NL8004759A - NEW SUBSTANCES WITH CARCINOSTATIC AND IMMUNO-STIMULATING EFFECTS, METHOD FOR THE PREPARATION THEREOF AND CARCINOSTATIC PREPARATIONS CONTAINING THESE SUBSTANCES. - Google Patents

Description

♦ * π

New substances with carcinostatic and immunostimulating effect, process for their preparation and carcinostatic preparations containing these substances.

The invention relates to a process for the preparation of new substances with carcinostatic and immunostimulating action, which consists of cultivating bacteria forming TF dust under anaerobic conditions, which belong to the Fusobacterium genus and collecting the formed substances from the culture or the supernatant thereof (this new substance is further referred to as TF substance), and refers to the obtained TF substances / further referred to as TF-100, 110, 120, 130 (1316, 132a, 132b, 133a, 133b, 1323 10 136), 1U0 and 1507 and relates to a carcinostatic preparation containing such a substance.

In recent years, as a drug for patients with various cancers, a drug has been widely used which enhances the immunological activity of the host and which achieves carcinostatic activity by immunological function. As anti-tumor agents used for such a drug, known compounds are obtained from organisms of different bacteria or cultures of different bacteria or polysaccharides obtained from fruit bodies of Basidiomycetes or cultured fungus bodies thereof. However, these anti-tumor agents are not yet satisfactory in their action, deleterious side effect and mode of production.

On the other hand, there are publications regarding an-organism and a supernatant obtained by culturing 8004759 2 bacteria to the Fusobacterium genus, e.g.

Fusobacterium K031-33, Fusobacterium fusiforais W-12, Fusobacterium girans 1012 and Fusobacterium nucleatum 1010 / Dental Surgery, 23, 322-333 (197 * 0 and 21_, 53 ^ -539 (1972) (Japanese) J. However, there is still 5 no research directed at the components obtained from a culture or the supernatant prepared by culturing bacteria belonging to the Fusobacterium genus and the pharmacological actions of the components.

We have now investigated the pharmacological activity of a supernatant obtained by culturing bacteria belonging to the Fusobacterium genus and removing the organisms from the culture, finding that a specific component is obtained from the supernatant, that has a carcinostatic effect; said component has virtually no effect on inhibiting the formation of a colony of cancer cells in a colony foam assay method and has no carcinostatic action by killing the cancer cells, but an indirect carcinostatic action by enhancing the host-borne anti tumor activity or the immunity of the host and using the promotion of immunity; we have also found that said component has very low toxicity and can also be obtained by treating the culture.

The object of the invention is to provide a method which consists in cultivating bacteria belonging to the Fusobacterium genus under anaerobic conditions and collecting new TF substances with carcinostatic and immunostimulating action from the culture obtained or the supernatant thereof .

Another object of the invention is to provide said TF substances.

Another object of the invention is to provide a carcinostatic preparation containing said TF substances.

As the bacteria used in the invention, all TF dust-forming bacteria belonging to it

Fusobacterium genus are used and e.g. Fusobacterium 8004759 * 3 * mieleatum "is preferably used. In practice, we have used Fusobacterium nucleatum TP-031 (FARM 5077; ATCC 3104-7) and the like.

The bacteriological properties of Fusobacterium 5 nucleatum TF-Q31 are as follows: (I) Form (1) Form of the cells: rod-shaped (Fig. 1) (2) Polymorphism of the cells: absent (3) Motility: absent 10 (U ) Spores: absent (5) Gram stain: Gram negative (6) Acid resistance: negative (II) Growing conditions in a culture culture medium (1) TF-a agar plate and wedge culture medium 15 external shape: round shape

Size: about 1 mm Outgrowth: hemispherical shape Structure: just like dewdrop Surface: smooth 20 Edges: smooth

Color: milky yellowish white Transparency: opaque (2) TF-a liquid culture medium

Degree of growth: vigorous 25 Turbidity: coagulum

Backlash: none

Growth of surface: none, no growth to a depth of 5 mm

Gas: none 30 (lil) Physiological properties (1) Hydrogen sulfur formation: + (2) Nitrates reduction: - (3) Butyric acid formation: + (U) Indole formation: + 35 (5) Urease: - (6 ) Catalase: - 8004759 u (T) Hydrolysis of starch: - (3) Behavior towards oxygen: anaerobic (9) Ammonia formation: + (10) Carbon dioxide formation: + 5 (11) Range of growth: pH 5 - 3.5, temperature

30 - i + 5 ° C

(12) Formation of gas from saccharides: L-arabinose (-), D-xylose (-), D-glucose (-), D-mannose (-), D-fructose (-), D-galactose (-) , 10 maltose (-), sucrose (-), trehalose (-), sorbitol (-), mannitol (-), inositol (-), glycerol (-), starch (-).

The new TF substances according to the invention are e.g. prepared in the following manner.

8004759 ♦ 5 r „

Culture of TF dust-forming bacteria belonging to the Fusobacterium genus -, ""> "" "- Ί

Culture or the supernatant liquid thereof

Hydrophilic r-j organic | Let it stand | solvent

Soluble - - Separation fraction x i —— j | Precipitate |

Phosphate buffer (sodium chloride) or more

Insoluble r-. "~ i fraction Γ- [Separation] \ Drying] <Water-soluble fraction - ^ - TF-100 ---- -

Outer L ·, ___ * Dialysis or "I."

solution P, ultrafiltration, (Drying) - Inner * • -—I] solution TF-110 - - _

Non-adsorbed __ ~ * Treatment with boar fraction ion exchanger ——

Phosphate buffer __ (sodium chloride)

Desalting | Sluate l I Drying I l -; - | I Concentration F - », TF-120 Desalination

ZZCZI

To dry

V

TF-130 (TF-131Ö, 132a, 132b, 133a, 133b, 1323, 136) 8004759 6

Culture or supernatant liquid thereof

Water soluble r-- mr,

fraction -2 - ^ Drying - ^ -IOO-100

Ι · »Ι · Μ ·· ^ Μ ^ ————— I ^ if -> | v ° 4iag T a 11 "" | | 5 ^ Mmea | barium salt. ...................

Divorce -> Solution \

Barium

Barium removal

Separation ............................. »Solid

Solution

Dialysis or ultrafiltration

Concentration |

Dry 'f TF-1U0 8004759 τ

Water-soluble * fraction_

Inner * ______

solution ^ »Drying ->? F-11Q

Treatment with a

Soluble fraction «....... quaternary ammonium salt

A quaternary ^ -Ψ ammonium complex

Solution with sodium chloride

Dissociation treatment

Washings

Soluble fraction

Hydrophilic organic solvent

Soluble fraction. Separation

Precipitation

To dry

T

TF-150 8004759> 8

The above preparation method is explained as follows: (1) Culture of bacteria.

The culture of bacteria belonging to the Fusobacterium genus is carried out using a conventional method of culturing anaerobic bacteria. I.e. a culture medium containing a nitrogen source, such as calf brain heart extracts, various peptone or the like; a vitamin source, such as a yeast extract or the like; an organic 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 sulfite, thioglycolate or the like, is adjusted to a pH of 6 - 8.5, preferably 7.2 - 3.2 and the bacteria are seeded on the culture medium, after which a stable state culture is carried out under anaerobic conditions at 35 - 2 ° C, preferably 36 - 38 ° C, for 1 - 5 days, preferably 2k - 72 hours. In particular, it is desirable to use the culture medium described in Table A (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 can be replaced by a heart infusion which is a calf heart extract, a bovine extract, a fish extract, a corn liquor or the like. Of the different peptones, proteose peptone and phyton peptone are not always necessary, and the trypticase peptone can be replaced by polypeptone.

When no agar is used, it is desirable to stir the culture.

Table A

Components of culture TF_a TF_C

medium (g / l)

Trypticase peptone 17 17 17

Phytonpepton 3 3 1.5

Proteose peptone 10 5 5

Brain heart infusion 35 17.5 35 Heart infusion - - 25

Yeast Extract 3 33 8004759 9 r

Sodium chloride 7.5 7.5 7.5

Glucose 666

Lactose 555 L-Cystine 0.25 0.5 0.5 5 Sodium sulfite 0.1 0.1 0.1

Thioglycolate 0.5 0.5 0.5

Agar 0 or 0.7 0 or 0.7 0 or 0.7 (2) Collecting a supernatant from the culture (removal of the organisms).

The organisms are removed from the culture obtained above to obtain a supernatant. For the removal of the organisms, a conventional method, such as e.g. centrifugation or filtration using a filter aid, such as Hyflo Super Gel, is used and in particular a centrifugation method is preferred from the viewpoint of performance, the degree of removal of the organisms and the yield of supernatant. The organisms are preferably removed in this step, but they can also be removed in the subsequent step (3).

20 (3) Collection of carcinostatic substance TF-100.

A hydrophilic organic solvent is added to the supernatant or culture obtained above and the precipitate formed is collected. At this time, the supernatant or culture is preferably adjusted to a pH of 2-7. The hydrophilic organic solvent consists e.g. from an alcohol, such as ethanol or methanol, a ketone, such as acetone, although alcohols, especially ethanol, provide the best results. The hydrophilic organic solvent is suitably added such that its concentration is 30 - 30 vol., Especially 50 - 70 vol. 5. After the addition of the hydrophilic organic solvent, the resulting mixture is allowed to stand at a low temperature, preferably about 5 ° C for several hours to several days to complete the formation of the precipitate. The precipitate thus formed is separated by conventional methods, such as decanting, centrifuging or filtering.

8004759 10

Subsequently, water in an amount of 10-15 times that of the precipitate obtained above is added to the precipitate / the water can be replaced by a phosphate buffer or a sodium chloride-containing phosphate buffer when targeting TF-120 or 130 (131c, 132a, 132b, 133a, 133b, 1323 or 13 (:), as mentioned below 7 and the formed water-insoluble materials are removed by conventional method, such as centrifugation or filtration, after which the water-soluble fraction is collected. the organisms are removed by the above methods The water-soluble fraction thus obtained is dried, eg by lyophilization, in order to obtain a carcino-static substance TF-100.

The TF-100 thus obtained has properties as listed in Table 3.

(¼) Collection of carcinostatic substance TF-110.

The water-soluble fraction obtained in step (3) is subjected to dialysis or ultrafiltration; or instead TF-100 is dissolved in a 10-15 times the amount of water 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 inner solution obtained on dialysis or ultrafiltration is collected and then dried to obtain a carcinostatic substance TF-110.

The FT-110 thus obtained has properties listed in Table B.

(5) Collection of carcinostatic substance TF-120.

The water-soluble fraction obtained in step (3) or, if necessary, the inner solution obtained by subjecting the water-soluble fraction to dialysis or ultrafiltration (the inner solution obtained in step (U), or a solution of a powder of TF -110 in a small amount of water, is treated with an ion exchanger. As the ion exchanger, a weakly basic ion exchanger or a weakly basic ion exchanger with a molecular sieve capacity is preferably used, 8004759 11 and suitable e.g. diethylaminoethyl-Senhadex A-50 (registered trademark). A column packed with an ion exchanger is equilibrated with e.g. a phosphate buffer having a pH of 8, after which the above-mentioned solution to be treated is passed through the column and the eluted solution is collected and then dried by lyophilization or the like; or instead the same buffer as above with the solution to be treated is placed in a vessel containing an ion exchanger and the contents are stirred and then filtered, after which the filtrate is dried; therefore, a carcinostatic substance TF-120 having the properties described below can be obtained. TF-120 can also be obtained by desalting the eluted solution or the filtrate, e.g. by dialysis using a cellophane tube or the like, using Sephadex G-25 '15 (registered trademark), or by ultrafiltration and then drying thereof.

The TF-120 thus obtained has the properties listed in Table B.

(6) Collection of carcinostatic substance TF-130.

A column containing the adsorbed components, from which TF-120 has been removed by the treatment according to step (5) above, is treated with a phosphate buffer, preferably a phosphate buffer with a sodium chloride concentration of 0.1 - 0.6 mol and a pH of 8 (the phosphate buffer having a sodium chloride concentration of 0.1 - 0.6 mole is further referred to as a 0.1 - 0.6 mole sodium chloride phosphate buffer); or instead the water-soluble fraction from step (3) is subjected, if necessary, to dialysis or ultrafiltration and then asm treatment with an ion exchanger using a 0.1-0.6 molar sodium-30-chloride-phosphate buffer, in order to obtain a fraction which is not eluted with a sodium chloride phosphate buffer but which is eluted with a 0.1-0.6 molar sodium chloride phosphate buffer.

This method can be performed with either a column 35 system or a discontinuous system. The desired fraction thus obtained, which is eluted with the sodium chloride 8004759 * 12 phosphate buffer, is dried by lyophilization or the like in order to obtain a carcinostatic substance TF-130. The eluate-containing desired fraction, eluting with the above sodium chloride phosphate buffer, can be desalted, e.g. by dialysis using a cellophane tube, molecular sieving using Sephadex G-25 or ultrafiltration and then drying.

The term "TF-130" as used herein refers collectively to desired eluted fractions obtained by treating the water-soluble fraction obtained in the above step (3) or, if necessary, the inner solubilizing component obtained by subjecting the water-soluble fraction to dialysis or ultrafiltration, with an ion exchanger, these fractions not being eluted with a sodium chloride-free phosphate buffer but eluting with a 0.1-0.6 molar sodium chloride-phosphate buffer. TF-130 more particularly includes e.g. the following groups.

(i) A fraction that is not eluted with a phosphate-20 buffer free of sodium chloride, but is eluted with a 0.6 molar sodium chloride phosphate buffer in the above ion exchange treatment / fraction is designated TF-13167.

(ii) A fraction that is not eluted with a 0.1 molar sodium chloride phosphate buffer, but is eluted with a 0.2 molar sodium chloride phosphate buffer in the above ion exchange treatment / fraction is designated TF-132a and b; the TF-132a is obtained by washing an ion exchanger containing the above adsorbed component with a 0.1 molar sodium chloride phosphate buffer, treating the ion exchanger with a 0.2 molar sodium chloride phosphate buffer and then collecting the fraction which eluting with the 0.2 molar sodium chloride phosphate buffer; and the TF-132b is obtained by treating the water-soluble fraction obtained in the above step (3), or the inner solution component (TF-110) obtained in the above step (U) with an ion exchanger /.

8004759 13 r (iii) Sen fraction not eluted with a 0.2 molar sodium chloride phosphate buffer but eluted with a 0.3 molar sodium chloride phosphate buffer in the above ion exchange treatment / the fraction is designated 5 TF-133a and b; the a and b have the same meaning as defined in the case of resp. TF-132a and.

(iv) A fraction that is not eluted with a 0.1 molar sodium chloride phosphate buffer but is eluted with a 0.3 molar sodium chloride phosphate buffer in the above ion exchange treatment / the fraction is designated TF-13237.

(v) A fraction that was not eluted with a 0.5 molar sodium chloride phosphate buffer but eluted with a 0.6 molar sodium chloride phosphate buffer in the above ion exchange treatment / the fraction is designated TF-136 /.

Since these fractions TF-1316, 132a, 132b, 133a, 133b, 1323 and 136 have common properties in the following respects, the substances with the common properties are referred to as TF-130. D.v.z. that the TF-130 materials obtained in the manner described above have the following properties: (a) Gray-white-light brown powder.

(b) It inhibits the proliferation of murine Ehrlich ascites tumor, Ehrlich solid tumor and Sarcoma-180 carcinomas and has an immunostimulating effect.

(c) It is soluble in water and insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether.

(d) It has no sharp melting point, starts to decompose at 110 ° C and clearly decomposes above 200 ° C.

(e) The infrared absorption spectrum obtained with a KBr tablet method shows absorption bands near 3600-3200, 2960-2930, 1670-16U0, 1550, 1UUO-1380, 12U0, 111 * 0-1000 and 820 cm-1.

35 - (f) The ultraviolet absorption spectrum of the aqueous solution shows a strong absorption at the absorption edge and 8004759 11+ shows an absorption peak in the region of 256-280 nm.

(g) It is positive in Molic reaction, phenol sulfuric acid reaction, anthron sulfuric acid reaction, indole hydrochloric acid reaction and Lowry-Folin's reaction and negative in the ninhydrin reaction.

(H) Elemental analysis runs C 27.5 - 39.3 / e; H 3.2-5.755; N 2.8-6355.

(i) The saccharide content, measured by a phenol-sulfuric acid method, is about 19.0 - 61 +, 0.0 wt.% (expressed as 10 in glucose), and the protein content, measured by Lovry-Folin's method is in the vicinity of 6.0 - 28.0 wt. 3 (expressed in calf serum albumin).

The above ion exchanger treatment is further explained below. As the ion exchanger, preference is given to a weakly basic ion exchanger or a weakly basic ion exchanger with molecular sieve capacity, and thus e.g. diethylaminoethyl-Fephadex Λ-50 suitably used in the above step (5) and the method below can be carried out with a discontinuous system.

(I) A 0.6 molar sodium chloride phosphate buffer, preferably having a pH of about 3, is passed through a column of an ion exchanger containing the adsorbed component, where the solution to be treated is passed in the above tran (5), and the eluted fraction is collected, optionally concentrated and desalted by dialysis or ultrafiltration and then dried by lyophilization or the like, to obtain a carcino-static TF-1316.

The TF-1316 thus obtained has the properties listed in Table B.

(Ii) -1) A 0.1 molar sodium chloride phosphate buffer, preferably having a pH of about 3, is passed through a column of an ion exchanger containing the adsorbed component, where the solution to be treated in previous tran (5) is passed to wash the column, then a 0.2 molar sodium 35 chloride phosphate buffer, preferably a pH of about 8, is passed through the column and the eluted fraction is passed through 8004759 4 * r, optionally concentrated Desalted by dialysis, ultrafiltration or the like and then dried by lyophilization or the like to obtain a carcinostatic substance TF-132a.

The PF-132a thus obtained has the eggshell as shown in Table B.

(ii) -2) Methods for collecting the fraction which is not eluted with a 0.1 nol sodium chloride phosphate buffer but eluted with a 0.2 nol sodium chloride 1Q phosphate buffer in the ion exchanger treatment are carried out on the in the following manner by using the water-soluble fraction obtained in the above step (3) or the inner solution obtained in the above step (10) to obtain TF-132b.

