JPH0365353B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to heat-denatured deoxyribonucleic acid RD-01 having antitumor activity obtained from the cells of a microorganism belonging to the genus Propionibacterium. It has been known for a long time that bacteria belonging to the genus Propionibacterium have physiological activities such as antitumor effects and adjuvant effects. Attempts are being made to clinically apply bacterial cells. However, the cells of this microorganism have various side effects, and in order to alleviate these side effects, attempts have been made mainly to separate the active substance from the cell wall. Examples of active substances derived from cell walls include the above-mentioned cell wall skeleton and polymeric mucopeptides prepared from cell walls, but those with high antitumor activity also have strong side effects, and those with weak side effects have antitumor effects. It has the disadvantage that the activity is not necessarily high. The present inventors previously investigated various antitumor active substances obtained from Mycobacterium tuberculosis, and found that heat-denatured deoxyribonucleic acid isolated from a cell-free extract obtained by crushing Mycobacterium tuberculosis has a strong host-mediated anti-tumor effect. Patented for the first time discovering that it has tumor activity and extremely low toxicity and antigenicity (Japanese Unexamined Patent Application Publication No. 139096/1983)
has been applied for. Furthermore, the present inventors conducted a diligent search for antitumor active substances based on their experience with Mycobacterium tuberculosis in order to find novel antitumor active substances in bacterial cell extracts of bacteria belonging to the genus Propionibacterium. As a result, heat-denatured deoxyribonucleic acid prepared from Propionibacterium bacteria using a method similar to that used for Mycobacterium tuberculosis showed high antitumor activity, extremely low toxicity, and almost no pyrogenicity or antigenicity. After confirming this, the present invention was named heat-denatured deoxyribonucleic acid RD-01, and the present invention was completed. Hereinafter, the substance of the present invention is often simply referred to as heat-denatured deoxyribonucleic acid, modified deoxyribonucleic acid, deoxyribonucleic acid, or the present substance. The deoxyribonucleic acid obtained by the present invention has strong host-mediated antitumor activity similar to the substance derived from Mycobacterium tuberculosis, and its action is similar to that of the substance derived from animal cells. Antitumor effect based on direct damage to cells (Science, 149 , 997,
1965, Cancer Research 27 , 2342, 1967,
Proceedings of the National Academy of
Science 61 , 207, 1968). The substance of the present invention, its production method, and its strong host-mediated antitumor activity were first revealed by the present inventors. That is, the present invention provides heat-denatured deoxyribonucleic acid which has antitumor activity through heat denaturation.
The present invention relates to RD-01 and its salts, and more particularly to heat-denatured deoxyribonucleic acid RD-01 having antitumor activity obtained from Propionibacterium bacteria, as well as its production method and antitumor agent. Furthermore, the present invention relates to a method for producing heat-denatured deoxyribonucleic acid RD-01 having antitumor activity, which comprises heating a nucleic acid fraction obtained by centrifuging a crushed product of Propionibacterium bacteria. 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. The present invention relates to a method for producing heat-denatured deoxyribonucleic acid RD-01 having antitumor activity. Furthermore, the present invention provides deoxyribonucleic acid-containing substances about
This is a method for producing heat-denatured deoxyribonucleic acid RD-01 having antitumor activity, which is characterized in that it is made to have antitumor properties by heating to 80 to 120°C. Furthermore, the present invention provides heat-denatured deoxyribonucleic acid RD which has antitumor activity through heat-denaturation.
It is an antitumor agent containing -01 as an active ingredient. The microorganism used in the present invention is a bacterium belonging to the genus Propionibacterium, and suitable examples include those described in Virgies, Manual of Determinative Bacteriology, 8th edition, No. 634.
The known Propionibacterium acnes described on pages 641 to 641
acnes) ATCC 11829, Propionibacterium granulosum
granulosum) ATCC 25564, Propionibacterium avidum (Propionibacterium
avidum) ATCC 25577, but preferably a strain that has high antitumor activity as a whole and has low pathogenicity. In the present invention, the above-mentioned bacterial strain can be cultured by a conventional anaerobic culture method, and the bacterial cells can be disrupted by a conventional method. For example, nitrogen sources include meat extract, peptone, phyton, yeast extract, etc., carbon sources include glucose, reducing agents include cysteine hydrochloride, sodium thioglycolate, potassium phosphate, magnesium sulfate, etc.
