ ) yielded an O-alkylated oligosaccharide allitol to produce FAB-MS The analysis showed that Man (including Man) ₆ (Ara) ₆ (Glu) (Gal) and (Man) ₉ (Ara) ₉ (Glu) (Gal), which are present in abundant amounts from the simple structure (Man) ₂ , , Various combinations of Ara, Glu and Gal are shown.

According to the above analysis results, it can be seen that the complex polysaccharide compound tubercine-5 of the present invention is a complex polysaccharide compound having a molecular weight of up to 7000 in which mannose, arabinose, glucose and galactose are glycoside-linked with straight or branched chains.

The tubercin-5 is present as a compound of various molecular weights according to the degree of synthesis of various sugars, in terms of preventing side effects such as hypersensitivity reactions, the largest molecular weight of the complex polysaccharide compound should be 7000 or less. The composite polysaccharide compound tubercin-5 of the present invention mainly consists of polysaccharides having a molecular weight of 2500 to 3500, and includes those having a molecular weight of 5000 to 7000.

Tubose-5, a pure polysaccharide material prepared from M. tuberculosis cells as described above, improves clinical symptoms and prolongs survival in cancer patients, including lung cancer, gastric cancer and cervical cancer. In addition, while acting to reduce or eliminate cancer tissue, there is no side effect such as hypersensitivity reactions and LD ₅₀ is so high that it can not be compared with the effective amount can be seen as a safe anticancer drug.

The anticancer composition comprising the tubercine-5 of the present invention as an active ingredient is a subcutaneous injection according to a known method by adding a pharmaceutically acceptable conventional excipient such as a carrier, a diluent, a filler, an anti-agglomerating agent, a lubricant, a flavoring agent, It may be prepared as a pharmaceutical preparation such as an intravenous injection, and may be administered parenterally as necessary. Dosage may be used in an amount of 0.001 to 1 μg, preferably 0.01 to 0.5 μg per kg of body weight per day for subcutaneous injection, and can be adjusted according to various conditions such as the patient's symptoms, age, sex, and weight.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are merely illustrative, and the scope of the present invention is not limited only to these.

Example 1

Preparation of Toversin-5

M. tuberculosis (H ₃₇ R _V : ATCC 25618) was added to Sauton medium [Sauton, B., Sur la nutoition minterale du tuberculux, Res. 8, Intern. Cong. Appl. Chem., 19, 267-269 (1912)] and incubated for 4 weeks at 37 ℃. Aqueous dilution of 300 g of cultured cells with about 20 times of water was heated in an autoclave (15 1b, 120 ° C) for 30 minutes to extract polysaccharides and protein complexes, followed by centrifugation at 3,500 ~ 4,500rpm for 30 minutes. The liquid was separated. The supernatant was filtered out, and the filtrate was passed again through a Schaumerene filter to completely remove the bacterial body. Sulfosalicylic acid (sulphosalicylic acid) was added to the filtrate at 10% concentration to remove the tan precipitate (protein) by centrifugation, and the supernatant was put in dialysis semipermeable membrane, soaked in pure water for 2-3 days, and sulfosalicylic acid was exuded into pure water. Concentrate the colloidal solution in the semipermeable membrane, add secondary phosphotungstic acid at 7% concentration, centrifuge at 3,500 rpm for 20 minutes, and then add the supernatant to the dialysis semipermeable membrane. Soak for 3 days to remove inorganic components. Ethanol was added in an amount of about 5 times to the dialysis solution, and the precipitate obtained by standing at 4 ° C. for 24 hours was washed twice with an ethanol-water (1: 3) mixture. Subsequently, extraction was repeated three times with phenol, ether, and ethanol in 20 mM Tris-HCl buffer (pH 8.0) -5 mM EDTA to remove incorporated lipids and the like. Finally, the solution was eluted with distilled water through a column (1.2 × 65 cm) filled with Bio-Gel P4 (Bio-Gel P4: Bio-Rad Laboratories, USA) to remove inorganics to obtain 100 mg of the complex polysaccharide compound of the present invention. It was.