15 i.e. that the column packed with the ion exchanger is equilibrated with a 0.2 - 0.3 molar sodium chloride phosphate buffer, preferably a pi of 3, after which the above 0.2 - 0.3 molar sodium chloride phosphate buffer of the water-soluble fraction 20 or the inner solution component obtained in the above step (3), 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 above 0.2 - 0.3 molar sodium chloride phosphate buffer and the eluted solution and washings are combined. The ion exchanger treatment can be performed several times. Then, a solution prepared by diluting the above eluted solution with a phosphate buffer, so that the sodium chloride concentration is 0.1 molar, is passed through a column in which the ion exchanger is equilibrated with a 0.1 molar sodium chloride phosphate buffer preferably having a pH of about 8, and the fraction eluted with the above 0.1 mole sodium chloride phosphate buffer is removed, then the above 0.2 mole sodium chloride phosphate buffer is passed through column 35 to obtain an eluate. In the relevant example of the above ion exchanger treatment, 8004759 16 a fraction that is not adsorbed on an ion exchanger equilibrated with the above 0.2 molar sodium chloride phosphate buffer is adsorbed on an ion exchanger equilibrated with the above 5 mentioned 0.1 molar sodium chloride phosphate buffer, washed with the above 0.1 molar sodium chloride phosphate buffer, and then eluted with the above 0.2 molar sodium chloride phosphate buffer to obtain an eluate. One may also use a method in which the fraction adsorbed on an ion exchanger equilibrated with the above 0.1 M sodium chloride phosphate buffer above is collected in reverse, and then a method for separating this fraction from a fraction which is adsorbed on an ion exchanger equilibrated with the above 0.2 molar sodium chloride chloride phosphate buffer is run and a fraction eluted with the above 0.2 molar sodium chloride phosphate buffer is collected.

Then, the eluate obtained in the manner described above is optionally concentrated and desalted by dialysis, ultrafiltration or the like and then dried by lyophilization or the like to obtain carcinostatic substance TF-132b.

The TF-132b thus obtained has the properties listed in Table B.

(Iii) -1) The inner solution component obtained in previous step (U) is passed through a column of an ion exchanger equilibrated with a phosphate buffer, preferably having a pH of about 8, and a 0.2 sodium chloride phosphate molar buffer, preferably having a pH of about 8, is passed through this column to wash the column and then a 0.3 sodium chloride phosphate buffer, preferably having a pH of about 3, is passed through this column or instead thereof, the above 0.3 molar sodium chloride phosphate buffer is passed through the column treated in the previous step (ii) -1) and still contains the remaining adsorbed component. The eluted fraction is collected, if desired, concentrated and desalted by dialysis, ultrafiltration or the like, and then dried by lyophilization or the like to obtain a carcinostatic substance T133a.

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

(iii) -2) The fraction that is not eluted with a 0.2 molar sodium chloride phosphate buffer to eluted with just a 0.3 nolar sodium chloride phosphate buffer in the ion exchanger treatment is collected in the following manner, which is the same as 10 in the above step (ii) -2) to obtain TF-133b.

I.e. that the column having just been ion exchanger is equilibrated with a 0.3 nolar sodium chloride phosphate buffer, preferably at a pH of about 8, after which the above 0.3 molar sodium chloride phosphate buffer of the above step (3 ) obtained water-insoluble fraction or the inner solution component obtained by subjecting the water-soluble fraction to dialysis or ultrafiltration, is passed through the colon and the eluted solution is collected. If necessary, the column is washed with the above 0.3 molar sodium chloride phosphate buffer, and the eluted solution and washings are combined. The ion exchanger treatment can be performed several times. Then, a solution prepared by diluting the above eluted solution with a phosphate buffer such that the sodium chloride concentration is 0.2 molar is passed through a column in which the ion exchanger is equilibrated with a 0.2 molar sodium chloride phosphate buffer with preferably a pH of about 8, and the fraction eluted with the above 0.2 molar sodium chloride phosphate buffer above is removed, after which the above 0.3 nol sodium chloride phosphate buffer is passed through the column to obtain an eluate. In the relevant example of the above ion exchanger treatment, a fraction that is not adsorbed on an ion exchanger equilibrated with the above 0.3 molar sodium chloride phosphate buffer above is adsorbed on an ion exchanger equilibrated 8004759 13 with the above 0.2 molar sodium chloride phosphate buffer, washed with the above 0.2 molar sodium chloride phosphate buffer and then eluted with the above 0.3 molar sodium chloride phosphate buffer to obtain an eluate. It is also possible to use a method in which, conversely, a fraction adsorbed on an ion exchanger equilibrated with the above 0.2 molar sodium chloride phosphate buffer is collected, and then a method for separating this fraction from a fraction that is adsorbed on an ion-exchanger equilibrated with the above 0.3 molar sodium chloride phosphate buffer, and a fraction eluted with the above 0.3 molar sodium chloride phosphate buffer is collected.

Then, the eluate obtained in the manner described above is optionally concentrated and desalted by dialysis, ultrafiltration or the like, and then dried by lyophilization or the like in order to obtain the desired carcinostatic substance TF-I33b.

The TF-133b thus obtained has the properties listed in Table B.

(iv) The water-soluble fraction obtained in the above step (3) or the internal solution component obtained in the above step {h} is formed in a 0.3 molar sodium chloride phosphate buffer preferably having a pH of about 25 and the resulting buffer is passed through an ion exchange column pre-equilibrated with a 0.3 molar sodium chloride phosphate buffer, preferably with a pH of about 8. The eluted solution is adjusted by adding a phosphate buffer such that the sodium chloride concentration is 0.1 molar and then passed through an ion-exchange column previously equilibrated with a 0.1 molar sodium chloride phosphate buffer which preferably has a pH of about 8 and then the above 0.3 molar sodium chloride phosphate buffer is passed through that column, after which the eluted the fraction is collected, if desired, concentrated and desalted by dialysis, ultrafiltration or and then 8004759 19 r is dried by lyophilization or the like to obtain a carcino-static substance TF-1323.

TF-1323 reed thus obtained has the properties listed in Table B.

(V) The inner solution component obtained in the above step (H) is passed through a column of an ion exchanger equilibrated with a phosphate buffer which preferably has a pH of about 8 and a 0.5 molar sodium chloride phosphate buffer which preferably has a pH of about 8 is passed through that column to wash the column and then a 0.6 molar sodium chloride phosphate buffer which preferably has a pK of about 8 is passed through that column or alternatively, the above 0.5 molar sodium chloride phosphate buffer is passed through the column treated in the above step (ii) -2) and still contains the remaining absorbed component, after which the above 0.6 molar sodium chloride phosphate buffer is passed through the 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 TF-136.

The TF-136 thus obtained has the properties listed in Table B.

(7) Collection of carcinostatic substance TF-1U0.

Barium ions are obtained from the culture or the supernatant liquid thereof obtained in the above step (1) or (2), or from the water-soluble fraction in the above step (3) or from the solution by dissolving TF-100 in water, added to form a barium salt, then the precipitate is collected and then subjected to a method of removing the barium and the water-soluble fraction is collected and then subjected to dialysis or ultrafiltration to obtain TF-1UO . More specifically, an aqueous barium hydroxide solution, preferably a 0.1-0.5 molar aqueous barium hydroxide solution, is added to the culture or supernatant thereof as obtained in 8004759 above step (1) or (2) or the water soluble fraction as obtained in step (3) above or to an aqueous solution obtained by dissolving TF-100 in a 10-15 times greater amount of water, and the pH of the resulting mixture is preferably adjusted to 10 - 13 in order to form a barium salt. A precipitate is deposited by adding a barium hydroxide solution and the amount of barium ions added is preferably adjusted so that the concentration of the total barium ions to the final volume of the mixture is 0.005-0.1 molar. The mixture thus obtained is filtered off and a method of separating 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 method to obtain a solution. The precipitate which has separated and been removed is again washed with an aqueous sodium sulfate solution and the washings are combined with the above solution. The solution thus obtained is subjected to dialysis, ultrafiltration or the like in order to remove the excess sodium sulfate or lower molecular substances and the resulting solution is then dried by lyophilization or the like in order to obtain a carcinostatic substance TF-lko.

The TF-1kO thus obtained has the properties as stated in Table B.

(3) Collection of carcinostatic substance TF-150.

The water-soluble fraction obtained in the above step (3) or the inner solution obtained in the above step (k) is treated with a quaternary ammonium salt and the formed precipitate 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 a quaternary ammonium salt which can conventionally be used herein, any complex with just a polysaccharide, and particularly preferred examples thereof, are cetyl-pvridinone chloride, cetyl-trimethyl ammonium bronide. The treatment with a quaternary ammonium salt is preferably carried out in the presence of a buffer, such as a borate buffer. The approximate description of the aforementioned quaternary ammonium salt treatment and the dissociation method using a solution containing sodium chloride is as follows. An aqueous solution of a 10 quaternary ammonium salt is e.g. added dropwise to the water soluble fraction obtained in the above step (3) or the inner solution as obtained in the above step (k) while keeping the pH slightly basic and the resulting mixture becomes 10 mm. stirred for several hours at 0 - 50 C, 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 molar sodium chloride and a small amount of a quaternary ammonium salt, and then dissociated with a buffer, preferably a 20 0 Borate buffer containing 0.5 molar sodium chloride and a small amount of a quaternary ammonium salt in order to obtain a soluble fraction, and then the soluble fraction is preferably adjusted to pH 2-6. Then, a hydrophilic organic solvent, preferably an alcohol, is added to the soluble fraction, so that the concentration thereof is finally 50 - 90 vol. #, preferably 70 - 90 vol. # and the mixture thus obtained is left for several hours to 90 hours at 0 - 30 ° C, after which the deposited precipitate is collected in order to obtain a carcinostatic substance 30 TF-150.

The TF-150 thus obtained has the properties listed in Table B.

8004759 22 I 'j \

"I

H Λ I H

t i Ρ β H H

• H +3 -r-C 0 »> s X

(I) p Ρ Ρ X X

X Js P P o x p P P x! ο ρ · η cd p cd + J n Ό cd · Η X Ο d Λ X ω ο 6 Ρ ω (η ο "X! η 4! Ο Ρ Η Η Ο - = ε = Η cd Ρ- Ο" ft + 3 Ρ: X ο Ρ p Ο Cd · C ra Ρ Ρ ο Ρ Ρ Ρ ο ο X -ρ ο -ρ ai Η X Ρ Η Ο X ft Ρ Ρ Ο X Ο χ ο ο ε Ρ u Ρ ο

<D

'ϋ ρ ο cd cd ai ai o ρ 03 C Ti Ό Ρ Ό co Ό xxaic xcai x> - pp bs o o u o ρ u o x aio icdcd

P * β .H x p P np.rCNp fl.H x cd P

• H Ρ Η X O O Ρ Η · Η S Ρ H O g Ρ P

X O p P N X # 3 · Η Ρ Ρ X O Ρ X

Pi Ρ X X η P Ρ X Ε X Ρ X X U N P

ai χ W M p g X W w χ (Η Ρ Ρ · Η E

£ · Η Η · Η · Η ·> -3 O · Η X Hi 3 Ή X Ρ Ρ -Ρ X Ρ X Ρ £ Ρ W β g Ρ cd PP <D "-3W ρ cd χ ρ pcd ra XX> X Ο X t * Ε X> * β, Γ3 Ο ο ρ χ ρ ρ s = ρ ρ χ ρ ra poop ο ο ο χ ο ο ο χ Ρ = • Η X * Η g Ρ g * Η X Ε · Η X g ρ ε χ to χ χ 3 ο Ε χ χ Ρ ρ χ χ 3 ο ρ ε ο ο cd χ Ε · Η ο cd χ ο ci d ρ g φ · η η η cd ρ 3 · ρ Ρι ο · ρ ρ 3 ε ρ ο ρ ο w χ ρ bo ρ ο ra be ρ ο ra χ ο ρ bo X Ο ft ft Ο PP ft ft Ο X Ρ ft ft O Ρ O Ρ Ρ Ρ · Η X Ρ Ρ · Η XX Cd X «Η X Ρ X Ρ · Η

CH Ε 1) Η · Η ρ X Ο Η X X X Ο Η X X Ο X

Ρ Ν Ο Ο Μ X Ρ NOU Ρ NOUraPXP

Ra ppraPPp ρ Ρ ra ρ ρ ppraPPPP

ft Q ft d ^ ί Ω pi d «I ρ Ω ft Cd!> UX> Ρ XX · Η * 1 0 · Η Ρ • Η | 3 Ρ Η 1 X Ρ ρ ra χ ρ Ο XX ζ ε ε ε X · Η Ο Ρ • Η Ρ · Η Ο X Ο Η Ρ, Ρ Ρ ft &

X

ο ω Ρ Ρ ω ρ \ ο ρ • Η · Η Ο Ο Ο «- CV1

Μ X Ο> - CM CX CO

ü r— r— t “r— r—

PI lil I

P ft ft ft ft ft ft Eh E ^ E-I EH E-1 8004759

/ I

<23 o u w ρ. o +3 c

m · Η 4) bS

tj o m c W M-t 4j cd Ti Λ! fö cd ^ u & xi v a (do ^ <u · η ö H ^ H <U «} λ \ · Η ^ 'Ö η Λ Α Ö α

S Ö W 0) V

Cj · ι-Ί £ h “d · Η (1) t £ = = = = = ω 2? 2 d = h d ^ s 2

o +3 E

S O D'Tr (U HS · Η · Η +3 o -p ^ μ

> l! aj O

(ü <U O 2 N 'h Ë Ë <u · η 3 E

O H +3 · Η fit a fii cn „_! M on on cj vo o o co on on on on -3 · ir \ I- Γ" r- r- r- <- r—

I I I I I I I

fiL & L fc fc k h P = H

IPi IH H EH EH ^ EH

8004759 2k j i j

si I a I

3 1) I <D I 4) 4) I

in · Η Ο Λ · Η Ο Λ · Η Ο "4J + J Ο Ο +3000 +300 ο α, ΛΟ-ίΐ- ftvo- = ro ftvo-d-

φ ρ rovo ρΓγοό ο Pi COO

ρ. θ'- Ο t- r- O '- w wei · well to e I «· 4J t— P CÖ O O'- ioo flip o * -

• h I cö i> ¢ -.4- I cö> ¢ - = 1- «J> £ - · £ I

+3 s of EO'-ocjE

Cl ü 4_3 CJ + Ϊ -P Γ- r- 4J + D r— x— O

M • Sm ^ ζτΗ in ^ S-l ^ 0 <D 3 * «Ό <D 0 * Λ <v 0 * ** Ο

02 ^ φ 2 O O C \] (1) 000 <D 0 Ο O CVJ

0 (u xj ^ oncoco iJr ^ onvo & & rococo

CjJtJ Ο 0Π O Cn o? 0 gojoj'-c Weir »- gajcM ^ c ϋ Λ B-diiaj r 3 · ϋιι p ti ι ι 4) O + J Ρι Ο O p <oouooo ai | 3 ÖV04 0 +3 eve« · + 3Cvc: ~ ro

Is s U.H Cs-4- O O · Η ON COr- O · Η 0-4-0 Π5 <U OJ'-O 4) 04 «-! 0J ° 1 (^ p, c i— P- Ö d Cl fl * -

S-i 23 W (U "* I K 4) *« «O W 4)" "I

cy TdOCO TJCO -Ö ooo (- | —- +3 CO Lf \ -4 + 3CCLAC -PCO LTN-d- 4> cd cm m <- 4) cd cj l / n cm 4Jujojirv «- s, a ro T - i- te fl: co - co S + 3COT - »- v ö 'd ca <u -ö O)

Η 4) 4) O

4J Η HO

& +3 +3 O

cö a c o

E- (O O OJ

bC II m h H +3 4) 4) o <u> a o>

Pi 0 0 4) 4) 600 tt t> +3 4) d § ^ ° P, 4) Pl Ό ~ ~ = m · Η 4) · Η

bO +3 4> P

c o> • H (o 4) 05

Tl Pi bD · Η 4) tw C +3 Η O Λ +3 4) o e n ·ι-3 tö

O 4) Ή P

flflx c 4) ft & Λ

O

you)

S 4) 'd O

bo -h o ° o rz w

• H +> o r- CM co CO

w S 7 7 7 J J

IS £ iï IS S & 8004759 25 ί I; I i

I I

I I

i dad

iH

£ Ο) 4) 13 a -ο ΰ -n =; a) χ} <υ ^ ^ rs <u · o

Hi! 4) 4) O 4) 4) CP

, _j r-1 Η Ο * H Ή O

O .p .p O 4-3 4-> O

^ £ 3 Cj CO £ h CJ "

£ Ο O <M O O CJ

> a) a * h «U a ^ + j <a <u -p <v <v t) ¢) Ö O> Ö C '> 0 0 4) 0 0 4)

b £> O b3 to O isO

0)> - 0 0) «- C

P CJ O, 0--0 0) Yh · Ό <1) S-i * »- = ♦ H 4) Η · Η 4) · -p 4) Ρ +) ί) ί Ο> 4)>

Cd 4) to cd 4) tü

- - - - - £ -i tC · Η in tC-H

dn a -P 4-1 O +5 ο Λ o si 4) O 4) 4) n cd ί) π d 4) Η f-l 4) Η Sh Ο, α fi p!

J3 Cd, £> CO

CM CO CO CJ Ό O O

CO CO CO CO CO _3- LTN

T T I I I I, 1

i = i t) & H

EH Eh Eh Eh Eh Eh Eh 8004759 *

2C

\ / \ i c ~

CM

n T j P o K C C P co C C P s Cl la P o C 3

I ^ X-rC I

Ο -P -P O · C H

c i '«a>« - g X <3 S VO P Pt

O CO

ρ c ί a td M!> 4) Pt

P

P ------- 4) in »: o o p o o! £ p Pt t ~ o c o I pt

3 · "CO

O £! > o

VO · on C H

CJ O> 1Λ β Ë

P I

C O O O * G ro co c t— H> Pt CO 00 jr è _ • η Ρ ή Ρ Ρ Ρ

5 +5 C CC C C

4) ρ. i) cc CC CC CC CC

, ο Ρ ίο ρ c cc cc cc cc CO-H 4J> CJ!> 4>> 4)!> 4)> E ~ * MP · η · η · η ή · η, a!) pp pp pp pp pp fe £ CP Ρ, P CC Ρ. P p. c c- c r-ι c ccs ccs ccs ccs ces

OPS: -'- 'OCG OCG OCC OCG OCC

> u - «-> S tc p wp ui jz d p ω p

CH OC PO P O P CM PO PO

cosc'- 'cd o vo cco cd ω c- cd ai vo cd a> co

>.-I 3 Ή P CU P CV! P 0J P CJ P CM

t »Ρ Μ X 4! O <U 42 Φ

C P n C PCI PCI PCI PCI PCI

C o o S · γ—! · »-Ί» f-i »c · α

P 42 H _ C VO C VO C CO CVP CO

H pj C-ι o- cd C LA cd C la CCVD dl C 1Λ CCt—

O W O <aj 4) CM <5) CM <41 CM <42 CM <42 CM

I CM I CM «- IC-I VD

0J "CM" O "CV" CO "

CVi "LTV" IXV "CO" LA "CO

<l) - ^ _ = r Pt CM pr CM

P S

c ------ C I CM I CM I LA 1-3 · I t— p Cd ^ CM "IA" LA "00" LA "

.-J £ Vï. "LA" LA "LA" PT "LA

cd --- Pd "-3" Pf CO Pt

POP

JH JQ --------- - ||> .- - ll I ................. ............. I '"" II II ".............

