JPS5933227A - Antitumor substance rd-01, its preparation and pharmaceutical preparation containing the same - Google Patents

Abstract

NEW MATERIAL:The thermally denaturated deoxyribonucleic acid RD-01 having antitumor activity developed by thermal denaturation, and its salt. The sodium salt of the substance has the following physical and chemical properties: appearance, white powder; melting point, no distinct melting point; elementary 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%; molecular weight, 30,000-1,000,000; solubility, soluble in water, insoluble in ethanol, methanol, ether and acetone; base composition (%), guanine 33.4, adenine 17.2, cytosine 32.5, thymine 16.9; effect of enzymatic treatment, loses antitumor activity with deoxyribonuclease I but keeps the activity with ribonuclease T2; etc. USE:An antitumor agent exhibiting antitumor activity via the immune reaction of the host. PROCESS:The disintegrated cell of bacteria belonging to Propionibacterium genus is centrifuged, and the resultant nucleic acid fraction is heated at 80-120 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides an anti-1III! The present invention relates to heat-denatured deoxyribonucleic acid RD-01 having tumor activity.

Bacteria belonging to the genus Propionibacterium have antitumor effects,
It has been known for a long time that it has physiological activities such as adipant action, and in recent years, research has been conducted on the cell wall skeleton of this bacterium and its physiological activities. Attempts are being made to apply this method.

However, the cells of this one-year-old organism 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.

An example of a heavy substance derived from a cell wall is the above-mentioned Tomari 1iW.
Examples include polymeric mucopeptides prepared from wall skeletons and cell walls, but these also have the disadvantage that products with antitumor activity have strong side effects, and products with weak side effects do not necessarily have high antitumor activity.

Previously, the present inventors investigated the antitumor active substances obtained from Mycobacterium tuberculosis, and found that heat-denatured deoxyribonucleic acid isolated from radioactive vesicle extract obtained by crushing Mycobacterium tuberculosis has a strong host-mediated effect. Anti-rII! It was discovered for the first time that it has s activity and extremely low toxicity and antigenicity, and was patented (Special Patent, H, 1986-2', U (51i 9).
) was reviewed once.

In addition, the present invention also includes the use of cell extracts of bacteria belonging to the genus Propionbacterium, based on the experience of tuberculosis, in order to find antitumor active substances. As a result of our intensive search for tumor active substances '1 I↓, we found that heat-denatured deoxyribonucleic acid prepared from Propionbacterium urinosa by the same method used for Mycobacterium tuberculosis was also found. It was confirmed that the substance of the present invention exhibited strong antitumor activity, 111 cases were extremely low, and had almost no pyrogenicity or antigenicity. To name and complete the present invention.

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. Deoxyliponuclear r'i! obtained by the present invention! 2 has strong host-mediated antitumor activity similar to the substance derived from tuberculosis 1, and its action is similar to that of the substance derived from tuberculosis 1.
Deoxylipo nuclear enemy temple tumor micro 11i derived from animal micro 1 Mutsumi known so far! ! 5science, 14'4.997.196
5゜Cancer Research? centre,
2342. 1967, Proceedings o
fthe National Academy of
5science fil, 207. It is 93i force compared to 1968).

The substance of the present invention, its production method, and its strong host-mediated anti-J 4 viscosity have been thoroughly clarified by the present inventors.

That is, the present invention relates to a heat-denatured deoxyribonucleic acid R1)-(moon and its()) salt which has anti-tumor activity by heat denaturation, and more specifically relates to an anti-tumor salt obtained from Propionibacterium jMII bacterium. 7 with activity
JI I:! ~'J & deoxyliponucleic acid RD-01
It also relates to 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 disrupted product of Propionibacterium genus bacteria. be.

Furthermore, as a method for separating nucleic acid fractions, the present invention adds a nucleic acid flocculant to a cell-free extract, and extracts 1 fraction from the soluble fraction juice of the resulting precipitate.
- Heat-denatured deoxyribonucleic acid RD-01 with I-S activity, characterized by having 4! (Koho (related to C).

Furthermore, +: The invention is equivalent to 1 guest and 1 song (0 minutes 141 is the modulus [
7), 1(1) Add a nucleic acid precipitating agent to the extract of 111 rivers of water, dialyze the 17 molar volume against water or saline, heat the mixture, and extract from the I-clear. Separation 4-
'4J゛Soru Anti) 11 1. ．． The present invention relates to a method for producing heat-denatured deoxyribonucleic acid Rr)-01 having 3 activities.