Example 2

[Structural Analysis of Tuversin-5]

For the analysis of the constituent monosaccharides of Toversin-5 prepared in Example 1, it was dissolved in water to a concentration of 500 μg / ml, and the standard room and blank test solution of various monosaccharides as control bacteria, Oxford Glycosystems (UK) It was carried out according to the method for separating and quantifying the constituent monosaccharides from the polysaccharides developed in

Perversin-5 is then per-O-methylated, followed by acid hydrolysis, reduction, acetylation and per-O-trimethylsilyl (TMS) methylglycosides, and separation of each monosaccharide using a GLC-MS system. Confirmed. A fluorescence ionization detector (FID) was used for quantitative analysis of each TMS-methylglycoside. As a result, the glycosyl residues and ratios present in the tubercine-5 complex polysaccharide and the types and relative ratios of the monosaccharides constituting the polysaccharide were shown in Tables 1 and 2 below.

Analysis of the polysaccharide showed an arabinomannan structure, which is similar to that of other tuberculosis bacteria. In addition, with the fact that 4-binding Mannopyranose (Manp) residues are present, it has been found that arabinose and mannose are present in large quantities, followed by glucose and galactose.

Subsequently, 10 mg of Toversin-5 was dissolved in 6N hydrochloric acid, left at 70 ° C. for 24 hours to hydrolyze, and then neutralized with 6N sodium hydroxide solution, followed by high-speed anion exchange chromatography using Bio-LC equipped with a pulse current detector. Photography (HPAEC) was performed.

The column uses a Dionex CarboPac PA (4 × 250mm) column with a CarboPac PA guard column (3 × 25mm) .The detector uses a gold-coated electrode on Dionex's pulsed amperometric detector (pad). Used. The sample injection loop was 26 μl and the pulse voltage of the pad was E = 0.05 V, t = 480 ms, E = 0.60 V, t = 120 ms, E = -80 V, t = 60 ms, and the response time of the detector was 1 second. . The eluate was used with 1M sodium acetate (eluent 1), 200 mM or 375 mM sodium hydroxide (eluent 2), and distilled water (eluent 3). The eluent gradient program was as follows:

(1) Program A:

Eluate 1 at 0% to 5% and 45% to 50%

50% eluent 2 (200 mM NaOH) from 0 to 45 minutes

(2) Program B:

Eluent 1 is 5% to 0 minutes, 15% to 5 minutes, 25% to 20 minutes, 35% to 45 minutes, 45% to 65 minutes

Eluent 2 (375 mM NaOH) was kept constant at 1 ml / min for 40% flow rate from 0 to 65 minutes.

As the standard solution, various known carbohydrate standards were prepared and injected into the solution, and the obtained peak area was calibrated by dividing into μg or μmol.

FIG. 1 shows the results of acid hydrolysis of the tubercine-5 of the present invention and analysis by Bio-LC equipped with a pulse current detector. As shown in Figure 1, it was confirmed that the tubercin-5 is a mixture of monosaccharides and polysaccharides of various lengths, it was possible to identify the polymerization of carbohydrates depending on the elution position. It was aliquoted into four kinds of fractions and the analysis continued.

Each fraction of arabinomannane fractionated as described above is 1 ml [ HO], 1 μl of acetone was added as an internal standard, and analyzed by NMR (Bruker ACE-300). Acetone signal is In C-NMR, δ31.4 In H-NMR, it was identified as δ 2.04. 2 shows the arabinomannan fractions 1, 2, 3, and 4 contained in tubercin-5 of the present invention. As a result of H-NMR analysis, it was possible to confirm the presence of the sugar chain residues in fractions 1 and 3 (the small arrow at the bottom in 1 and 3 means the signal identified as H-1 of β-glucosyl residue). Different anomer protons for each polysaccharide signal in the range δ 5.0-4.5, corresponding to α- or β-furanosyl and α-pyranosyl residues.