Ot> IIP IO I CM IO ICO

SH "" VO "CV" <- "O" 00 "® tj H" LA »1—" O "Pt" σ \

HG '—- O ΓΟ LAPt LA-3- O CO Pt CO

33 CD O CO COCO CO CO

_

O

P.

P.

CD

P

O

Onion c

0) O VO O

bO · Η O O O - CM

O P O * - CM CO CO

S3 O i— t— r— <- r— cd i ll i I i

Gh pL4 pti E- * EH EH Eh ^ 8004759 i 27 i i i i j

+3 -P

u u s s "2" 2 T3 Ό Jc Φ & Τ3

S S Ö S S C S

S S S S S o> So SS S S SC

ft SSSS ur) SO SSSSS ® <u> o os og <u> o> a>> H —1 · Η · Η SC · Η SC · Η · Η · Ρ O -P +3 -P + 1 +3. Η -Ρ · Η -Ρ-Ρ + -3 + 1 Ρ> -Ρ, α & s ft S _ ft ο ft at <u f. S F- ’b =

S SSS SSS S S co S S co J-.3S SSS S S S

£ h OSS OSS O ®CJ O <1> CM OSS OSS OSS

CflrQ ta S3 W T) M'S WrQ U3 .Ω 01 & tc ^ o, g cm +3 s I -gsi rO c β ° -5 -, .9

H di ¢¢ 0 S <D Ό SO SO S <D VO S 0) VC S JU VO

O S CM Ό OJ xi O χ | in Ό CM Ό CM Ό OJ

> 0) 4! <D O t— Ο O VO OJ S3 j ®

S t) S I ϋ s I Ό M CM "Swcj Td s I TiSI 'SSI

<U · Η · Η * H * ri · Η r · S m SCO SSS SSS S VO C m S S VO SStA SOS S <D S S S in B S 1Λ S S in

<3 d) CM <<U CM «ΙΟί» <3 <u> <o CM <3 0) CM «3 <U CM

(γ * \ I h_ j3 * I I m I CM

• Pi JL «J-« CO »C" O "CO"

CO "" ivo "in -VO -VO

W, | A J · m CM m irv ^ _ I ηι I Vf) I Ο I in I -S ’I VO JL“ Λ "σ \« CM "O" I O "" IA "in -Pi -co i r · *

^ prj 0O CO CO CO

• —T {

Iir, I Ό I in I + f I VO I ° i lin ο »* - * ON" in "Ο" O "00 nvn" CO "On" CM "CO" -S '-o \ con t — n on nt · - co cmcm n. co

into a CM CM CM CO

m S XI CO

oo co n cm vo ο o i- η n co n -s · in I i— r— T— 1— t—

ii I I I j I I

PH ft ft pt, fc ft E-t E + c-ι EH & -1 £ * 8004759 i 23 4) Ό f \ «* · χ Ο Ο +3 OJ cvi

<υ ιη LTS

Β C I C I C

cd o cd ο ö J · ί> CM ι — I> CM Η Ι> ο - * ε -a · ε c CG + 3 + 3 +3 4) CM ^ Λ Ο Μ "Ο h Η to 3 OM) 3 OVO 3 Ο ε ι os cxd t— 3® t- 3-3 · a itn ι ο ο ι ο η o ο ε ο o co hS 0) I CM 4) I CM 0) I ΙΛ

χ; 'Ö · MO' Ö · MO Ό · I

+3 O>>> O

a cm cc cc c -3

<VO M t> 4) M> «M!> OO

♦ I 0 * K 'Ö c

00 CM O C

ox: in co co CM +3 I -3Γ 1Λ 14) CO Cl C Cl O Ë eö * - CO cd cdo> - = t rH> VO ι-M t »> -3" Η X, 3 8 00e h O ε fC α) O +3 * +3 +> 8 + 3

Td CG h O O h "O h h'O

H cd CM CcO M3 3 ova Co Coo (0X1 to 3 00 t— 3 VC C- C VO 3 -3 · C \ Ό α I Ê X) l X co 1 χ <- χ oo 1

cd 4) H C I O O O

EnCGO 4) 00 4) 1 «- 4) 1 4) I CM

co X · VD T3 · LTV X · x · t— bC 4) CTs>>>>

Η 1¾ -3 "C * C C · c c · c · C

O M> 4) M!> O m> m> 4)> u ï o o § a § § § o

t »4)> C!>> OH

g 4) H VO H

a +3 »+3 +3 ε +3

h O H Sh * H U U - O

0 3 co ε 3 ΙΑ g 30 3 O 00

VO COO C CM 3 vo C-3CO

CM XiO X 0Π ΙΛ X <- X 00 I

I CM C— O

CTn 4) I co 4) I 4) I CM

X · I X · I X · X · t—> 0 t> (A l> 3 »

C · O C · t- G · G · C

___M i> VO__I — I t> 3 —__ M> __ M!> 4)

C

4) & S "

X

0 to

G

4) 4) VO CD

bD · Η O o 0 »- CM

• H +3 O CM 00 00 1¾ o r- t- »-> - ^

cd ι I I I I

M fc fc fe & 4

Em Eh Eh Eh 8004759 29 j 0 ö · Η ra +>> ρ,

Η You

Ρ g Ο

Ρ W

2 ° & Ρ OJ (6 ρ-ί <υ 0 ι be -d · c> · Η Ti c · ip c Μ! »Rara O P3 c

0 IA

H 00 SS S c p ci c cd cd cd cd ft cd o h cd>> r * i> P ί> ΙΛ g>

H H H H O \ p H

^, PË PS PS Ρ β ra PO ρ E

B P P U U & p »o p po PO PO PO cd POO Pc

HP LA p CM PlA P LA p O> - p LA

0 P2 P2 -3 "pi * - pi · - 0) p LA I P2 · -

B ÜO O

Cd 0 I (Dl (Dl (Dl C (DIIA 01

Eh Ti ♦ Τί · Ti · Ti ♦ .h p Ti · cO Ti ·. "_>>>!> PC>> ö. · Ö · ö · C * 0 cd c · C c · Η H i> H> H!> M> O P2 M> 0 M>

O

> u $ t * o o o S S S c e ö i ö ra ra ra ra ra ra o h ra

>> h>, ί>,> t> o s> P

S H H LA 0 PB p Ρ β Ρ β p po p

_ b "° h P» H H p "O Th" H

po pop- po po ps Poo ρ o g

PLA Ρ O C \ Pt— p Ό P PO> - PO

P2 * “P (Π I P3 * - P *“ p O pi OO I pir-O

o on o vo 01 HI I 01 01 01 0O \ 0IO 0 I «-

Ti · Ti «vo tJ · O · Til τί · σ \ τί · ι. > _ ί »_ _>> o>> oc · c · cc · c · c -ra · c * cc · o H> H> 0 H> H> H LA H> 0 Η> t- & ra pi ro cj co oo oj vo oo on on on on on la f— »” t "“ t— r "* r ·

1 J I I I I I

fe fe B B B

E- * E- * Eh E-ι En E- (En 8004759

V

30 * i r λ λ f *

P P -P P

c c c c 0 0 0 0 p p p p ft C — I <i — i C— »

r * H M M pH

03 03 03 03 'ti τ) x! t) Ό τΡ Ό Ό • ι-i · Η · Ρ · Η

Ë B B E

CO 03 CO CO

Ο Ο ο ο

r — I H rH H

B ft P P. p.

CO O 0 0 OP -P -P +3 VO 03 α) φφ CM Si · Λ JC Si c K C ft c c c +! cd S = «d si si ·

• K>> ·> ·> C

E la C C ft

co p vc p ft p ft -P B

P P PE PE P

to CC C C 3 ΙΛ O C 03 PO PC Pvc

p ο o VO P Ό rC

ed "IIC

E (DO 03 CO 030 (DO) 0 Ό V0 p I «si c co cc cc ccm

U M (A H 0) MO) HVO

ft -f r> ψ> φ>

OP -P P -P

-PC C C C

(0 ο ο 0 O

a · η p p p p 0) ft ft ft ft

ιΗ Ο I — I I — I ft I — I

0) ft 03 03 03 03 Ο ^ 'Ο τΡ Ό Ό ed Ό τ3 Ό Ό

Eh C · η ft ft ft

03 B E E E

b £ bO cn co co co

ft O E O O O O

O E C ft ft ft ft> P ft ft ft Pi

P 03 Ο Ο Ο Ο O

03> CM

t> CM -P -P -P PC

tsD 03 03 03 43 ft

Si Λ · si si si E

O c = K C ft C C C in ed B «3 si ed Ό>> ·> ·>

LA C C C

P VO P ft p ft P 03

P P B P E P

3c c c co C 03 CO CO Cvo

ο o VO Si VO Si I

"I I CO

030 03 CO 030 (DC1 '")

Tp VO Tp CO Tp pT Ό I *

C CO C C CC CO

MOO 1—1 03 M 03 MCA

C

0) ft &

-C

C3

CO

C

03

M

ft 03 VO ed

ft ft Ο Ο Ο 1- CM

P O'- CM CA CA.

S3 «- * - * - * -« -

CD II I I I

P ft ft ft 1 ft ft ft Eh Eh Eh Eh Eh 8004759 I'i '31 ί i ι Λ Λ Λ ft Λ

YY +3 +3 -P -P -P -P

P C P C P C C

o o o o o o o in U U f *} * ^ .5-1 4-i in <U 4-1 4h <+ - i * i- <

Η H f — I Η Η ι — I M

(U (1) I) P P P P

d τί Ό d) Ό Ό Ό * 0> d 'd di τ2 τ3 t?

• H · (- * · Η · Η -Η * H

s s e s s s w to m h w m ca ο ο ο ο ο o o rH ί — I ί — 1 ί — I <-! <-! r ~ i P. P- P P p · p £ - o o o o o o o • -PG-P +3 -P +3 4-3 4-3

0) -H (U P P P P P

42 S 42 · 42 · 42 42 42 -P · cc p Ö W Ρ m Ρ · Η P P c c '5

cd la cj s PS eo · cd cd PS

> ι ί>>> c> ·> τ> _

LA CJ CJ Ö PC

4J_d- +3 O +3 ΙΛ -PS 4-3 · Η -Ρ · Η -P LA

P U U P PS PS pi cqpp g P pp go g g Λ 2¾ d P d P d P d LA PO d O d -3- M 42 42 42 42 42 O 42 0 42

¢) «*» P (I P

42 <UC \ (DO 4) 0 Ρ P 4) CJ 4) 0 4) 0) ö tj tn -do d) co -d "d -s · di -3 ·" d

Eh ί ί I **

POJ PO pco Po PP PP P £ J

by M CO 1-4 0 MOO Η η M 4) M4) MOO

rH

____________ ^ A A A A Λ ft ft

p 4-3 -P +3 +3 P P -P

(UP P P P p P P

> 0 oooooo PPPPP p. 54 (p ip O-ι Oh 4-, ip (p Μ H t — I Μ M r — I ί i 4) 0) 4) 4) 4) 4) 4) di> 0 d ! 43 di dl dl di di di 'd di d)' d • Η · Ρ · Η · Η · γ4 · Η · Η s s ε s ε s s μ ω η tn κ w w Ο ο Ο ο ο ο ο Μ ί—! Μ Μ Μ Μ Μ

Pi CU Pi Pi Pf ΡρΡ- Ο ο ο ο ο ο ο Ρ · Ρ ρ Ρ ΡΡΡ Ρ 4) Ρ 4) 4) 4) 4) · Ρ 4) 4) 42 · Η 42 42 42 β · 42S 42 42 ^ Ρ Ρ Ρ Ρ · Η Ρ Ο Ρ Ρ · Ρ

CD Ο CD · CD PS PO P PS

i> crs> P i> ·>> 1> ·>

I · Η Ρ O CJ Ρ O

poo PS Ρ · Η -P LA pp · p-p p ia f4p U U £ PI P PS 21 d do d d co dp d d co

dp d la do d p do do dP

42P ο 42 (a 42 42 420 42 p 1 p «i p

4) t— PP p Os PP PO PO PP

df co d) "dp to" dp dp Ό I A A I ft

PO pco PP PO pco PP PCJ

MiO HCO HP M CO MCJ MP MCO

42 cd 42 co CU to ¢ 0 CJ 0 0 0 CO <0 CO CO CO P LA 1 8004759

I II I I I

& i pti fe fc, [ii fc ^ EH E-C El S-ciE-i

. I

32, ί

i I

c

• H

ί — I

0

fe (U

1 * H

P + + + + +> d 0 (1) J h

rH

O

E 0) i rH +5 Ο ϋ + + + + +

El d ö <u

Hi £ (

cT

(- · 7 a o Ο Ή

P

DEO + + + + + • h p d

pD P CJ QJ

U <u rH d a) <u, u ------- d i-ι _ +

E- 2 C

<D K

FCC H OJ

H «K 0 O I · Η

> HP

U Ο O

(I) d d + + + + +> <U 0)

Jh

Λ <D

CJ "H

W P

• HO + + + + + H d O 0) '

S U

ω c

• H

SH 0 E Ή

! >> H

+ O + I 1 I I

d d

• Η O

a u_____ c'e o a & a o m

C

a> <u Ό d

fee · η o o o r- CJ

• Hp o «- cj n ro HO r- t-« - r- r-

d I ί I ί I

> -i Η Η Η H

Ch Eh Eh E-ι Eh Eh 8004759 + - 33 + + + + + + + + + + + + + +

P3 I-I

0) + + + + + + + s

M

0 -----

YOU

<You

> + + + + + + + + + + + + + +

I I I I I 1 I

fi> «3 & co CJ on on c \ j ό o o tn on on on on jt ir> 1 8004759

I I I I I I

fc fc fe (i, fri E-ι E- »E- <E-ι £ -i 3k ---, -1 '^ 0 0) +3. ΐβ α> -> —1 S cd <u oj fa2 Ό P O ^

3 O) P

! h a «_« 'HE o o o c o

<13 P A A A A A

a 3 to on oj co co co

4J.D- cvj oj r- CM OJ

• Hi — I £ 3 cd * h · · · · · H cd (d cd cd cd 0) S 0 ü O V V ü p 3 &.

ri a I t I I t I

Cd G) fa "; £ a in co oo ¢) 15» «» o ·> * faOCO O CO · * C co 4J ¢) w OJ <- ON <~ • H> tZ Η P · · · · · h cd oj cd cd cd cd ® a ϋ gu υ ο ο o

rH

<u §

EH P

tS

w d I-I £ -i

O Tj -P

> a> <u · -

Sh 63 6¾¾.

d) P '- ^ ί> Ή 0 3 (1) (1) c o o o o

J 1 A A A A A

(uoo vo cn on o pr p ε λ ho · vo t- tfv vo

H 0) P

cd bO 0) · · · · ·, Ü ë cd cd «3 cd cd 0) * * u o o o b3 G) πΙ-

0) cn O I I I I I

ΐ o to • H U CM o o o c o a a a a a

cd I — I I Ο O VO LA LTV

xi fcfl rl Pf ΓΟ tA CO LA

Ü o o w ö · · · · "cd H a> cd td ed cd cd CQCdO-l o o o o o ö a) (¾ 8 * Λ

O

tfl 0 0) 0) vo cd

fafl · Η O O O * - OJ

• H P Ο Γ- cm CO CO

w u

cd I I I I I

h [xj Pp Ip fe & h EH Eh E "1 E- <8004759 35 Ο ο σ \ oooo λ λ η« "Λ CO ¢ 0 CM t— CM OJ -3- CM CM CM» “*“ m • · · · · ·: Cö tö cö cö cd cd ® uooo 'uuoiill I l 1 IA ooooo

»Λ Λ r» O

IA CM CM * - ^ JTJ ^ T- CM i- * “CM r ~ tö Cö cö cö ώ o-ίο 0 Ü O o CJ ou ffl I — I a> 3 --------- Μ r —I

O

£ la O LA o O O ° tvi ft P λ »> Λ"

> IA IA - =! ITt O LTV LA

P _d- CO CM CM CO * - LA

cö cö aJ cö tö cd co 5 o O O o o o

1 I i I I I I

for o o o o o o

CO \ C CN ON ON LA O

CM CM * -> “·“ · * ... ···· cd cd cd cd cd cö oj 0 o o o a o o, a cö. & rn CM _ CM CO; CO CM vo o o co CO CO CO CO J- A 1 8004759 i \ έ έ fe έ é Λ '“

V

36

In Table B, for the Sephadex G-50, denoted by a column of 50 mm diameter x 600 mm, distilled water was used as the eluent; for the Sephadex G-200 (trademark), denoted "* i", an Out mm diameter x 500 mm column 5 was used for TF-110, 120, 132a, 133a, 136 and 1k0, and a 21 mm diameter x b00 mm column for TF-1316, 132b, 133b, 1323 and 150, and as eluent all used a 0.1 molar phosphate buffer with a pH of 7 and to obtain the high power liquid chromatogram designated "** i" a 7.9 mm diameter x 600 mm x 2 column of TSK-GEL G300SW (trade name) was used and the elution was performed at room temperature at a flow rate of 0.8 ml / min. by using a 0.1 molar phosphate buffer with a pH of 7 as the eluent.

The pharmacological action of the present carcino-15 static substances TF-100, TF-110, TF-120, TF-1316, F-132a, TF-132b, TF-133a, TF-133b, TF-1323, TF- 1U0 and TF-150 are as follows: (I) Action of TF substances on cell viability (colony formation assay).

Based on the method of J.H. Kim et al. / Cancer Res.

25, 698 (1965) J, HeLa S-3 cells were grown in Eagle's MEM supplemented with 10 # calf serum for 3-5 days at 37 ° C, COg incubator, after which the culture was removed and a PBS (- ) solution (an aqueous solution containing 8.0 g / l sodium chloride, 0.2 g / l potassium chloride, 1.15 g / l monobasic sodium phosphate and 0.2 g / l dibasic potassium phosphate) in which 0.01 wt. # ethylenediamine tetraacetic acid and 0.1 wt% trypsin were added to the cells adhering to the glass. The cells were removed from the glass surface of the culture flask and dissociated into individual cells by pipetting, and then the number of cells was counted microscopically. The cell suspension was diluted with

Eagle's MEM, supplemented with 20 # calf serum so that it can contain 200 cells / ml. 1 ml of the cell suspension was seeded on Petri dishes and grown in a CO2 incubator at 37 ° C for 2b hours, after which the obtained supernatant from Example I (1), described below, was added to the cell suspension so that the concentration 1/16 - 1/128. Each of the test compounds was added to each of the 1 ml 8004759 f 37 aliquots of the cell suspension cultured for 2 h at 3 ° C and the HeLa cells were cultured for 7 days in each case, culture medium was removed , after which the cultivator.