Furthermore, the present invention provides that deoxyribonucleic acid contains about 80 to
It is characterized by heating it to 120C to make it have antigen J4 properties.
This is the Oo°1 construction method.

Further, the present invention provides an anti-tumor agent containing heat-denatured deoxyribonucleic acid RD- (an anti-tumor agent as an active ingredient) which has anti-tumor activity by heat modification.

Microorganisms used in the present invention? The snout belongs to the genus Propionii. 1. Although it is a cocoon, it is a suitable cocoon.The well-known Gropioninocle TC listed in Old Shiteha, Birshis, Manual e Obtain Native Bacteriology 8th Edition, No. 634-6 Old White, jl:
02tri7'B av i Pain m1%-Do-nono-like is kicked out, but it is still anti-111j with all bacterial cells! Strains with tumor activity (c4+ <) and weak pathogenicity are preferred.

In the present invention, the culture of I-6 Hakubi strain is usually (1)
Anaerobic culture '11 method can be used, and 1. Method of crushing the shell 4r Ordinary method ('(2) may be used. For example, as a nitrogen source, meat extract, peptone, phyton, fermented mushroom extract, as a carbon source, glucose blue, reduction 91j
and 1. What about salt hasystine, sodium thioglycolate, etc., and phosphorus L'16? It is preferable to use a medium to which potassium, magnesium sulfate, sodium chloride, etc. are added.

The culture is preferably left standing at 30 to 40°C for 2 to 15 days. 14] The resulting +11 bodies were sprouted by centrifugation, etc., washed with saline water, etc., and then directly used for the 5th 1st dyeing crop (・koi・?su;ji・,・()) <is four, tl, ba,
After phenol or weak heat sterilization, the next J・t
It may be attached to h.

The obtained Kokutai was mixed with water or a suitable mild liquid for 9 minutes, and placed in a Dyno-Mil (Dyno-Mil) while cooling with water.
l) Or French Pre, <f (How to crush 1llll bacterial cells and crush 1i% empty body 1
Centrifuge this back suspension to obtain a cell-free extract.The conditions for centrifugation are as follows. , ordinary centrifugal force that can remove cell wall fractions etc. as precipitates may be used, for example 5,
000 xg.

Centrifuge for 10 minutes or more and obtain the supernatant.

In the present invention, the extract of B. elegans 11.
1°1A7 flat fluid is centrifuged to remove unbroken bacterial cells, partially disrupted 1:11 cells, and 1:1 cells that form in front of the cell wall fraction.

Collecting the nucleic acid fraction from the cell-free extract: 1.
Filura 111 blood extract is treated with 1ba grafting agent and nucleic acid 1+, +! How to obtain i minutes (e.g. Marm
ur ) method, phenol method, etc. (hereinafter referred to as direct organic solvent method) and δ+III, ie 4111, and add nucleic acid i, Nf:song in the evening to prepare the precipitate containing the nucleic acid fraction.
The method to be applied is "one-chop function".

Interviewed Isosolvent method i, 1 small scale (usually 17 or less) of the substance of the present invention! This is a method suitable for I.I., and the resulting R, D nucleic acid fraction (hereinafter referred to as R, D nucleic acid fraction) usually contains impurities such as antigenic polysaccharides and proteins. (After removing as many impurities as possible through mental methods, etc.)
The substance of the present invention can be obtained by heat-treating the purified deoxyliponuclei r-1!2 fraction, or by first heat-treating the RD nucleic acid fraction, followed by centrifugation, precipitation, etc.
The substance of the present invention is obtained by removing the +1 substance.

Rr) The heat treatment conditions for the 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 door 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, and reduce the rate of dissolution at one time. It is an object of the present invention to obtain a substance of the present invention that has enhanced toxity, high antitumor activity, and low toxicity and side effects.

Uj (extraction of 111 cells) night (method of precipitating the nuclear i''i7 fraction by adding the normal 1 pseudocarboxylic agent, nucleic acid) [11 cells can be reduced, so this invention can be carried out on an Otake scale. This is a suitable method for obtaining substances.