3 shows the arabinomannan fractions 1 and 3 contained in tubercin-5 of the present invention. Identification of Soluble Aribinomannan of M. tuberculosis previously studied by C-NMR analysis [Daffe, M., Brennan, P.J. And in M. McNeil, J. Biol. Chem., 265, 6734-6743 (1990)], were able to obtain the resonance values corresponding to the non-reducing ends of the arabinane chain. That is, the terminal t-β-Araf (C-1, δ 101-102) is bound to position 2 of α-Araf (C-1, δ 106-108), and this disaccharide is also found in the residue of α-Araf. It can be seen that it is bound to positions 3 and 5. Furanoid properties of the remaining Ara and Man residues were also identified at the anomer carbon resonance site (δ-107-109). That is, nuclear magnetic resonance data of C-1 of t-β-Araf and 2-binding α-Araf, followed by arabinose residues and C-2, and C-5 of t-β-Araf are described in the above literature. Already announced, this is very similar. For furanosyl residues, information on 2-, 3-, and 5-bound Araf residues and 5-bound Galf residues are available from existing data, and most of the C-1 resonances appearing between δ108 and 109. It can be seen that this corresponds to the α-Araf / β-Galf unit [Gorin, PAJ And M. Mazulek, Carbohydrate Res., 48, 171-186 (1976); Joseleau, J. P., Chambat, G., Vignon, M. and F. Barnoud, Carbonydrate Res., 58, 165-175 (1977); And Mizutani, K., Kasai, R., Nakamura, M. and O. Tanka, Carbohydrate Res., 185, 27-38, (1989)]. Of the Soluble Arabinomannan Fraction from the Cell Wall Components of M. tuberculosis The similar C-NMR results indicate that each fraction has the same structural motif. As a result, it was confirmed that these arabinomannans have at least three structures:

① 5-binding α-Araf residue

A branched (3 → 5) -binding α-Araf unit substituted with a 5-binding α-Araf residue at a branched position, and

③ (3 → 5) -linked α-Araf units substituted with two disaccharides β-Araf- (1 → 2) -α-Araf

For more accurate structural analysis of the arabinomannan component of tubercin-5, the per-O-metalated arabinomannan component was treated with 1 M CFCOOH at 75 ° C. for 1 hour and partially acid hydrolyzed, followed by NaB [ Per-O-alkalized oligosaccharide Altitol was prepared from oligosaccharide allitol obtained by reduction to H] and analyzed by FAB-MS.

FIG. 4 shows the results of cationic FAB-MS analysis of olidinealtitol contained in tubercin-5. Or it can be seen that the strong molecular ions of M + NH, this heterogeneity shows various forms of Man and Ara. Where 408 is (Man), 612 is (Man), 932 is (Man) (Ara), 1862 is (Man) (Ara), and 2270, which is present in abundant quantities, is (Man) (Ara (Glu) (Gal), and 2882 has the structure (Man) (Ara) (Glu) (Gal).

The structure of Toversin-5 obtained from the above results is shown in Table 3 below.

In Table 3 below, the sugar chain containing glucan, ie, a glucose residue or galactose, may be provided in the X site, and the largest molecular weight among the tubercin-5 of the present invention is 7,000, and most between 2,500 and 3,500, and 5,000 to 7,000. The following is also included.

In Table 3, Ara ---, Ara --- in 2270 and 2882 are indications that the arabinose residue is continuously, and the polysaccharide having a molecular weight of 3000 or more is present in the X region with glucose and galactose. Polysaccharides having a molecular weight of 3500 or more are present in less than 10% of the total polysaccharides.

Toxicity Test

[Acute Toxicity Test in Mice and Rats]

Toversin-5 prepared in the above example was dissolved in 0.9% physiological saline in an amount of 2 mg / ml, and various doses in 6-week-old mice (ICR mice; 20-25 g) and rats (SD rat; 150 g) After administration by intravenous injection or subcutaneous injection (single or continuous administration), survival was observed and the results are shown in Table 4.