It is preferable to use a medium supplemented with sodium chloride or the like. The culture is preferably left at 30 to 40°C for 2 to 15 days. The obtained bacterial cells are collected by centrifugation, washed with physiological saline, etc., and then subjected to the next operation, or, if necessary, sterilized with phenol or mild heat, and then subjected to the next operation. It may be attached to The obtained bacterial cells are thoroughly mixed with water, preferably an appropriate buffer, and crushed with a Dyno-Mill or French press while cooling on ice to obtain a suspension of crushed bacterial cells. obtain. This suspension is centrifuged to obtain a cell-free extract, and the centrifugation conditions are as follows:
Ordinary centrifugal force that can remove partially crushed 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 suitable for obtaining the substance of the present invention in small quantities (usually 1 g or less), and the resulting nucleic acid fraction (hereinafter referred to as RD 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 or the like is heat-treated to obtain the substance of the present invention, or
The RD nucleic acid fraction is first heat-treated, and then impurities are removed by centrifugation, precipitation, etc. to obtain the substance of the present invention. The heat treatment conditions for the RD nucleic acid fraction and the purified deoxyribonucleic acid fraction are the same as those for the preparation method described below, so they will be described in detail later. The purpose of heating in the present invention is to adjust the molecular weight distribution of the substance of the present invention, change the double-stranded structure to a single-stranded structure, increase the efficiency of removing impurities, facilitate formulation, increase solubility during use, It is an object of the present invention to obtain a substance of the present invention that has high antitumor activity and low toxicity and side effects. 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
It is called the RN-1 fraction. All operations for obtaining the RN-1 fraction are performed under cooling to prevent unnecessary decomposition and denaturation of the nucleic acid fraction. The cooling temperature is preferably 0 to 10°C. Two methods are possible to obtain the substance of the invention from the RN-1 fraction. One method is to separate the nucleic acid fraction from the RN-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 RN-1 fraction. This is a method for separating the substance of the present invention. In the former method, first, the degree of absorption of the RN-1 fraction into the RN-1 fraction and the pH and salt concentration of the aqueous solvent are adjusted, and then the RN-1 fraction is separated by centrifugation or filtration.
A soluble fraction containing deoxyribonucleic acid is separated from one fraction. The substance of the present invention can be obtained by separating the deoxyribonucleic acid fraction from this soluble fraction by precipitation, column chromatography, electrophoresis, and, if necessary, ribonuclease treatment, followed by heat treatment. The preferred concentration of the RN-1 fraction when separating the soluble fraction from the RN-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 RN-1 fraction adjusted in this way is usually
When centrifuged at 20,000xg for 5 minutes or more, a clear soluble fraction containing a nucleic acid fraction is obtained as the 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 purified substance of the present invention can be obtained by further subjecting the thus obtained deoxyribonucleic acid fraction to ribonuclease treatment followed by heat treatment, or by heat treatment followed by ribonuclease treatment, but the heating conditions are common to the latter method. Therefore, it will be explained in detail later. In the latter method, the RN-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. For example, when the aqueous solvent is strongly acidic or alkaline, the heating temperature is not limited to 80 to 120°C, and a temperature around room temperature is sufficient. Furthermore, when the aqueous solvent is alkaline, it may be advantageous in that ribonucleic acid can be removed from the substance of the present invention by appropriately selecting the pH, heating temperature, and time. As described above, by selecting appropriate heating conditions, it is possible to obtain a suspension containing heat-denatured deoxyribonucleic acid with 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 RN-1 fraction in this manner, the fraction 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 include a precipitation method using a nucleic acid flocculant, a fractionation method using an organic solvent, a column chromatography method, or a combination of electrophoresis method and a ribonuclease treatment method. The substances of the present invention obtained by these methods can be used as they are as original materials for preparations, or can be freeze-dried to form dry powders. 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 66%. The transition temperature Tm of the substance of the present invention does not show a clear value, and analysis by hydroxyapatite column chromatography indicates that the substance of the present invention is a denatured single-stranded deoxyribonucleic acid. Some of the substances obtained by the method shown in this specification contain trace amounts of ribonucleic acid, protein, sugar, etc. in addition to heat-denatured deoxyribonucleic acid.