The I'L nucleic acid used in this method, ), is removed from a normal nuclear f'! ! ! ii)
+T stop, but f father (Lini 1 - agglomerated il used at crosspiece)
11 for low-molecular aggregation in the sense that l can be easily removed.
is preferred. A suitable evening 11. , l-, polyvalent metal cations such as manganese chloride, 1h manganese chloride, magnesium monide, aluminum sulfate, water-soluble basic antibiotics such as streftomycin, or the like are prohibited. . Nuclear;! Attack r'ie (:' Prefectural Agent's 1st History Month) Law can be selected by 11% due to its force 1, but for example, for polyvalent metal cations, it is 0, 1 to 10%, favorable 01-3
%, antibiotic nonnfHO. o] ~10''

Procedure: None, p+++ 11 = Add a nucleic acid flocculant to the extract, and centrifuge or pass through the resulting precipitate.
: After a minute, a suspension containing the nucleic acid fraction is obtained. Stiffness: i? Dialysis is suitable for removing nucleic acid 7j & fruit from the J riii solution.

In this case, the 4-free water solvent for convenience is preferably a buffered solution having an appropriate ionic strength and a pH around neutrality in order to increase the removal efficiency of the nucleic acid flocculant. For example, i
: 0. 1-IM NaCQ-containing phosphate buffer, 0
．． 1~IMKCQ citrate buffer, 01~1M NaCQ
．． Examples include a sodium carbonate buffer containing 0.1 to 1M NaCρ containing tris-hydrochloride (+).

After the suspension containing the nucleic acid fraction is dialyzed against a salt-containing buffer, the suspension is further dialyzed against water to remove salts. The suspension containing the dialyzed nucleic acid fraction is hereinafter referred to as 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. Cooling temperature is 0
Preferably, the temperature is between 10°C and 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, R IJ-1 +ll+
i Adjust the degree of sweat absorption and the pH and salt concentration of the aqueous solvent [7 times, centrifugation fraction 141] [or extract the soluble fraction containing deoxylipo-cough cloud from the RN-1 fraction by a method such as filtration. Pilf the minute.

From this soluble fraction, a deoxyribonucleic acid fraction is separated by precipitation, Calano chromatography, electrophoresis, and if necessary, ribonuclease treatment.
When heat treated, the film of the present invention can be reduced by -1. RN-1
Suitable RN- for separating soluble fraction from fraction l/ifF
The concentration of one fraction is 1-15 mg/mQ. The pH of the aqueous solvent is preferably around neutrality, and is usually adjusted strictly to a pH range of 5 to 8 with a base or buffer. The ionic strength of the aqueous solvent (the ionic strength) affects the removal efficiency of precipitates during centrifugation, etc., and is usually preferably 0 or 1 or more. It's okay.

The RN-1 fraction thus adjusted is usually 20, former) Ox
After centrifugation in fZ for 5 minutes or more, a clear soluble fraction containing a nucleic acid fraction is obtained as the supernatant. Thioxyribonucleic acid was extracted from this supernatant by a precipitation method using streptomycin, manganese chloride, cetyltrimethylammonium bromide, etc., a column chromatography method using Sepharose, etc., or an electrophoresis method using a vinyl chloride-vinegar ()-tavinyl copolymer solution tray. Fractions can be separated.

The above operations are preferably carried out at a temperature of total -CO 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 the same as in the latter method. Therefore, it will be explained in detail later.

In the latter method, the RN-1 fraction is heated without rinsing, but this heating operation is necessary to appropriately denature the nucleic acid fraction and to remove impurities' (11) to facilitate the subsequent separation of the substance of the present invention. The heating conditions are closely related to the anti-jerking paralysis activity of the substance of the present invention, as will be shown later, and there is an appropriate range of conditions.

Heating factors include the solute concentration during heating, the type and pH of the aqueous solvent, ionic strength, heating temperature, and heating time, and the appropriate range of conditions is as follows.

The solute concentration is 1 to 15 mf/mQ.

The aqueous solvent is water or salt water such as salt (ionic strength is 0)
．． 1 to 05) for 5 minutes to 120 minutes at 80°C to 120°C. If the aqueous solvent is 1 cup of buffer, it will be greatly affected by the pH of the buffer, and if the pH is around 19, 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, if the water bath medium is strong, 1 or strong alkali, the heating temperature is not limited to 80 to 12C (1'C), and even room temperature is sufficient. ,
If the aqueous solvent is alkaline, it may be advantageous in that ribonucleic acid can be removed from the substance of the present invention if the heating temperature and time are appropriately selected.

