JPH0366316B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides heat-denatured deoxyribonucleic acid having antitumor activity obtained from bacteria of the genus Mycobacterium.
Regarding MD-011. It is already well known that Mycobacterium tuberculosis has strong antitumor activity and adjuvant activity, and bacterial cell components that exhibit these activities have also been sought. As a result, cell wall-derived substances such as cell wall, cell wall skeleton, water-soluble adjuvant, and wax D have been reported as active fractions, and bacterial intracellular-derived substances such as arabinomannan and RNA have been reported as active fractions. The present inventors have been pursuing antitumor-active substances derived from Mycobacterium tuberculosis cells for some time, and in the process discovered for the first time that modified deoxyribonucleic acid of Mycobacterium tuberculosis has highly host-mediated antitumor activity. Specifically, the present inventors systematically investigated the antitumor activity of various components of Mycobacterium tuberculosis and found that the cell-free extract of Mycobacterium tuberculosis exhibits high antitumor activity. Since the antitumor activity is recovered from the cell-free extract as a precipitate, and the antitumor activity is increased rather than inactivated by heating this precipitate, the present inventors have determined that the antitumor activity is recovered from the cell-free extract of M. tuberculosis. After heating the antitumor active substance recovered by a nucleic acid flocculant, the antitumor active substance was further purified by precipitation, column chromatography, electrophoresis, etc., and the antitumor active substance was heat-denatured deoxyribonucleic acid. They discovered this substance, named it heat-denatured deoxyribonucleic acid MD-011, and completed the present invention. Hereinafter, this substance is often simply referred to as heat-denatured deoxyribonucleic acid, deoxyribonucleic acid, or this substance. Regarding the antitumor activity of nucleic acids derived from Mycobacterium tuberculosis, there has been a report on ribonucleic acids by Youmans et al. (Infection and Immunity 14 , 929, 1976).
However, there have been no reports regarding deoxyribonucleic acid. Reports on the antitumor effects of deoxyribonucleic acid derived from animal cells were made around 1965 (Science 149 , 997,
(1965), but their antitumor activity is low and species-specific, and their antitumor activity is often abolished by heating. Its mechanism of action was considered to be an action that causes tumor cell death by altering tumor cell metabolism or genetic control, that is, a direct damaging action on tumor cells.
(Cancer research 27 , 2342, 1967,
Proceedings of the National Academy of
Science 61 , 207, 1968) However, the present inventors investigated the antitumor spectrum of the substance of the present invention and found that, as shown later, line 10 Hepatoma, a guinea pig syngeneic tumor, and IMC carcinoma, a mouse syngeneic tumor, Lewis. Lang. The substance of the present invention exhibits high antitumor activity against tumor systems such as carcinoma (LLC) and EL-4 leukemia cells, regardless of the bacterial cells used as raw materials, and the toxicity and antigenicity of the substance of the present invention are extremely low. found. Furthermore, even when the substance of the present invention is cultured with tumor cells in vitro, almost no cell growth-inhibitory effect is observed, so it is thought that the substance of the present invention exhibits an antitumor effect through the immune response of the host. Furthermore, various studies were conducted on the immunological activity of the substance of the present invention, and it was found that the substance of the present invention exhibited a high level of natural killer cell activity enhancement effect in addition to killer T cell enhancing effect and macrophage activation effect. The activity-enhancing effect of natural killer cells by heat-denatured deoxyribonucleic acid was first discovered by the present inventors, and this activity-enhancing effect is at least one of the strong grounds for the antitumor action mechanism of the substance of the present invention. is considered to be presenting. In addition, the present inventors investigated the ability of the substance of the present invention to transform normal cells and tumor cells, and as shown later, under the conditions tested, the substance of the present invention did not transform normal and tumor cells. and that the substance of the present invention is derived from microorganisms that are taxonomically very distant from animal cells;
Since the deoxyribonucleic acid is denatured by heating, this possibility is considered to be extremely low. Based on the above various findings, the present inventors have completed the invention shown below. That is, the present invention provides heat-denatured deoxyribonucleic acid which has antitumor activity through heat denaturation.
The present invention relates to MD-011 and its salts, and more particularly to heat-denatured deoxyribonucleic acid MD-011 having antitumor activity obtained from Mycobacterium bacteria, and to its production method and antitumor agent. Furthermore, the present invention relates to a method for producing heat-denatured deoxyribonucleic acid MD-011 having antitumor activity, which comprises heating a nucleic acid fraction obtained by centrifuging a crushed Mycobacterium bacterium. Furthermore, the present invention provides a method for separating the nucleic acid fraction by adding a nucleic acid flocculant to a cell-free extract and separating it from the soluble fraction of the resulting precipitate. It concerns the manufacturing method. Furthermore, the present invention is characterized in that, as a method for separating nucleic acid fractions, a nucleic acid flocculant is added to a cell-free extract, the resulting precipitate is dialyzed against water or a salt solution, heated, and separated from the supernatant. This invention relates to a method for producing heat-denatured deoxyribonucleic acid MD-011, which has antitumor activity. Furthermore, the present invention provides deoxyribonucleic acid-containing substances about
This is a method for producing heat-denatured deoxyribonucleic acid MD-011 having antitumor activity, which is characterized by heating it to 80 to 120°C to make it have antitumor properties. Furthermore, the present invention provides heat-denatured deoxyribonucleic acid MD which has antitumor activity through heat denaturation.
It is an antitumor agent containing -011 as an active ingredient. The microorganism used in the present invention is a bacterium belonging to the genus Mycobacterium, and preferably Mycobacterium bovis
BCG, ATCC 19015), Mycobacterium tuberculosis H ₃₇ Ra
ATCC25177), Mycobacterium
smegmatis ATCC 607). A method for culturing microorganisms and a method for disrupting bacterial cells may be carried out according to conventional methods. For example, in the case of Mycobacterium bovis, a good culture can be obtained by statically culturing it for 1 to 8 weeks at a temperature of about 37°C using Sauton's medium or meat extract glycerin medium, which is then centrifuged or filtered. The bacterial cells can be obtained by The obtained bacterial cells are thoroughly mixed with water, preferably an appropriate buffer solution, and crushed with a DynoMill or French press while cooling on ice to obtain a suspension of crushed bacterial cells. This suspension is centrifuged to obtain a cell-free extract, but the centrifugation conditions are as follows:
Ordinary centrifugal force that can remove partially disrupted bacterial cells, cell wall fractions, etc. as sediment may be used, for example.
Centrifuge at 5000xg for 10 minutes or more to obtain the supernatant. In the present invention, the cell-free extract is a fraction obtained by removing as much of unbroken bacterial cells, partially crushed bacterial cells, cell wall fractions, etc. as possible from a suspension of crushed bacterial cells by centrifugation. . To obtain a nucleic acid fraction from a cell-free extract, there is a method to obtain a nucleic acid fraction by directly treating the cell-free extract with an organic solvent (for example, the Marmur method, the phenol method, etc., hereinafter referred to as the direct organic solvent method). An applicable method is to add a nucleic acid flocculant to a cell-free extract to obtain a precipitate containing a nucleic acid fraction. The direct organic solvent method is a method suitable for obtaining the substance of the present invention on a small scale (usually 1 g or less), and the resulting nucleic acid fraction (hereinafter referred to as D nucleic acid fraction) usually contains antigenic polysaccharides and Since it contains impurities such as proteins, the purified deoxyribonucleic acid fraction obtained after removing as many impurities as possible by density gradient centrifugation is heat-treated to obtain the substance of the present invention, or the D nucleic acid fraction is first heat-treated. The substance of the present invention is obtained by removing impurities by post-centrifugation, precipitation, or the like. The heat treatment conditions for the D nucleic acid fraction and the purified deoxyribonucleic acid fraction are the same as those for the preparation method described below, and will be described in detail later. The purpose of heating in the present invention is to adjust the molecular weight distribution of the present substance, increase impurity removal efficiency, facilitate formulation, increase solubility during use, have high antitumor activity, and reduce toxicity and side effects. The objective is to obtain the substance of the present invention with a low concentration. A method in which a nucleic acid flocculant is added to a cell-free extract to precipitate a nucleic acid fraction is a method suitable for obtaining the substance of the present invention on a large scale because the nucleic acid fraction can be concentrated. As the nucleic acid flocculant used in this method, any ordinary nucleic acid flocculant can be used, but a low molecular flocculant is preferable in the sense that the flocculant used in a later step can be easily removed. Suitable examples thereof include polyvalent metal cations such as calcium chloride, manganese chloride, magnesium chloride, and aluminum sulfate, water-soluble basic antibiotics such as streptomycin and kanamycin, or salts thereof. The amount of nucleic acid flocculant used can be selected appropriately depending on the type of nucleic acid flocculant, but for example, for polyvalent metal cations,
0.1-10%, preferably 0.1-3%, for antibiotics
It is appropriate to use 0.01 to 10%, preferably 0.1 to 1%, based on the extract. As for the operation, a nucleic acid flocculant is added to the cell-free extract, and the resulting precipitate is separated by centrifugation or other methods to obtain a suspension containing the nucleic acid fraction. Dialysis is suitable for removing the nucleic acid flocculant from this suspension. The aqueous solvent used in this case is preferably a buffer solution with an appropriate ionic strength and a pH around neutrality in order to increase the removal efficiency of the nucleic acid flocculant. For example, suitable ones include phosphate buffer containing 0.1-1M NaCl, 0.1-1M KCl citrate buffer, 0.1-1M
Sodium carbonate buffer containing NaCl, 0.1-1M
Examples include NaCl-containing Tris-HCl buffer. After the suspension containing the nucleic acid fraction is dialyzed against a salt-containing buffer, it is further dialyzed against water to remove salts, if necessary. The suspension containing the dialyzed nucleic acid fraction is hereinafter referred to as the N-1 fraction. The operation to obtain the N-1 fraction is
All operations were performed under cooling to avoid unnecessary degradation of the nucleic acid fraction.
Prevent degeneration. The cooling temperature is preferably 0 to 10°C. Two methods are possible to obtain the substance of the present invention from the N-1 fraction. One method is to separate the nucleic acid fraction from the N-1 fraction and then heat the obtained nucleic acid fraction to obtain the substance of the present invention, and the other method is to heat-treat the N-1 fraction. This is a method for separating the substance of the present invention. In the former method, first, after adjusting the content concentration of the N-1 fraction and the pH and salt concentration of the aqueous solvent, the N-1 fraction is
A soluble fraction containing deoxyribonucleic acid is separated from the fraction. The substance of the present invention can be obtained by separating a deoxyribonucleic acid fraction from this soluble fraction by a method such as precipitation, column chromatography, or electrophoresis, and subjecting it to heat treatment. The preferred concentration of the N-1 fraction when separating the soluble fraction from the N-1 fraction is 1 to 15
mg/ml. The pH of the aqueous solvent is preferably around neutral, and usually acids, bases, or buffers are used to
to 8. The ionic strength of the aqueous solvent affects the efficiency of removing precipitates during centrifugation, etc., and is usually preferably 0.1 or more, and if necessary, the ionic strength may be adjusted by adding salt, potassium chloride, or the like. The N-1 fraction adjusted in this way is usually 20,000xg
When centrifuged for 5 minutes or more, a clear soluble fraction containing a nucleic acid fraction is obtained as a supernatant. From this supernatant, the deoxyribonucleic acid fraction is separated by precipitation using streptomycin, manganese chloride, cetyltrimethylammonium bromide, etc., column chromatography using Sepharose, etc., or electrophoresis using vinyl chloride-vinyl acetate copolymer, etc. I can do it. All of the above operations are preferably carried out at 0 to 10°C. The substance of the present invention can be obtained by heat-treating the deoxyribonucleic acid fraction thus obtained, but since the heating conditions are the same as in the latter method, they will be described in detail later. In the latter method, the N-1 fraction is first heated, and the purpose of this heating operation is to appropriately denature the nucleic acid fraction and to denature impurities to facilitate the subsequent separation of the substance of the present invention. shall be. As will be shown later, the heating conditions are closely related to the antitumor activity of the substance of the present invention, and there is an appropriate range of conditions. Heating factors include solute concentration during heating,
The type of aqueous solvent, PH, ionic strength, heating temperature, and heating time are listed, and the appropriate condition range is as follows. A suitable concentration of solute is 1 to 15 mg/ml. The aqueous solvent is water or saline water such as common salt (ionic strength is