Therefore, the LD of Toversin-5 is at least 20,000 μg / kg when injected intravenously, at least 50,000 μg / kg when injected subcutaneously, and at least 20,000 mg / kg when injected intravenously and at least 50,000 mg / kg when injected subcutaneously. do. Thus, considering that therapeutically effective dosages for humans are between 0.001 and 1 μg, preferably between 0.01 and 0.5 μg per kg body weight per day, tubulin-5 is considered safe in terms of acute toxicity.

In addition, tubercine-5 was administered to mice and rats as described above, and the general condition was observed for 14 days, and the change in body weight was measured. On the 15th day, all animals were sacrificed for autopsy and then liver, kidney, spleen, stomach, The gross findings of the lungs and heart were observed, pathological specimens were prepared by immobilization in 10% formalin and microscopic observation was performed by hemitoxylin-eosin staining. As a result, no abnormalities were found in all cases.

[Effect test]

Tubosecin-5 alone and the combined effect of tubersine-5 and cyclophosphamide (cytosan) in mice

Sixty-eight white male mice (ICR mice; 20-25g) weighing 22g were divided into eight groups of eight dogs, separated and housed. Baylif's method for each animal [Baillif, RN, The solid phase of the Ehrlich ascites tumor in mice, Cancer Research, 15, 554-558 (1954)], 0.2 ml (1.4 × 10) of 10% Ehrlich ascites cancer cell fluid Cells) were inoculated subcutaneously on one back. Then, as shown in Table 6 below, a control group injected with 0.2 ml of 0.9% saline solution intraperitoneally, Tubosecin-5 was dissolved in 0.9% saline in an amount of 1 μg / ml and injected with subcutaneous subversin-5 Single administration group (1), tubercine-5 alone administration group (2), which was injected subcutaneously by dissolving toversin-5 in 0.9% physiological saline in an amount of 2 μg / ml, 0.2 ml of 25 mg of chemical anticancer drug cytosan per kg body weight Cytolic acid alone administration group (3) dissolved in water and injected into the abdominal cavity, 50 mg of cytoic acid per kg body weight dissolved in 0.2 ml of water and injected into the intraperitoneal cavity (4), 25 mg / kg body weight Administration group (5) with oxane intraperitoneal injection and 1 μg tubercin-5 subcutaneous injection, and administration group with 50 mg of cytoxane peritoneal injection and 1 μg tubercin-5 subcutaneous injection (5), and body weight kg Seven of the administration group (6) which combined 50 mg of cytotoxic acid peritoneal injection and 1 μg of tubercin-5 subcutaneous injection In the treated animals from each of the 3 days after the inoculation, each test solution. Toversin-5 was injected subcutaneously in the back of the animal, and cytoic acid was injected 6 times intraperitoneally.

Solid carcinoma developed in the stomach by migrating under the anesthesia on the 15th day after inoculation of the plural cancer cell fluids of the animals treated as described above was carefully cut out from the surrounding tissues, separated only from the cancer tissues, and the tissue fluid was washed with filter paper and weighed immediately. It was. The weight of cancer tissue of each animal group was calculated by the average value and the error, and the difference between the average value was verified by t-test. The results are shown in Table 5 below.

As shown in Table 5, the administration of the tubercin-5 alone and the cytotoxic acid alone showed a similar decrease in cancer tissues, but when the two were administered in parallel, the cancer accumulation was significantly reduced. From this, it can be seen that the use of the tubercine-5 of the present invention in combination with an existing anticancer agent is effective in treating cancer.

[Effective Test-Clinical Test]

(1) Screening effects of tubercin-5 on various cancer patients

Toversin-5 prepared in Preparation Example was dissolved in 0.9% physiological saline in an amount of 2 μg / ml and injected into the subcutaneous of various cancer patients every day, and their clinical injuries and various laboratory findings were continuously observed.

