NO155697B - PROCEDURE FOR THE PREPARATION OF PHYSIOLOGICALLY ACTIVE COMPOUNDS. - Google Patents

Description

This invention relates to a method for

position of new carcinostatic and immunostimulating TF-

substances that are TF-100, 110, 120, 130, (1316, 132a, 132b,

133a, 133b, 1323, 136), 140 and 150, under which Fusobacterium nucleatum TF-031 (ATCC 31647) is grown anaerobically

conditions in a culture medium containing nitrogen-

sources, carbon sources, vitamin sources, reducing agents and inorganic

ganic salts and possibly additionally agar and set on

a pH value from 5 to 8.5, adds the culture or super-

the supernatant a hydrophilic organic solvent, separates from the thus formed precipitate and then collects the water-soluble fraction of the precipitate, after which the following treatment (1), (2), (3) or (4) is optionally carried out,

(1) dialyze or ultrafilter the water-soluble

fraction and then isolates a product fraction from the internal solution and optionally, treats this product fraction with an ion exchanger and isolates an adsorbed product fraction or a non-adsorbed product fraction, or adds product

the fraction isolated from the internal solution a quarter-

near ammonium salt, collects the formed precipitate, then carries out a dissociation treatment and isolates a product fraction from the treated liquid,

(2) treating the water-soluble fraction with an ion exchanger and

isolates the adsorbed fraction or the non-adsorbed fraction

faction,

(3) adds barium ions to the water-soluble fraction

(in which case the culture or supernatant may be used instead of the water-soluble fraction), separate from the barium salt formed,

removes the barium from the salt, then dialyzes or ultrafil-

filters the water-soluble fraction and isolates a product

fraction ion from the internal solution,

(4) adds a quaternary to the water-soluble fraction

ammonium salt, collects the formed precipitate, dissociation

processes this and then isolates a product fraction from the treated

traded liquid.

In recent years, there has been widespread use of, as a drug for patients with various types of cancer, a drug that includes increasing the immunological function of the host and achieving a carcinostatic effect with the help of the immunological function. As antitumor agents used for such a drug, there are known components obtained from organisms of various bacteria or cultures of various bacteria or polysaccharides obtained from fruiting bodies of Basidiomycetes or cultured fungal bodies therefrom. However, these antitumor agents are not yet satisfactory with regard to effect, harmful side effects and manufacturing method.

On the other hand, an organism and an overflow fluid obtained by cultivating bacteria belonging to the genus Fusobacterium, for example Fusobacterium K031-3B, Fusobacterium fusifomis W-12, Fusobacterium girans 1012 and Fusobacterium nucleatum 1010 [Dental Surgery, 2_3' 322 -333

(1974) and 21, 534-539 (1972) (Japanese)]. However, no investigation has yet been made with respect to the components obtained from a culture, or its supernatant fluid, produced by cultivating bacteria belonging to the genus Fusobacterium, and the pharmacological activities of the components.

The present inventors have investigated the pharmacological activity of a superfluid obtained by culturing bacteria belonging to the genus Fusobacterium, and removing the organisms from the culture, to find that a specific component obtained from the superfluid has a carcinostatic activity, that said component essentially has no effect on inhibiting the formation of a colony of cancer cells by a colony-foaming test method, and that it has no carcinostatic activity to kill the cancer cells, but an indirect carcinostatic activity by increasing the host-mediated anti-tumour activity or the immunity of the host and utilization of the aid in the immunity, and that said component has very low toxicity and can also be obtained by treating the culture.

Advantages of this invention will be apparent from the following description.

The bacteriological characteristics of Fusobacterium nucleatum TF-031 are as follows:

(In shape

(1) Shape of the cells: coil-shaped (fig. 1) (2) Polymorphism of the cells: absent

(3) Mobility: absent

(4) Spores: absent

(5) Gram stain: Gram negative

(6) Acid-resistant: negative

(II) Growth conditions in a culture medium

(1) TF - an agar plate and slant culture medium Outer shape: a round shape

Size: approx. 1 mm

Swelling: hemispherical shape Structure: dewdrop-like Surface: smooth

Edges: smooth

Colour: milky yellowish white Transparency: cloudy

(2) TF - a liquid culture medium

Degree of growth: vigorous

Turbidity: coagulate

Explanation: none

Growth on surface: none, no growth until one

depth of approx. 5 mm Gas: none

(III) Physiological properties

(1) Production of hydrogen sulphide: +

(2) Reduction of Nitrates:-

(3) Production of butyric acid: +

(4) Production of indole: +

(5) Urease:-

(6) Catalase:-

(7) Hydrolysis of Starch:-

(8) Behavior towards oxygen: anaerobic

(9) Production of ammonia: +

(10) Production of carbon dioxide.: +

(11) Growth range: pH 5-8.5, temperature 30-45°C (12) Production of gas from saccharides: L-arabinose (-), D-xylose (-), D-glucose (-), D-mannose (-), D-fructose (-), D-galactose (-), malt sugar (-), sucrose (-), trehalose (-), sorbitol (-), mannitol (-), inositol ( -),

glycerol (-), starch (-).

The new TF substances according to this invention are produced, for example, in the following way.

The above production process is illustrated as follows:

(1) Cultivation of bacteria.

The cultivation of bacteria belonging to the genus Fusobacterium is carried out by a standard method for the cultivation of anaerobic bacteria. That is, a culture medium containing a nitrogen source, such as calf-brain-heart extracts, various peptones or the like, a vitamin source, such as yeast extract or the like, an inorganic salt, such as sodium chloride or the like, a carbon source , such as glucose, lactose or the like, a reducing agent, such as L-cystine, sodium sulphite, thioglycollate or the like, is adjusted to a pH of 6 to 8.5, preferably 7.2 to 8.2, and the bacteria are inoculated onto the culture medium, after which steady-state cultivation is carried out under anaerobic conditions at 35 to 42°C, preferably 36 to 38°C, for 1-5 days, preferably 24-72 hours. It is particularly desirable to use the culture medium described in table 1 (hereinafter referred to as TF culture medium). However, the brain-heart infusion, which is a calf-brain-heart extract, is not always necessary as a carbon source, and it can be replaced by a heart infusion which is a calf-heart extract, a beef- extract,.a fish extract, corn casting fluid or the like. Among the various peptones, proteose-peptone and phyton-peptone are not always necessary, and trypticase-peptone can be replaced by polypeptone.

When agar is not used, it is desirable to stir the culture. (2) Collection of an supernatant fluid from the culture (removal of the organisms).

The organisms are removed from the culture obtained above for

to get an overflowing fluid. For the removal of the organisms, a conventional method can be used, e.g. centrifugation and a filtration method using a filter aid, such as Hyflo Super Cel, and in particular it is advantageous to have a centrifugation method with a view to work operations, provided by the removal of the organisms and yield of the overflowing fluid. The organisms are preferably removed in this step, but they can be removed in the subsequent step (3).

(3) Collection of carcinostatic substance TF-100.

A hydrophilic organic solvent is added to the supernatant obtained above or to the culture, and the resulting supernatant is collected. At this point, the supernatant fluid or culture is advantageously adjusted to a pH of 2 to 7. The hydrophilic organic solvent includes, for example, alcohols, such as ethanol, methanol and the like, and ketones, such as acetone and the like, although alcohols, especially ethanol, gives the best; results. The hydrophilic organic solvent is added so that its concentration can be 30 to 70% by volume, and especially 50 to 70% by volume. After the addition of the hydrophilic organic solvent, the resulting mixture is allowed to stand at a low temperature, preferably at about 5°C, for several hours to several days to complete the formation of the precipitate.

The thus formed precipitate is separated by usual methods, such as decantation, centrifugation, filtration and the like.

Then water is added in an amount of 10-15 times the amount for the precipitation obtained above, to the precipitation [the water can be replaced with a phosphate buffer or a phosphate buffer containing sodium chloride when TF-120 or 130 (1316, 132a, 132b, 133a, 133b, 1323 or 136) is intended to be obtained as discussed below] and the water-insoluble material formed is removed by a conventional method, such as centrifugation, filtration or the like, after which the water-soluble fraction is collected. When the culture is used, the organisms are removed by the above methods. The water-soluble fraction thus obtained is dried by lyophilization or the like to obtain a carcinostatic substance TF-100.

The thus obtained TF-100 has the properties shown in table 2.

(4) Collection of carcinostatic substance TF-110.

The water-soluble substance obtained in (3) above is subjected to dialysis or ultrafiltration, or alternatively TF-100 is dissolved in water in an amount 10-15 times that of TF-100 and the resulting solution is subjected to dialysis or ultrafiltration . Low molecular weight substances such as amino acids and inorganic materials are removed by the above treatment. The internal solution obtained by dialysis or ultracentrifugation is collected and then dried to obtain a carcinostatic substance TF-110.

The TF-110 thus obtained has the properties shown in table 2.

(5) Collection of carcinostatic substance TF-120.