As described above, by selecting appropriate heating conditions, it is possible to obtain a suspension containing heat-denatured deoxyribonucleic acid with good antitumor I9 activity and low antigenicity, which is the object of the present invention, as will be shown later. I can do it.

The heating conditions shown here are based on the material of the present invention! i12 method ■1
Due to certain factors, it is applicable to any of the preparation methods presented herein.

After heating the RN-1 fraction in this manner, the fraction is cooled and the nephroliths or precipitates are removed by a method prior to filtration to obtain a clear solution containing the substance of the present invention. Molecules of the substance of the present invention can be easily isolated from this solution using conventional methods. Suitable examples include a precipitation method using a nuclear condensing agent, a fractionation method using an organic solvent, a column chromatography method, an electrophoresis method, etc., combined with a bonuclease treatment method. Ru.

The substance of the present invention obtained by these methods can be used as it is as a base material for preparations, or it can be freeze-dried to form a dry powder.

The molecular weight of deoxyribonucleic acid in the substance of the present invention is distributed between about 30,000 and 1 million, including guanine, cytosine (GC,
The content is approximately 66%. The transition temperature Tm of the substance of the present invention is
It does not show a clear value, and when analyzed by hydroxyapatite column chromatography, it is the present invention! I'a
This means that it is a denatured single-stranded deoxyribonucleic acid.

Among the materials obtained by the method described in this specification, in addition to heat-denatured thioxyribonucleic acid, there are traces of ribonucleic acid, protein 2. There are also things that contain sugar, etc., but all of them can be considered as contaminants, and if necessary, they can be removed by sucking the leaves into the petals.

Next, the physicochemical properties of the In)-AI obtained by the Kinutashi process in Example 1 were investigated, and the results are shown below.

Note that the purified product of 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-AI (by Na salt) (1) Elemental analysis (%): C: 26.18 -: 31
31 H:4. (12-498N:]2.11-13.
89 P: 8.13-9.18 Na: 3.02-4
．． 28 (2) Molecular weight: 30,000 to 1,000,000 molecules - The distribution map follows the trace shown in Figure 1.

(3) - point: Does not show a clear melting point.

(4) Ultraviolet absorption spectrum: Figure 2 (as shown.

(5) Infrared absorption spectrum: 11i1 shown in FIG.

(6) Solubility in solvents: Soluble in water, insoluble in ethanol, methanol, alcohol, 5-1-thyl, aceton.

(7) Colored anti-1 core A) Orcinol reaction S RN fractionated by STS method
1 cloud 1 life for A fraction.

B) Diphenylamine reaction: ii,! on the DNA fraction fractionated by the s′rs method. I live.

C) Ninhydrin reaction: Negative at a concentration of 10 ttfl/mρ of this substance.

1)) Anthrone reaction: this substance 1011f//m,
The concentration of Q is negative.

(8) Distinguish between basic, 1°, and neutral The pH of the aqueous solution of the two substances is 6.5 to 7.5.

(9) Color of substance: White powder 00) Characteristics: Solubility is imparted by heat treatment, and it has high tumor activity, and denaturation and antigenicity are extremely low.

(11) Base composition diagram) Nigeanin: 33.4
Adenine: 17.2 Cytosine: 32.5 Chimi /:
t6. c+ (The method is based on chemical analysis) OQ When the two enzyme-treated substances are treated with deoxyribonuclease l ('DNase l), anti-+1tt
t JSi 4 is gone, but Ribonozo Rease T2 (
Treatment with RNase 'r2) does not change the antitumor properties.

Analysis by column chromatography (formerly known as hydroxyabatai) shows a single pattern.

(1.0 Transition width 1 stop ('rm): A clear transition temperature Tm cannot be determined from the measured fusion curve.

The fact that the main body of the anti-++IJi tumor 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 deoxylipo-bale fraction W4 enzyme (DNase I), the antitumor activity disappears; Even if strawberries were treated with ribonucleic acidase (RNaseT), anti-q'lj
41; This is clear from the fact that the Il activity does not change.

When the substance of the present invention is used in the form of an injection, it is preferable. The present invention'proboscis'μ
f can be used alone or as a commonly used additive, or can be applied as a solution by adding 11 agents, or as a co-dissolved lyophilized RF solution.