All of these can be considered to be contaminants, and if necessary, they can be removed by further purification. Next, RD- obtained by purification in Example 1
We investigated the physical and chemical properties of 01 (hereinafter referred to as RD-A1) and present the results. Note that the purified product of RD-01 (hereinafter referred to as RD-A2) obtained in Example 2 also exhibits similar physical and chemical properties. Physical and chemical properties of this substance, heat-denatured deoxyribonucleic acid RD-A1 (by Na salt) (1) Elemental analysis (%): C: 26.18-31.31 H: 4.02-4.98 N: 12.11-13.89 P: 8.13-9.18 Na: 3.02～4.28 (2) Molecular weight: 30,000～1,000,000 Using protein as a marker, Sepharose CL-
According to the gel filtration method using 6B, it is distributed from 30,000 to 1,000,000 with a peak around 220,000. (The molecular weight distribution map is shown in Figure 1). (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 (as shown in Figure 2). (5) Infrared absorption spectrum: around 780cm ^-1 , 1090
It has characteristic absorption bands of nucleic acids around cm ^-1 , around 1230 cm ^-1 , and around 1700 cm ^-1 (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) Basic, acidic, and neutral: The pH of an aqueous solution of this substance is 6.5 to 7.5. (9) Color of the substance: White powder (10) Characteristics: It is made soluble through heat treatment, has high tumor activity, and has extremely low toxicity and antigenicity. (11) Base composition (%): Guanine: 33.4 Adenine: 17.2 Cytosine: 32.5 Thymine: 16.9 (method is based on chemical analysis) (12) Enzyme treatment: Antitumor properties are abolished when this substance is treated with deoxyribonuclease (DNase). but ribonuclease T ₂ (RNaseT ₂ )
The antitumor properties do not change with treatment. (13) When analyzed by hydroxyapatite column chromatography, it has a single-stranded structure. (14) 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-dissolving lyophilized 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, for IMC carcinoma, which is a syngeneic tumor in mice, 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 to inhibit tumor engraftment (suppression activity). When administered into tumor tissues engrafted in the body, the antitumor activity (regression activity) was equivalent to or higher than that of the bacterial cells that were the raw material. In addition, 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 tumor growth 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 determined by the 50% lethal dose _LD50 value per kg body weight when administered intravenously to mice.
It is extremely low as it is over 500mg. 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. 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 Production of the substance of the present invention from the soluble fraction of a suspension obtained using streptomycin Heart infusion medium (manufactured by Eiken Chemical Co., Ltd.) 25g Glucose 5g Sodium thioglycolate 0.5g Water was added to adjust the pH to 1, and the pH was 6. Adjust from .8 to 7.2. Propionibacterium avidum ATCC 25577 was inoculated into a Rubin with a rubber stopper containing a medium with the above composition, and after the air in the Rubin was replaced with nitrogen, it was statically cultured at 37°C for 5 days. After the culture was completed, phenol was added to the culture at a final concentration of 5% and left at room temperature for 2 days. Bacterial cells were collected from the obtained culture by centrifugation and washed three times with physiological saline to obtain wet bacterial cells. 100 g of the wet bacterial cells were suspended in 100 ml of 10 mM phosphate buffer (PH7.0), crushed with a Dynomill under ice cooling, and centrifuged at 20,000 x g for 20 minutes to obtain a supernatant cell-free extract. . Streptomycin sulfate was added to this extract to a final concentration of 0.3%, stirred thoroughly and allowed to stand overnight at 4°C. It was suspended in a buffer solution (PH7.0). This suspension was packed into a cellophane tube and dialyzed against the same buffer, followed by dialysis against distilled water to obtain 180 ml of a suspension containing the nucleic acid fraction (hereinafter referred to as this suspension).
RN-1) was obtained. The solid concentration of this suspension was 3.5 mg/ml, and the yield was 0.63% based on wet bacterial cells. Next, NaCl was added to the entire amount of RN-1 to a final concentration of 0.9%, stirred, and heat denatured (100℃,
60 minutes), and then centrifuged at 20,000xg for 20 minutes to obtain a supernatant. After adding NaCl to this supernatant to a final concentration of 0.4M and dissolving it, cetyltrimethylammonium bromide (hereinafter referred to as CTAB)
It is called. manufactured by Tokyo Kasei Co., Ltd.) with a final concentration of 0.2%.
(W/V), stirred thoroughly, and left at room temperature for 30 minutes. The generated precipitate was collected by centrifugation, dissolved in 40 ml of 1M NaCl solution, and then added with an equal volume of chloroform-isoamyl alcohol (24:1).
A mixed solution was added, shaken, and centrifuged to obtain an aqueous layer. After repeating this operation two more times, 3 times the amount of 99.5% ethanol was added to the resulting aqueous layer, stirred, and allowed to stand overnight at 4°C. The generated precipitate was collected by centrifugation, dissolved in 30 ml of distilled water, and then dialyzed against distilled water to obtain a purified nucleic acid solution. After neutralizing this solution with 1N NaOH, freeze-dry it to obtain a dry specimen.