0.1 to 0.5) at 80℃ to 120℃ for 5 minutes to 120 minutes.If the aqueous solvent is a buffer solution, it will be greatly affected by the pH of the buffer solution; if the pH is around neutral, the heating temperature will be high; In acidic or alkaline conditions, it is preferable to set the heating temperature to a low temperature and shorten the heating time. As described above, by selecting appropriate heating conditions, it is possible to obtain a suspension containing heat-denatured deoxyribonucleic acid having high antitumor activity and low antigenicity, which is the objective of the present invention, as will be shown later. The heating conditions shown here are important factors in the method of preparing the substance of the present invention, and are applicable to any of the preparation methods shown herein. After heating the N-1 fraction in this manner, it is cooled and the precipitate is removed by centrifugation or filtration to obtain a clear solution containing the substance of the present invention. The substance of the present invention can be easily separated from this solution using conventional methods. Suitable examples thereof include precipitation methods using nucleic acid flocculants, fractionation methods using organic solvents, column chromatography methods, electrophoresis methods, and the like. The substance of the present invention obtained by these methods can be used as it is as a raw material for pharmaceutical preparations, or it can be freeze-dried into a dry powder, and if necessary, a solution containing the substance of the present invention can be made into a dry powder. The substance of the present invention is purified by deproteinization treatment and the like. The substance of the present invention is deoxyribonucleic acid denatured by heating, as is clear from its physicochemical properties, and its content is 60% in the lyophilized product of the substance of the present invention.
~100%. Although this content varies depending on the preparation method and analysis method, heat-denatured deoxyribonucleic acid is the main component of the substance of the present invention obtained by any of the preparation methods shown in this specification. The molecular weight of deoxyribonucleic acid in the substance of the present invention ranges from about 30,000 to 1,000,000, and the guanine and cytosine (GC) content is about 60%. The transition temperature Tm of the substance of the present invention does not have a clear value, which means that the substance of the present invention is a modified deoxyribonucleic acid. Some of the substances obtained by the method shown in this specification contain ribonucleic acids, proteins, sugars, etc. in addition to heat-denatured deoxyribonucleic acids, but these can only be considered contaminants, and their respective contents are contains less than 30% ribonucleic acid, less than 10% protein, and less than 10% neutral sugar.
Less than 10%. These can be removed by further purification. Next, C-B obtained by purification in Example 12
We investigated the physical and chemical properties of -16 and present the results. The purified products of C-B-14 obtained in Example 10 and C-B-15 obtained in Example 11 also exhibit similar physical and chemical properties. This substance, heat-denatured deoxyribonucleic acid MD-011
Physical and chemical properties (by Na salt): (1) Elemental analysis (%): C: 25.57-29.75 H: 3.80
~4.75 N: 12.67~13.47 P: 7.30~7.91 Na:
8.12-11.59 (2) Molecular weight: 30,000-1,000,000 The molecular weight distribution map is as shown in Figure 1. (3) Melting point: Does not show a clear melting point. (4) Ultraviolet absorption spectrum: As shown in Figure 2. (5) Infrared absorption spectrum: As shown in Figure 3. (6) Solubility in solvents: Soluble in water, insoluble in ethanol, methanol, ether, and acetone. (7) Color reaction A Orcinol reaction: Negative for RNA fractions fractionated by STS method. B Diphenylamine reaction: Positive for DNA fraction fractionated by STS method. C. Ninhydrin reaction: Negative at a concentration of 10 μg/ml of this substance. D Anthrone reaction: Negative at a concentration of 10 μg/ml of this substance. (8) Distinction between basic, acidic, and neutral: The pH of an aqueous solution of this substance is 6.5 to 7.5. (9) Color of substance: White powder (10) Properties: Antitumor properties are imparted through heat treatment. (11) Base composition (%): Guanine: 30.4 Adenine: 16.9 Cytosine: 30.8 Thymine: 21.9 (method is based on chemical analysis) (12) Enzyme treatment: When this substance is treated with deoxyribonuclease I (DNase I), it exhibits antitumor properties. However, treatment with ribonuclease T ₂ (RNaseT ₂ ) does not change the antitumor activity. (13) Transition temperature (Tm): A clear transition temperature Tm cannot be determined from the measured melting curve. The fact that the main body of the antitumor activity exhibited by the substance of the present invention is heat-denatured deoxyribonucleic acid means that when the substance of the present invention is treated with deoxyribonuclease (DNaseI), the antitumor activity disappears; This is clear from the fact that the antitumor activity does not change even after treatment with (RNaseT ₂ ). When the substance of the present invention is used as an antitumor agent, it is preferably used in the form of an injection. The substance of the present invention is
It can be applied alone or with commonly used additives and excipients as a liquid preparation, or a co-dissolved freeze-dried preparation. The substance of the present invention can also be applied as an oil-in-water or water-in-oil emulsion. The amount and route of administration of the substance of the present invention are selected as appropriate, but the amount used is preferably 0.01 to 100 mg per kg of body weight, and administration routes include intradermal, subcutaneous, intravenous, intraperitoneal, and intratumoral administration. The substance of the present invention exhibits high antitumor activity against various tumor types in guinea pigs and mice. For example, IMC carcinoma, a syngeneic mouse tumor, Lewis.
Lang. For carcinoma (LLC), EL-4 leukemia cells, P-815 mastocytoma, etc., the substance of the present invention is dissolved in physiological saline and brought into contact with the tumor cells, and then implanted into the animal body. In terms of suppression of engraftment (suppression activity) or antitumor activity (regression activity) when administered into tumor tissue that has engrafted within an animal body, it showed an antitumor activity equal to or higher than that of the bacterial cells that are the raw material. . Furthermore, for line 10, which is a syngeneic tumor in guinea pigs, by intratumoral administration of the substance of the present invention in the form of water-in-oil droplets, the substance of the present invention not only suppresses the growth of the primary tumor, but also suppresses the growth of the tumor in the regional lymph nodes. Metastasis was also suppressed. Furthermore, the substance of the present invention can also be administered by dissolving it in physiological saline and administering it to a site different from the tumor tissue.
It showed antitumor activity against IMC carcinoma. The acute toxicity of the substance of the present invention is extremely low since the 50% lethal dose LD ₅₀ value per kg of body weight when administered intravenously to mice is 1 g or more. Regarding the antigenicity of the substance of the present invention, an anaphylaxis test and a delayed allergy test using guinea pigs revealed that the substance of the present invention is extremely safe. Other pyrogenic, painful, and inflammatory properties of the substance of the present invention,
Granulation-forming properties are also extremely low compared to the raw bacterial cells.