The substance of the present invention can also be applied as an emulsion of the oil-in-water type, water-in-oil type, or water-in-oil type.

The needle and route of administration of the substance of the present invention should be selected appropriately, but the first use is the body! li 0.01 to 100 per kg
m1i+ is preferred, and the route of administration is subcutaneous, intravenous,
Intra-abdominal IK administration and na ++ (-! intra-internal administration) are performed.

The substance of the present invention exhibits antitumor effects against individual tumor systems in guinea pigs and mice. For example, for IMC rutinoma, which is a syngeneic tumor in mice, the substance of the present invention is brought into contact with frostbitten cells (dissolved in physiological saline), and then implanted into the animal body to suppress tumor engraftment (zabrenosion activity). Alternatively, the anti-tumor activity (regression activity) achieved by engrafting in an animal body and administering it into tumor tissue is equivalent to or higher than that of the bacterial cells used as the raw material. In addition, for line 10, 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 primary tumor. It also inhibited tumor metastasis.

Furthermore, the substance of the present invention also exhibited anti-IIIE J-bow activity against IF and IC chart tumors when dissolved in physiological saline and administered to a site different from the tumor tissue.

The acute activity of the substance of the present invention is determined by intravenous administration to mice, which results in 50% lethality per kilogram of body weight.The LD5o value is 500.
+n'/ is extremely low.

・Also, regarding the η21 trillion property of the present substance, an anaphylaxis test using guinea pigs was carried out, i1% 91
F. The present invention was confirmed by one 71i11 allergy test.
The substance was found to be extremely safe.

Pyrogenic, painful, and inflammatory properties of the material invented by the ribs.

The granulation-forming properties, etc., were also lower than those of the 11 raw materials, and it was found through divine testing that it was so good that it did not cause any problems in its application as a regular medicine.

The use of the substance of the present invention against cancerous tumor cells
c! When the inhibitory effect of the present invention was investigated, the substance of the present invention did not exhibit any inhibitory effect on the growth of +1 lil Jlf! The immunological activities of the substances of the present invention were investigated individually because they were thought to have a positive effect on mice.As a result, the substances of the present invention had a strong effect on killing T cells in mice and a macrophage-hosting effect, as well as an effect on macrophages. Ral Killer Thin 11ti
71! :・1・−(.

Based on the divine knowledge that the enhancing effect is small (2..1), the substance of the present invention is considered to be extremely useful as an anti-inflammatory agent.

Below is the invention I! IA of the present invention's product 'C'↓: The usefulness as a tumor agent was tested (shown by measurement) using a practical example of the production method of fI/).

Example 1 Suspension 1 obtained using streptomycin
Manufacture of the substance of the present invention from one soluble component...-Toinfusion base (manufactured by Eiken Chemical) 257 glucose
57 Thioglycol+! Add 0.5 g of sodium chloride to make I and Q, and adjust the pH to 6.8 to 7.2.

Propionibacterium avidu
m) ATCC25577 was inoculated into a lupine with a rubber stopper containing a medium with the above composition, and the culture was statically cultured for 5 days at 37°C with the air in the lupine replaced with nitrogen. After the culture was completed, phenol was added to the culture to give a final concentration of 5%, and the culture was cooled and left at room temperature for 2 days. From the culture obtained: eyebrow body by 111F for 1 faith! , 1st recommendation, period t”E 41
Rinse three times with water for a while to obtain a wet field; missing. This wet bacterial body 1 f
l (l fl to Ioom(!'s 10 m~・■ Phosphoric acid loose Mfi I ((pr(7,o)! lWi +1
ij L fc V&, crushed in a dyno mill under water cooling, 2
11. ', +t:lII IIi'2 extraction chant r4
I was. Add 1 word of streptomycin sulfate to this eruption fluid.
After cooling to a temperature of 0.3% and stirring thoroughly,
・I 'CI'-・Apparatus overnight and collect the generated precipitate by centroid separation, Y), 10 m containing 0,5 M NaCQ
M phosphate Fj? "l'fir liquid (pfl 7. (1
). This W5j 7@i night is a cello bunch =
--bui/(packed and same loose: Iti Dialyzed against deer, then nuclear i + 12un with a'1lrL against distilled water
Suspension containing i minutes K 18 (1 mQ.

(hereinafter this suspension is referred to as RN-1).