Obtained 145mg. 90mg of this product was added to 0.05M acetate buffer (PH
4.5) After dissolving in 10 ml, 200 U of ribonuclease T ₂ (manufactured by Sankyo Co., Ltd.) dissolved in 2 ml of the same buffer was added and reacted at 37° C. for 22 hours. Equal volume of chloroform-isoamyl alcohol (24:1) to the reaction solution
After adding the mixed solution and shaking, the mixture was centrifuged to obtain an aqueous layer. After repeating this operation, the entire amount of the aqueous layer was loaded onto a Sephadex G-100 (manufactured by Pharmacia Fine Chemicals) column with a diameter of 2.5 cm and a length of 90 cm, which had been previously washed with 0.5 M ammonium hydrogen carbonate solution. After elution, a fraction containing deoxyribonucleic acid was obtained as the first eluted fraction.
This fraction was dialyzed against water, neutralized with NaOH, and then lyophilized to obtain 67 mg of the substance of the present invention. This product is hereinafter referred to as RD-A1. The content (purity) of deoxyribonucleic acid in RD-A1 was determined by the diphenylamine method (Biochemical Journal 62 , 315, 1956) after hydrolysis with 5% perchloric acid.
It was 98.8% when quantified by. Example 2 Production of the substance of the present invention by the marmer method: Propionibacterium granulosam ATCC25564
250 g of wet bacterial cells obtained under the same culture conditions in the medium of Example 1 were suspended in 7 times the volume of 10 mM phosphate buffer, crushed with a Dynomill under ice cooling, and then crushed at 20,000 x g.
A cell-free extract was obtained by centrifugation for 20 minutes. From this extract, Marmur (Journal of
A crude deoxyribonucleic acid fraction was obtained by the method (Molecular Biology 3 , 208, 1961). This fraction is 0.9
After dialyzing against a saline solution, the mixture was heated at 100°C for 60 minutes. After cooling, 55 mg of the sample obtained by dialysis against distilled water, neutralization, and freeze-drying was purified by ribonuclease T ₂ treatment on the same scale as in Example 1 to obtain 31 mg of the substance of the present invention. Below is this
It is called RD-A2. The deoxyribonucleic acid content of RD-A2 was analyzed in the same manner as in Example 1 and was found to be 98.1%. Example 3 10 mg of liquid RD-A1 was dissolved in 10 ml of PBS minus solution (manufactured by Eiken Chemical Co., Ltd.), and Nyukrypore No. 20
(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 4 Freeze-dried preparation 10 mg of RD-A1 was dissolved in 10 ml of distilled water for injection, and then 500 mg of mannitol was added and dissolved, followed by sterilization using Nycrypore No. 20.
The obtained liquid was aseptically dispensed into vials in 1.5 ml portions and then freeze-dried to obtain a freeze-dried agent of the substance of the present invention. Example 5 Emulsion agent RD-A1, 4 mg was dissolved in 0.5 ml of physiological saline,
Next, Drakeol 6-VR (Drakeol 6-VR,
(manufactured by Pennsylvania Retihing 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 of the following products (manufactured by Kasei Chemical Co., Ltd.) and using a connected injection needle. Test Example 1 Antitumor activity against mouse IMC carcinoma The antitumor activity of the substance of the present invention against mouse IMC carcinoma was investigated. The test system is as follows. 5 × 10 ⁵ IMC carcinoma cells were intradermally transplanted into CDF1 male and female mice, and 0.1 ml of the preparation prepared according to the method of Example 3 was injected into the tumor every other day for a total of 6 times from the first day after transplantation. Administered intravenously. 35 after transplant
The tumor was excised on the same day, and its weight was measured. The results are shown in Table 1.

[Table] Student's t-test and Fitscher's test were used to test for significant differences in the frequency of occurrence of average tumor weight and number of cured cases, including the tests described below. 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
used BCG vaccine (manufactured by Nippon BCG Company). Physiological saline was used as a control. Test Example 2 Antitumor effect of strain 2 guinea pigs on line 10 hepatoma The substance of the present invention in strain 2 guinea pigs line
The antitumor effect against 10 hepatomas was investigated. 1×10 ⁶ line 10 hepatoma tumor cells were intradermally transplanted into strain 2 guinea pigs, and 7 days after transplantation, 0.1 ml of the preparation prepared by the method shown in Example 3 was injected into the tumor. was administered. Seventy days 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 2.

[Table] Test Example 3 Antitumor activity of a substance treated with a nucleolytic enzyme 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. AD-A1 is a deoxyribonuclease DNase
(manufactured by Sigma Chemicals), the resulting decomposition product was treated with chloroform to remove DNase, and then Sephadex G10 (Pharmacia Fine
The product was desalted using a column (manufactured by Chemicals) and lyophilized to obtain a nuclease-treated product of the substance of the present invention. The antitumor activity of the obtained treated product was tested in the same manner as in Test Example 1, and the results shown in Table 3 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 4 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 solution of RD-A1 or RD-A2 in physiological saline (1 mg/ml) was intraperitoneally administered to 8-week-old C ₅₇ BL/6 female mice in an amount of 0.3 ml per mouse.