5 were washed with Hank's solution and the cells were fixed with 10 wt.% Ethanol and then washed with 100 wt.% Ethanol. After the ethanol was removed, the cells were stained with a C-imsa staining solution, the colonies were counted and the cell viability was calculated.

The results are reported in Table 0 and Table D. The cell viability in the tables was calculated by the following equation. The cell viability of every cell? is shown as an average value of 5 repetitions.

15 Number of colonies in the case

Cell viability. PVf. f ° eÏ ~, * 100

Number of Columns in the case

of an untreated group Table C

Effect of the supernatant 20 on cell viability____

Cell viability {%)

Dilution (ml / ml) -1-1 p-~ -1 - 8 hours 72 hours 96 hours 1/128 79.6 66, h 80.1; 25 1/61; 30.0 7.9 5.8 1/32 000 1/16 000

Comparison 100 100 100 _, _____ J__ 4.

8004759 38

Table D

Effect of the TF substances on cell viability

Substance Concentration Cell Viability __ (^ ug / al) __ (J) _ 10 99, + TF-100 50 97,! + __100__95 ^ 9_ 10 9 ^, 5 TF-110 50 38.9 __100__87 ^ 3_ 10 98, 8 TF-120 50 91.2 __100__83.3_ 10 96.7 TF-132a 50 97, ^ __100__21 * 9_ 10 96.9 TF-133a 50 95,! + __100__88j8_ 10 95.9 TF-136 50 96,! + __100 I_9iii ± _ i 10 98.5! 50 98.6 TF-11 + 0 | 100 98.6 | 200 I 96.8 i 500 93 + + j_i_ 8004759 39

As can be seen from this test, the TF substances have a very low cytoxide effect on HeLa cells compared to the supernatant.

(ïl) Immunostimulating action.

Four ICR stem mice were used for each group. Sik of the test substances was dissolved in 0.2 ml of physiological saline and then administered to the mice intraperitoneally.

2b Hours after the administration, 0.2 ml of a carbon suspension prepared by mixing 1 ml of Perikan Drawing Ink 17 Black with 2 ml of physiological saline containing 3 parts by weight of gelatin was injected intravenously into the tail of the mice and 1, 5, 10 and 15 min after the injection, 0.02 ml of blood were removed from the web using a hematocrit capillary, coated with heparin and immediately diluted and hemolyzed with 1.6 ml of a 0.1 wt. aqueous sodium carbonate solution. This suspension was subjected to colorimetry at 675 nm and the phagocyte index, i.e. K value, was determined using the equation of Halpern et al.

The mice in the control group were only administered 0.2 ml of physiological saline.

log C. - log C

K - f — j—

t - tQ

where = carbon powder in blood at time tQ, and C = 25 carbon in blood at time tQ.

The results are shown in Tables E, F and G.

8004759 bo

Table E

Substance Dosage Average of K values (mg / kg) 10 0.035 1 + 0.01) +5 T ”-100 x '50 0.0o05 + 0.0302 5 0.0901 + 0.0213 TF_11 ° 20 0.1025+ 0.0025 5 0.0718 + 0.0152 TP-120 20 0.0559 + 0.0197 5 0.0832 + 0.0193 TF'1316 20 0.1001 + 0.02b6 5 0.1102 + 0.0203 TT ? - 1 0 20 0.0809 + 0.0296 5 0.0351 + 0.0057 ^ 20 0.0769 + 0.017) + 5 0.0959 + 0.0087 TF_13ü 20 0.1027 ± 9.015) +

Comparison - 0.0300 + 0.002H

8004759 hi

Table F

Substance Dosage Average of K values _______ (ng / kg) ______ TF_132b 5 0.1190 + 0.0051 20 0.1073 + 0.0031 5 0.0550 + 0.0020

rnF_ 10TL

J 20 0.1099 + 0.0075 5 0.1399 + 0.0011 πητ_iςη J 20 0.0577 + 0.0098 5 0.1 ^ 00 + 0.0075 TF "1323 20 0.0665 + 0.0126

Comparison - 0.0236 + 0.0035

Table G

Substance Dosage Average of K values (mg / kg) 1 0.0691 + 0.0156 TF-H0 5 0.09116 + 0.0139 20 0.0815 + 0.0200

Comparison - 0.0379 + 0.001 * 8

As can be seen from Tables E, F and G, the groups to which each of the TF substances of the invention were administered show activation of retinal lendothel macrophages as compared to the control group and the cell immunity of the normal mice was increased.

(lil) Carcinostatic effect.

(a) Anti-tumor action against Ehrlich ascites tumor.

Ehrlich ascites tumor cells were inoculated intraperitoneally into ICR stem mice (female, 6 weeks old) in an amount of 1 x 10 4 cells per mouse. Subsequently, each of the 8004759 1 * 2 test substances was dissolved in 0.2 ml of physiological saline and then administered intraperitoneally to the rizzards repeated once a day for J days from the first day after inoculation with the tumor cells, or once a day at the first day and 5 from the third to the seventh day for 6 days in total.

In the case of the test substances from Table J, each was administered to the mice once a day on the first day and from the third through the seventh day for 6 days in total after inoculation with the tumor cells. Comparative group 10 was administered 0.2 ml of physiological saline in the same manner as described above.

The results are reported in Tables H and J.

Number of survival days of the mice in a group to which the 15 T / c-substance was administered _ 1Q0 / ~ Number of survival days of the mice in the comparison group 8004759

1 A

43 ü 0) I μ 0) Η be a _ g τ— τ— τ— r- ^ Η Ή * -Κ * * CJ 0) Ρ Ρ

§ S WW ΙΛ ΙΑ ΙΑ VO VO VO SO VG VO VO VO

<P

l \ N ^ ^ ^ ^ a; on o w on o wwo «- o« - o on j- on o> ί ο ö o r-1 ’O Cβ Ö +3 0) C>« 5 0) <H *

WO W AA O J-OOO «- VO IA O - E— WO

O ^ Ό O _ =} Os O Λ ^ O W o ΓΟ o VOOC— O

T — T— r- t— T — 1 * r— r— T— r— τ— r— t— r— t— t— t—

E- <^ / \ Λ A / SA .A A A A A

H Ö: (Ö 4) ρ ω

§0 T- CA ° Os ΓΟ on w CM 00 W CO W CO VO CO W

rrj (Q «« Ο ”Λ" "" "" "" "" "" aa so · - 'cor- rw «- o \ os wc— j- oo wt— os vo wh H-Htd + | + | + | +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +! +1 +1

Jo · §§j * 3 · - vo o ia ο λ M co co on on w w co t— w

qJ Tj (H A Λ Λ A A AAA A Λ Λ * 1 AAAA

EH.Hfc oco -g- W VO c— t— t— o co on c · - comost-

So on r- won'- w w * - W'-W'w w w «- $ g A A aA A a Λ Λ Λ Η 63 Λ! to ö Β ω - Ρ ίώ cö G (30 Ρ · Η rt ö Ö

* 8.3 * V t LAO ^ ^ ^ VO VO VO

£ ρ ο * 1 * * XXI χ χ χ, χχχ, 00) 00 00 00 “Q, Ο Ρ t— ί— ΙΑ Τ- ΙΑ ΙΑ Ο *“ ΙΑ Ο r- r— ί cö π) Ο Ο ο! w οη Ο * 4 w ί on οη Ιη «- r ~ τ— Ο I I ι. ι · Ρ! χ <Pl, j jF <CQ Eh EH E-f E- <: 8004759) +1+ cvi

«- r- CM CM -K

* * * * o o o c VO VO VO Ό ΙΛ ΙΛ ΙΛ in in in in c c

v x v N. \\ N, VX, -4 H

• —I I — I

r- cn t ~ o ocjo cj- ^ rc <—coojo.p-covco <u <uouud ^ 00 N 4J -P 0) c cj cj _ cj oc ή tz cct: g ά • H - * ^ T5 .g - vc m 0 t- t - 0 vc 00 0 co vo in c 00 vo ccuc; <u CO f—, G O CV1CCC CO T- 0 CJf— COO t- * PJ Ο O r-4 · Η t +

, -, - f—, - t—, -, - 1— CJ "-« - 1— * - * - '- CJ CJ "- I — i + J-P

/ \ AA A A A AAA AAA g $ c d c cö d

tg 0 r— · H

d P, P.

r-4 Cd Cfi W

cj ^ 5 S

ςΗ G d d

C_ | CJ -P -P

be cj on r- m co ο η ο; ί)

HinvD (MOJCj ', ovcOLnoj · »« "cj» m · »> - cj τί Ό Q λλλλ λ f * n e * w * r— fj O λΟ λγ— * 4- ^

> VO in CO OJ in> ”*“ CV -i Cl «- t—> - CJ, - CO r— 1— <H G G

0 +! +1 +1 +1 +1 +1 +! +! +! +1 +1 +1 +1 +! +1 +1 +1 +1 m ^ ^ o co o c \ i o co m 0000 o cj co co -G -g o o dcö ·, *, ·. * *> «, #. ^ o - "» ** «g ό ό cioint— CMOJt— inoco j rc p co on co _g c- · <u CMmojr- OJ CO r— PO, G <- CJ CO CO> - (VI Μ r- 11 4) o

A AA A AA A A A A AA

G LTV C

aj co _g Ό

O CJ CJ Cd Ό 'C

r *

r — I

p. P · G O O CJ

H to HO

----- add CJ · Η · Η ί »Η Η CJ CJ CJ r-l Ό Ό d d d

VO O VO VD VO OO VO VO VO C— C— t— CJOO

> 00

«« «I« «I X X I: X X X I XXX I O CJ CJ

I 3 £> 1 - mo mo mo t »- in 0 * -ino cj

«- CVI 0» - * - Η O CJ

P- I H a Q

----- 1 «- CvJ

VO J3, JD i CO HK · * «! - * <: VO <- OJ ί 00 I CJ * CO CO CO 'CO i 00

T— r— <- t— I r- -P

1> 1 1; I O

lx, fe & H t *. 1 c

Eh Eh Eh & H j EH 3 8004759!, C

"J

j j: j ί; c

<D I tsS

a> · η at 3 ε ί-ί cd cd -Ρ -Ρ ^ CG Κ C ΙΑ ΙΑ CÖfDCÖ ir \ <- I— LA> - ΙΛ ΙΛ ΙΛ ΙΛ <-Ρ ό \ <D \ ^ ^ ^ I \ - Ρ OLACMPtCtiPtOCOCALAO ^> η ° C „~ Η 'ê' ο ^ ρ ¢) Si

β> S

cd Φ £.

<Η ^ * to

C.

CD -— «CO Ο CO CO Ο C‘ OC Μ O 4J

_ ^ T t “" * C \ t * · * O i-O Ό CO O f "-

r- j s_,, _ f—, -, - t— «- f— r— * - · p-J

AAAA / SAA 0 β __ cd o 2 2 ggc β ►- cd C _, cd co --- ^ co β bDbO η ιλ h “-ί Pt t— Ai Ο nj a ί" »>« pt η «ί · .

1-3 Η · Η Ό C \ ί— «Ν'— CA LA Pt Ο *“ κ ί-I 'S φ +! +1 +1 +1 +1 + ΐ +! +! +1 + ί Ο Ό ί — I "" -.ρβ M3 C LA Pt C% V3COCJ Ο e g 4) *> "" "Λ" "" "w

Eh (U> r ^ t -'- cOLA Ο CM ΙΑ CA Cd co CM CM CO CM CO CO CO i- A λ A A A A A g o to --- β <u * 5

to S

, ¾ -0 SC β β 'ε S ω -' H b £ 'VOOVOVO ΌΌνο Ό td β β to -ρ · ρ «« «« ί x * x ί α> β β β ^ Η β 0) ΙΑ «- ΙΑ Ο ΙΑ Ο Ο Ο β Cd · Η *>> “Τ-ΙΑ Η ο Td ο ^ CT +3 Ο 2? ο ο ο> β, β · ρ ο Ο)

rH

r—! <-1-- * ο ο

Pt ΙΑ <η τ- * - -ρ ° i rI ο +3 El, Bh ο

00 tH s-c S

8004759 t / - 40

As can be seen from Tables H and J, the greeting to which also the ïF substances of the invention were administered underwent the life-prolonging action compared to the control group.

5 (b) Anti-tumor activity against Ehrlich solid tumor.

(1) Ehrlich tumor cells were transplanted subcutaneously in the armpit of ICR stem mice (female, 6 weeks old) at U x 10 6 cells per mouse. Subsequently, each of the test substances was administered to the mice once a day 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 to the seventh day with a total of 6 days. The control group was administered 0.2 ml of physiological saline in the same manner as described above. The weights of the 15 tumors were determined on the 11th or 11th day after the transplantation of the tumor cells. The tumors' weights were determined by the largest diameter (a) mm and the smallest diameter (b) mm using calipers, and calculate from the following equation: 20 a χ, 2

Wt, of the tumor = —-- (mg)

The results are shown in Table K.

8004759 h7 i i

τ— '- CJ

M * K CM * «-O’-ο VO ΙΑ O CM -3- CO O r O ~ 2

CM on CM O 0--3-0 VO -3- -3- O t— VO - «O

1— * - «-« -

-P

Λ

YOU

tl · Η H £

«Ö {£ · -. 0-, 0-0 3 ont - 3- r-OCMVO VC Ο O VC

«Ö! H fjC« - C \ «- IA VO IA -3- VO O on O -« -3 "oo Q___ η η o>, tr Λ l>» «« "" "" ggt— 1—, - Ln cm «- on LT \ -3" -3 "G \ VO 3V -3" CT, ¢) 3 o -p

X

ω

Al ^ w to 'c S (U -ί «-Ή b3

0 tóCVQVOVO VOVO VO VO VO VOVOVO

"tóöëë" xxi xxxi XX. xi EH "H CÖ 0" _ in Cd · Η O O O CMC «- LA O« -ir \ o 0 -3 la ο o «- K -P 0« - «-

0 0 0 Q Λ -P

bO

O

• H

O

• Η Η H

3 Cd Cd ® 0 cd cd 5 O Ö c g + j m Ο O tn 0

3 4J -P 3 -P

3 0 «Η 3« 3 ω ’Γ1

cd 3 ^ <d U 3 M

> 0 0 cd 0 0 0> ft -P ft> ft 0 cd cd 3 cd cd o N fn p O Ih U k · «- = -P -P A -p -p + j • Η 3 3 3 3 3 3

3: Η H CQ Μ M H

vo ^ g o «- cj on o on on on l ·)« - «-« - 7

01 I I I

+3 ft ft ft ft

CO EH EH eh EH I

8004759

Jt ί + 8 CM oj cu + (+ (+ (OJ CO Ο t— ΙΛ VO o COO VO Ο -3 CO O O co ΙΛ Ο A .3 CO Ο 1— 1— t— • ·

__ G G

“O 0 rH rO i — I r — I 0 0 ü O U h O O g g CO t— CO 00 00 CV CO IA * -OVO 03 -3 co a co <- vo cu vc co o +3 p

Λ Λ i »A fi A P ri ft ft« V

rococo A -3- (O A A CO CO O ', 0 0 T3 Ό

ft CC

0 0} H>> 0 O 0 0 0 · Η · Η

EH +3 P

0 C

bC PP

H C C

o 3 d ^, —1 i — i fH ft ft 0 ω ω

ï> CC

cd c c u P +3 C— t— VOVOVO \ £> VO Ό 00

O O

KXIXXKIXXXI

cd cd

AO »- A O t-AO CC

* ”*” BC tjC

0 0 O O

---- (U 0)

O O

H - -rr 0 0 C 0 0

O m m O O

+3 0 0 • H 0 0 ft ft C C 0 0 0 0 0

ft>? * O O

0 0 0 i — t i — 1? H k h 0 0 +3 +3 +3 0 0 C G C ft ft Μ Μ M 0 0 m w

«- CM

<0 & + <tt CO VO co co co co

* “V— 4J

II I o ft ft ft o

& c E-ι I E-ι S

8004759% 1 * 9 (2) Ehrlich tumor cells were transplanted subcutaneously into the armpit of ICR stem mice (female, 6 weeks old) at U x 10 cells per mouse. Each of the test substances was then administered to the mice, which were divided into 5 groups for intravenous administration, intraperitoneal administration, subcutaneous administration and intramuscular administration, at a dose of 10 mg / kg or 20 mg / kg once a day on the first day. and from the third to the seventh day for 6 days in total, after the transplantation of the tumor cells. The comparative group was administered 0.2 ml of physiological saline in the same manner as described above. The weights of the tumors were determined on the 13th day after the transplantation of the tumor cells.

The results are as shown in Table L.

8004759 50 & S.

O t— O MO MO O M3 <- * - t— MO O

\ -a-ο c \ iw-iwin-ï \ oj-o

Eh t- u 0 a 3 -P - ~ - bG Td ^ f — i a; -p co «-> - - = f cj cm t- vc m cm e ··

3 i CO * “CCCCCCCOIACOMOCMCM

• η .η co co <- <- cm> - co cm -a · co mo £ £ <u <u c te te! --h H to 5h &

0) SO! OOCOOOOO

, JmbO T- t-CNJ'-OJ'-CM'-rjl O 8 H d'- '<ü

.O

Cd £ h to c

• H

Ö <u

• H H

J cd Jh φ cd · Η o e cd

• P m and o H

3 0 -P 3 c φ Φ · η c o cd ö ö k cd and i> <u φ Φ cd 3>> o * -p ε Φ cd cd cd El cd N ^ Sh O in

O +3 -P -p & -P

• H ö ö ö 3 ö

OS W Μ M CO M

cd cd

CM CO

co <o <tH «- i- O 1 1 -p CO EH Eh 8004759 ♦ *

As shown in Tables Z and L, a tumor antifouling activity is observed in ds growth in which each of the TF substances of the invention was administered.

(c) Anti-tumor action tar Sarcoma-13l carcinomas.

Sarcoma-120 carcinomas were transplanted intraneritically to ICR stems (female, 6 weeks old) at 1 x 105 cells per mouse. Subsequently, each of the test substances was administered intraperitoneally to the mice at a dose of 1 mg / kg, 5 mg / kg or 10 ng / kg repeated once a day from the first 10 days to the seventh day for a total of 7 days, 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. The comparative green was administered 0.2 ml of physiological saline in the same manner as described above. The results are shown in Table M.