The solids concentration of this suspension is 3.5 m f / , +1
The yield was 0.63% per wet bacterial cell.

Next, RN=1 total 1・TeNaCJ! Inlet concentration is 0.9
%, stir, and add to the solution (+00
'C.

60 minutes) you, 20. t) OOxf, cool for 2 t + 111 minutes and drain the supernatant. This” - NaCQ in Qing
Strings dissolved by adding to a final concentration of 0.4M,
Cetyltrimethylammoniumpromide (hereinafter referred to as C'FA13-J-0 Song Shij (manufactured by Kasei Co., Ltd.) was added to a final concentration of 0.2% (W/V), stirred thoroughly, Vortex for 30 minutes. Precipitate formed:
I・((J double due to heart separation, I Ai NaCQ liquid 4
Waves dissolved in 0 mR, caustic iii: mixture of nochloroform-isoamyl alcohol (211:1)? , followed by shaking and centrifugation to obtain an aqueous layer. After repeating this operation two more times, the aqueous layer was filled with three times the amount of 99.
5% ethanol was added, stirred, and left at 4°C overnight. The generated precipitate was centrifuged and distilled water 30+
After dissolving in nfl, it was dialyzed against distilled water to obtain a purified nucleic acid solution. This solution was lyophilized to 145 + nf/
f, 14 suda. 90 m7 of ice crystals 11 0.05 M vinegar
1221jHj7(t (pH71,5) 10 m
After dissolving in fl, dissolve in 2 mρ of the same buffer or cool 200 U of Bonuclease T2 (manufactured by Sankyokai).
The reaction was carried out at 37°C for 22 hours. Reaction H'ly, jr chloroform-isoamyl alcohol (24:
]) was added and shaken, followed by centrifugation to obtain an aqueous layer. This operation is repeated for the entire stage of Mizuichiso ();
゛, in advance (,), 5 hl charcoal, 1 g ammonium hydrogen, washed overnight / 2.5 cm, j〈
90cm long seven-float Denox G-100 (Pharrn
aciaFine Chemicals'fl: The mixture was eluted overnight and the first eluted fraction contained deoxyribonucleic acid. This fraction can be used with water (t7T', then Na0H-
After neutralization, the product of the present invention was frozen 1 and 4 times and dried.
I got fl.・This is called r<D-AI.

RD-AI deoxyribonucleic acid negative III: (purity)
was hydrolyzed with 5% perchloric acid and the diphenylamine method (Biochemical Journal 6).
2.315. [156) when it is 7, it is 98.
8: It was cooked in a pot.

Actual sister example 2 Preparation of the substance of the present invention by the Marmer method: Probionibacterium red granicolosa 11 (Propion
il) acterium granulosam) AT
CC255fi71 was cultured in the same culture medium as in Example 1 under the same conditions.
(lrr1M phosphorus 1゛throwing'': f!1''I'+ Suspended at night, then crushed in a dyno mill under ice cooling, 20.■)OX
The cell-free extract was obtained by centrifugation at 7°C for 20 minutes. Extract, night to 17-mer (he Iarmur, Jo
Urnal of, □, Iolecular Bi++
The todeoquine ribonucleic acid fraction was obtained by the following method. This fraction is 0.9'X;
After dialysis against a saline solution, the mixture was heated at 100° C. for 00 minutes. Dialyzed against cooled steamed WI water, freeze-dried at 11°C (in addition, 55m7 of the standard sample was treated with ribonuclease r2 on the same scale of 1'>Q as in Example 1, processed into leaves, Invention 'I' (3+mg) was obtained.Hereinafter, this product will be referred to as '4HRD-A2.When analyzed in the same manner as the thioxyliponuclear r/p of RD-A2 and the case I, it was found to be 98.
．． That's 1%.

Example RD-AI, 10, NFL total 10m, PRS of Q
Bath in minus liquid (manufactured by Eiken Chemical Co., Ltd.) and use Nuclebore N.

20 (manufactured by Nuclepore)
” Over L Ta.

The obtained P solution was aseptically dispensed in 1.5111 (!) portions into vials to obtain a liquid preparation of the substance of the present invention.

Practical hm Example 4 Lyophilized preparation RD-AI, 10m7 was dissolved in 10rrJ of distilled water for injection, then 500m? Add mannitol and dissolve 7c.
) b, Niko Krivoa - NO20 on the moon” 5#, F,
1 Meat F' A I.