Peritoneal exudate cells were collected 48 hours later. The number of cells in the collected solution was reduced to 2 with RPMI1640 medium supplemented with 10% fetal bovine serum.
After adjusting the concentration to ×10 ⁶ cells/ml, the cells were transferred to a plastic shear dish and incubated at 37°C for 90 minutes in a 5% carbon dioxide atmosphere to obtain adherent cells and non-adherent cells on the shear dish. 5 × 10 ⁵ cells of each cell and 1 × 10 ⁴ mouse leukemia cells YAC-1 labeled with ⁵¹ Cr.
Added 10% fetal bovine serum at 37℃ under 5% carbon dioxide atmosphere
Culture was performed for 4 hours using RPMI1640 medium. 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 5 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. RD-A1 was then 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 5.

[Table] Test Example 6 Acute Toxicity 10 5-week-old ddY male mice per group (average weight
Dissolve RD-A1 in physiological saline and weigh 1
500 mg per kg was administered intravenously. 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 500.
It is considered to be more than mg/Kg. Test Example 7 Antigenicity RD-A1 dissolved in physiological saline was intradermally administered to six Hartley female guinea pigs (average weight 348 g) per group at a dose of 1 mg per animal three times a week for a total of six times to sensitize them. . RD- after 2 weeks from the last sensitization date
A1 was dissolved in physiological saline and administered intravenously at a dose of 10 mg or 2 mg per kg body weight. 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 anaphylactic shock at all at the above dose.

[Brief explanation of drawings]

FIG. 1 shows the elution pattern of the substance of the present invention by Sepharose CL-6B 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 RD-01 and its salts, which have antitumor activity through heat-denaturation and exhibit the following physicochemical properties (based on Na salt). (1) Elemental analysis (%): C: 26.18-31.31 H: 4.02-4.98 N: 12.11-13.89 P: 8.13-9.18 Na: 3.02-4.28 (2) Molecular weight: 30,000-1 million Sepharose using protein as a marker CL−
According to the gel filtration method using 6B, it is distributed from 30,000 to 1,000,000 with a peak around 220,000. (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 , around 1230 cm ^-1 , and around 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 10 μg/ml of this substance. D) Anthrone reaction: Negative at a concentration of 10 μg/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) Characteristics: It becomes soluble through heat treatment, has high antitumor activity, and has extremely low toxicity and antigenicity. (11) Base composition (%): Guanine: 33.4 Adenine: 17.2 Cytosine: 32.5 Thymine: 16.9 (method is based on chemical analysis) (12) Enzyme treatment: Antitumor properties are abolished when this substance is treated with deoxyribonuclease (DNase). but ribonuclease T ₂ (RNaseT ₂ )
The antitumor properties do not change with treatment. (13) When analyzed by hydroxyapatite column chromatography, it has a single-stranded structure. (14) Transition temperature (Tm): A clear transition temperature Tm cannot be determined from the measured melting curve. 2. The heat-denatured deoxyribonucleic acid RD-01 according to claim 1, which is obtained from Propionibacterium. 3 Using Propionibacterium bacteria that can produce RD-01 by heating and denaturing the nucleic acid fraction obtained from the cell-free extract obtained by centrifuging the crushed product, the crushed product is subjected to these treatments. 1. A method for producing heat-denatured deoxyribonucleic acid RD-01 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. The manufacturing method according to any one of claims 3 to 5, characterized in that the heat denaturation is carried out at about 80° to 120°C. 7 Add a nucleic acid flocculant to the cell-free extract obtained by centrifuging the crushed material, dialyze the resulting precipitate against water or a salt solution, heat denaturation, and separate RD-01 from the supernatant. 1. A method for producing heat-denatured deoxyribonucleic acid RD-01 having antitumor activity, which comprises using a Propionibacterium bacterium capable of producing a combustible molecule, and subjecting its crushed product to these treatments. 8. The manufacturing method according to claim 7, characterized in that the heat denaturation is carried out at about 80°-120°C. 9. An antitumor agent containing heat-denatured deoxyribonucleic acid RD-01, which has anti-tumor activity through heat denaturation, as an active ingredient. 10. The antitumor agent according to claim 9, wherein the heat-denatured deoxyribonucleic acid RD-01 is obtained from Propionibacterium.