8004759 52 e o

IN

0) .r- 3 H £

cd Φ -K * * -K

-P P

C m in cd <U VO VO VO VO VO VO VO VO VO VO VO VO in m * - <+3

l \ X V ^ X X V ^ X ^ V N X V

I-1 A

<u c \ i «-oo p- on * - o on on cm o in u \ c>

0 C Φ H

cd c P 0)

C!> C <u

<H

* O ^ C Ο v- C CO on CM O t- cm vo ο O Q o

T- »- c in p- * - c in p- p- o c o C

C- (<- ^ t— 1— 1— v— t— r— τ— i— r— T- * * - r— 0 ^ ΓΟ ’-

A A A A A AA A AA

d ö ed a) p to on d (d * cö ό ο o on id ta ,,, tö'- '+! +1 +! rrj £ * Η · η rf co cm on cm on ma cj cm on o cm ο ο o ^ 71 (yy ^ f \ * sna η λ is f> r is Λ is r> fs λ ^ d ΰ m - c \ oc— »- cs oc— co c \ cj (\ ip γΜ-οη cm cm cm <- on cm cm> - on cm cm - p- p- * -

“S Φ ^ A A A A AAA A A

a3 1)>

Eh Ο O

........ 11 "" ................... · * *

00 I

p ο \ o to -p g

- H

td Ö VO VO VO VO MO VO VOVOVO C— Ρ ω P 0)

C C ίώ K K * I «« «I K X * I« «I

, J j $ £ * i-i cd · η o in * - o in <- o in * - on Φ a * - * - - ^ w P a>

O (U -H

Q λ n cd cd cd p CM on vo cm on on on on

o 7 7 I I

P P =, CO Eh Eh Eh Eh 8004759 53 OJ <-

* CVJ HK OJ

+: +:

O O O IA O Q O

t— t— t— t-1-τ- LA LA LA

'n \ X V. ^ V \ V N \ N \ X

O O t— O VO LA O LA l3- O -3-1-0 r · «- d c ¢) © 'ö

r-H rH d Η Η CÖ Φ Φ P COW

d d Φ Ο Ο Ο

_ +3 Ρ C

φ 0) Φ Ν Ό Ό Φ d - d C Φ

VC VC VO Ο ΙΑ Ο C τ- VC Ο <- t— Ο CÖCÖtC

CO CC VD Ο LA -3-0 Ο> - σ t— C— Ο r-i »CM C! CM r- CJ pj - CM OJ »- CM« - * - fc £ A A * A A A A A A A A. «S .¾ 3

P -P Φ cö d H

SI P 4y H

d d 0 I — I Cti CÖ> C - ------ ί—! —1 rO i — t Ï-U d CÖ W W · Η = H LA CO d d COCO VO VO VO "ΙΛ A ON λ LA CÖ d d

Λ * ΛΛΛ Γ- Λ Λ Λ £ \ J Λ ^ Et GJ

η o o vo co omen <- a oi co> - oj ρ ρ τί> +1 + | +] +! +1 +! +! +1 +1 +! +1 + i +1 φ φ φ d Ό Τί> Φ Ο Ο CO Ο Ό CO C \ C0 Ο 03 Ο Φ

ί \ Λ ** ί> Λ * »*» Λ • '•' «'CÖCÖl — I

OOt— -3- 3 · CJ Μ CO Ο -3 * C3 -3--3- d G d -3- -3- CO v- CO CO »- CM CO» - 00 CM * - Φ AAAAAAAA .A $ $ §

Tl Ti Φ φ Φ Ό P P W W C LA Ο Φ CO -3- d £ 0 φ Φ> T) T5

____OJ

& c- + 0 0 w ta μ h d ö cö

• H -H

f — I r-i »- t— t— t— t-t— t — t— VO VO ΦΦ + Ό Tj

XXXI XXI X X I XXI d d H

Ο Ο Φ O LA «- LA * - LA» ~ LA »- 0 is 0

r- Φ φ O

& JO N

bet „____Q Q 1-3

«- CM

£> CO VO * «

CO CJ - VO

CO 00 00 CO

I— 1, -, - 4-3 I I I 10

lx, Pl lx, fc O

Eh Eh Eh Eh 5 8004759

As can be seen from Table M, the groups to which the TF substances of the invention had been administered had anti-tumor activity compared to the control group.

(d) Anti-tumor enrichment against 3-16 melanomas. p (1) B-16 Meianoma tumor cells were transplanted subcutaneously in the armpit of BDF stem mice (male, 7 weeks old) in an amount of 1 x 10 cells per mouse. Subsequently, TF-133a was administered intraperitoneally to the mice at a dose of 5 mg / kg or 10 mg / kg once a day on the first day and from 10 the third day through the seventh day for a total of 6 days after transplantation. of the tumor cells. In the same manner as above, 0.2 ml of physiological saline were administered to the control group and 20 mg / kg 5-FU to the group for the 5-FU administration.

The results are shown in Table N.

Table N

Substance Dosage (mg / kg x Average tumor T / C {%) the number of weight (g) _services_______________ 5x6 U, 0? 70 20 TF-133a 10 x 6 2.52 i * 3 5-FU! 20 x 6 U, 21 72 i

Comparison - 5j8 ^ 100 25 ___: -

As shown in Table N, the groups administered with TF-133a had apparently tumor growth inhibitory activity, compared to the control group, and the groups administered with 5 mg / kg and 10 mg / kg TF-133a had equal or 30 more pronounced tumor growth inhibitory activity compared to the group administered with 5-FU.

(2) The transplantation of B-16 meianoma tumor cells and the administration of TF-132a, TF-133a, 5-FU and physiological saline were performed in the same manner as above in 35 (1). On the 21st day after transplantation of the tumor cells, the mice were dissected and the metastasis of the meianoma tumor cells 8004759 55 * to the lungs was assessed.

The results are shown in Table 0.

Table 0

Substance Dosage (mg / kg x Average on) Percentage the number of metastases metastatic services inhibition (5) 5x6 16.0 h8 TF-132a 10 x 6 9.36 69 10 5x6 9.3 70 TF-133a 10 x 6 6.7 73 5-FU 20 x 6 2U, 8 20 15 Comparison - 31.0 0 y

As shown in Table 0, in the groups administered TF-132a and TF-133a, the metastasis of B-16 melanoma markedly inhibited tumor cells, compared to the green administered 5-FU.

(IV) Acute toxicity.

LD ^ for mice is shown in Table P.

80 0 4 759 - r po

Table?

Substance λτ1 method of administration LD wjï-z) TT-100 Intraperitoneal 500 TF-110 Intravenous> 250 Intraperitoneal> 1000

Intravenous> 250 TF-120 Intraperitoneal> 1000

Intravenous 300 intraperitoneal> 500

Intravenous 300 133a intraperitoneal> 500

Intravenous 300 Intraperitoneal> 500 TF-132b Intravenous> 100 TF-133b Intravenous> 200 TF-lk0 Intravenous> 100 TF-150 Intravenous> 200 TF-1323 Intravenous> 200 TF-131Ö Intravenous> 200 ___! _ 8004759 I 4 4 51

As shown in the above pharmacological tests, the TF substances of the invention are useful as carcinostatic agents which are expected to act against various cancerous conditions and are expected to have significant activity against certain solids. cancer types. All TF fabrics of the invention have excellent performance, although TF-130, especially 132a, 132b, 133a, 133b and 1323 are preferred for several reasons.

The TF substances of the invention can be used after they have been incorporated into various pharmaceutical prenarates, such as oral drugs, injections, steroids or the like, although they are preferably used in the pharmaceutical form of an injection. When used as oral medicaments, the oral medicaments may contain various excipients and are formed into capsules, tablets, powders or granules. When used as injections, the injections can be any subcutaneous injection, intramuscular injection or intravenous injection and they are used in the form of a suspension, a solution or a powder, which is dissolved in use. The injections can contain a local anesthetic.

The dosage of the TF substances of the invention is suitably selected depending on the circumstances of a patient, although it is generally desirable to administer them in an adult dosage of 0.01 - 50 mg / kg once daily or several times a day and in terms of the mode of administration, they are preferably administered by subcutaneous, intramuscular or intravenous injection or injection into the affected part.

The invention is further illustrated in the examples below and the accompanying drawing, of which Fig. 1 shows a microscopic photograph showing the shape of Fusobacterium nucleatum TF-031 used in the invention, Fig. 2 shows an infrared absorption spectrum of the carcinostatic substance TF -100, 35 with the wave * number in cm-1 along the horizontal axis and the light transmission in% along the vertical axis. Fig. 3 shows an ultraviolet 8004759 58 ♦.

absorption spectrum of that substance with units in nm along the horizontal axis; Fig. U gives an elution pattern obtained from that substance being subjected to gel filtration using Sephadex G-50 with the number of volume-5 units in ml along the horizontal axis and along the vertical axis (OD 260 nm ^ 20nm ^ X ^ and in the graph - OD 2Ê0nm and x - x OD 620nm, Fig. 5 shows a high power liquid chromatogram with the time in min along the horizontal axis in which the solid line is UV 220nm and the dotted line IJV is 20 nm, Fig. 6 shows an infrared absorption spectrum of the carcinostatic substance TF-110 with the wave number in cm ^ along the horizontal axis and the light transmission in Fig. 7 along the vertical axis shows an ultraviolet absorption spectrum of that substance. with the units in nm along the horizontal axis, Fig. 8 shows an elution pattern obtained when that substance is subjected to gel filtration using Sephadex G-200 with along the vertical axis on the left (OD U90 - ^ ^ gQnra ^ and on the back along the vertical axis (O.D 2c0 nm) and along the horizontal axis the number of ml where x-x-x O.D is 260 nm in the graph, O.D

Is 20 ^ 90nm and represents 0-0-0 O.D 620 nm; Fig. 9 shows a high power liquid chromatogram of that substance with the time in minutes along the horizontal axis in which the solid line represents UV 220 nm and the dotted line UV 260 nm; Fig. 10 shows an infrared absorption spectrum of the carcinostatic substance TF-120, in which the wave number in cm 1 is shown along the horizontal axis and the light transmission in% along the vertical axis; Fig. 11 shows an ultraviolet absorption spectrum of that substance with the number of units in nm along the horizontal axis; Figure 12 shows an elution pattern obtained when that substance was subjected to gel filtration using Sephadex G-200 along the left vertical axis (OD ^ 90 nin £ 20nm ^ sn right vertical axis (OD 2FC0 nm) and along the horizontal axis the number of ml where in the graph xxx is OD 260 nm, OD is U90 nm and 0-0-0 ÖD is 620 nm, Fig. 13 shows a high power liquid. chromatogram of that substance in which the time is plotted in min. along the horizontal axis and shows the solid line UV 220 nm and the 8004759 59 * dotted line UV 2Ê0 nm; fig. 1 ^ + shows an infrared absorption spectrum of the carcinostatic substance TF-316 with along the horizontal axis the wave number in cn "1 and along the vertical axis the light transmission in FIG. 15 shows an ultraviolet absorption spectrum of that substance just along the horizontal axis the number of units in nm; FIG. 16 shows an elution pattern obtained when that substance was subjected to gel filtration using v n Sephadex G-200 with the left-hand side along the vertical axis (O.D bpO nm260nnP and lan®s the horizontal-focal axis the number of ml where x-x-x O.D is 260 nm and O.D 390 nm; Fig. 17 shows a high power liquid chromatogram of that substance with the time in min along the horizontal axis with the solid line UV being 220 nm and the dotted line UV 260 nm; Fig. 18 shows an infrared absorption spectrum of the carcionostatic substance TF-132a 15 with the wave number in cm 1 along the horizontal axis and the light transmission in% along the vertical axis, Fig. 19 shows an ultraviolet absorption spectrum of that substance with the number of units in nm along the horizontal axis; Fig. 20 shows an elution pattern obtained when that substance was subjected to gel filtration 20 using Sephadex G-200 with the left side along the vertical axis (OD 1 + 90 µgQnm ^ and the right side along the vertical axis (OD). 260 nm), along the horizontal axis, the number of ml where in the graph xxx OD is 260 nm, OD 1 + 90 nm and

o-o-o O.D is 620 nm; Fig. 21 shows a high power liquid chromatogram of that substance with the time in min along the horizontal axis with the solid line UV 220 nm and the dotted line UV 260 nm; Fig. 22 shows an infrared absorption spectrum of the carcinostatic substance TF-132b with the 'wave' number in cm -1 along the horizontal axis and the light transmission 30 in% along the vertical axis, Fig. 23 shows an ultraviolet absorption spectrum of that dust with units in nm along the horizontal axis; Figure 2b shows an elution pattern obtained when that substance was subjected to gel filtration using Sephadex G-200 with the number of ml along the horizontal axis and along the vertical axis (OD 260 h ^ pg ^) with graph xxx OD

260 nm and O.D 1 + 90 nm; Fig. 25 is a high power flow 800475960

substance chromatogram of that substance with the time in min along the horizontal axis, with the solid line LP / 220 nm and the dotted line representing UV 260 nm; Fig. 26 shows an infrared absorption spectrum of the carcinostatic substance TF-133a with the wave number in 0111-1 along the horizontal axis and the light transmission in% along the vertical axis. Fig. 27 shows an ultraviolet absorption spectrum of that substance in the horizontal axis the units in nm; Fig. 28 shows an elution pattern obtained when that substance was subjected to gel filtration using Sephadex G-200 with the number of ml along the horizontal axis and along the left vertical axis (0.D 1 + 90 nng20nm ^ en ^ an®) s rec5: lter vertical axis 0.D 260 nm where xx 0.D 260 nm, - OD 1 + 90 nm and 0-0 OD 620 nm in the graphs; Fig. 29 shows a high power liquid chromatogram of that substance represents the time in minutes along the horizontal axis, the solid line being üV 220 nm and the dotted line representing UV 260 nm; Fig. 30 shows an infrared absorption spectrum of the carcinostatic substance TF-133b with the wave number in the horizontal axis. cm and * along the vertical axis the light transmission in%. Fig. 31 shows an ultraviolet absorption spectrum of the substance with the units in nm along the horizontal axis; Fig. 32 shows an elution pattern obtained when the substance was subjected to gel filtration with using Senhadex G-200 with along the ho horizontal axis the number of ml and along the vertical axis O.D 26θητη and U90 nm where in the graph x-x-x O.D 260 nm and O.D

1 + 90 nm; Fig. 33 shows a high power liquid chromatogram of the substance with the time along the horizontal axis in min. where the solid line UV is 220 nm and the stubble line TTV is 269 nm; Fig. 3 * + shows an infrared absorption spectrum of the carcinostatic substance TF-1323 in which the wave number in cm-1 is plotted along the horizontal axis and the light transmission in% along the vertical axis; Fig. 35 shows an ultraviolet absorption spectrum of the substance with the number of units in nm along the horizontal axis; fig.

36 shows an elution pattern obtained when the substance was subjected to gel filtration using Sephadex G-200 with the number of ml along the horizontal axis and along the 8004759 4 6ι vertical axis OD 20 ma and 190 nm with the graph xxx OD 260 ma and OD is 190 nm; Fig. 3T shows a high power liquid chromatogram of the substance with the time in min along the horizontal axis, with the solid line UV 220 nm and the dotted line UV 260 nm; Fig. 33 shows an infrared absorption spectrum of the carcinostatic substance TF-136 with the wave number in cm-1 along the horizontal axis and the light transmission in% along the vertical axis; Fig. 39 shows an ultraviolet absorption spectrum of the substance with units in nm along the horizontal axis; Fig. 10 shows an elution pattern obtained when the substance is subjected to gel filtration using Sephadex G-200 with the number of ml along the horizontal axis and O.D. along the left vertical axis. I90 nm and 620 nm and along the right vertical axis O.D 260 nm where x-x O.D 260 nm, .-. O.D 190 nm 15 and 0-0 O.D 620 nm display; Fig. 11 shows a high power liquid chromatogram of the substance with the time in min along the horizontal axis with the solid line UV being 220 nm and the dotted line UV 260 nm; Fig. 12 shows an infrared absorption spectrum of the carcinostatic substance TF-10 with the wave number cm "* 1 along the horizontal axis and the light transmission in% along the vertical axis. Fig. 13 shows an ultraviolet absorption spectrum of the substance along the horizontal axis. axis units in nm, Fig. 11 shows an elution pattern obtained when the substance was subjected to gel filtration using Sephadex G-200 with along the left vertical axis OD 190 nm and along the right vertical axis OD 260 nm, along the horizontal axis is the number of ml where in the graph xxx is OD 260 nm and OD -90 nm, Fig. I5 shows a high power liquid chromatogram of the substance with the time in min along the horizontal axis at which the 30 solid line UV is 220 nm and the dotted line is UV 260 nm, Fig. 16 shows an infrared absorption spectrum of the carcinostatic. * -1 substance TF-150 with the wave number cm along the horizontal axis and the light transmission in% along the vertical axis. 1? T shows an ultraviolet absorption spectrum of the substance with the number of units in nm along the horizontal axis; Fig. 18 shows an elution pattern obtained when the substance was subjected to gel filtration 8004759 62

just using Sephadex G-200 with the number of ml along the horizontal units and along the vertical axis O.D 260 nm and 1 * 90 nm where x-x-x O.D 260 nm and O.D

il-90 nm; and Figures 1 + 9 show a high power liquid chromato-5 gram with time along the horizontal axis in min. where the solid line UV is 220 nm and the dotted line is UV 260 nm.

Example I

(1) In each of 15 2 1 culture bottles, each with a screw cap, 2 1 were placed in a culture medium containing 3I + g 10 trypticase peton, 6 g phyton peptone, 20 g proteose peptone, 70 g of a brain-heart infusion, contains 6 g of yeast extract, 15 g of sodium chloride, 12 g of glucose, 10 g of lactose, 0.5 g of L-cystine, 0.2 g of sodium sulfite, 1.0 g of sodium thioglycolate and 1.1 µg of agar and the culture medium is set to a pH of 7. The culture medium was 15 under a pressure of 1.2 at. Sterilized at 120 ° C for 15 min, warmed in a boiling water bath for 20 min and then immediately cooled with water, followed by a culture solution of Fusobacterium nucleatum TF-031 previously prepared by placing it in a culture medium to culture with the same composition as above, inoculated into the above water-cooled culture medium was seeded in sterile conditions in an amount of 100 ml per culture bottle and under stable culture in an incubator at 37 ° C for 1 + 8 hours. Upon completion of the culture, the culture was centrifuged to remove organisms for 20 min at 5 ° C at a rate of 1000 rpm. The amount of the supernatant obtained was about 27 liters.

(2) To the supernatant obtained under (1), 1 + 0 1 ethanol was added with stirring at 5 ° C and the resulting mixture was allowed to stand in a room at low temperature until the amorphous precipitate had completely settled. Then the mixture was centrifuged for 15 min at 5 ° C and 6000 rpm and the precipitate was collected, washed with ethanol and then dried under reduced pressure to obtain 60 g of crude powder.

(3) 20 g of the crude powder obtained under (2) were dissolved in 120 ml of water and the water-insoluble materials formed at this time were removed by centrifugation at 7500 rpm for 10 min, after which the thus water-soluble fraction obtained was combined with the washings obtained by washing the water-insoluble materials twice with 60 ml of water each time and the resulting solution was dried under reduced pressure to obtain 1 g of a white-grayish light brown powder of TF-100.