The water-soluble fraction obtained in (3) above, or, if necessary, the internal solution by subjecting the water-soluble fraction to dialysis or ultrafiltration (the internal solution obtained in (4) above), or a solution of a powder of TF -110 in a small amount of water, treated with an ion-exchanger. As ion exchanger, a weakly basic ion exchanger or a weakly basic ion exchanger which has the ability to act as a molecular sieve is preferably used, and for example suitable diethylaminoethyl Sephadex A-50 (registered trademark from Pharmacia Co., Ltd.) is used. A column packed with an ion-exchanger is brought

in equilibrium, for example with a phosphate buffer that has a ph of approx. 8, after which the above solution to be treated is passed through the column, and the eluted solution is collected and then dried by lyophilization or the like, or alternatively the same buffer as above and the solution to be treated are placed in a vessel containing an ion exchanger, and the contents are stirred and then filtered, after which the filtrate is dried, and with this a carcinostatic substance TF-120 can be obtained which has the properties described later. TF-120 can also be obtained by desalting the eluted solution or filtrate, for example by dialysis using a cellophane tube or the like, using Sephadex G-25 (registered trademark of Pharmacia Co., Ltd.) or by ultrafiltration, and then drying the same.

The TF-120 thus obtained has the properties shown in table 2.

(6) Collection of carcinostatic substance TF-130.

A column containing the adsorbed components, from which TF-120 has been removed by the treatment in (5) above, is treated with a phosphate buffer, preferably a phosphate buffer having a sodium chloride concentration of 0.1 to 0.6 M and an arrow of approx. 8 (hereafter a phosphate buffer having a sodium chloride concentration of 0.1 to 0.6 M is referred to as a 0.1-0.6 M sodium chloride phosphate buffer), or alternatively the water-soluble fraction becomes obtained in (3) above if necessary subjected to dialysis or ultrafiltration, and then to a treatment with an ion exchanger using a 0.1 - 0.6 M sodium chloride-phosphate buffer, to obtain a fraction which not eluted with a phosphate buffer that is free of sodium chloride, but eluted with a 0.1-0.6 sodium chloride-phosphate buffer.

This method can be carried out either by a column system or by a batch system. The desired fraction thus obtained, which is eluted with the sodium chloride-phosphate buffer, is dried by lyophilization or the like in order to obtain a carcinostatic substance TF-130. The eluate containing the desired fraction eluted with the aforementioned sodium chloride-phosphate buffer can, for example, be desalted by dialysis using a cellophane tube, by molecular sieving using Sephadex G-25 or by ultrafiltration, and then dried .

The term "TF-130" used herein refers collectively to desired eluted fractions obtained by treating the water-soluble fraction obtained in (3) above, or if necessary the internal solution component obtained by subjecting the water-soluble fraction to dialysis or ultrafiltration, with an ion-exchanger, which fractions are not eluted with a phosphate buffer free of sodium chloride, but are eluted with a 0.1-0.6 -\ sodium chloride-phosphate buffer. TF-130 concretely includes, for example, the following factions. (i) A fraction which has not been eluted with a phosphate buffer free of sodium chloride, but has been eluted with a 0.6 M sodium chloride-phosphate buffer in the above ion-exchange treatment [the fraction is designated as TF -1316]. (ii) A fraction which has not been eluted with a 0.1 M sodium chloride-phosphate buffer, but has been eluted with a 0.2 M sodium chloride-phosphate buffer in the above ion-exchange treatment [the fraction is designated as TF-132a and b; TF-132a is obtained by washing an ion exchanger containing the aforementioned adsorbed component with a 0.1 M sodium chloride phosphate buffer, treating the ion exchanger with a 0.2 M sodium chloride phosphate buffer, and then collecting the fraction eluted with the 0.2 M sodium chloride-phosphate buffer; and'TF-132b is obtained by treating the water-soluble fraction obtained in (3) above or the inner solution component (TF-110) obtained in (4) above, with an ion exchanger]. (iii) A fraction which has not been eluted with a 0.2 M sodium chloride-phosphate buffer, but has been eluted with a 0.3 M sodium chloride-phosphate buffer in the above ion-exchange treatment [the fraction is denoted TF -133a and b; a and b have the same meanings as defined for the case of TF-132a and b respectively]. (iv) A fraction which has not been eluted with a 0.1 M sodium chloride-phosphate buffer, but has been eluted with a 0.3 M sodium chloride-phosphate buffer in the above ion-exchange treatment [the fraction is denoted TF- 1323]. (v) A fraction which has not been eluted with a 0.5 M sodium chloride-phosphate buffer, but has been eluted with a 0.6 M sodium chloride-phosphate buffer in the above ion-exchange treatment [the fraction is denoted TF- 136].

Since these TF-1316, 132a, 132b, 133a, 133b, 1323 and

136 have common properties in the following respects, the substances with common properties are denoted by TF-130. That is to say, TF-130 obtained in the manner described above has the following properties:

(a) Greyish white - light brown powder.

(b) It inhibits the proliferation of, in mice, Ehrlich ascites tumor, Ehrlich solid tumor and Sarcoma-180 carcinoma, and has an immunostimulatory activity. (c) It is soluble in water and is insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether. (d) It has no clear melting point, begins to decompose at approx. 110°C, and decomposes remarkably above 200°C.

(c) Its infrared absorption spectrum obtained with

a KBr table method has absorption bands in the vicinity of 3600-3200, 2960-2930, 1670-1640, 1550, 1440-1380, 1240, 1140-1000 and 820 cm<-1. >(f) The ultraviolet absorption spectrum of its aqueous solution shows a strong absorption at the absorption edge, showing an absorption peak near 256-280 nm. (g) It is positive by Molisch reaction, phenol-sulphuric acid reaction, anthrone-sulphuric acid reaction, indole-hydrochloric acid reaction and Lowry-Folin's reaction, and negative by ninhydrin reaction.

(h) Elemental analysis values:

C: 27.5-39.8%, H: 3.2-5.7%, N: 2.8-6.3%. (i) Its saccharide content measured by a phenol-sulphuric acid method is approx. 19.0 - approx. 64.0% by weight (expressed by glucose), and its protein content measured by Lowry-Folin's method is in the vicinity of 6.0 - 28.0% by weight (expressed by calcium-serum-albumin).

The above ion-exchange treatment is explained more specifically below. As the ion exchanger used in the following methods, a weakly basic ion exchanger or a weakly basic ion exchanger capable of molecular sieve is preferred, and diethylaminoethyl Sephadex A-50 used in (5) above is, for example, suitably used, and the following method can be carried out by a batch system. (i) A 0.6 M sodium chloride-phosphate buffer preferably having a pH of about 8, is passed through a column of an ion exchanger containing the adsorbed component through which the solution to be treated has passed at (5) above, and the eluted fraction is collected, possibly concentrated and desalted, by dialysis, ultrafiltration or the like, and then dried by lyophilization or the like to obtain a carcinostatic substance TF-1316.

The thus obtained TF-1316 has the properties shown in table 2. (ii)-l) A 0.1 M sodium chloride phosphate buffer which preferably has a pH of approx. 8, is passed through a column of an ion exchanger containing the adsorbed component through which the solution to be treated has passed in (5) above, to wash the column, after which a 0.2 M sodium chloride-phosphate buffer preferably having a pH of approx. 8 is passed through the column, and the eluted fraction is collected, optionally concentrated and desalted by dialysis, ultrafiltration or the like, and then dried by lyophilization or the like to obtain a carcinostatic substance TF-132a.

The TF-132a thus obtained has the properties shown in Table 2. (ii) -2) Methods for collecting the fraction that has not been eluted with a 0.1 M sodium chloride-phosphate buffer, but has been eluted with a 0.2 M sodium chloride-phosphate buffer in the ion-exchange treatment is carried out in the following manner using the water-soluble fraction obtained in (3) above or the internal solution in (4) above, to obtain TF-132b. That is, the column packed with the ion exchanger is brought into equilibrium with a 0.2-0.3 M sodium chloride-phosphate buffer which preferably has a pH of approx. 8, after which the aforementioned 0.2-0.3 M sodium chloride-phosphate buffer in the water-soluble fraction obtained in (3) above or the internal solution component obtained by subjecting the water-soluble fraction to dialysis or ultrafiltration is passed through the column, and the eluted solution is collected. If necessary, wash the column with the aforementioned 0.2-0.3 f4 sodium chloride-phosphate buffer, and the eluted solution and washes are combined. The ion exchange treatment can be carried out several times. Then a solution, prepared by diluting the aforementioned eluted solution with a phosphate buffer so that the sodium chloride concentration can be 0.1 M, is passed through a column in which the ion exchanger is brought into equilibrium with 0.1 M sodium chloride -phosphate buffer which preferably has a pH of approx. 8, and the fraction eluted with the aforementioned 0.1 M sodium chloride-phosphate buffer is removed, after which the aforementioned 0.2 M sodium chloride-phosphate buffer is passed through the column to obtain an eluate. In the concrete example of the above-mentioned ion-exchange treatment, a fraction that has not been adsorbed on an ion-exchanger is brought into equilibrium with the aforementioned 0.2 M sodium chloride-phosphate buffer, collected, adsorbed on an ion-exchanger brought into equilibrated with the aforementioned 0.1 M sodium chloride-phosphate buffer, washed with the aforementioned 0.1 M sodium chloride-phosphate buffer and then eluted with the aforementioned 0.2 M sodium chloride-phosphate buffer to obtain an eluate. A method can also be used whereby, on the other hand, the fraction that has been adsorbed on an ion exchanger brought into equilibrium with the aforementioned 0.1 M sodium chloride-phosphate buffer is collected, and where a method is then carried out for the separation of said fraction from a fraction that has been adsorbed on an ion exchanger brought into equilibrium with the aforementioned 0.2 M sodium chloride-phosphate buffer, and a fraction that has been eluted with the aforementioned 0.2 H sodium chloride-phosphate buffer, is collected.