It has been found through various tests that this level is not a problem for ordinary pharmaceutical applications. When the cell growth inhibitory effect of the substance of the present invention on various tumor cells was investigated, the substance of the present invention showed almost no cell growth inhibitory effect. From this, it is thought that the substance of the present invention exhibits an antitumor effect through the immune response of the host, and therefore various immunological activities of the substance of the present invention were investigated. As a result, the substance of the present invention showed an effect of enhancing killer T cells in mice, an effect of activating macrophages, and an effect of enhancing natural killer cell activity. Furthermore, the present inventors investigated the ability of the present substance to transform normal and tumor cells, taking into account that although the present substance has been denatured by heating, it is deoxyribonucleic acid, which is a genetic factor. From the results shown later, it is considered that the possibility of transformation by the substance of the present invention is extremely low. Based on the above various findings, the substance of the present invention is considered to be extremely useful as an antitumor agent. EXAMPLES The method for producing the substance of the present invention will be illustrated below by Examples, and the usefulness of the substance of the present invention as an antitumor agent will be illustrated by Test Examples. Example 1 Preparation of the substance of the present invention by the Marmer method: Glycerin 50ml Citric acid 2g Meat extract 15g Ammonium citrate 0.05g K ₂ HPO ₄ 1g MgSO ₄ 7H ₂ O 1g Add water to 1 and adjust the pH to 7.0 to 7.2. Adjust. Mycobacterium bovis BCG, ATCC19015 was statically cultured in a meat extract glycerin medium with the above composition at 37℃ for 3 weeks, and the culture solution was centrifuged. 500 g of wet bacterial cells were suspended in 7 times the amount of 10 mM phosphate buffer. After making it cloudy, crush it with a Dyno Mill under ice cooling, and crush it at 20000xg for 20 minutes.
Centrifugation was performed for a minute to obtain a cell-free extract. Marmur, Journal of Molecular
A crude deoxyribonucleic acid fraction was obtained by the method (Biology 3 , 208, 1961). This fraction was added to Seph Arrows 6B.
(manufactured by Pharmacia Fine Chemicals) A deoxyribonucleic acid fraction obtained as an excluded volume fraction was concentrated and purified by density gradient centrifugation using cesium chloride to obtain a purified deoxyribonucleic acid fraction. After dialyzing this fraction the final concentration is 0.9
%, and heated at 100°C for 60 minutes.
After cooling, the mixture was dialyzed against distilled water and lyophilized to obtain 8.4 mg of the substance of the present invention. Hereinafter, this product will be referred to as BM-1. Deoxyribonucleic acid content (purity) in B-M-1
is hydrolyzed with 5% perchloric acid followed by diphenylamine method (Biochemical Journal 62 , 315, 1956).
It was 93.3% when quantified by . Example 2 Production of the substance of the present invention from the soluble fraction of a suspension obtained using streptomycin: Mycobacterium bovis BCG, ATCC19015 was statically cultured at 37°C for 3 weeks in the meat extract glycerin medium of Example 1, and the culture solution Wet bacterial cells obtained by centrifuging
Suspend 3.3Kg in 7 times the volume of 10mM phosphate buffer (PH7.0), crush with a Dynomill under ice cooling, and shake at 20,000xg.
Centrifugation was performed for 20 minutes to obtain 21 cell-free extracts. After adding 63 g of streptomycin sulfate to this extract and stirring thoroughly, it was allowed to stand at 4°C overnight, and the resulting precipitate was collected by centrifugation and diluted with 10 mM phosphate buffer (PH7.0) containing 0.5 M NaCl. suspended in. This suspension was packed in cellophane tubes and dialyzed against the same buffer, and then dialyzed against distilled water to obtain suspension 8 (hereinafter referred to as this suspension) containing the nucleic acid fraction.
NB-1) was obtained. The solid concentration of this suspension was 10 mg/ml, and the yield was 12% based on dry bacterial cells. Next, 200 ml of 8.1% NaCl solution was added to NB-1200 ml, stirred, and centrifuged at 20,000 xg for 30 minutes to obtain a supernatant. After adding and dissolving common salt to the supernatant to a final concentration of 0.4M, add cetyltrimethylammonium bromide (hereinafter referred to as CTAB).
manufactured by Tokyo Kasei Co., Ltd.) with a final concentration of 0.2% (w/v)
The mixture was stirred thoroughly and left at room temperature for 30 minutes. The generated precipitate was collected by centrifugation, and 1M
Equal volume of chloroform after dissolving in 60 ml of NaCl solution.
A mixture of isoamyl alcohol (24:1) was added, shaken, and centrifuged to obtain an aqueous layer. After repeating this operation two more times, three times the amount of
99.5% ethanol was added, stirred, and left at 4°C overnight. The generated precipitate was centrifuged, dissolved in distilled water, and then dialyzed against distilled water to obtain a purified nucleic acid solution. Add salt to this solution to make the salt concentration 0.9
%, heated at 100°C for 60 minutes, dialyzed against distilled water, and lyophilized to obtain 93 mg of the substance of the present invention.
Hereinafter, this product will be referred to as CB-1. The deoxyribonucleic acid content of C-B-1 was analyzed in the same manner as in Example 1 and was found to be 95.2%. Example 3 Production of the substance of the present invention from the soluble fraction derived from Bacterium smegmatis: Mycobacterium smegmatis, ATCC607, was statically cultured in the meat extract glycerin medium of Example 1 at 37°C for 4 weeks, and the culture solution was centrifuged to obtain the substance obtained. wet bacterial cells
500 g was treated in the same manner as in Example 2 to obtain suspension 1 containing the nucleic acid fraction, and water was added to adjust the solid concentration of this suspension to 10 mg/ml. The yield at this time was 15% based on dry bacterial cells. This suspension is shown below.
It is called NS-1. NS-1 was replaced with NB-1 of Example 2.
110 mg of the substance of the present invention was obtained by processing in the same manner as described below.
Hereinafter, this product will be referred to as C-S-1. Example 4 Production of the substance of the present invention from the soluble fraction by column chromatography using Sepharose: After adjusting the solid concentration of NB-1 and NS-1 obtained in Examples 2 and 3 to 5 mg/ml, Each soluble fraction was obtained by centrifugation for hours. 50 ml of each of these soluble fractions was added to a Sepharose Cl solution with a diameter of 5 cm and a length of 90 cm.
-6B (manufactured by Pharmacia Fine Chemicals) column to obtain a fraction containing deoxyribonucleic acid as an excluded volume fraction. These fractions were dialyzed against water, heated at 100°C for 60 minutes, dialyzed, and freeze-dried.
190 mg and 210 mg of each of the substances of the present invention were obtained. Hereinafter, these will be referred to as C-B-2 and C-S-2. For example, the deoxyribonucleic acid content of C-B-2 is
When analyzed by the method shown in Example 1, it is 85.9%. Example 5 Production of heated supernatant from nucleic acid-containing suspension: NB-1 obtained in Example 2 was heated under the conditions shown in the table below, and then centrifuged at 20,000xg for 20 minutes to obtain a supernatant. This supernatant was dialyzed against distilled water and then freeze-dried to obtain an intermediate containing the substance of the present invention.