Example II

The solution obtained in Example I (3), which was prepared by combining the water-soluble fraction, free from the water-insoluble materials, with the washings obtained by washing the water-insoluble materials, was subjected to ultrafiltration through a hollow fiber type ultrafiltration system 15 (membrane No. HI-1), and the inner solution was lyophilized to give 10 g of a gray-white-light brown powder of TF-110.

Example III

10 g of the powder obtained in Example II were dissolved in a small amount of water and the resulting solution was applied to a column filled with diethylaminoethyl-Sephadex A-50 equilibrated with 0.025 molar phosphate buffer with a pH of 8 and the eluted solution was collected. Further, 2.5 L of the same phosphate buffer 25 as above were passed through the column and the resulting eluted solution 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), was concentrated under reduced pressure and then was subjected to lyophilization to obtain VfO mg of a gray-white-light brown powder of TF-T20.

Example IV

2 g of the powder obtained in Example 1 (2) were dissolved in 2k ml of water, and the water insoluble materials were removed by centrifugation at 7500 rpm for 10 min.

The water-soluble fraction was dialyzed overnight against 8004759 6b distilled water at 5 ° C using a cellophane tube and the inner solution was concentrated under reduced pressure. Then, the concentrated solution was applied to a column packed with 150 ml diethylaminoethyl-5 Sephadex A-50, which had been previously equilibrated with a 0.025 molar phosphate buffer with a pH of 8, and the column was washed with 600 ml of a 0.025 molar phosphate buffer with a pH of 8 was passed therethrough, after which a 0.025 molar phosphate buffer with a sodium chloride concentration of Q, c mol and a pH 10 of 8 was passed through the column to obtain an eluate. The eluate was concentrated to about 100 ml under reduced pressure, dialyzed against distilled water at 5 ° C, concentrated again under reduced pressure, desalted using a Sephadex G-25 column and then lyophilized to yield 1 + 70 mg of gray white light brown TF-1316 were obtained.

Example V

The solution as described in Example 1 (3), which was prepared by combining the water-soluble fraction free of water-insoluble materials with the washings obtained by washing the water-insoluble materials, was dialyzed overnight against distilled water at 5 ° C. 0 using a cellophane tube and the inner solution was concentrated under reduced pressure. The concentrated solution was then applied to a column that had been made with 50 g of diethyl-25 aminoethyl-Sephadex A-50, which had been previously equilibrated with a 0.025 molar phosphate buffer with a PI of 8 and 2 L of a 0.025 molar phosphate buffer with a pH of 8 and 2.5 l of a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.1 molar and a pH of 8 were passed successively through the column, after which 2.5 l of a 0.025 molar phosphate buffer with a sodium chloride concentration of 0 , 2 moles. and a pH of 3 was passed through the column to elute the active agent adsorbed on the column and the resulting eluted solution was desalted using a hollow fiber type ultrafiltration-35 system (membrane no., HI-1) and then subjected to lyophilization, giving 600 mg of gray-white-light brown powder of TF-132a 8004759 65.

Example VI

In 1.2 L of a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.3 mol. and a pH of 3, 5 HU, 2 g were dissolved from the stock obtained in Example 1 (2), and the suspended insolubles were removed by filtration using Hyflo Super Cel and the resulting filtrate was applied to a column with a diameter 33 cm. packed with 500 ml diethylaminOethyl-Sephadex A-50 previously equilibrated with a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.3 mol. and a pH of S and the eluted solution was collected. The column was washed with a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.3 mol. and a pH of 8 and the washings 15 were combined with the above eluted solution to give 1.6 µl of solution. The solution was repainted on an 8 cm diameter column with HO ml of diethylaminoethyl-Sephadex A-50 previously equilibrated with a 0.025 molar phosphate buffer with a sodium 20 chloride concentration of 0.3 mol. and a pH of 8, affording 1.63 L of eluted solution. The column was washed with a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.3 mol. and a pH of 8 to give 1.6H 1 washes. The eluted solution and washings were combined and a 0.025 molar phosphate buffer with a pH of 8 was added thereto, yielding H 98 L of a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.2 mol. and a pH of 8 was obtained. The buffer solution thus obtained was applied to a column with a diameter of 8 cm, packed with 500 ml of diethylaminoethyl-Senhadex A-50, which had been equilibrated beforehand with a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.2 mol. and a pH of 8, and the column was washed with 2 L of a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.2 mol. and a pH of 8.1 To 7 L solution prepared by combining the washings with the eluted solution, a 0.025 molar phosphate 8004759 66 buffer with a pH of 3 was added to give 1U 1 of a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.1 mol. and a υΗ of 8. The eluted solution thus obtained was applied to an 8 cm diameter column packed with 500 ml diethvlaminoethyl-Sephadex A-50, which had been previously equilibrated with a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.1 mol. and a pH of 8, and the eluted solution was removed. The column was washed with 2 L of a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.1 mol. and a pH of 3, after which 2 L of a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.2 mol. 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 active carbon and then lyophilized to yield 330 mg of gray-white-light brown TF-132b powder.

Example VII

10 g of the powder obtained in Example II were dissolved in a small amount of water and the resulting solution was applied to a column which had been dipped with diethylaninoethyl-Sephadex A-50, which had been previously equilibrated with a 0.025 molar phosphate buffer. with a pH of 3, Then 5 liters of a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.2 mol. and a pH of 8 passed through the column, after which 2.5 L of a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.3 mol. and an πίί of 8 is passed through column 30 to elute the active substance adsorbed on the column, and the resulting eluted solution is concentrated under reduced pressure. The concentrated solution was desalted using a cellophane tube, completely desalted using Sephadex G-25 and then lyophilized to yield 600 mg of gray-white light brown powder of TF-133a.

8004759 67

Example VIII

In 1.2 l of a 0.025 molar phosphate buffer, just a sodium chloride concentration of 0.3 mol. and an nH of 8 bh, 2 g of the powder obtained in Example 1 (2) were dissolved and the 5 insoluble materials suspended were removed by filtration using Hyflo Super Cel, after which the resulting filtrate was applied to a 33 cm column diameter, packed with 500 ml diethylaminoethyl-Sephadex A-50, which had been previously equilibrated with a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.3 mol. and a pH of 3, and the eluted solution was collected. The column was washed with a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.03 mol and a pH of 8, and the washings were combined with the above eluted solution to give 1.67 L of solution. The solution was re-applied to an 8 cm diameter column packed with bo ml of diethylaminoethyl-Sephadex A-50 previously equilibrated with a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.3 mol. and a pH of 3, whereby 1.68 L of eluted solution was obtained. The column was washed with a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.3 mol. and a pH of 8, giving 1.6h of 1 washes. The eluted solution and washings were combined and a 0.025 molar phosphate buffer having a pH of 8 was added thereto, yielding 1 + .98 L of a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.2 mol. and a pH of 8. The eluted solution thus obtained was applied to an 8 cm diameter column packed with 500 ml diethylaminoethyl-Sephadex A-50 which had been previously equilibrated with a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.2 mol. and a pH of 3, and the column was washed with a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.2 mol. and a pH of 8, after which 2 l of a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.3 mole. and a pH of 8 was passed through the column and the eluted solution was collected. The eluted solution was concentrated 8004759 63 and desalted using an ultrafiltration system (filter used: Toyo Ultrafilter UK-10), further desalted using a Sep'nadex G-25 column, decolorized with active carbon and then lyophilized yielding 590 mg of a gray-white light brown powder of TF-133b.

Example IX

(1) 20 g of the crude powder obtained in Example 1 (2) were dissolved in 120 ml of water, and the water-insoluble materials formed at this time were removed by centrifugation at 7500 rpm for 10 min, after which the thus obtained water-soluble fraction and washings, obtained by washing the water-insoluble materials twice with 60 ml of water, were combined, and then subjected to ultrafiltration using a whole fiber type ultrafiltration (membrane No. HI- 1) and the inner solution was subjected to lyophilization to give 10 r. gray white-light brown mother were obtained.

(2) In 1 L of a 0.325 nolar phosphate buffer with a sodium chloride concentration of 0.3 mole. and an oH of 3, 25 g of a powder obtained by the treatment described under (1) above were dissolved and the resulting solution was applied to a column with a diameter of 33 cm, packed with 500 ml diethylaminoethyl-Sephadex A-50 which had previously been equilibrated with a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.3 mol. and a pH of 3. The column was washed correspondingly with a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.3 mol. and a pH of 8, and the eluted solution and washings were combined to give 1.6 L of mixture. The mixture was applied to a column with a diameter of 3 cm, contacted with 100 ml of diethylaminoethyl-Sephadex A-50, which was likewise equilibrated with a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.3 mol. and a pH of 8, whereby 1.65 L of eluted solution were obtained.

The eluted solution was combined with 1.7 L washes, 8004759

V

£ 9 J.

by washing the column with a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.3 mol. and a pH of 3, and the resulting mixture was diluted with 0.025 molar phosphate buffer having a pH of 8 to prepare 10.35 L of a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.1 mol. and a pH of 8.

Then, the buffer solution thus obtained was applied to an 8 cm diameter column packed with 500 ml diethylaminoethyl-Sephadex A-50 which had been previously equilibrated with a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.1 mol. and a pH of 8, and the column was washed with 2 L of a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.1 mol. and a pH of 8, after which 2 liters of a 0.025 molar phosphate buffer with a sodium chloride-15 concentration of 0.3 mol. and a pH of 8 was passed through the column and the eluted solutions were collected. The eluted solution thus obtained was desalted and concentrated using an ultrafiltration system (filter used: Toyo Ultrafilter UK-10) and further desalted on Sephadex G-25, decolorized with active carbon and then subjected to lvophilization to yield 1.65 g of greyish white. TF-1323 light brown powder were obtained.

Example X.

10 g of the powder obtained in Example II were dissolved in a small amount of water and the resulting solution was applied to a column packed with diethylaminoethyl-Sephadex A-50, which had been previously equilibrated with a 0.025 molar phosphate buffer with a pH of 8. Then 6 L of a 0.025 molar phosphate buffer with a sodium-30 chloride concentration of 0.5 mol. and a pH of 3 is passed through the column, after which 2.5 l of a 0.025 molar phosphate buffer with a sodium chloride concentration of 0.6 mol. and a? H of 8 was passed through the column to elute the active substance adsorbed on the column, and the resulting solution passed was desalted using a hollow fiber type ultrafiltration system (membrane No. HI-1), under reduced pressure concentrated 8004759 το and then subjected to lyophilization to yield 130 mg of gray-white-light brown powder of TF-136.

Example XI

15 g of the crude powder obtained in Example 1 (2) were dissolved in 200 ml of water and the water-insoluble materials were removed by centrifugation at 7500 rpm for 10 min, after which the water-soluble fraction thus obtained was adjusted to pH 10 by adding a 0.2 molar aqueous barium hydroxide solution dropwise thereto. A white precipitate was formed by the addition of the aqueous barium hydroxide solution. Earium ions were added so that the concentration of the total barium ions in the final volume was 0.01 mol, then the solution was stirred for 30 min and the precipitate was collected by filtration. The precipitate of a barium salt thus obtained was suspended in 10 ml of 10 wt.% Aqueous sodium sulfate solution, stirred sufficiently and then filtered and the filtrate was collected. The precipitate was resuspended, stirred and then filtered in the same manner as above, once with 10 ml of a 10 wt% aqueous sodium sulfate solution and twice with 5 ml aliquots of that solution and each filtrate was collected. The residual precipitate was then washed twice with 10 ml of water each, and then filtered to obtain washings. The above collected filtrate and washings were combined, dialyzed against distilled water, concentrated under reduced pressure and then lyophilized to yield 0.5 g of a grayish white-light brown powder of TF-1U0.

Example XII

32 g of the powder as obtained in Example 1 (2) were dissolved in U50 ml of water and the suspended insoluble materials were removed by filtration using Hyflo Super Cel, after which the obtained filtrate was dialyzed overnight against running water using of a cellophane tube. To the dialyzed solution was added dropwise 80 ml of a 20 wt.% Aqueous cetylpridinium chloride 8004759

V

solution added with stirring while adding 200 ml of a 10 wt% borate buffer (? H 9> 0) and the resulting mixture was stirred at room temperature for 30 min, after which the precipitate was collected by filtration. The precipitate 5 was suspended in 150 ml of a 0.1 wt. 5 'borate buffer (pH 9.0) containing 0.1 mol. sodium chloride and 0.2% by weight of cetylpyridinium chloride and the resulting suspension was stirred for 30 minutes. Then, the precipitate was collected from the suspension by filtration and resuspended in 150 ml of a borate-10 buffer (pH 9.0) of the above composition and the suspension thus obtained was stirred for 30 min, after which the precipitate was collected by filtration . The collected precipitate was suspended in 150 ml of a 0.1 wt% borate buffer (Pi 9.0) containing 0.5 mole. sodium chloride and 0.25 cetylpyridinium-15 chloride and the resulting slurry was stirred and dissociated for 30 min. Then the precipitate was separated from the slurry by filtration to obtain a soluble fraction. Then, the precipitate separated by filtration was again subjected to the same dissociation method as above in order to obtain a soluble fraction. The two soluble fractions were combined and the resulting soluble fraction was adjusted to a pH of 3 to 10 with 10% hydrochloric acid and ethanol was added thereto so that its concentration was finally 30 vol. amount. The resulting solution was allowed to stand at 5 ° C overnight and the formed precipitate was collected by filtration to yield 5.6 g of TF-150 gray-white-light brown powder.

Preparation examples.

Preparation Example 1 Each vial was filled with 1 mg or 5 mg of one of the powders of TF-100, 110 and 120 as obtained in Examples I, II and III. The powders are dissolved in sterilized saline, a solution containing 0.5 * lidocaine or the like when used, and then used as injections.

35 Preparation example 2

Each vial was filled with 1 mg or 5 mg of one of the 8004759 72 powders of 27-1316, 132a and 132b as obtained in Examples 17, 7 and 71. The powders are dissolved in sterilized physiological saline, a solution containing 3, 5 contains lidocaine, or the like when used and then used as injections.

5 Preparation example 3

A vial was charged with 1 mg or 5 mg of the powder of TF-133a obtained in Example VII. The powder is dissolved in a sterilized physiological saline solution, a solution containing 0.5% lidocaine or the like, in use and then used as an injection.

Preparation example h

A vial was packed with 1 mg or 5 mg of the powder of IFF-133b obtained in Example VIII. The powder is dissolved in sterilized physiological saline, a solution containing 0.5% lidocaine, or the like, when used and then used as an injection.

Preparation example 5

A vial was packed with 1 mg or 5 mg of the powder of TF-1323 obtained in Example IX. The powder is dissolved in sterilized physiological saline, a solution containing 0.5% lidocaine or the like, when used and then used as an injection.

Preparation example 6

Each vial was packed with 1 mg or 5 mg of one of the 25 powders TF-136, 1bo and 150 obtained in Examples X, XI and XII. The powders are dissolved in sterilized physiological saline, a solution containing 0.5% lidocaine, or the like, when used and then used as injections.

8004759

Claims (46)