The eluate, if any, obtained in the manner described above is then concentrated and desalted by dialysis, ultra-

in

filtrerina or the like, and then dried by lyophilization

or the like to obtain a carcinostatic substance TF-132b.

The TF-132b thus obtained has the properties shown in Table 2. (iii)-l) The internal solution component obtained in (4) above is passed through a column of an ion exchanger which is brought into equilibrium with a phosphate buffer which preferably has a pH of approx. 8, and a 0.2 M sodium chloride-phosphate buffer which preferably has a pH of approx. 8 is passed through said column to wash the column, and then a 0.3 M sodium chloride-phosphate buffer is passed which preferably has a pH of approx. 8, through said column, or alternatively the aforementioned 0.3 M sodium chloride-phosphate buffer is passed through the column which has been treated in (ii)-l) above and still has the remaining adsorbed component. The eluted fraction is collected, optionally concentrated and desalted by dialysis, ultrafiltration or the like, and then dried by lyophilization or the like to obtain a carcinostatic substance TF-133a.

The TF-133a thus obtained has the properties shown in Table 2.

(iii)-2) The fraction which has not been eluted with a 0.2 M sodium chloride-phosphate buffer, but which has been eluted with a 0.3 M sodium chloride-phosphate buffer during the ion-exchange treatment, is collected on the following method, which is the same as in the above (ii)-2) to obtain TF-133b. That is, the column packed with the ion exchanger is brought into equilibrium with a 0.3 M sodium chloride-phosphate buffer which preferably has a pli of approx. 8, after which the aforementioned 0.3 M sodium chloride phosphate buffer in the water-soluble fraction obtained in (3) above or the internal solution component obtained by subjecting the water-soluble fraction to dialysis or ultrafiltration is passed through the column , and the eluted solution is collected. If necessary, the column is washed with the aforementioned 0.3 M sodium chloride-phosphate buffer, and the eluted solution and washings are combined. The ion-exchange treatment can be carried out several times. Then a solution prepared by diluting the aforementioned eluted solution with a phosphate buffer so that the sodium chloride concentration can be 0.2 M is passed through a column in which the ion exchanger has been brought into equilibrium with a 0.2 M sodium chloride-phosphate buffer which preferably has a pH

of approx. 8, and the fraction that has been eluted with the pre-

said 0.2 M sodium chloride-phosphate buffer is removed, after which the aforementioned 0.3 sodium chloride-phosphate buffer is passed through the column to obtain the eluate. In the concrete example of the above ion-exchange treatment, a fraction that has not been adsorbed on an ion-exchanger brought into equilibrium with the aforementioned 0.3 M sodium chloride-phosphate buffer, collected, adsorbed on an ion-exchanger brought into equilibrated with the aforementioned 0.2 M sodium chloride-phosphate buffer, washed with the aforementioned 0.2 M sodium chloride-phosphate buffer, and then eluted with the aforementioned 0.3 M sodium chloride-phosphate buffer to obtain an eluate. A method can also be used whereby, on the other hand, a fraction that has been adsorbed on an ion exchanger brought into equilibrium with the aforementioned 0.2 M sodium chloride-phosphate buffer is collected, and where a method for separating said fraction is then carried out from a fraction adsorbed on an ion exchanger equilibrated with the aforementioned 0.3 M sodium chloride-phosphate buffer, and a fraction eluted with the aforementioned 0.3 M sodium chloride-phosphate buffer are collected.

Then, optionally, the eluate obtained in the manner described above is concentrated and desalted by dialysis, ultrafiltration or the like, and then dried by lyophilization or the like to obtain the desired carcinostatic substance TF-133b.

The TF-133b thus obtained has the properties shown in

table 2.

(iv) The water-soluble fraction obtained in (3) above

or the internal solution component obtained in (4) above, is prepared in a 0.3 M sodium chloride-phosphate buffer which preferably has a pH of approx. 8, and the resulting buffer is passed through an ion-exchange column which has previously been brought into equilibrium with a 0.3 M sodium chloride-phosphate buffer which preferably has a pH of approx. 8. The eluted solution is adjusted by adding a phosphate buffer so that the sodium chloride concentration can be 0.1 M, and is then passed through an ion-exchange column that has previously been brought into equilibrium with a 0.1 M sodium chloride phosphate buffer which preferably has a pH of approx. 3, and then the aforementioned 0.3 M sodium chloride-phosphate buffer is passed through said column, after which the eluted fraction is collected, optionally concentrated and desalted by dialysis, ultrafiltration

or the like, and then dried by lyophilization or the like to obtain a carcinostatic substance TF-1323.

The TF-1323 thus obtained has the properties shown in

table 2.

(v) The internal solution component obtained in (4) above is passed through a column with an ion exchanger brought into equilibrium with a phosphate buffer which preferably has a pH of approx. 8, and a 0.5 M sodium chloride-phosphate buffer which preferably has a pH of approx. 8, is passed through said column to wash the column, and then a 0.6 M sodium chloride-phosphate buffer is passed which preferably has a pH of approx. 8, through said column, or alternatively the aforementioned 0.5 M sodium chloride-phosphate buffer is passed through the column, which has been treated in (ii)-2) above and still has the remaining adsorbed component, after which the aforementioned 0.6 M sodium chloride- phosphate buffer is passed through said column. The eluted fraction is collected, optionally concentrated and desalted by dialysis, ultrafiltration or the like, and then dried by lyophilization or the like to obtain a carcinostatic substance TF-136.

The TF-136 thus obtained has the properties shown in

table 2.

(7) Collection of carcinostatic substance TF-140.

Barium ions are added to the culture or its supernatant fluid obtained in (1) or (2) above or to the water-soluble fraction obtained in (3) above or to the solution obtained by dissolving TF-100 in water, to form a barium salt, after which the precipitate is collected, and then subjected to a process to remove the barium, and the water-soluble fraction is collected, and then subjected to dialysis or ultrafiltration to obtain TF-140. Specifically explained is an aqueous barium hydroxide solution, preferably a 0.1-0.5 1

aqueous barium hydroxide solution, added to the culture or its supernatant fluid obtained in (1) or (2) above or to the water-soluble fraction obtained in (3) above or to an aqueous solution obtained by dissolving TF-100 in water in an amount of approx. 10-15 times that of TF-100, and the pH of the resulting mixture is preferably adjusted to 10-13 to form a barium salt. A precipitate is deposited by the addition of a barium

hydroxide solution, and the added amount of barium ions is preferably adjusted so that the concentration of the total barium ions in the final volume of the mixture can be 0.005 to 0.1 M. The mixture thus obtained is filtered, and a method of separation of the barium from the obtained barium salt is carried out. The barium is preferably removed by adding the barium salt to sodium sulfate, preferably a 5-15% aqueous sodium sulfate solution, stirring the resulting mixture and then performing a filtration process to obtain a solution. The precipitate which has been separated and removed is again washed with an aqueous sodium sulfate solution, and the washing liquids are combined with the above solution. The solution thus obtained is subjected to dialysis, ultrafiltration or the like, to remove the excess of sodium sulfate or low molecular weight substances, and the resulting solution is dried by lyophilization or the like to obtain a carcinostatic substance TF-140.

The TF-140 thus obtained has the properties shown in

table 2.

(8) Collection of carcinostatic substance TF-150.

The water-soluble fraction obtained in (3) above or

the internal solution obtained in (4) above is treated with a quaternary ammonium salt, and the precipitate formed is collected, and then subjected to a dissociation process using a solution containing sodium chloride, after which a hydrophilic organic solvent is added to the soluble fraction,

and the precipitate thus formed is collected and then dried to obtain TF-150. As the quaternary ammonium salt that can be used here, any complex with a polysaccharide can usually be used, and particularly preferred examples thereof include cetyl-pyridinium chloride, cetyl-trimethyl-ammonium bromide, etc. The treatment with a quaternary ammonium salt is preferably carried out in presence of a buffer such as a borate buffer or the like. A concrete description of the above-mentioned treatment with a quaternary ammonium salt and the dissociation process using a solution containing sodium chloride is as follows. For example, an aqueous solution of a quaternary ammonium salt is added dropwise to the water-soluble fraction up-

reached in (3) above or the internal solution obtained in (4) above while keeping the pH slightly basic, and the resulting mixture is stirred at 0-50°C for 10 minutes to several hours, after which the deposited precipitate is collected. The precipitate is washed several times with a buffer, preferably a 0.1% borate buffer, containing 0.1 M of sodium chloride and a small amount of a quaternary ammonium salt, and then dissociated with a buffer, preferably a 0.1% borate buffer , containing 0.5 M of sodium chloride and a small amount of a quaternary ammonium salt to obtain a soluble fraction, and then preferably the soluble fraction is adjusted to a pH of 2-6. A hydrophilic organic solvent, preferably an alcohol, is then added to the soluble fraction so that its concentration can finally be 50 to 90% by volume, preferably 70 to 90% by volume, and the mixture thus obtained is allowed to stand at 0 to 30°C for several hours to 90 hours, after which the deposited precipitate is collected to obtain a carcinostatic substance TF-150.