[Table] *Example 6 in which no clear supernatant was obtained Production of the substance of the present invention from heated supernatant using various precipitants: NB-1,1 obtained in Example 2 and 1.8% saline solution After stirring thoroughly, the mixture was heated at 100° C. for 60 minutes, and centrifuged at 10,000×g for 20 minutes to obtain a supernatant (hereinafter this supernatant will be referred to as S-3). Various precipitants shown in Table 2 were added to this supernatant, the mixture was stirred, and the mixture was allowed to stand at room temperature for 30 minutes, and the resulting precipitate was collected by centrifugation. If necessary, this precipitate was dissolved in distilled water by adding a small amount of alkali, dialyzed against distilled water, and freeze-dried as it was, or treated with an organic solvent in the same manner as in Example 2, followed by dialysis and freeze-drying.

[Table] Example 7 Production of the substance of the present invention from heated supernatant by electrophoresis method: S-3 obtained in Example 6 was dialyzed against distilled water and lyophilized, and 44 mg was added to 0.05M borate buffer. (PH7.4) and electrophoresed while cooling using vinyl chloride-vinyl acetate copolymer (manufactured by BDH Chemicals) as a carrier. After electrophoresis, the copolymer was cut out as a 1 cm wide block and eluted with 0.1M saline.
Fractions exhibiting ultraviolet absorption at 260 nm were collected, dialyzed against distilled water, and lyophilized to obtain 19 mg of the substance of the present invention. Hereinafter, this product will be referred to as CB-12. Example 8 Production of the substance of the present invention from heated supernatant by ion-exchange resin method: S-3 obtained in Example 6 was dialyzed against distilled water, and 20 ml of it was added to lysine sepharose with a diameter of 2.2 cm and a length of 5.2 cm. (manufactured by Pharmacia Fine Chemicals) After loading the column, the column was thoroughly washed with 0.15M Tris-HCl buffer (PH7.6) containing 0.1M NaCl, and then
The fraction obtained by elution with the same buffer containing 0.15M NaCl was dialyzed against distilled water and lyophilized to obtain 9 mg of the substance of the present invention. Example 9 Production of the substance of the present invention using CTAB from heated supernatant derived from BCG bacteria: An equal amount of 1.8% saline was added to NB-1,1 obtained in Example 2, stirred, and then incubated at 100°C for 60 minutes. Heated for minutes.
After cooling, this suspension was centrifuged at 10,000xg for 20 minutes. NaCl was added to the resulting supernatant to give a final concentration of 0.4M, and after stirring, CTAB was added at 0.2% (w/v).
Stir thoroughly, then stir at room temperature for 30 minutes.
It was left standing for a minute. The generated precipitate was collected by centrifugation and dissolved in 400 ml of 1M saline. This solution was treated with an organic solvent and subjected to ethanol precipitation in the same manner as in Example 2, and then dialyzed against distilled water and freeze-dried to obtain 1.04 g of the substance of the present invention. Below, this is C-B-
It is called 13. The deoxyribonucleic acid content of C-B-13 is 86.6% when analyzed by the same method as in Example 1. Example 10 Production of the substance of the present invention using CTAB from the heated supernatant derived from P. nigra: 1.8% in the same volume as 1500 ml of NS-obtained in Example 3
After adding saline and stirring, the mixture was treated in the same manner as in Example 9 to obtain 0.68 g of the substance of the present invention. Hereinafter, this product will be referred to as CB-14. Example 11 Production of the substance of the present invention from heated supernatant derived from humanoid bacteria: Glycerin 50ml Citric acid 2g L-aspartic acid 2g Iron ammonium citrate 0.05g K ₂ HPO ₄ 1g MgSO ₄・7H ₂ O 1g ZnSO ₄・7H ₂ O 0.01g CaCl ₂・2H ₂ O 0.5mg CuSO ₄・5H ₂ O 0.1mg Add water to 1 and adjust the pH to 7.0 to 7.2. Mycobacterium tuberculosis H ₃₇ Ra,
ATCC25177 in Sorton's modified medium with the above composition.
A final concentration of 5% was added to cultures grown for 4 weeks at °C.
Phenol was added thereto and stirred, left to stand overnight, and then centrifuged to collect wet bacterial cells and washed with physiological saline. 100 g of this wet bacterial body was treated in the same manner as in Example 2 up to obtaining NB-1 to obtain a suspension containing the nucleic acid fraction.
Obtained 200ml. The solid concentration of this suspension was set to 10 mg/
After adjusting the volume to ml, the same treatment as in Example 9 was carried out to obtain 310 mg of the substance of the present invention. Hereinafter, this product will be referred to as CB-15. The deoxyribonucleic acid content of C-B-15 was 80.9% when analyzed by the method of Example 1. Example 12 Production of the substance of the present invention from heated supernatant derived from BCG bacteria using CTAB and ribonuclease: 94 mg of C-B-13 obtained in Example 9 was dissolved in 10 ml of 0.05 M acetate buffer (PH4.5). After that, add the same buffer 2
Ribonuclease T ₂ (Sankyo co.
200 U (manufactured by Manufacturer, Inc.) was added thereto, and the mixture was allowed to react at 37°C for 22 hours.
An equal amount of a mixture of chloroform and isoamyl alcohol (24:1) was added to the reaction solution, which was then shaken and centrifuged to obtain an aqueous layer. The entire aqueous layer was pre-washed with 0.05M ammonium bicarbonate solution. Diameter: 2.5cm, length: 90mm
cm Sephadex G100 (Pharmacia Fine
Chemicals) column and eluted with the same solution, a fraction containing deoxyribonucleic acid was obtained as the first eluted fraction. This fraction was dialyzed against water, neutralized with sodium hydroxide, and then lyophilized to obtain 67 mg of the substance of the present invention. Below, this thing is C-
It is called B-16. The deoxyribonucleic acid content of C-B-16 is as shown in Example 1.
When analyzed using the same method as above, it is 96.5%. Example 13 Liquid: Dissolve 10 mg of C-B-13 in 10 ml of PBS minus solution (manufactured by Eiken Kagaku Co., Ltd.) and add Nyukrypore N020.
(manufactured by Nuclepore). The obtained liquid was aseptically dispensed into vials in 1.5 ml portions to obtain a liquid preparation of the substance of the present invention. Example 14 Freeze-dried preparation: 10 mg of C-B-13 was dissolved in 10 ml of distilled water for injection, and then 500 mg of maltose was added and dissolved, followed by sterilization using Nuclepore N020. The obtained liquid was aseptically dispensed into vials in 1 ml portions and lyophilized to obtain a lyophilized preparation of the substance of the present invention. Example 15 Emulsion: 4 mg of C-B-13 was dissolved in 0.5 ml of physiological saline, and then Drakeol 6-VR (Drakeol6-VR,
(manufactured by Pennsylvania Refining Company) and Arlacel A (manufactured by Atlas Chemical Industries)
A water-in-oil emulsion was obtained by adding 0.5 ml of a 8.5:1.5 mixture and using a connected injection needle. Test Example 1 Antitumor effect on mouse IMC carcinoma: The antitumor effect of the substance of the present invention on mouse IMC carcinoma was investigated. The test system is as follows. 5 × 10 ⁵ IMC carcinoma cells were intradermally transplanted into CDF ₁ female mice, and 0.1 ml of the preparation prepared by the method of Example 12 was administered every other day six times in total from the first day after transplantation. , administered intratumorally. 35 after transplant
The tumor was excised on the same day, and its weight was measured. The results are shown in Table 3.