1. Process for the preparation of substances having a carcinostatic and immunostimulating effect, characterized in that TF-anaerobic bacteria belonging to the Fusobacterium genus are cultivated under anaerobic conditions and the substances are collected from the culture or the supernatant thereof. 2. Process according to claim 1, characterized in that the TF-forming bacteria belonging to the Fusobacterium genus are cultivated under anaerobic conditions and a hydrophilic organic solvent is added to the culture or the supernatant thereof, after which the said substances are added with carcinostatic and immunostimulating action from the precipitate formed. 3. Process according to claim 1 or 2, characterized in that the water-soluble fraction of the precipitate is removed from the precipitate and then dried. Process according to claim 2 or 3, characterized in that the precipitate formed is collected, the water-soluble fraction of the precipitate is subjected to dialysis or ultrafiltration and the substances with carcinostatic and immunostimulating effect are recovered from the inner solution. 5. Process according to claim 2 or 3, characterized in that the precipitate formed is collected, the water-soluble fraction of the precipitate is subjected, if necessary, to dialysis or ultrafiltration and then treated with an ion exchanger and the non-adsorbed fraction is collected. 6. Process according to claim 2 or 3, characterized in that the precipitate formed is collected, the water-soluble fraction of the precipitate is subjected, if necessary, to dialysis or ultrafiltration, and then treated with an ion exchanger and the substances with carcinostatic and immunostimulating effect are isolated from the adsorbed fraction. 7. Process according to claim 6, characterized in that the adsorbed fraction is treated with a 0.6 molar sodium-35 chloride-phosphate buffer and the eluted fraction is collected. Process according to claim 6, characterized in that a fraction which is not eluted with a 0.1 molar sodium chloride phosphate buffer but is eluted with a 0.2 sodium sodium chloride phosphate buffer is collected from the adsorbed fraction. 9. Process according to claim 6, characterized in that a fraction which is not eluted with a 0.2 molar sodium chloride phosphate buffer, but which is eluted with a 0.3 molar sodium chloride phosphate buffer, is collected. 10. Process according to claim 6, characterized in that 10 sen collects a fraction which is not eluted with a 0.1 molar sodium chloride phosphate buffer, but eluted with a 0.3 molar sodium chloride phosphate buffer. 11. Process according to claim 6, characterized in that a fraction which is not eluted with a 0.5 molar sodium chloride phosphate buffer, but eluted with a 0.6 molar sodium chloride phosphate buffer is collected. 12. Process according to claim 2, characterized in that the precipitate formed is collected, if necessary subjecting the water-soluble fraction of the precipitate to dialysis or ultrafiltration, a 0.2-0.3 molar sodium chloride phosphate buffer of the water-soluble fraction. of the precipitate or a 0.2-0.3 molar sodium chloride phosphate buffer of the inner solution component, obtained by dialysis or ultrafiltration, and then treated with an ion exchanger equilibrated with a 0.2-0, 3 molar sodium chloride phosphate buffer; collects a fraction that has passed through the ion exchanger; then adjust this eluted solution such that its sodium chloride concentration is 0.1 mole. and then treated with an ion exchanger equilibrated with a 0.1 molar sodium chloride phosphate buffer to be adsorbed thereon; and then collects a fraction that is not eluted with a 0.1 molar sodium chloride phosphate buffer, but eluted with a 0.2-0.3 molar sodium chloride phosphate buffer. Process according to claim 2 or 3, characterized in that the precipitate is collected, if necessary subjecting the water-soluble fraction 8004759 of the precipitate to dialysis or ultrafiltration, a 0.3 molar sodium chloride phosphate buffer of the water-soluble fraction of the precipitate or a 0.3 molar sodium chloride phosphate buffer of the inner solution component, obtained by dialysis or ultrafiltration, and then treated with an ion exchanger equilibrated with a 0.3 molar sodium chloride phosphate buffer; collects a fraction that has passed through the ion exchanger; then adjust the sodium chloride concentration of this eluted solution to 0.2 mol. and then treating the solution with an ion exchanger equilibrated with a 0.2 molar sodium chloride phosphate buffer to be absorbed thereon, then a fraction that is not eluted with a 0.2 molar sodium chloride phosphate buffer, but is eluted with 0.3 molar sodium chloride phosphate buffer. 1¾. Process according to claim 1, characterized in that barium ions are added to the culture or the supernatant thereof to form a barium salt, a hydrophilic organic solvent is added to the culture or the supernatant thereof and the precipitate formed is collected and then adding barium ions to the water-soluble fraction of the precipitate thus collected, or adding barium ions to a solution of a powder obtained by drying the water-soluble fraction of the precipitate, to form a barium salt; the barium salt formed collects and then removes barium therefrom; and the water-soluble fraction is collected and subjected to dialysis or ultrafiltration. 15. Process according to claim 1, characterized in that a hydrophilic organic solvent is added to culture 30 or the supernatant thereof; collects the precipitate formed; the water-soluble fraction of the precipitate, or the inner solution component, obtained by subjecting the water-soluble fraction to dialysis or ultrafiltration, treating with a quaternary ammonium salt, collecting the precipitate formed and then subjecting it to a dissociation method using a solution containing sodium chloride contains; a hydrophilic organic 8004759 Tc organic solvent adds to the resulting insoluble fraction to form a precipitate, and the precipitate thus formed collects. 16. A method according to any one of the preceding claims, characterized in that a culture medium is used for cultivating the bacteria, which contains nitrogen sources, carbon sources, vitamin sources, reducing agents and inorganic salts, and if desired also agar. 17. The method according to any one of claims 1-15, 10, characterized in that a culture medium is used for cultivating bacteria containing trypticase penton, iyton peptone, proteose peptone, a brain heart infusion (or heart infusion), yeast extract, sodium chloride, glucose, lactose, L-cystine, sodium sulfite and thioglycolate, and optionally also agar. 18. Method according to one or more of the preceding claims, characterized in that the culture is carried out at 35 - 2 ° C for 1 - 5 days in a culture medium adjusted dat'-a pH of 6.0 - 3.5. 19. A method according to claim 13, characterized in that the cultivation is carried out at a temperature of 36-38 ° C for 1-3 days in a culture medium set at a pH of 7.2-8.2. A method according to any one or more of the preceding claims, characterized in that as bacteria belonging to it Fusobacterium genus uses Fusobacterium nucleatum. Process according to one or more of the preceding claims, characterized in that an alcohol is used as the hydrophilic organic solvent added asm the culture or the supernatant. Process according to one or more of the preceding claims, characterized in that the hydrophilic organic solvent is added to the culture or the supernatant in a solvent concentration of 30-70 mol%. 23. Process according to one or more of claims 6-22, 35, characterized in that an ion exchanger is used which uses a weakly basic ion exchanger or a weakly basic ion exchanger with a molecular sieve capacity. 2h. Process according to one or more of claims 6-23, characterized in that a 0.1-0.6 molar sodium chloride phosphate buffer is used to elute the fraction adsorbed on the ion exchanger. Process according to one or more of claims 7 to 2k, characterized in that the pH of the phosphate buffer containing sodium chloride, which is used for treating the adsorbed fraction, is adjusted to 3. A method according to any one of claims J-2k, characterized in that the fraction subjected to the ion exchange treatment is concentrated, desalted and dried. Process according to one or more of Claims 15 to 26, characterized in that cetyl-15 pyridinium chloride is used as the quaternary ammonium salt. 28. Process according to one or more of claims 15 to 27, characterized in that the treatment with the quaternary ammonium salt is carried out in the presence of a borate buffer, 29. Process according to one or more of claims 15 to 28, 20, characterized in that the sodium chloride-containing solution is a borate buffer containing sodium chloride and a quaternary ammonium salt. 30. Method according to one or more of claims 6-29, characterized in that Fusobacterium nucleatum is inoculated in a culture medium which is adjusted to a pH 7-2-2.8 and trypticase peptone, phytonpeptone, proteosepentone, a brain heart infusion (or a heart infusion), yeast extract, sodium chloride, glucose, lactose, L-cystine, sodium sulfite and thioglycolate and optionally also agar, and then grown under anaerobic conditions at 36-38 ° C for 1 - 3 days; adding an alcohol to the culture or supernatant at a concentration of 50-70 vol. collects the precipitate formed, subjects the water-soluble fraction of the precipitate to dialysis or ultrafiltration, if necessary, and then treats with a weakly basic ion exchanger or a weakly basic molecular sieve ion exchanger, in order to remove the unadsorbed fraction 8004759 • A; a fraction that is eluted does not remove a 0.1 or 0.2 molar sodium chloride phosphate buffer from the adsorbed fraction; and then collecting a fraction eluted with a 0.3 molar sodium chloride phosphate buffer, concentrating, unsalting and then drying. A method according to any one of claims 6 to 29, characterized in that Fusobacterium nucleatun is inoculated on a culture medium at 7.2 - 3.2, containing trynticasenenton, phytonpepton, proteosepepton, a brain-heart infusion ( or a heart infusion), yeast extract, sodium chloride, glucose, lactose, L-cystine, sodium sulfite and thioglycolate and optionally agar, and then grown under anaerobic conditions at 36 -38 ° C for 1-3 days; add an alcohol to the culture or its supernatant in a concentration of 50 to 70 vol. *; the precipitate formed collects, if necessary subjects the water-soluble fraction of the precipitate to dialysis or ultrafiltration and then a 0.2 - 0.3 molar sodium chloride phosphate buffer of the water-soluble fraction of the precipitate or a 0.2 - 0.3 molar inner chloride sodium chloride phosphate buffer obtained by dialysis or ultrafiltration treated with a weakly basic ion exchanger or a weakly basic molecular sieve ion exchanger equilibrated with a 0.2-0.3 molar sodium chloride phosphate buffer ; collects the fraction that has passed through the ion exchanger; adjust this eluted solution to a sodium chloride concentration of 0.1 - 0.2 mol. and then treated with a weakly basic ion exchanger or a weakly basic ion exchanger with a molecular sieve equilibrated with a 0.1-0.2 molar sodium-30 chloride phosphate buffer to be absorbed thereon; and collecting a fraction eluted with a 0.2 to 0.3 molar sodium chloride phosphate buffer from the adsorbed fraction, concentrating, desalting and then drying. 32. A method according to any one of claims 20, 30 or 31, characterized in that a strain of TF-031 is used as Fusobacterium nucleatum. 8004759 A .A- 33. Carcinastatic substance TF-100 with the following properties: (a) gray-white-light brown powder, (h) it inhibits the scattering of mice Ehrlich 5 ascites tumor and Ehrlich solid tumor and has an immuno-stimulatory action, (c) it is soluble in water and insoluble in methanol ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether, 10 (d) it has no sharp melting point, starts to decompose at about 110 ° C and decomposes markedly above 200 ° C, (e) it infrared absorption spectrum obtained with a KBr tablet method shows absorption hands in the vicinity of 3é00-3200, 2960-2930, 1670-161 * 0, 1550, 1 MO-1330, 121 * 0, 111 * 0-1000 15 and 820 cm ”\ ( f) the ultraviolet absorption spectrum of its aqueous solution shows a strong absorption at the absorption edge and shows an absorption peak in the range of 256-260 nm, (g) at a fractionating gel filtration (column: 50 mm diameter x 600 mm, eluent: distilled water) using Sephadex G. -50 (registered trademark), it has absorption bands at the elution volumes near the void volume -1 * 00, 1 * 30 - 530, 700 - 300 and 81 * 0 - 370 ml at the ultraviolet absorbance measurement at 260 nm , and at the elution volumes near 25 the void volume - 390 and 1 * 10 - 1 * 30 ml at the absorbance measurement at 620 nm by an anthron sulfuric acid method, (h) the high power liquid chromatogram (eluent: 0.1 molar phosphate buffer with a pH of 7, flow rate: 0.8 ral / min, at room temperature) obtained using TSK-GEL G300 Svi (trade name, column: 7.9 mm diameter x 600 mm x 2) shows peaks in near the solvent front, 38 -60 and 65 min. at the ultraviolet absorbance measurement at 220 nm and 260 nm, (i) it is positive in Molic reaction, phenol sulfuric acid reaction, anthron sulfuric acid reaction, indole hydrochloric acid reaction, Lowry - Folin's reaction and ninhydrin reaction, 8004759 33 (, j) basic analysis skills: n on oc; 7 ^ · ττ 1 · Ο ^ θ <*. η ^ ο - ζ '/ ^ 0 5 "- *" -32 3 · / "' 3" ♦ ·) »- J yC., - 1 y *« χ 3— - 1 (k) the saccharide content, measured net a phenol-sulfuric acid method "amounts to Ιϋ, Ο-1C, 3 parts", expressed as glucose, 5 and the protein content, measured according to Lovr, p-r'olin's method is 20.0-23.0 wt. , i, expressed as calving serum albunine, 3l Carcinostatic substance TF-110 with the following properties: (a) gri.jsvit-light brown powder, 10 (b) it inhibits the metastasis of mouse Shrlich ascites tumor and has an immunostimulating effect, (c) it is soluble in water and is insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether, 15 (d) it has no sharp melting point, begins to decompose, o. at 110 ° C and decomposes pronounced above 200 C, (e) it has an infrared absorption spectrum, obtained with a K3r tablet method with absorption bands in the vicinity of 3600-3200, 2960-2930, 1670-16I0, 1550, 1110-1380, 1210, nlO-1000 20 and 820 cm @ (f) ultra violet absorption spectrum of the aqueous solution shows strong absorption at the absorption edge and shows an absorption peak in the range of 256 - 260 nm, (g) when fractionated by gel filtration 25 (column: 11 ... mm diameter x 500 mm, eluent: a 0.1 molar phosphate buffer with a pH of 7) using Sephadex G-200 (registered trademark), it has absorption bands at the elution volumes near the void volume - 380 and 600 -920 ml at the ultraviolet absorption shade at 260 nm, at the 30 elution volumes near the void volume - 380, 120 - 520 and 630 - 760 ml for the absorbance measurement at I90 nm with a phenol-sulfuric acid method and at the elution volumes near the void volume - 380, 120 - 520 and 620 - 760 ml at the absorbance measurement at 620 nm with an anthron sulfuric acid method, 35 (h) the high power liquid chromatogram (eluent: 0.1 molar phosphate buffer with a pH of 7, flow rate: 8004759 0.8 ml / min. at room temperature) obtained using TSK-GEL G30Q SW (trade name, column: 7.9 mm diameter x 600 mm x 2) shows peaks near the oolosmid front, 38 - 60 and 65 min at the ultraviolet absorbance measurement at 220 nn 5 and 260 nm, (i) it is positive in Molic reaction, phenol-sulfuric acid reaction, anthron-sulfuric acid reaction, indole hydrochloric acid reaction and Lowry-Folin's reaction and negative in ninhydrin reaction, (j) elemental analysis values 10 C 35.9-1 + 1.0%, H U, 5-5.2%; NU, 2 - 5.2%, (k) the saccharide content, measured by a phenol-sulfuric acid method, is 30.0 - 69.0% by weight, expressed as glucose, and the protein content, measured by a Lowry-Folin1s method. is 18.0-22.0 wt / l, expressed as calving serum albumin. 35. Carcinostatic substance TF-120 with the following properties: (a) gray-white-light brown powder, (b) it prevents the metastasis of mouse Ehrlich ascites tumor and has an immunostinulating effect, 20 (c) it is soluble in water and is insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether, (d) it has no sharp melting point, starts to decompose at 110 ° C and decomposes markedly above 200 ° C, 25 (e) it has an infrared absorption spectrum, obtained by a KBr tablet method with absorption bands in the vicinity of 3600-3200, 2960-2930, 1670-16 ^ 0, 1550, 1380-1360, 100 µl-1000 and 820 cm -1, (f) the ultraviolet absorption spectrum of its aqueous 30 solution shows strong absorption at the absorption edge and shows an absorption peak in the neighborhood of 263-272 nm, (g) upon fractionation by gel filtration (column: hk mm diameter x 500 mm, eluent: a 0.1 molar phosphate buffer with a pH of 7) using Sephadex G-200 35 ver shows the absorption bands at the elution volumes near the void volume - 325 and 775 - 875 ml at the ultraviolet 8004759 32 absorbance measurement at 26 µm; at the elution volumes near the void volume - 360, 3'6θ - U3o and 510 - JoO ml at the absorbance measurement at U90 nm using a phenol-sulfuric acid method; and at the elution volumes near the void volume - 380, 5 b20 - 520 and 620 - 60 ml at the absorbance measurement at 620 nm by an anthrcn sulfuric acid method, (h) its high power liquid chromatogram (eluent: a 0.1 molar phosphate buffer with a pH of 7, flow rate: 0, 3 ml / min at room temperature) obtained using TSK-GEL G300 SW (column: 7.9 mm diameter x 600 mm x 2) shows peaks near the solvent front, 38 - 60 min at the ultraviolet absorption measurement at 220 nm and 260 nm, (i) it has a positive folic reaction, phenol sulfuric acid reaction, anthron sulfuric acid reaction, indole hydrochloric acid reaction and lovry-olin's reaction and a negative ninhvdrine yn «pf1 λ * - · - 'x i (j) elemental analysis values: c 35.1 - 0.20; H h, 5 - 5.56; 7 2.9-3.16.20 (k) the saccharide content, measured by a phenol-sulfuric acid method, is 5c, 0 - 73.0% by weight, expressed as glucose and the protein content, measured by Lowry-Folin's method is 9.0-13.0 wt.%, expressed as calving serum albunine. 36. Carcinostatic substance TF-130 with the following properties: (a) gray-white-light brown powder, (b) it prevents the metastasis of mouse Ehrlich ascites tumor, Ehrlich solid tumor and Sarcoraa-130 carcinomas and has an immunostimulating effect, (C) it is soluble in water and it is insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether, (d) it has no sharp melting point, begins to decompose at 110 ° C and decomposes markedly above 200 ° C, 35 (e) the infrared absorption spectrum, obtained with a KBr tablet method, shows absorption bands near 8004759 33 3cQ0-3200, 296Ο-293Ο, ΙόΤΟ-Ιο ^ Ο, 1550, lUl + QT 3β0, 12to, 11 to-1000 and 320 cm-1, (f) the ultraviolet absorption spectrum of its aqueous solution shows a strong absorption at the absorption edge and 5 shows an absorption peak in the range of 256-280 na (g) it has a positive lolic reaction , phenol-sulfuric acid reaction, ant'nron sulfuric acid reaction, indo hydrochloric acid reaction and Lowry-Folin's reaction and a negative ninhydrin reaction, (h) elemental analysis values: 10 C 27.5 - 39> 3 $; H 3.2 - 5.72; : i 2.3 - 6.35, (i) the saccharide content, measured by a phenol-sulfuric acid method, is 19.0 - 6b, 0 wt. 5, expressed as glucose and the protein content, measured by Lowry-Folin's method. is 6.0-28.0 wt. 5, expressed as calving serum albumin. 