The TF-150 thus obtained has the properties shown in table 2.

In Table 2, for the Sephadex G-50 marked "*", a column of 50 mmø x 600 mm and distilled water was used as eluent; for Sephadex G-200 (registered trademark of Pharmacia Co., Ltd.) marked "<**>" a column of 44 mmø x 500 mm was used for TF-110, 120, 132a, 133a, 136 and 140, and a column of 21 mmø x 400 mm for TF-1316, 132b, 133b, 1323 and 150, and as eluent a 0.1 M phosphate buffer with a pH of 7 was used for all, and when obtaining a liquid chromatogram, labeled ''<***>" with high performance, a 7.9 mm x 600 mm x 2 column of TSK-GEL G300SW (trade name of Toyo Soda Co., Ltd.) was used, and the elution was carried out at room temperature with a flow rate of 0.8 ml/min using 0.1 M phosphate buffer with a pH of 7 as eluent.

The pharmacological activities of the present carcinostatic substances TF-100, TF-110, TF-120, TF-1316, TF-132a, TF-132b, TF-133a, TF-133b, TF-1323, TF-140 and TF-150 are as follows: (I) 'Effect of TF substances on cell viability (colony formation test).

Based on the method of Kim. J. H. et al. [Cancer Res. 25, 698 (1965)], HeLa S-3 cells were cultured in eagle's MEM supplemented with 10% calf serum for 3-5 days at 37°C, CO 2 ~ incubator, after which the culture was removed, and a PBS( -)solution (an aqueous solution containing 8.0 g/l of sodium chloride, 0.2 g/l of potassium chloride, 1.15 g/l of sodium phosphate, monobasic, and 0.2 g/l of potassium phosphate, dibasic) containing 0.01 wt% ethylenediaminetetraacetic acid and 0.1 wt% trypsin were added to the glass-adherent cells. The cells were stripped from the glass surface of the culture bottle, and were dissociated into single cells by pipetting, and then the number of cells was counted with a microscope. The cell suspension was diluted with eagle's MEM supplemented with 20% calf serum so that it could contain 200 cells per ml. After 1 ml of the cell suspension was inoculated onto petri dishes and cultured in CC^ incubator at 37°C

for 24 hours, the supernatant fluid obtained in Example 1 (1), which is mentioned later, was added to the cell suspension so that the concentration became 1/16 to 1/128. And to each of the 1 ml portions of the cell suspension cultured at 37°C for 24 hours,

each of the test substances was added, and the HeLa cells were in each case cultured for 7 days. After cultivation, the culture medium was removed, after which the culture dishes were washed with Hank's solution, and the cells were fixed with 70 wt% ethanol, and then washed with 100 wt% ethanol. After the ethanol had been removed, the cells were stained with a Gimsa stain solution, after which the colonies were counted, and the cell viability was calculated.

The results are. shown in tables 3 and 4. The cell viability in the tables was calculated from the following equation. The cell viability for each test substance is indicated with an average value of 5 reproductions.

As is clear from this example, the TF substances produced here have a very low cytotoxic effect on

HeLa cells, compared to the superfluid.

(II) Immunostimulatory activity.

Four mice of the ICR strain were used in each group. Each of the test substances was dissolved in 0.2 ml of a physiological saline solution, and was then administered intraperitoneally to the mice. 24 hours after the administration, 0.2 ml of a carbon suspension was prepared by mixing 1 ml of Perikan drawing ink 17 black (manufactured by Gunter-WagnerCo., Ltd.) and 2 ml of a physiological saline solution containing 3 wt% gelatin , intravenously injected into the mouse tail, and 1, 5, 10, and 15 minutes after the injection, 0.02 ml of the blood was collected from the eye socket using a hematocrit capillary coated with heparin, and was immediately diluted and hemolyzed with 1.6 ml of a 0.1% by weight aqueous sodium carbonate solution. This suspension was subjected to colorimetry at 675 nm, and the phagocytotic index, namely the K value, was determined from the equation of Halpern et al.

The mice in the comparison group were administered 0.2 ml of a physiological saline solution.

where CQ = carbon-powder content in blood at time t, and C = carbon-powder content in blood at time t.

The results are shown in tables 5 to 7.

As is clearly evident from tables 5-7, presented

the groups to which each of the manufactured TF substances were administered, the activation of reticuloendothelial macrophages compared with the comparison group, and the cellular immunity of the normal mice was increased.

(III) Carcinostatic activity.

(a) Antitumor activity against Ehrlich ascites tumor.

Ehrlich ascites tumor cells were intraperitoneally inoculated into mice (females, 6 weeks old) of the ICR strain in an amount of 1 x <1>0^ cells per mouse. Then, each of the test substances was dissolved in 0.2 ml of a physiological salt solution, and then administered intraperitoneally to the mice once a day repeatedly for 7 days from the first day after the inoculation of the tumor cells, or once a day on the first day and from there the third day to the seventh day for a total of six days. As for the test substances, in Table 9, each of them was administered to the mice once a day on the first day and from the third day to the seventh day for a total of six days after the inoculation of the tumor cells. To the comparison group, 0.2 ml of a physiological salt solution was administered in the same way as above.

The results are as shown in tables 8 and 9.

As can be seen from tables 8 and 9, it took place with

the group to which each of the manufactured TF substances was administered, a life-prolonging effect, compared to the comparison group.

(b) Antitumor activity against Ehrlich-- f aststof f tumor .

(1) Ehrlich tumor cells were subcutaneously transplanted in the armpit of mice of the ICR strain (females, 6 weeks old) in an amount of 4 x 10 cells per mouse. Then, each of the test substances was administered to the mice once a day repeatedly for 7 days from the first day after the transplantation of the tumor cells, or once a day on the first day and from the third day to the seventh day for a total of 6 days. To the comparison group, 0.2 ml of a physiological salt solution was administered in the same way as described above. The weights of the tumors were determined on the 11th or 14th day after the transplantation of the tumor cells. The weights of the tumors were determined by measuring the longest diameter (a) mm and the shortest diameter (b) mm using calipers, and calculating from the following equation:

The results are shown in table 10.

(2) Ehrlich tumor cells were transplanted subcutaneously at the armpit of mice of the ICR strain (females, 6 weeks old) in an amount of o 4 x 10 6 cells per mouse. Then, each of the test substances was administered to the mice divided into groups for intravenous administration, intraperitoneal administration, subcutaneous administration and intramuscular administration, with a dose of 10 mg/kg or 20 mg/kg once a day on the first day and from the third day to the seventh day, for a total of 6 days, after the transplantation of the tumor cells. To the comparison group, 0.2 ml of a physiological salt solution was administered in the same way as above. The weights of the tumors were determined on the 13th day after the transplantation of the tumor cells.

The results are shown in table 11.

As is clear from tables 10 and 11, observed

there was a tumor growth-inhibiting effect in the groups to which each of the manufactured TF substances was administered.

(c) Antitumor activity against Sarcoma-180 carcinoma. Sarcoma-180 carcinoma was transplanted intraperitoneally into mice of the ICR strain (females, 6 weeks old) in an amount of 1 x 10~* cells per mouse. Then, each of the test substances was administered intraperitoneally to the mice at a dose of 1 mg/kg, 5 mg/kg or 10 mg/kg once a day repeatedly from the first day to the seventh day, a total of 7 days, after the transplantation of the tumor cells, or once a day now on the first day and from the third day to the seventh day, for a total of 6 days. To the comparison group, 0.2 ml of a physiological salt solution was administered in the same way as above.

The results are shown in table 12.

As is clearly evident from Table 12, the groups to which each of the manufactured TF substances was administered had antitumor activities, compared to the comparison group.

(d) Antitumor activity against B-16 melanoma.

(1) B-16 melanoma tumor cells were transplanted subcutaneously at the armpit of mice of the BDF strain (males, 7 weeks old) in an amount of 1 x 10 cells per mouse. Then, TF-133a was intraperitoneally administered to the mice at a dose of 5 mg/kg or 10 mg/kg once a day on the first day and from the third day to the seventh day, a total of 6 days, after the tumor transplantation - cells. In the same way as above, 0.2 ml of a dysiological salt solution was administered to the comparison group, and 20 mg/kg of 5-FU to the group for 5-FU administration.

The results are shown in table 13.

As is clear from Table 13, the groups to which TF-133a was administered had a clear tumor growth inhibitory activity, compared to the comparison group, and the groups to which each of 5 mg/kg and 10 mg/kg of TF- 133a was administered, had similar and more remarkable tumor growth inhibitory activity, respectively, compared to the group to which 5-FU was administered.