As a result, Toversin-5 was effective from as early as 40 days to as late as several years. The effects of tubercin-5 on 100 patients (64 males; 36 females) who were followed up among various cancer patients treated between 1985 and 1995 are shown in Table 6 below.

As shown in Table 6, 19 out of 100 patients with various cancers showed some tumor deterioration. Symptoms improved in 53 and no response in 28.

(2) Lung Cancer: Life Extended

A 71-year-old male patient was diagnosed as inoperable with late epidermoid carcinoma and underwent chemotherapy, but continued to deteriorate various symptoms (cough, etc.) and tolerate more chemotherapy than 2.0 μg of tubercin-5. Treatment with subcutaneous injection once daily for 40 days improved symptoms. Stopping tubercin-5 injections as they determined that they were almost ill, and all previous symptoms recurred, and symptoms were improved by subcutaneous injection of 2.0 μg of tubercin-5 daily.

(3) lung cancer: pain is gone

A 64-year-old male patient who was diagnosed with late lung cancer (small cell type infiltrated to the head and ribs) and chemotherapy, but despite 4 months of treatment, various symptoms continue to worsen and chest pain disappears. Was not. Subcutaneous injection of 2.0 μg of tubercin-5 once daily resulted in loss of chest pain and improvement of symptoms, and survival for 7 months without any pain.

(4) stomach cancer: life prolonged

A 68-year-old male who was diagnosed with inoperable gastric cancer and temporarily treated with 5-FU and Chinese medicine, but the symptoms continued to worsen. It improved, pain was relieved, and tolerated with non-narcotic painkillers (Tylenol). It is endured in the progression of cancer until 10 months after the start of Tobus-5 administration.

(5) Gastric cancer: When cancerous tissue is reduced

A 72-year-old male patient was diagnosed with tubular adenocarcinoma, but had advanced age, advanced cancer tissue, severe lung tuberculosis, and no chemotherapy. As a result of administration of 2.0 μg of tubercin-5 once daily to subcutaneous injections for 40 days, all symptoms such as abdominal pain, impaired passage, and indigestion improved and became pre-onset. Stopping tubercin-5 injections after the cancer was judged to be better, and after 7 days, all previous symptoms recurred, and again, 2.0 μg of tubercin-5 was injected every day for 40 days to improve symptoms and discontinue treatment. . Compared with the initial diagnosis, the size of the cancer tissues was reduced, and no symptoms of cancer appeared after about 10 months of discontinuation.

(6) cervical cancer: cancerous tissue disappeared

A 58-year-old female patient was diagnosed with inoperable terminal cervical cancer, but the cancer was so widespread that she had ascites and no treatment was possible. 2.0 μg of tubercin-5 was administered once daily for six months, and then twice a week, after which all symptoms improved and returned to normal state (carcinoma withdrawal). Fifteen years later, he is alive with 2.0 μg of tubercin-5 administered twice a week subcutaneously.

In view of the above various test results, it can be seen that the tubercine-5 of the present invention can be used as an effective anticancer agent with little toxicity or side effects.

Claims (4)

Complex polysaccharides having a molecular weight of 7,000 or less, isolated from Mycobacterium tuberculosis, mannose, arabian, glucose and galactose as essential constituent monosaccharide components, and these monosaccharides glycosided in straight or branched chain. As a complex, a polysaccharide mixture having a sugar structure of the following structural formulas (1) to (10) upon acid hydrolysis: Wherein Man is a mannose residue, Ara is an arabinose residue, --- is a continued arabinose residue, and X is glucose or galactose. The mixture of claim 1 wherein the molecular weight is between 2,500 and 3,500. Mycobacterium tuberculosis was incubated, the aqueous suspension of the cultured cells was heated under pressure, and then filtered and separated, and the filtrate was added to sulfosalicylic acid to remove protein precipitates, and then phosphotungstic acid was again removed. Adding to remove the protein precipitate, and then precipitated with ethanol, and extracting the precipitate with ethanol and ether in a buffer solution, respectively. An anticancer agent composition comprising the complex polysaccharide mixture of claim 1.