15 37 · Carcinostatic substance TF-1316 with the following properties: (a) gray-white-light brown powder, (b) it prevents the metastasis of mouse Ehrlich ascites tumor, Ehrlich solid tumor and Sarcoma-130 carcinomas and has an immunostimulating effect, (c) it is soluble in water and it is insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether, (d) it has no sharp melting temperature, starts to decompose at 110 ° C and decomposes markedly above 200 ° C, (e) the infrared absorption spectrum obtained with a KBr tablet method exhibits absorption bands in the region of 36ΟΟ-32ΟΟ, 296Ο-293Ο, 1670-16 ^ 0, 1550, 1UU0-13S0, 12to, 11to-1000 and 820 cm ”^ , 30 (f) the ultraviolet absorption spectrum of its aqueous solution shows a strong absorption at the absorption edge and shows an absorption peak in the range of 256 - 260 run, (g) upon fractionation by gel filtration (column: 21 mm diameter x too ml; eluent : a 0.1 molar phosphate buffer me at a pH of 7) using Sephadex C-200, it has an absorption band at the elution volumes in the vicinity of the empty 8004759 - 31+ volume - IcO ml at the ultraviolet absorbance measurement at 200 nm; and an absorption band at the elution volumes near the void volume - 160 ml at the absorbance measurement at I + 90 nm with a phenol-sulfuric acid method, 5 (h) its hocg power liquid chromatogram (eluent: a 0.1 molar phosphate buffer with a pH of T, flow rate: 0.8 ml / min at room temperature) obtained using TSK-GEL G30Q SW (column: 7.9 mm diameter x 600 ram x 2) shows peaks near the solvent front and 10 1 + 0 - Co min. At the ultraviolet absorbance measurement at 220 nm and near the solvent front and 1 + 0 - 60 min. At the ultraviolet absorbance measurement at 260 nm, (i) it has a positive Molical reaction, phenolic sulfuric acid reaction, anthron sulfuric acid reaction, indole hydrochloric acid reaction and Lowry-Folin's reaction and negative ninhydrin reaction, (j) elemental analysis values: C 30.0-3I +, 3 / i; H 3.3-1 +, 1 + fS; M! + ,? - 5.1%, (k) the saccharide content, measured by a phenol-sulfuric acid method, is 35.0 - 50.0% by weight, expressed as glucose, and the protein content, measured by Lowry-Folin's method, is 10.0 - 23.0 wt. 3 expressed as calving serum albumin. 3-S. Carcinostatic substance 7? -132a with the following properties: (a) gray-white-light brown powder, 25 (b) it inhibits the metastasis of mouse Ehrlich ascites tumor, Ehrlich solid tumor and Sarcoma-130 carcinomas and has an immunostimulating effect, (c) it is soluble in water and it is insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether, (d) it has no sharp melt count, begins to decompose at 110 ° C and decomposes markedly above 200 ° C, ( e) the infrared absorption spectrum, obtained by an XBr tablet method, shows absorption bands in the vicinity of 35 3600-3200, 2960-2930, 1670-16I + 0.1550, 11 + 1 + 0-1380, 121 + 0.11 1 * 0 -1000 and 820 on-1, 8004759 2 / 3c (f) the ultraviolet absorption spectrum of si. In aqueous solution shows a strong absorption at the absorption edge and shows an absorption peak in the region of 270 - 2B0 nm, (g) when fractionated by gel filtration (column: 1 + 1 + mm by-5 cut x 500 mm; eluent: a 0.1 mo phosphate buffer (pH 7) using Sephadex G-200, it exhibits absorption bands at the elution volumes near the void volume - 3 ^ 0, 600-700 and 720-880 ml at the ultraviolet absorbance measurement at 2βθ nm; at the elution volumes in the vicinity of the void volume - 3l + 0, 3 ^ 0 - 530 and 720 - 900 ml at the absorbance measurement at 1 + 90 nm by a phenol-sulfuric acid method; and at the elution volumes near the void volume - 3 ^ 0, and 3I + O - 58Ο ml at the absorbance measurement at 620 nm by an anthron sulfuric acid method, 15 (h) are high power liquid chromatogram (eluent: a 0, 1 molar phosphate buffer with a pH of 7 »flow rate: 0.8 ml / min at room temperature) obtained using TSK-GEL G300 SW (column: 7.9 mm diameter x 600 mm x 2) shows peaks near the solvent front, 30, 38 - 60 and 65 min. 20 at the ultraviolet absorption measurement at 220 nm and near the solvent front, 62 and 65 min. at the ultraviolet absorption measurement at 260 nm, (i) it has a positive Molic reaction , phenol sulfuric acid reaction, anthron sulfuric acid reaction, indole hydrochloric acid reaction and Lowry-Folin's reaction and negative ninhydrin reaction, (j) elemental analysis values: C 34.8 - 39.8%; H i +, 5 - 5.75; N 2.8 - 3.65, (k) the saccharide content, measured by a phenol-sulfuric acid method, is 55.0 - 61 +, 0 wt. 5 expressed as glucose and the protein content, measured by Lowry-Folin * s method is 18.0-28.0 wt. 5, expressed as calving serum albumin. 39 · Carcinostatic substance TF-132b with the following properties: (a) gray-white-light brown powder, 35 (b) it hinders the metastasis of mouse Ehrlich ascites tumor, Ehrlich solid tumor and Sarcoma-180 carcinomas and has 8004759 36 an immunostimulating effect, (c) it is soluble in water and it is insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether, 5 (d) it has no sharp melting point, begins to decompose at 110 ° C and decomposes markedly above 200 ° C, (e) the infrared absorption spectrum obtained with a KBr tablet method shows absorption bands in the vicinity of 3600-3200, 2960-2930, 1670-161 * 0.1550, 11 * 1 * 0-1380, 121 * 0, 10 111 * 0-1000 and 820 cm-1, (f) the ultraviolet absorption spectrum of its aqueous solution shows a strong absorption at the absorption edge and shows an absorption peak in the vicinity of 265-280 nm, (g) when fractionated by gel filtration 15 (column: 21 mm diameter x 1 * 00 ram; eluent : a 0.1 M phosphate buffer with a pH of 7) using Oephadex G-200, it shows a weak absorption band at the elution volumes near the void volume - 150 ml the ultraviolet absorption measurement at 260 nm and an absorption band at the 20 elution volumes near the void volume - 150 ml at the absorbance measurement at 1 + 90 nm with a phenol-sulfuric acid method, (h) its high power liquid chromatogram (eluent: a 0.1 moiety phosphate buffer with a pH from 7; flow rate: 0.3 ml / min. at room temperature) obtained using TSK-GEL G300 SW (column: 7.9 mm diameter x 600 mm x 2) showing peaks near the solvent front, 36-37 and 1 * 3 - 50 min. at the ultraviolet absorption measurement at 220 nm and has very low peaks near the solvent front, 32 - 39 and 1 * 5 - 52 min. in the ultraviolet absorption measurement at 30 260 nm, (i) it has a positive Molic reaction, phenol sulfur -acid reaction, anthron-sulfuric acid reaction, indole hydrochloric acid reaction and Lowry-Folin's reaction and a negative ninhydrin reaction, (j) elemental analysis values: 35 C 35.3-39.52; H 1 +, 5 - 5.62; N 2.8-5.5 ^ 8004759 37 (k) the saccharide content, measured by a phenol-sulfuric acid method, is 23.6-1 + 5.5% ev.% Expressed as glucose and the protein content, measured just Lowry-Folir.1's method is 15.5-28.0 wt.% Expressed as calving serum albumin. 5 bo. Carcinostatic substance FF-133a with the following properties: (a) gray-white-light brown powder, (b) it hinders the metastasis of mouse Ehrlich ascites tumor, Ehrlich solid tumor and Sarcoma-180 carcinomas and has an immunostimulating effect, (c) it is soluble in water and it is insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether, (d) it has no sharp melting point, begins to decompose at 110 ° C and decomposes markedly above 200 ° C, ( e) the infrared absorption spectrum obtained with a KBr tablet method exhibits absorption bands in the vicinity of 3600-3200, 2960-2930, 1o70-16U0, 1550, lUU0-13S0, 12Uo, 11U0-1000 and 820 cm-1, 20 (f) the ultraviolet absorption solenoid of its aqueous solution shows a strong absorption at the absorption edge and shows an absorption peak in the neighborhood of 253 - 262 nn, (g) when fractionated by gel filtration (column: bb mm diameter x 500 mm; eluent: a 0 , 1 molar phosph phate buffer with a pH of 7) using Sephadex G-200, it has absorbance bands at the elution volumes in the vicinity of the void volume - 300 and 630-9 ml at the ultraviolet absorbance measurement at 260 nm; an absorption band at the elution volumes in the vicinity of the void volume - U20 ml at the absorbance measurement at ^ 90 nm using a phenol-sulfuric acid method; and an absorption band at the elution volumes close to the void volume - b20 ml at the absorbance measurement at 620 nm by an anthron sulfuric acid method, (h) its high power liquid chromatogram (eluent: 35 a 0.1 molar phosphate buffer with a pH of 7, flow rate: 0.8 ml / min at room temperature) obtained using 8004759 38 TSK-GEL G300 SW (column: 7.9 mm diameter x 100 mm x 2) shows peaks near the solvent front, 38 and 50 min. At the ultraviolet absorption measurement at 220 nm and near the solvent front, 50 - 60 and 62 min. At the ultraviolet absorption measurement at 260 nm, (i) it has positive Molic reaction, phenol-sulfuric acid reaction, anthron - sulfuric acid reaction, indole hydrochloric acid reaction and Lowry-Folin's reaction and negative in ninhydrin reaction, (j) elemental analysis values: 10 C 28.0 - 36.6 $; H 3.5 * 5.1 $; NH, 5 - 6.33, (k) the saccharide content, measured by a phenol-sulfuric acid method, is 26.0 - 35-0 wt. 3, expressed as glucose, and the protein content, measured by Lowry-Folin's method, is 22.0-28.0 wt. 3, expressed as calving serum albumin. 15 Onion. Carcinostatic substance TF-133b with the following properties: (a) gray-white-light brown powder, (b) it prevents the metastasis of mouse Ehrlich ascites tumor, Ehrlich solid tumor and Sarcoma-180 carcinomas and has an immunostimulating effect, (c) it is soluble in water and it is insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether, (d) it has no sharp melting point, begins to decompose at 110 ° C and decomposes markedly above 200 ° C, ( e) the infrared absorption spectrum obtained with a KBr tablet method exhibits absorption bands in the vicinity of 36ΟΟ-32ΟΟ, 296Ο-293Ο, 1670-16UO, 1550, 1UU0-1380, 12U0, 11HO-1000 and 820 cm "1.39 (f) the ultraviolet absorption spectrum of its aqueous solution shows a strong absorption at the absorption edge and shows an absorption peak in the region of 270-280 nm, (g) when fractionated by gel filtration (column: 21 mm diameter x H00 mm; eluent: a 0 1 molar phosphate buffer with a pH of 7) using Senhadex G-200, it has an absorption band at the elution volumes in the 8004759 t. t 39 near the void volume - 173 ml with the ultraviolet absomtis measurement at 260 nm and an absorption band at the elution volumes near the void volume - 153 ml with the absorbance measurement at 1 + 90 nn with a phenol-sulfuric acid method, 5 (h) its high power liquid remat o'ram (closing agent: a 0.1 molar phosphate buffer with an nH of 7; flow rate 0.8 ml / min at room temperature), obtained using TSK-GEL G300 GW (column J, 9 mm diameter x 50 mm x 2) exhibits peaks near the solvent front, 1 + Q - 50 min at the ultraviolet absorbance measurement at 220 nn; and near the solvent front 33 - 39 and 52 min. in the ultraviolet absorbance measurement just 260 nn, (i) it has a positive Molic reaction, phenol sulfuric acid reaction, anthron sulfuric acid reaction, indole hydrochloric acid reaction and Lovry-Folin's reaction and negative ninhydrin reaction, (j) elemental analysis values: C 31.1 - 33.5 / ¾; H 3.9-5.2S; II 3,1+ - 1 +, 7 ^, (k) the saccharide content, measured by a phenol-sulfuric acid method, is 19.0 - 2l +, 5 wt. 5, expressed as glucose and the protein content, measured with Lovry- Folin's method is 12.9-22.9 J, expressed as calving serum albumin. 1 + 2, Carcinostatic substance TF-1323 with the following properties: (a) gray-white-light brown powder, 25 (b) it prevents the metastasis of mouse Ehrlich ascites tumor, Ehrlich solid tumor and Sarcoma-180 carcinomas and has an immunostimulating effect, (c) it is soluble in water and it is insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether, (d) it has no sharp melting point, begins to decompose at 110 ° C and decomposes markedly above 200 ° C, (e) the infrared absorption spectrum obtained with a KBr tablet method shows absorption bands in the vicinity of 35 3600-3200, 2960-2930, 1670-161 + 0.1550, 11 + 1 + 0-1380, 121 + 0.140 -1000 and 820'cm "1, 8004759 Λ (f) the ultraviolet absorption spectrum of its aqueous solution shows a strong absorption at the absorption edge and shows an absorption peak in the vicinity of 265-280 na (g) when fractionated by gel filtration 5 (column: 21 mm diameter x 1 * 00 mm; eluent: a 0.1 molar phosphate buffer with a pH of 7) using Senhadex G-200, it shows a weak absorption band at the elution volumes near the void volume - 16Ο ml at the ultraviolet absorbance measurement at 260 nm and an absorption band at the 10 elution volumes near the void volume - 150 ral at the absorbance measurement at 1 * 90 nm with a phenol-sulfuric acid method, (h) its high power liquid chromatogram (eluent: a 0.1 molar phosphate buffer with a pH of 7 ; flow rate: 0.8 ml / min. at room temperature) obtained using TSK-GEL G300 SW (column: 7.9 mm diameter x 600 mm x 2) shows peaks near the solvent front, 36, and 1 * 3 - 50 min at the ultraviolet absorption measurement at 220 nm; and peaks near the solvent front, 36 and 50 min at the ultraviolet absorbance reading at 260 nm, 20 (i) it has positive Molic reaction, phenol sulfuric acid reaction, anthron sulfuric acid reaction, indole hydrochloric acid reaction and Lowry-Folin's reaction and negative ninhydrin reaction, (j) elemental analysis values: C 29.9 - 39 Λ *} H 3.9 - 5.6 *; N 2.8 - 5, W, 25 (k) the saccharide content, measured by a phenol-sulfuric acid method, is 19.0 - 25.0, injected as glucose and the protein content, measured by Lowry-Folin's method, is 11.0 - 17.0 $, expressed as calving serum albumin. 1 * 3. Carcinostatic substance TF-136 with the following properties: (a) gray-white-light brown powder, (b) it prevents the metastasis of mouse Ehrlich ascites tumor, Ehrlich solid tumor and Sarcoma-180 carcinomas and has an immunostimulating effect, 35 ( c) it is soluble in water and it is insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and 8004759 ✓ diethyl ether, (d) it has no sharp melt count, starts to decompose at 110 ° C and decomposes markedly above 200 ° C, (e) the infrared absorption spectrum, obtained Tiet a 5 KBr tablet method exhibits absorption bands near 3600-3200, 2960-2930, 16TO-16UO, 1550, 11 + 40-1330, 12 ^ 0, 11 ^ 0-1000 and 320 ca ”1, (f) the ultraviolet absorption spectrum of its aqueous solution shows strong absorption at the absorption edge and 10 shows an absorption peak in the neighborhood of 25 ° C - 2 ° C nm (g) when fractionated by gel filtration (column: hk mm diameter x 500 ram; eluent: a 0.1 molar phosphate buffer with a pH of 7) using Senhadex 0-200, it shows absorption bands at the elution volumes in the vicinity of 5 * 9-930 ml 15 at the ultraviolet absorbance measurement at 260 nn and all fractions have a low absorption band at the absorbance measurement at k '-) 0 nm with a phenol-sulfuric acid method and at the absorbance measurement at c20 nm with an anthron-sulfuric acid method, (h) its high power liquid chromatogram (eluent: 20 a 0.1 molar phosphate buffer with a pK of 7; flow rate: 0.3 ml / min at room temperature) t using TSK-GEL G30Q 3W (column: 7.9 mm diameter x 600 mm x 2) shows peaks near the solvent front, 23 - bQ and ^ 2 - 60 min in the ultraviolet absorbance measurement at 220 nm and peaks near the solvent front and ^ 2 - 60 min. at the ultraviolet absorption measurement at 260 nm, (i) it has a positive Molecular reaction, phenol-sulfuric acid reaction, anthron-sulfuric acid reaction, indole hydrochloric acid reaction and Lowry-Folin1s reaction and negative inhydrin reaction, 30 (j) elemental analysis values: C 27.5 - 32.6 $; H 3.2 - k.0 $; N 5.0 - 6.1 $, (k) the saccharide content, measured by a phenol-sulfuric acid method, is 19.0 - 30.0% by weight, expressed as glucose and the protein content, measured by Lowry-Folin's method. is 6.0 -35 12.0 wt.%, expressed as calving serum albumins. 8004759 1 + 1 +. Carcinostatic substance TF-1 ++ 0 has the following properties: (a) gray-white-light brown powder, (b) it prevents the metastasis of mouse Ehrlich ascites 5 tumor and has an immunostinulating effect, (c) it is soluble in water and it is insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether, (d) it has no sharp melting point, starts to decompose at 210 ° C and decomposes markedly above 280 ° C, (e) the infrared absorption spectrum, obtained with a KBr tablet method, absorption bands near 36OO-3200, 2960-2930, 1670-161 + 0, 1550, 1U0-1380, 121 + 0, 111 + 0-1000 and 320 cm-1, 15 (f) exhibit the ultraviolet absorption spectrum of its aqueous solution shows a strong absorption at the absorption edge and shows an absorption peak in the range of 255 - 260 m, (g) upon fractionation by gel filtration (column: 1 + 1 + mm diameter x 500 ml; eluent: 0, 1 molar phosphate buffer with a pH of 7) using Sephadex G-200, it shows absorption bands at the elution volumes near the void volume - 300, 520 - 900 and 900 - 1000 ml at the ultraviolet absorbance measurement at 260 nm and at the elution volumes near the void volume - 500, 650 - 850 and 850 - 1000 ml at the absorbance measurement at 1 + 90 nm with a phenol-sulfuric acid method, (h) its high power liquid chromatogram (eluent: a 0.1 molar phosphate buffer with a pH of 7; flow rate: 0.8 ml / min, at room temperature) obtained using TSK-GEL G300 SW (column: 7.9 mm diameter x 600 ram x 2) shows 30 peaks near the solvent front and 1 + 0 - 60 min. in the ultraviolet absorbance measurement at 220 m and 260 nm, (i) it has a positive lolic reaction, phenol sulfuric acid reaction, anthron sulfuric acid reaction, indole hydrochloric acid reaction and Lowry-Folin's reaction and negative ninhydrin reaction, 35 (j basic analysis values: C 22.0 - 28.0 $; H 3.0 - 3.5 $; N 5.0 - 6.5 *, 8004759 (k) the saccharide content, measured using a phenol sulfuric acid method, is 5.0 - 15.0% by weight, expressed as glucose, and the protein content, measured with Lowry- Folin's method is 23.0 - 32.0% by weight, expressed as calving serum albumin. 5 1 + 5. Carcinostatic substance TF-159 has the following properties: (a) gri.jswit-light brown powder, (b) it hinders the metastasis of mouse Ehrlich ascites tumor and has an immunostimulating effect, 10 (c) it is soluble in water and it is insoluble in methanol, ethanol, acetone, benzene, chloroform, ethyl acetate and diethyl ether, (d) it has no sharp melting point, begins to decompose at 11 ° C and decomposes markedly above 200 ° C, (e) the ultraviolet absorption spectrum of the aqueous solution shows a strong absorption at the absorption edge and shows an absorption peak in the region of 255-29 nm, (f) the infrared absorption node obtained with a KBr tablet method shows absorption bands in the vicinity of 20 3600-3200, 2960-2930, 167Q-16.-0, 1550, 1U0-13S0, 121 + 9, 111 + 0-1000 and 820 cm-1, (g) when fractionating by gel filtration (column: 21 mm diameter x 1 + 00 mm; eluent: a 0.1 molar phosphate buffer with a pH of 7.0) using Behadex 0—200, it has 25 absorption bands at the elution volumes near the void volume - 100 and 100 - 100 ml in the ultraviolet absorbance measurement at 260 nm and an absorption band at the elution volumes near the void volume - 150 ml in the absorbance measurement at 1 + 90 nm with a phenol-sulfuric acid method, 30 (h) are high power liquid chromatogram (eluent: a 0.1 molar phosphate buffer with a mean of 7.0; flow rate: 0.8 ml / min. at room temperature), obtained using ns ^ / an TSK-GEL G3GQ SW (column: 7.9 nin diameter x 600 mm x 2) shows peaks near the solvent front, 32 and 1 + 8 - 50 min. 35 at the ultraviolet absorbance measurement at 220 nm and near the solvent front, 32 and 1 + 8 - 50 min. at the ultraviolet 8004759 v V 'i * · Oh absorption measurement at 26o nm, (i) it has a positive Molecular reaction, phenol-sulfuric acid reaction, anthron- sulfuric acid reaction, indole hydrochloric acid reaction and Lowry-Folin's reaction and negative ninhydrin reaction, 5 (j) elemental analysis values: C 31.0-33055; E 4.0-4.42; N 2.8 - 3.2%, (k) the saccharide content, measured by a phenol-sulfuric acid method, is 1 + 0.0 - 55.0 wt. 2, expressed as glucose and the protein content, measured by Lovry- Folin's method is 10 7.0-14 found 2, expressed as calving serum albumin. Carcinostatic preparation, characterized in that it is a carcinostatic substance TF-100, 110, 120, 130, 1316, 132a, 132b, 133a, 133b, 1323, 136, 140 and 150 according to claims 33, 34, 35, 36 , 37, 33, 39, 40, 41, 42, 43, 44 or 45, in a form suitable for administration. 47. A carcinostatic preparation according to claim 46, characterized in that the carcinostatic substance consists of? F-132a, 132b, 133a, 133b or 1323, respectively. claim 38, 39, 0, 41 or 42. 48. A method of treating cancer in a warm-blooded mammal including man, characterized in that a carcinostatic composition according to claim 47 is used. 8004759