(2) The transplantation of B-16 melanoma tumor cells and the administration of TF-132a, TF-133a, 5-FU and a physiological salt solution were carried out in the same manner as in (1) above. On the 21st day after the transplantation of the tumor cells,

the mice were dissected, and the metastasis of the melanoma tumor cells on the lungs was observed.

The results are shown in table 14.

As is clear from Table 14, in the groups to which each of TF-132a and TF-133a was administered, the metastasis of B-16 melanoma tumor cells was remarkably inhibited, compared to the group to which 5-FU was administered.

(IV) Acute toxicity.

LD,.„ for mice is shown in Table 15.

50

As is clear from the above-mentioned pharmaco-tests, the produced TF substances are

useful as carcinostatic agents, they can be expected to have activities against various cancerous diseases and can be expected to have remarkable activities against especially solid cancers. All the TF substances according to this invention have excellent effects, although TF-130, especially, 132a, 132b, 133a, 133b and 1323 are preferred when various points are considered.

The TF substances that are produced here can be an-

are turned after they have been shaped into various pharmaceutical forms such as oral drugs, injections, suppositories or the like, although they are preferably used in the form of a pharmaceutical injection. When used as oral medications, the oral medications may contain various inert shaping agents, and may be shaped as capsules, tablets, powders or granules. When used as injections, they can be any of subcutaneous injections, intramuscular injections, and intravenous injections, and they can be used in the form of a suspension, a solution, or a powder that dissolves upon use. The injections may contain a local anesthetic.

The dose of the produced TF substances

are properly selected depending on the condition of the patient, although it is usually desirable to administer them at a dose for an adult of 0.01 to 50 mg/kg once a day or several times a day, and with regard to the mode of administration, then it is preferably administered by subcutaneous, intramuscular or intravenous injection or by injection into the affected part.

This invention is further explained below with reference to examples and the accompanying drawings, where fig.

1 shows a microscopic photograph showing the form of Fusobacterium nucleatum TF-031 used in this invention, fig. 2 shows an infrared absorption spectrum of the carcinostatic substance TF-100, fig. 3 shows an ultraviolet absorption spectrum of said substance, fig. 4 shows an elution pattern obtained when said substance was subjected to gel filtration using Sephadex G-50, fig. 5 shows a high performance liquid chromatogram, FIG. 6 shows an infrared absorption spectrum of the carcinostatic substance TF-110, fig. 7 shows an ultraviolet absorption spectrum of said substance, fig. 8 shows an elution pattern obtained when said substance was subjected to gel filtration using Sephadex G-200, fig. 9 shows a high performance liquid chromatogram of said substance, fig. 10 shows an infrared absorption spectrum of the carcinostatic substance TF-120, fig. 11 shows an ultraviolet adsorption spectrum of said substance, fig. 12 shows an elution pattern obtained when said substance was subjected to gel filtration using Sephadex G-200, fig. 13 shows a high performance liquid chromatogram of said substance, fig. 14 shows an infrared absorption spectrum of the carcinostatic substance TF-1316, fig. 15 shows an ultraviolet absorption spectrum of said substance, fig. 16 shows an elution pattern obtained when said substance was subjected to gel filtration using Sephadex G-200, fig. 17 shows a liquid chromatogram with high performance of said substance, fig. 18 shows an infrared absorption spectrum of the carcinostatic substance TF-132a, fig. 19 shows an ultraviolet absorption spectrum of said substance, fig. 20 shows an elution pattern obtained when said substance was subjected to gel filtration using Sephadex G-200, fig. 21 shows a high performance liquid chromatogram of said substance, fig. 22 shows an infrared absorption spectrum of the carcinostatic substance TF-132b, fig. 23 shows an ultraviolet absorption spectrum of said substance, fig. 24 shows an elution pattern obtained when said substance was subjected to gel filtration using Sephadex G-200, fig. 25 shows a liquid chromatogram with high performance of said substance, fig. 26 shows an infrared absorption spectrum of the carcinostatic substance TF-133a, fig. 27 shows an ultraviolet absorption spectrum of said substance, fig. 28 shows an elution pattern obtained when said substance was subjected to gel filtration using Sephadex G-200, fig. 29 shows a high performance liquid chromatogram of said substance, fig. 30 shows an infrared absorption spectrum of the carcinostatic substance TF-133b, fig. 31 shows an ultraviolet absorption spectrum of said substance, fig. 32 shows an elution pattern obtained when said substance was subjected to gel filtration by

use of Sephadex G-200, fig. 33 shows a liquid chromatogram with high performance of said substance, fig. 34 shows an infrared absorption spectrum of the carcinostatic substance TF-1323, fig. 35 shows an ultraviolet absorption spectrum of said substance, fig. 36 shows an elution pattern obtained on said substance subjected to gel filtration using Sephadex-200, fig. 37 shows a liquid chromatogram with high performance for said substance, fig. 38 shows an infrared absorption spectrum of the carcinostatic substance TF-136, fig. 39 shows an ultraviolet absorption spectrum of said substance, fig. 40 shows an elution pattern obtained when said substance was subjected to gel filtration using Sephadex G-200, • fig. 41 shows a liquid chromatogram with high performance of said substance, fig. 42 shows an infrared absorption spectrum of the carcinostatic substance TF-140, fig. 43 shows an ultraviolet absorption spectrum of said substance, fig. 44 shows an elution pattern obtained when said substance was subjected to gel filtration using Sephadex G-200, fig. 45 shows a liquid chromatogram with high performance of said substance, fig. 46 shows an infrared absorption spectrum of the carcinostatic substance TF-150, fig. 47 shows an ultraviolet absorption spectrum of said substance, fig. 48 shows an elution pattern obtained when said substance was subjected to gel filtration using Sephadex G-200 and fig. 49 shows a high performance liquid chromatogram of said substance.

Example 1

(1) In each of 15 two-liter culture flasks, each provided with a screw cap, 2 liters of a culture medium containing 34 g of trypticase peptone, 6 g of phyton peptone, 20 g of proteose peptone, 70 g brain-heart infusion, 6 g yeast extract, 15 g sodium chloride, 12 g glucose, 10 g lactose,

0.5 g L-cystine, 0.2 g sodium sulfite, 1.0 g sodium thioglycolate and 1.4 g agar and the culture medium was adjusted to a pH of 7. The culture medium was sterilized under 1.2 atm. at 120°C for 15 minutes, was heated in a boiling water bath for 20 minutes, and then immediately water-cooled, after which a pre-culture test of Fusobacterium nucleatum TF-031, previously prepared by cultivation in a culture medium of the same composition as

above, was inoculated into the above-mentioned water-cooled culture medium under sterile conditions in an amount of 100 ml per culture bottle, and was subjected to stable cultivation in an incubator at 37°C for 48 hours. After cultivation was complete, the culture was centrifuged to remove organisms at 4000 rpm. at 5°C for 20 minutes. The amount of overflowing fluid obtained was approx. 27 litres. (2) To the overflow fluid obtained in (1) above was added 40 liters of ethanol with stirring at 5°C, and the resulting mixture was left at low room temperature until the amorphous precipitate was completely deposited. The mixture was then centrifuged at 6000 rpm. at 5°C for 15 minutes, and the precipitate was collected, washed with ethanol and then dried under reduced pressure to obtain ca. 60 g of uncleaned powder. (3) In 120 ml of water was dissolved 20 g of the crude powder obtained in (2) above, and the water-insoluble materials formed at this time were removed by centrifugation at 7500 r.p.m. for 10 minutes, after which the water-soluble fraction thus obtained was combined with the washing liquids obtained by washing the water-insoluble materials twice with 60 ml portions of water, and the resulting solution was dried under reduced pressure to obtain 14 g of grayish white-light brown powder of TF-100.

Example 2

The solution described in Example 1-(3), which was prepared by combining the water-soluble fraction free from the water-insoluble materials with the washing liquids obtained by washing the water-insoluble materials, was subjected to ultrafiltration using a hollow fiber type ultrafiltration system (membrane no .HI-1: manufactured by Asahi Kasei Kogyo Kabushiki Kaisha), and the internal solution was subjected to lyophilization

to obtain 10 g of grayish white-light brown powder of TF-110.

Example 3

In a small amount of water, 10 g of the powder obtained in Example 2 was dissolved, and the resulting solution was added to a column filled with diethylaminoethyl Sephadex A-50 equilibrated with a 0.025 M phosphate buffer with a pH of 8, and the eluted solution was collected. Furthermore, 2.5 liters of the same phosphate buffer as above was passed through the column, and the eluted solution obtained was combined with the previously collected eluted solution, after which the resulting solution was desalted using a hollow fiber type ultrafiltration system (membrane No. HI-1), concentrated under reduced pressure and then subjected to lyophilization to obtain

470 mg of greyish white-light brown powder of TF-120.