Nko. 4S et al., 5 Hz) 1.5 tn
Qi''tsu;'!t1.l 1'White ((After dispensing into a vial, freeze-drying the present invention r↓d) to obtain a freeze-dried agent.

Real sister 1 evening IJ, 5 emulsion i'1llRD-
AI, 4 mW to 11.5 m (j cow jψf”JE”
'5A solution (・(solution 11jjr, then Drakeol 6-
VR (Drakenl 6-VR.

Pen5ilvania Retihinq Con]
pany) and Arlacel A,
At1as Chemical Industry
A water-in-oil emulsion was obtained by adding 0.51ηQ of 8.5:1.5 /M solution (manufactured by S.S.) using a connected syringe needle.

ir”(!,)92 ?!I 1 Mouse Llv+c
:/7 Rutinoma K
The antitumor activity against J-J was investigated.

The test system is as follows.

CDF female mouse ノ) ironically: 5 x 105 pieces ノ) r1
Sirc rutinoma ++; 0.1m per
ρ was administered intratumorally. Transfer 1@Test, Test 3 Antitumor activity of the substance treated with nucleolytic enzyme The main substance of the anti-+7 tumor activity of the substance of the present invention was investigated by treating the substance of the present invention with a whole nuclear J-wave degrading enzyme.

The test and test method are as follows.

AD-AI was treated with the enzyme DNase [ribonucleic acid degrading enzyme DNase [
(manufactured by Sigma Chemicals), the resulting decomposition product was treated with chloroform to remove DNase, and then treated with Sephadex G 10 (manufactured by Pharmaci
a (manufactured by Fine Chemicals) column, desalted, and lyophilized to obtain a nuclease-treated product of the substance of the present invention. The anti-liver tumor activity of the obtained treated product was tested in the same manner as Test Example 1, and the results shown in Table 3 were obtained.

Table 3: Antitumor activity of nuclease-treated product against mouse IMC rutinoma T/C is the percentage ratio of the average tumor weight of the administration group to the average tumor weight of the control group.

Physiological saline was used as a control, and BCG vaccine was used as BCG.

Test Example 4 Natural killer ('NK) cell activity enhancing effect The activity enhancing effect of the substance of the present invention on NK cells was investigated.

The test method is as follows.

Physiological saline, RD-Al or ID: RD-A 2 physiological saline tolerance: tm dissolution (Imzo, /mff) was administered to 8-week-old C57BL/[-i +'llf injection mice per mouse. Peritoneal exudate cells were collected 18 hours after intraperitoneal administration of .3 mfl. 2×10″ cells/m of mt+bacteria in the collected solution in RPMI 640 medium supplemented with 10% beef tallow
After adjusting ffK, transfer to a plastic petri dish and heat at 37°C.
, ink-bating for 90 minutes under 5'X carbon dioxide atmosphere,
Adhesive fine TLF on petri dish: 2, non-adherent 1 cow 411
I got a cell. 5 x 10 cells of each cell and 51 Cr-labeled mouse leukemia cells YA C-1, IXl, 0' were added to chilled RPMT164.0 medium containing 10% beef tallow broth at 137°C in a 5% carbon dioxide atmosphere. The cells were cultured for 4 hours.

The radioactivity of 51Cr released in the culture supernatant was measured using an autogamma counter (manufactured by Packard), and the cytotoxic effect of peritoneal exudate cells on YAC-1 cells was investigated.

No. 4 NK, l-111 cell l-divination enhancement effect test example 5
The inhibitory effect on L1al cell proliferation was investigated.

The test and test system 1d are as follows.

8J x 10' i-plane YAC-] cells and +5
．． 4 x 10' F M3 A threads 11] cells were each suspended in RPMIi640 medium supplemented with 10% beef tallow and pure f65, and 1 m of 241 Morima cultured RD-AI was cultured at 37°C under a 5% carbon dioxide atmosphere. The culture was continued further under the same conditions. @After the start of feeding 2
The number of viable cells was measured by trypan fluorescent staining every 4 hours.The results are shown in Table 5.

Test Example 6 Acute Toxicity Group 10 5-week-old ddY male mice (average size 41L)
231F), R+)-A 1 was dissolved in physiological saline and administered intravenously at 500 mfi'/kg 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% lethality rate (LD) for intravenous administration of the substance of the present invention, o, is 5.
It is thought to be more than 0.00 mzo/kg.