【table】

[Table] Student's t-test and Fischer's test were used to test significant differences in the frequency of occurrence of average tumor weight and number of cured cases, including the tests described below. Each was used.
T/C is the percentage ratio of the average tumor weight of the treated group to the average tumor weight of the control group. BCG is
BCG vaccine (manufactured by Nippon BCG Company) was used. Physiological saline was used as a control. Test Example 2 Antitumor activity of heated supernatant: The antitumor activity of supernatants obtained by heating suspensions containing nucleic acid fractions under various conditions was investigated. The test system is as follows. After mixing 0.5 ml of Hank's solution containing 1×10 ⁷ IMC carcinoma cells/ml and 0.5 ml of physiological saline in which the sample was dissolved at a concentration of 2 mg/ml, 0.1 ml of the mixture was added to the skin of a CDF ₁ female mouse. transplanted inside. On the 25th day after transplantation, the tumor was removed and its weight was measured. As a control, the same treatment was performed using only physiological saline. The test was also conducted using 10 mice per group. The results are shown in Table 4.

【table】

[Table] Test Example 3 Antitumor effect of the substance of the present invention obtained from heated supernatant using various precipitants: Antitumor effect of the substance of the present invention obtained from heated supernatant using various precipitants against IMC carcinoma I looked into it. The test was conducted in the same manner as Test Example 2, and the results shown in Table 5 were obtained.