Example 4

In 24 ml of water, 2 g of the powder obtained in Example 1-(2) was dissolved, and the water-insoluble materials were removed by centrifugation at 7500 rpm. for 10 minutes. The water-soluble fraction was dialyzed overnight against distilled water at 5°C using a cellophane tube, and the internal solution was concentrated under reduced pressure. Then the concentrated solution was added to a column filled with 150 ml of diethylaminoethyl-Sephadex A-50, which had previously been equilibrated with a 0.025 M phosphate buffer with a pH of 8, and the column was washed by passing through the 600 ml of a 0.025 M phosphate buffer with a pH of 8, followed by a 0.025 M phosphate buffer with a sodium chloride concentration of 0.6 M and a pH

of 8 was passed through the column to obtain an eluate. The eluate was concentrated under reduced pressure to approx. 100 ml, dialyzed against distilled water at 5°C, concentrated again under reduced pressure, desalted using a column of Sephadex G-25, and then subjected to lyophilization to obtain 470 with grayish white-light brown TF-1316.

Example 5

The solution described in Example 1-(3) which was prepared by combining the water-soluble fraction free of water-insoluble materials and the washing liquids obtained by washing the water-insoluble materials was dialyzed overnight against distilled water at 5°C using a cellophane tube, and the internal solution was concentrated under reduced pressure. The concentrated solution was then added to a column filled with 50 g of diethylaminoethyl Sephadex A-50, which had previously been brought

1 equilibrium with a 0.025 M phosphate buffer with a pH of 8, and

2 liters of a 0.025 M phosphate buffer with a pH of 8 and 2.5

liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.1 M and a pH of 8 were successively passed through the column, after which 2.5 liters of a 0.025 M phosphate

buffer with a sodium chloride concentration of 0.2 and a pH of 8 was passed through the column to elute the active substance adsorbed on the column, and the obtained eluted solution was desalted using a hollow fiber type ultrafiltration system (membrane No. HI-1 ), and then subjected to lyophilization to obtain 600 mg of grayish white-light brown powder of TF-132a.

Example 6

In 1.2 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8, 44.2 g of the powder obtained in Example 1-(2) were dissolved, and the suspended insoluble materials were removed by filtration using

addition of Hyflo Super Cel, after which the resulting filtrate was fed to a column with a diameter of 33 cm which was filled with 500 ml of diethylamino-ethyl-Sephadex A-50, which had previously been brought into equilibrium with a 0.025 .M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8, and the eluted solution was collected. The column was washed with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M pg at a pH of 8, and the washings were combined with the above eluted solution to obtain 1.67 liters of a solution. Loose-

The mixture was again added to an 8 cm diameter column filled with 400 ml diethylaminoethyl Sephadex A-50, which had previously been equilibrated with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8 to obtain 1.68 liters of an eluted solution. The column was washed with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8 to obtain 1.64 liters of washing liquids.

The eluted solution and washings were combined, and a

0.025 M phosphate buffer with a pH of 8 was added thereto to

obtain 4.98 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.2 M and a pH of 8. The buffer solution thus obtained was applied to a column with a diameter of

8 cm which was filled with 500 ml of diethylaminoethyl Sephadex A-50, which had previously been equilibrated with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.2 M and a pH

of 8, and the column was washed with 2 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.2 M and a pH of 8. To 7 liters of a solution prepared by combining the washings with the eluted solution, a 0.025 M phosphate buffer with a pH of 8 was added to obtain 14 liters of a 0.025

M phosphate buffer with a sodium chloride concentration of 0.1 M

and a pH of 8. The thus obtained eluted solution was fed to a column with a diameter of 8 cm which was filled with 500 ml of diethylaminoethyl Sephadex A-50, which had previously been brought into equilibrium with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.1 M and a pH of 8, and the eluted solution was removed. The column was washed with 2 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.1 fl and a pH of 8, after which 2 liters of a 0.025 phosphate buffer with a sodium chloride concentration of 0.2 M and a pH of 8 was passed through the column and the eluted solution was collected. The eluted solution was concentrated and desalted using an ultrafiltration system (filter used: Toyo Ultrafilter UK-10), further desalted using a Sephadex G-25 column, decolorized with activated carbon and then subjected to lyophilization to obtain 880 mg of grayish white-light brown powder of TF-132b.

Example 7

In a small amount of water, 10 g of the powder obtained in Example 2 was dissolved, and the resulting solution was fed to a column filled with diethyl-aminoethyl-Sephadex A-50 which had previously been equilibrated with 0.025 M phosphate buffer with a pH of 8. Then 5 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.2 M and a pH

of 8 was passed through the column, after which 2.5 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8 was passed through the column to elute the active substance adsorbed on the column, and the obtained eluted solution was concentrated under reduced pressure. The concentrated solution was desalted using a cellophane tube, completely desalted using Sephadex G-25 and then subjected to

lyophilization to obtain 600 mg of grayish white-light brown powder of TF-133a.

Example 8

In 1.2 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8, 44.2 g of the powder obtained in Example 1-(2) were dissolved, and the suspended insoluble materials were removed by filtration using Hyflo Super Cel, after which the resulting filtrate was fed to a 3 3 cm diameter column filled with 500 ml diethylaminoethyl Sephadex A-50, which had previously been equilibrated with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8, and the eluted solution was collected. The column was washed with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH

of 8, and the washings were combined with the aforementioned eluted solution to obtain 1.67 liters of a solution. The solution was again added to a column with a diameter of 8 cm which was filled with 40 ml of diethylaminoethyl-Sephadex A-50 which had previously been brought into equilibrium with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8, to obtain 1.68 liters of an eluted solution. The column was washed with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8 to obtain 1.64 liters of washing liquids. The eluted solution and washings were combined, and a 0.025 M phosphate buffer with a pH of 8 was added to obtain 4.98 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.2 M and a pH of 8. The eluted solution thus obtained was fed to a column with a diameter of 8 cm which was filled with 500 ml of diethylamino-ethyl-Sephadex A-50 previously equilibrated with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.2 M and a pH of 8, and the column was washed with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.2 M and a pH of 8, after which 2 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8 was passed through the column and the eluted solution was collected. The eluted solution was concentrated and desalted using an ultrafiltration system (filter used: Toyo Ultrafilter

UK-10), further desalted using a Sephadex G-25 column, decolorized with activated carbon and then subjected to lyophilization to obtain 590 mg of grayish white-light brown powder of TF-133b.

Example 9

(1) 20 g of the unpurified powder obtained in example 1-(2) was dissolved in 120 ml of water, and the insoluble materials formed at this time were removed by centrifugation at 7500 rpm. for 10 minutes, after which the water-soluble fraction thus obtained and the washing liquids obtained by washing the water-insoluble materials twice with 60 ml portions of water were combined, and then subjected to ultrafiltration using a hollow fiber type ultrafiltration system (membrane No. HI-1 ), and the internal solution was subjected to lyophilization to obtain

10 g of greyish white-light brown powder.

(2) In 1 liter of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8, 25 g of the powder obtained by the treatment in (1) above was dissolved, and the resulting solution was added to a column with a diameter of 33 cm which was filled with 500 ml of diethylaminoethyl-Sephadex A-50 which had previously been brought into equilibrium with a

0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8. The column was similarly washed with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8, and the eluted solution and washes were combined to obtain 1.6 liters of a mixture. The mixture was fed to a column with a diameter of 8 cm

which was filled with 400 ml of diethyl-aminoethyl-Sephadex A-50 which was similarly equilibrated with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8

to obtain 1.65 liters of an eluted solution. The eluted solution was combined with 1.7 liters of the washings obtained by washing the column with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8, and the resulting mixture was diluted with a 0.025 M phosphate buffer with a pH of 8 to prepare 10.35 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.1 M and a pH of 8.

Then the buffer solution thus obtained was applied to a column with a diameter of 8 cm which was filled with 500 ml of diethylaminoethyl Sephadex A-50 which had previously been brought into equilibrium with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.1 M and a pH of 8, and the column was washed with 2 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.1 M and a pH of 8, after which 2 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8 was passed through the column, and the eluted solutions were collected. The eluted solution obtained was desalted and concentrated using an ultrafiltration system (filter used: Toyo Ultrafilter UK-10), and further desalted on Sephadex G-25, decolorized with activated carbon, and then subjected to lyophilization to obtain 1.65 g with grayish white-light brown powder of TF-1323.

Example 10

In a small amount of water, 10 g of the powder obtained in Example 2 was dissolved, and the resulting solution was applied to a column filled with diethylaminoethyl Sephadex A-50 previously equilibrated with a 0.025 M phosphate buffer with a pH of 8. Then 6 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of

0.5 M and a pH of 8 passed through the column, after which 2.5

liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.6 M and a pH of 8 was passed through the column to elute the active substance adsorbed on the column, and the resulting run-through solution was desalted using a hollow fiber type ultrafiltration system (membrane No. HI-1), concentrated under reduced pressure and then subjected to lyophilization to obtain 130 mg of grayish white-light brown powder of TF-136.