Test Example 7 Antigen I/I:.

RD-AI dissolved in physiological saline was administered to 6 Hartley strain guinea pigs (average weight 348S).
[mS'] per animal was intradermally administered 3 times a week for a total of 6 times to sensitize. RD-A after 2 weeks from the last sensitization date
Dissolve I in physiological saline and add 10m per 1cg of body weight.
f or 2n11 was administered intravenously. The toxicity of the substance of the present invention was investigated by observing the behavior of guinea pigs before and after 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 the drawings] Figure 1 shows the elution diagram of the substance of the present invention by Sepharose CL-fi R column chromatography, Figure 2 shows the ultraviolet absorption spectrum of the intermediate 'tji, and Figures 3 and 3 show the same. Showing the infrared absorption spectrum of a substance'3-8 Akito Mitsu, Mitsui Toatsu Chemical Co., Ltd.
Meng 1 Koku 0 10 20
, JO word, test tube lead r water) 薯 2 Concave z2θ z4θ 26θ zgθ Jθθ J〃Wavelength (ylm)

Claims (1)

[Scope of Claims] 1. Heatable deoxylipo-nucleate RD-01 and its salts which exhibit anti-tumor activity through heat denaturation and exhibit physicochemical properties (due to Na salt) of 1. (1) Elemental analysis (%): C: 26.18-31.3
1) 1:4.02-4.98N:12.11-13.
89P: 8.13-9.18Na: 3
．． 02-4.28 (2) Molecular weight: 30,000-1,000,000 molecules Y (distribution diagram is 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, ethanol. Insoluble in methanol, ether, and acetone. (7) Coloration Reaction A) Orcinol reaction: R fractionated by STS method
Negative for NA 1ifti. B) D fractionated by diphenylamine reaction 5STS method
Positive for NA fraction. C) Ninhydrin reaction: this substance] (ltt?/mQ
- Negative for concentration. D) Anthrone reaction: This substance I Ofif//mQ) Negative at illuminance. (8) Distinguish between basic, neutral, and neutral The pH of the aqueous solution of the two substances is 6.5 to 75. (9) Material: White powder 00) Characteristics: It becomes soluble through heat treatment, has high antitumor activity, and has extremely low toxicity and antigenicity. (11) Base composition diagram) Nigeanine: 33.4 Adenine: 17.2 Cysyn: 32.5 Thymine:
+ 6.9 (method is based on chemical analysis) (+2 When two enzyme-treated substances are treated with deoxyribonuclease l (DNase l), anti-llll1l
! Although the tumor property disappears, ribonuclease T2 (RNas
In the case of eT2), the anti-tumor activity did not change. (Analysis of hydrogizoapatite by column chromatography shows that it has a single-stranded structure. (b) Transition temperature (Tm): From the measured melting curve, there is a clear transition temperature] ゛m (σ1. 2.C that was caused by Propionibacterium spp.
Heat-denatured deoglybonucleic acid RD-01° according to claim 1, which is
711: Heat-denatured thioxyribonucleic acid RD-01 having anti-ulcer activity characterized by heat-denaturing a nucleic acid fraction obtained from a cell extract.
The method of construction. 4. The production method according to claim 3, characterized in that the method for separating the nucleic acid fraction is to treat the cell extract with an organic solvent and divide it into aliquots. 5. As a method for separating a nucleic acid fraction, 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 Scope No. 3. 6. Propi, 1 piece of J. nibacterium genus bacteria
41. Obtained by separating the bundle stones. A nucleic acid flocculant is added to the wireless 1 blood extract, and the resulting precipitate is mixed with 1 part water or salt.
Heat-denatured deoxyribonucleic acid RD- (Production method of 11. Approximately 80° to 120° to the nucleus
Heat-denatured deoxyribonucleic acid RD-0 having anti-tumor jM activity, characterized in that it has anti-tumor properties when heated to C.
1 manufacturing method. 8. An anti-tumor agent containing heat-denatured deoxyribonucleic acid 1''19RD-01 as an active ingredient, which is made by heat-denaturing anti-III4 to have tumor activity. 9. Heat-denatured deoxyribonucleic acid RD-01 or Propionibacterium spp. (1!lπ"
The antigenic tumor agent according to claim 8.