【table】

[Table] Test example 4 Mouse, Lewis. Lang. Antitumor effect on carcinoma: Mouse of the substance of the present invention, Lewis. Lang. The antitumor effect on carcinoma was investigated. The test system is as follows. 5 × 10 ⁵ Lewis cells intradermally in a C57BL/6 female mouse. Lang. Carcinoma (Lewis Lung)
Carcinoma) cells were transplanted, and after transplantation 1, 4, 8,
On the 11th, 15th, and 18th days, 0.1 ml of the preparation prepared by the method of Example 13 was administered intratumorally at each time. On the 26th day after transplantation, the tumor was removed, its weight was measured, and the number of metastatic foci in the lungs was also examined. The results are shown in Table 6. Physiological saline was used as a control, and BCG was
BCG vaccine was used.

[Table] Test Example 5 Antitumor effect on mouse EL-4: The antitumor effect of the substance of the present invention on mouse EL-4 leukemia cells was investigated. The test system is as follows. Mix 0.5 ml of Hank's solution containing 1×10 ⁶ cells/ml of EL-4 leukemia cells and 0.5 ml of physiological saline in which the sample was dissolved at a concentration of 2 mg/ml, then add 0.1 ml of the mixture.
was intradermally transplanted into C57BL/6 female mice, and the number of surviving mice 37 days after transplantation was determined. As a control, the same treatment was performed using only physiological saline.
BCG used BCG vaccine. The results are shown in Table 7.

【table】

[Table] Test Example 6 Antitumor effect of strain 2 guinea pig on line 10 hepatoma: Strain 2 guinea pig line of the substance of the present invention
The antitumor effect against 10hepatoma was investigated. Patoma tumor cells were transplanted intradermally into strain 2 guinea pigs into line 10 of 1×10 ⁶ cells, and 0.1 ml of the preparation prepared by the method shown in Example 15 was added to the tumor on the 7th day after transplantation. Administered intravenously. On the 80th day after transplantation, we examined the number of surviving cases, the number of cured cases, and the presence or absence of metastasis to regional lymph nodes. As a control, a sample prepared in the same manner using physiological saline was used. The results are shown in Table 8.

[Table] Test Example 7 Antitumor activity of a substance treated with a nuclease: The substance of the present invention was treated with a nuclease to examine the substance of the antitumor activity of the substance of the present invention. The test method is as follows. C-B-13 is deoxyribonuclease
After treatment with DNaseI (manufactured by Sigma Chemicals) or ribonucleic acid degrading enzyme RNaseT ₂ (manufactured by Sigma Chemicals), the resulting decomposition product was treated with chloroform to remove DNaseI or RNaseT ₂ , and then subjected to a Sephadex G10 (manufactured by Farmacia Fine Chemicals) column. The product was desalted and freeze-dried to obtain a nuclease-treated substance of the present invention. The antitumor activity of the obtained treated product was tested in the same manner as Test Example 1, and the results shown in Table 9 were obtained.

T/C is the percentage ratio of the average tumor weight of the treated group to the average tumor weight of the control group. Physiological saline was used as a control, and BCG was
A vaccine was used. Test Example 8 Activity-enhancing effect on natural killer (NK) cells: The activity-enhancing effect of the substance of the present invention on NK cells was investigated. The test method is as follows. Physiological saline, a physiological saline solution of C-B-13 or C-B-16 (1 mg/ml) was administered to 8-week-old C ₅₇ BL/6.
0.3 ml per mouse was intraperitoneally administered to female mice, and peritoneal exudate cells were collected 48 hours later. The number of cells in the collected solution was adjusted to 2 x ¹⁰⁶ cells/ml using RPMI1640 medium supplemented with 10% fetal bovine serum, then transferred to a plastic shear dish, incubated at 37°C for 90 minutes in an atmosphere of 5% carbon dioxide, and then placed in a shear dish. Adherent and non-adherent cells were obtained. 5 x ¹⁰ cells each and ⁵¹ Cr
Mouse leukemia cells YAC-1 labeled with 1×10 ⁴
The cells were cultured for 4 hours at 37°C under a 5% carbon dioxide atmosphere using RPMI1640 medium supplemented with 10% fetal bovine serum. The radioactivity of ⁵¹ Cr released in the culture supernatant was measured using an autogamma counter (manufactured by Packard), and the cytotoxic effect of the peritoneal exudate cells on YAC-1 cells was investigated.

[Table] Test Example 9 Direct inhibition of cell proliferation Effect of the present substance on YAC-1 mouse leukemia cells
The direct inhibitory effect on cell proliferation of FM3A mouse breast cancer cells was investigated. The test system is as follows. 8.4×10 ⁴ YAC-1 cells and 15.4×10 ⁴
Each FM3A cell was supplemented with 10% fetal bovine serum.
The cells were suspended in RPMI1640 medium and cultured for 24 hours at 37° C. under a 5% carbon dioxide atmosphere. After that, CB-13 was added to a final concentration of 1 mg/ml, and the culture was further continued under the same conditions. The number of living cells was measured by trypan blue staining every 24 hours after the start of culture. The results are shown in Table 11.