Example 11

15 g of the crude powder obtained in Example 1-(2) was dissolved in 200 ml of water, and the water-insoluble materials were removed by centrifugation at 7500 rpm. for 10 minutes, after which the water-soluble fraction thus obtained was adjusted to pH 10 by dropwise addition thereto of a 0.2 M aqueous barium hydroxide solution. A white precipitate was deposited upon the addition of the aqueous barium hydroxide solution. Barium ions were added so that the total concentration of barium ions in the final volume was 0.01 M, after which the solution was stirred for 30 minutes and the precipitate was collected by filtration. The thus obtained precipitate of barium salt was suspended in 10 ml of a 10% by weight aqueous sodium sulfate solution, was sufficiently stirred and then filtered and the filtrate was collected. The precipitate was again suspended, stirred and then filtered in the same manner as above, once with 10 ml of a 10% by weight aqueous sodium sulfate solution and twice with 5 ml portions of said solution and each filtrate was collected. Furthermore, the remaining precipitate was washed twice with

10 ml portions of water and then filtered to obtain the washing liquids. The above collected filtrate and washings were combined, dialyzed against distilled water, concentrated under reduced pressure and then subjected to lyophilization to obtain

0.45 g of greyish white-light brown powder of TF-140.

Example 12

32 g of the powder obtained was dissolved in 450 ml of water

in Example 1-(2), and the suspended insoluble materials were removed by filtration using Hyflo Super Cel, after which the filtrate obtained was dialyzed overnight against running water using a cellophane tube. To the dialyzed solution, 80 ml of a 20% by weight aqueous cetylpyridinium chloride solution was added dropwise with stirring, while 200 ml of a 10% by weight borate buffer (pH 9.0) was added thereto, and the resulting mixture was stirred at room temperature for 30 minutes, after which the precipitate was collected by filtration. The precipitate was suspended in 150 ml of a 0.1 wt% borate buffer

(pH 9.0) containing 0.1 M sodium chloride and 0.2 wt% cetylpyridinium chloride, and the resulting suspension was stirred for 30 minutes. Then the precipitate was collected from the suspension by filtration, and again suspended in 150 ml of a borate buffer (pH 9.0) of the above composition, and the suspension thus obtained was stirred for 30 minutes, after which the precipitate was collected by filtration. The collected precipitate was suspended in 150 ml of a 0.1 wt% borate buffer (pH 9.0) containing 0.5 M sodium chloride and 0.2% cetylpyridineJ

chloride, and the resulting suspension was stirred and dissociated for 30 minutes. Then the precipitate was separated from the suspension by filtration to obtain a soluble fraction. Furthermore, the precipitate separated by filtration was again subjected to the same dissociation process as above to obtain a soluble fraction. The two soluble fractions were combined, and the resulting soluble fraction was adjusted to pH 3-4 with 10% hydrochloric acid, after which ethanol was added thereto so that its final concentration could be 80 vol.l. The resulting solution was left overnight at 5°C, and the precipitate formed was collected by filtration to obtain 5.6 g of grayish white-light brown powder of TF-150.

Claims (31)

1. Procedure for the production of new carcinostatic or immunostimulating TF substances which are TF-100, TF-110, TF-120, TF-130, TF-1316, TF-132a, TF-132b, TF-133a, TF-133b , TF-1323, TF-136, TF-140 and TF-150, characterized in that Fusobacterium nucleatum TF-031 (ATCC 31647) is grown under anaerobic conditions in a culture medium containing nitrogen sources, carbon sources, vitamin sources, reducing agents and inorganic salts and possibly additionally agar and adjusted to a pH value of 5 to 8.5 , adds a hydrophilic organic solvent to the culture or supernatant, separates from the resulting precipitate and then collects the water-soluble fraction of the precipitate, after which the following treatment (1), (2), (3) or (4) is optionally carried out, (1) dialyze or ultrafilter the water-soluble fraction and then isolate a product fraction from the internal solution and optionally treat this product fraction with an ion exchanger and isolate an adsorbed product fraction or a non-adsorbed product fraction, or add the product fraction isolated from the internal solution a quaternary ammonium salt, collects the formed precipitate, then carries out a dissociation treatment and isolates a product fraction from the treated liquid, (2) treating the water-soluble fraction with an ion exchanger and isolating the adsorbed fraction or the non-adsorbed fraction; (3) adding barium ions to the water-soluble fraction (wherein the culture or supernatant may be used instead of the water-soluble fraction), separating from the barium salt formed, removing the barium from the salt, then dialyzing or ultrafiltering the water-soluble fraction and isolating a product fraction from the internal solution, (4) adding a quaternary ammonium salt to the water-soluble fraction, collecting the formed precipitate, dissociating it and then isolating a product fraction from the treated liquid. 2. Method according to claim 1 for the production of TF-100, TF-110, TF-120, TF-130, TF-1316, TF-132a, TF-132b, TF-133b, TF-1323 and TF-136, characterized by cultivating Fusobacterium nucleatum TF-031 (ATCC 31647) under anaerobic conditions in a culture medium that contains nitrogen sources, carbon sources, vitamin sources, reducing agent and inorganic salts and possibly additionally agar, and setting the pH value to 5 to 8.5, adding a hydrophilic organic solvent to the culture or supernatant, which thereby separates from the formed precipitate, and then collects the water-soluble fraction of the precipitate, after which the following treatment (1) or (2) is optionally carried out: (1) dialyze or ultrafilter the water-soluble fraction , isolates a product fraction from the internal solution and optionally treats this product fraction with an ion exchanger and isolates an adsorbed product fraction or a non-adsorbed product fraction, (2) treats the water-soluble fraction with an ion-exchanger and isolates an adsorbed product fraction or a non-adsorbed product fraction. 3. Method according to claim 2 for the production of TF-100, characterized in that the water-soluble fraction of the precipitate is taken out from the precipitate and then dried. 4. Method according to claim 2 for the production of TF-110, characterized in that the precipitate formed is isolated; the water-soluble fraction of the precipitate is dialyzed or ultrafiltered and the substances that have carcinostatic and immunostimulating activities are isolated from the internal solution. 5. Method according to claim 2 for the production of TF-120, characterized in that the precipitate formed is isolated; the water-soluble fraction of the precipitate, if necessary, is dialyzed or ultrafiltered, and then treated with an ion exchanger; and the unadsorbed fraction is isolated. 6. Method according to claim 2 for the production of TF-130, TF-1316, TF-132a, TF-132b, TF-133b, TF-1323 and TF-136, characterized in that the precipitate formed is isolated; the water-soluble fraction of the precipitate is, if necessary, dialyzed or ultrafiltered, and then the fraction isolated from the internal solution is treated with an ion exchanger and the adsorbed product fraction is isolated. 7. Method according to claim 6 for the production of TF-1316, characterized in that the adsorbed fraction is treated with a 0.6 M sodium chloride-phosphate buffer; and the eluted fraction is collected. 8. Method according to claim 6 for the production of TF-132a and TF-132b, characterized in that a fraction which has not been eluted with a 0.1 M sodium chloride-phosphate buffer, but which has been eluted with a 0.2 M sodium chloride-phosphate buffer, is isolated from the adsorbed fraction. 9. Method according to claim 6 for the production of TF-133a and TF-133b, characterized in that a fraction which has not been eluted with a 0.2 M sodium chloride buffer, but has been eluted with a 0.3 M sodium chloride-phosphate buffer, is isolated. 10. Method according to claim 6 for the production of TF-1323, characterized in that a fraction which has not been eluted with a 0.1 M sodium chloride-phosphate buffer, but has been eluted with a 0.3 M sodium chloride-phosphate buffer, is isolated. 11. Method according to claim 6 for the production of TF-136, characterized in that a fraction which has not been eluted with a 0.5 M sodium chloride-phosphate buffer, but has been eluted with a 0.6 M sodium chloride-phosphate buffer, is isolated. 12. Method according to claim 2 for the production of TF-132b, characterized in that the precipitate formed is isolated; the water-soluble fraction, if necessary, is dialyzed or ultrafiltered, a 0.2 M sodium chloride-phosphate buffer is prepared from the water-soluble fraction of the precipitate or a 0.2 M sodium chloride-phosphate buffer from the internal solution component obtained by the dialysis or ultrafiltration, and it is then treated with an ion-exchanger; a fraction which has been passed through the ion exchanger is isolated; this eluted solution is then adjusted so that its sodium chloride concentration becomes 0.1 M, and then treated with an ion exchanger brought into equilibrium with a 0.1 M sodium chloride-phosphate buffer to be adsorbed thereon; and then a fraction is isolated which adsorbs at a 0.1 M sodium phosphate buffer but has been eluted with a 0.2 M sodium chloride phosphate buffer. 13. Method according to claim 2 for the production of TF-1323, characterized in that the precipitate formed is collected; the water-soluble fraction of the precipitate, if necessary, is dialyzed or ultrafiltered, a 0.3 M sodium chloride-phosphate buffer is prepared from the water-soluble fraction of the precipitate or a 0.3 M sodium chloride-phosphate buffer of internal solution component obtained by dialysis or ultrafiltration, and it is then treated with an ion exchanger equilibrated with a 0.3 M sodium chloride-phosphate buffer; a fraction which has been passed through the ion exchanger is isolated; the sodium chloride concentration of this eluted solution is then adjusted to 0.1 M, and the solution is then treated with an ion exchanger equilibrated with a 0.1 M sodium chloride phosphate buffer to be adsorbed thereon; and then a fraction is collected which adsorbs with a 0.1 M sodium chloride phosphate buffer but is eluted with a 0.3 M sodium chloride phosphate buffer. 14. Method according to claim 2 for the production of TF-133b, characterized in that the precipitate formed is collected; the water-soluble fraction of the precipitate, if necessary, is dialyzed or ultrafiltered, a 0.3 M sodium chloride-phosphate buffer is prepared from the water-soluble fraction of the precipitate or a 0.3 M sodium chloride-phosphate buffer of internal solution component obtained during dialysis or ultrasound the filtration, and it is then treated with an ion exchanger equilibrated with a 0.3 M sodium chloride-phosphate buffer; a fraction which has been passed through the ion exchanger is isolated; the sodium chloride concentration of this eluted solution is then adjusted to 0.2 M, and the solution is then treated with an ion exchanger equilibrated with a 0.2 M sodium chloride phosphate buffer to be adsorbed thereon; and then a fraction is collected which adsorbs with a 0.2 M sodium chloride phosphate buffer, but is eluted with a 0.3 M sodium chloride phosphate buffer. 15. Method according to claim 1 for the production of TF-140, characterized in that barium ions are added to the culture or its supernatant to form a barium salt, or a hydrophilic organic solvent is added to the culture or its supernatant, and the precipitate formed is isolated, and then barium ions are added to the water-soluble fraction of the precipitate which is thus isolated, the barium salt formed is isolated, and then treated to remove barium, and the water-soluble fraction is collected and dialyzed or ultrafiltered. 16. Method according to claim 1 for the production of TF-150, characterized in that a hydrophilic organic solvent is added to the culture or its supernatant; the formed precipitate is isolated, the water-soluble fraction of the precipitate, or the internal solution component obtained by dialyzing or ultrafiltering the water-soluble fraction is treated with a quaternary ammonium salt; the formed precipitate is isolated and then subjected to a dissociation process using a solution containing sodium chloride; a hydrophilic organic solvent is added to the obtained soluble fraction to form a precipitate, and the precipitate thus formed is isolated. 17. Method according to any one of claims 1 - 16, characterized in that the culture medium used for growing the bacteria comprises trypticase peptone, phyton peptone, proteose peptone, a brain-heart infusion (or heart infusion), yeast extract, sodium chloride, glucose, lactose, L-cystine , sodium sulphite and thioglycollate, and possibly agar. 18. Method according to any one of claims 1-16, characterized in that the cultivation is carried out at 36 to 38°C for 1 - 3 days in a culture medium adjusted to pH 7.2 - 8.2. 19. Method according to any one of claims 2-16, characterized in that the hydrophilic organic solvent added to the culture or its supernatant fluid is an alcohol. 20. Method according to any one of claims 2-16, characterized in that the hydrophilic organic solvent is added to the culture or its overflowing fluid in a solvent concentration of 30 - 70% by volume. 21. Method according to any one of claims 6-14, characterized in that the ion exchanger is a weakly basic ion exchanger or a weakly basic ion exchanger with molecular sieving action. 22. Procedure according to claim 6, characterized in that a 0.1 - 0.6 M sodium chloride phosphate buffer is used to elute the fraction adsorbed on the ion exchanger. 23. Method according to any one of claims 7 - 14 or claim 22, characterized by the atpHi phosphate buffer containing sodium chloride, which is used for treating the adsorbed fraction, is approx. 8. 24. Method according to any of claims 7-14 or claim 22, characterized in that the fraction that has been treated with ion exchange is concentrated, desalted and dried. 25. Procedure according to claim 16, characterized in that cetylpyridinium chloride is used as a quaternary ammonium compound. 26. Procedure according to claim 15, characterized in that the treatment with the quaternary ammonium salt is carried out in the presence of a borate buffer. 27. Procedure according to claim 15, characterized in that a borate buffer containing sodium chloride and a quaternary ammonium salt is used as a solution containing sodium chloride. 28. Method according to claim 6 for the production of TF-133a, characterized in that Fusobacterium nucleatum TF-031 (ATCC 31647) is inoculated into a culture medium adjusted to pH 7.2 - 8.2 and comprising trypticase peptone, phyton peptone, proteose peptone, a brain-heart infusion ( or a heart infusion), yeast extract, sodium chloride, glucose, lactose, L-cystine, sodium sulphite and thioglycollate and possibly agar, and then cultivated under anaerobic conditions at 36-38°C for 1 - 3 days; an alcohol is added to the culture or its supernatant at a concentration of 50-70% by volume: the precipitate formed is isolated; the water-soluble fraction of the precipitate, if necessary, is dialyzed or ultrafiltered, and then treated with a weakly basic ion exchanger or a weakly basic ion exchanger with molecular sieving action to remove the unadsorbed fraction; a fraction eluted with a 0.2 M sodium phosphate buffer is removed from the adsorbed fraction; and then a fraction that has been eluted with a 0.3 M sodium chloride-phosphate buffer is isolated, concentrated, desalted and then dried. 29. Method according to claim 6 for the production of TF-1323, characterized in that Fusobacterium nucleatum TF-031 (ATCC 31647) is inoculated into a culture medium adjusted to pH 7.2 - 8.2 and comprising trypticase peptone, phyton peptone, proteose peptone, a brain-heart infusion ( or a heart infusion), yeast extract, sodium chloride, glucose, lactose, L-cystine, sodium sulphite and thioglycollate and possibly agar, and then cultivated under anaerobic conditions at 36-38°C for 1 - 3 days; an alcohol is added to the culture or its supernatant at a concentration of 50-70% by volume: the precipitate formed is isolated; the water-soluble fraction of the precipitate, if necessary, is dialyzed or ultrafiltered, and then treated with a weakly basic ion exchanger or a weakly basic ion exchanger with molecular sieving action to remove the unadsorbed fraction; a fraction eluted with a 0.1 M sodium phosphate buffer is removed from the adsorbed fraction; and then a fraction that has been eluted with a 0.3 M sodium chloride phosphate buffer is isolated, concentrated, desalted and then dried. 30. Method according to claim 6 for the production of TF-132b, characterized in that Fusobacterium nucleatum is inoculated into a culture medium which is adjusted to pH 7.2 - 8.2 and which comprises trypticase-peptone, phyton-peptone, protease-peptone, a brain-heart infusion (or a heart infusion), yeast extract, sodium chloride, glucose, lactose, L-cystine, sodium sulphite and thioglycollate, or in a culture medium comprising such and agar, and then cultured under anaerobic conditions at 36 - 38°C for 1 - 3 days; an alcohol is added to the culture or the supernatant liquid in a concentration of 50 - 70% by volume; the precipitate formed is isolated; the water-soluble fraction of the precipitate, if necessary, is dialyzed or ultrafiltered, and then treated with a 0.2 M sodium chloride-phosphate buffer of the water-soluble fraction of the precipitate or a 0.2 M sodium chloride-phosphate buffer of the internal solution component obtained by dialysis or ultrafiltration, with a weakly basic ion exchanger or a weakly basic ion exchanger which can act as a molecular sieve, equilibrated with a 0.2 M sodium chloride-phosphate buffer; the fraction passed through the ion exchanger is collected; this eluted solution is adjusted to a sodium chloride concentration of 0.1 M, and then treated with a weakly basic ion-exchanger or a weakly basic ion-exchanger that has molecular sieve capability and is brought into equilibrium with a 0.1 M sodium chloride- phosphate buffer, to be adsorbed thereon; and a fraction which has been eluted with a 0.2 M sodium chloride phosphate buffer is isolated from the adsorbed fraction, concentrated, desalted and then dried. 31. Method according to claim 6 for the production of TF-133b, characterized in that Fusobacterium nucleatum is inoculated into a culture medium which is adjusted to pH 7.2 - 8.2 and which comprises trypticase-peptone, phyton-peptone, protease-peptone, a brain-heart infusion (or a heart infusion), yeast extract, sodium chloride, glucose, lactose, L-cystine, sodium sulphite and thioglycollate, or in a culture medium comprising such and agar, and then cultured under anaerobic conditions at 36 - 38°C for 1 - 3 days; an alcohol is added to the culture or the supernatant liquid in a concentration of 50 - 70% by volume; the precipitate formed is isolated; the water-soluble fraction of the precipitate, if necessary, is dialyzed or ultrafiltered, and then treated with a 0.3 M sodium chloride-phosphate buffer of the water-soluble fraction of the precipitate or a 0.3 M sodium chloride-phosphate buffer of the internal solution component obtained by dialysis or ultrafiltration, with a weakly basic ion exchanger or a weakly basic ion exchanger capable of acting as a molecular sieve, equilibrated with a 0.3 M sodium chloride-phosphate buffer; the fraction passed through the ion exchanger is collected; this eluted solution is adjusted to a sodium chloride concentration of 0.2 m, and then treated with a weakly basic ion-exchanger or a weakly basic ion-exchanger that has molecular sieve capability and is brought into equilibrium with a 0.2 m sodium chloride- phosphate buffer, to be adsorbed thereon; and a fraction which has been eluted with a 0.3 M sodium chloride phosphate buffer is isolated from the adsorbed fraction, concentrated, desalted and then dried.