[Table] Test example 10 Acute toxicity: 10 mice per group, 5-week-old ddY male mice (average body weight
23g), CB-13 was dissolved in physiological saline and administered intravenously at a dose of 1g per kg of body weight. The substance of the present invention did not inhibit body weight gain during the observation period of one week after administration, and there were no cases of death. From this result, the 50% lethal dose LD ₅₀ for intravenous administration of the substance of the present invention is 1
It is considered to be more than g/Kg. Test Example 11 Antigenicity: C-B-13 dissolved in physiological saline was administered to a group of 6 Hartley female guinea pigs (average weight 350 g).
Each animal was sensitized by intradermally administering 1 mg per animal three times a week for a total of six times. Two weeks after the last sensitization date C-
Dissolve B-13 in physiological saline and give 10mg per kg of body weight.
Alternatively, 2 mg was administered intravenously. The antigenicity of the substance of the present invention was investigated by observing the behavior of guinea pigs before and after the challenge, and it was found that the substance of the present invention did not induce anaphylaxis at all at the above dose. Test Example 12 Effect on the growth form of normal mouse cells: The effect of the substance of the present invention on the growth form of cultured normal cells was investigated using second-generation cultured cells derived from mouse fetal skin and muscle (fibroblasts). Ta. Monolayer cultures of primary cells from the skin and muscles obtained from 6 fetuses (19 days old) from a litter of SHN female mice mated at 10 weeks of age and pregnant were subjected to trypsinization to obtain fetal bovine serum 10%. % addition
The number of viable cells was adjusted to 6.2×10 ⁴ per ml of RPMI1640 medium (hereinafter abbreviated as RPMI medium), and second culture was performed at 37° C. in a 5% carbon dioxide atmosphere. After 5 hours of culture, fresh RPMI medium (control group) and C-B-13 were added.
RPMI at concentrations of 500μg/ml and 50μg/ml
Each medium was replaced with a medium (test group) and culture was continued for 15 days. During this time, the medium was exchanged three times, and the growth behavior of the cells and the presence or absence of morphological changes were observed under a microscope. As a result, the growth behavior and morphology of cells in the test group were the same as in the control group, and no abnormal cells were observed. Test Example 13 Effect of enhancing tumor cell xenobiotic conversion: The presence or absence of the substance of the present invention in enhancing tumor cell xenobiotic conversion was examined. That is, 1×10 ⁵ cells/ml of YAC-1 leukemia cells or P815 mastocytoma cells were cultured for 48 hours in the presence of the substance of the present invention (final concentration 1 mg/ml), and after thorough washing, the former were cultured as NK cells. activity, the latter being compared with the respective untreated cells as a target of macroage activity. The medium is RPMI1640 medium, culture conditions are 37℃, 5%
The experiment was carried out under a carbon dioxide atmosphere. As a result, no difference was observed in NK cell activity and macrophage activity between the groups treated with the substance of the present invention and those not treated. From this, it is considered that the substance of the present invention does not enhance target tumor cell foreignization.

[Brief explanation of drawings]

FIG. 1 shows the elution diagram of the substance of the present invention by Sepharose column chromatography, FIG. 2 shows the ultraviolet absorption spectrum of the same substance, and FIG. 3 shows the infrared absorption spectrum of the same substance.

Claims (1)

[Scope of Claims] 1. Heat-denatured deoxyribonucleic acid MD-011 and its salts, which have antitumor activity through heat denaturation and exhibit the following physicochemical properties (based on Na salt). (1) Elemental analysis (%): C: 25.57-29.75 H: 3.80-4.75 N: 12.67-13.47 P: 7.30-7.91 Na: 8.12-11.59 (2) Molecular weight: Using protein as a marker and Sepharose CL-6B According to the gel filtration method, it is distributed between 30,000 and 1,000,000, with a peak around 250,000. (3) Melting point: Does not show a clear melting point. (4) Ultraviolet absorption spectrum: 260nm for neutral aqueous solution
It has a maximum absorption wavelength near 230 nm and a minimum absorption wavelength near 230 nm. (5) Infrared absorption spectrum: around 780cm ^-1 , 1090
It has characteristic absorption bands of nucleic acids around cm ^-1 , 1230 cm ^-1 and 1700 cm ^-1 . (6) Solubility in solvents: Soluble in water, insoluble in ethanol, methanol, ether, and acetone. (7) Color reaction A Orcinol reaction: Negative for RNA fractions fractionated by STS method. B Diphenylamine reaction: Positive for DNA fraction fractionated by STS method. C Ninhydrin reaction: Negative at a concentration of 10μg/ml of this substance. D Anthrone reaction: Negative at a concentration of 10μg/ml of this substance. (8) Basic, acidic, and neutral: The pH of an aqueous solution of this substance is 6.5 to 7.5. (9) Color of substance: White powder (10) Properties: Antitumor properties are imparted through heat treatment. (11) Base composition (%): Guanidine: 30.4 Adenine: 16.9 Cytosine: 30.8 Thymine: 21.9 (method is based on chemical analysis) (12) Enzyme treatment: This substance has antitumor properties when treated with deoxyribonuclease I (DNase I). However, ribonuclease T ₂ (RNaseT ₂ )
The antitumor properties do not change with treatment. (13) Transition temperature (Tm): A clear transition temperature Tm cannot be determined from the measured melting curve. 2. Heat-denatured deoxyribonucleic acid MD-011 according to claim 1, which is obtained from a Mycobacterium genus. 3 These operations are carried out using bacteria of the genus Mycobacterium, which can produce MD-011 by heating the nucleic acid fraction obtained from the cell-free extract obtained by centrifuging the crushed product. A method for producing heat-denatured deoxyribonucleic acid MD-011 having antitumor activity. 4. The production method according to claim 3, wherein the nucleic acid fraction is separated by treating the cell-free extract with an organic solvent. 5. Claims characterized in that as a method for separating nucleic acid fractions, a nucleic acid flocculant is added to a cell-free extract, the resulting precipitate is dialyzed against water or a salt solution, and then separated from the soluble fraction. The manufacturing method described in paragraph 3. 6. Add a nucleic acid flocculant to the cell-free extract obtained by centrifuging the crushed product, dialyze the resulting precipitate against water or a salt solution, denature it by heating, and separate it from the supernatant to obtain MD. 1. A method for producing heat-denatured deoxyribonucleic acid MD-011 having antitumor activity, which comprises using Mycobacterium bacteria capable of producing MD-011 and carrying out these operations. 7. The manufacturing method according to any one of claims 3 to 6, wherein the heating temperature is about 80 to 120°C. 8. An anti-tumor agent containing heat-denatured deoxyribonucleic acid MD-011 as an active ingredient, which has anti-tumor activity through heat denaturation. 9. The antitumor agent according to claim 8, wherein the heat-denatured deoxyribonucleic acid MD-011 is obtained from Mycobacterium.