DK151639B - PROCEDURE FOR PREPARING TF-100 OR FRACTIONS WITH CARCINOSTATIC AND IMMUNOSTIMULATING ACTIVITY - Google Patents

Description

in

DK 151639 B

The present invention relates to a process for the preparation of TF-100 or fractions thereof, having carcinostatic and immunostimulatory activity, characterized by the characterizing part of claim 1.

In recent years, patients with various types of cancer have been used extensively to strengthen the host organism's immune system and produce a carcinostatic effect through the enhanced immune function. As antitumor agents in such drugs are known compounds derived from different bacteria or from cultures of different bacteria, or polysaccharides derived from the fruiting bodies of the genus Basidiomycetes or from cultured fungal organisms belonging to the same genus. However, these antitumor agents are not yet satisfactory in terms of efficacy, deleterious side effects or method of preparation.

On the other hand, there are descriptions of an organism and a supernatant fluid produced by the cultivation of bacteria belonging to the genus Fusobacterium, e.g. Fusobacterium K031-3B, Fusobacterium fusifomis W-12, Fuso-bactrium girans 1012 and Fusobactrium nucleatum 1010 [Dental Surgery, 23, 323-333 (1974) and 21, 534-439 (1972) 25 (Japanese)]. However, to date, no study has been conducted on the ingredients derived from a culture or its supernatant liquid prepared by cultivation of bacteria belonging to the genus Fusobacterium or the pharmacological effects of the ingredients.

30 The pharmacological action of the supernatant fluid obtained by growing bacteria belonging to a strain of the genus Fusobacterium namely Fusobacterium nucleatum TF-031 (FERM 5077, ATCC 31647 and the ferrous organisms of

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2 culture, and it has surprisingly been found that a specific component derived from the supernatant fluid has carcinostatic effect; that this component is substantially unable to inhibit the colony formation of cancer cells 5 by the colony formation assay and does not exhibit carcinostatic effect by killing the cancer cells, but that it has an indirect carcinostatic effect by increasing the host's own antitumor ability or host organism immunity and exploiting the immunity effect; and further, that this component is very little toxic and that it can be extracted by treatment of the culture.

The invention is described in more detail below.

Fusobacterium necleatum TF-031 (FERM 5077; ATCC 31647) has the following bacteriological properties.

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3 (I) Shape (1) Shape of cells: ten-shaped (Figure 1) 10 (2) Polymorphism attached to cells: Not found (3) Movement: Not found (4) Spores: Not found (5) Gram staining: Gram negative (6) Acid resistance: Negative 15 (II) Growth conditions in a culture substrate (1) TH-a agar plate and oblique agar substrate Outer shape: Round shape

Size: About 1 mm

Arches: Hemisphere 20 Structure: As a dewdrop

Surface: Smooth

Edges: Smooth

Color: milky, yellowish-white

Transparency: Opaque 25 (2) TF-a Liquid Growth Substrate Growth Scope: Strong

Blur: Coagulate

Precipitation: Nothing

Surface growth: None, no growth until a depth of approx. 5 mm

Gas: None

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4 (III) Physiological properties (1) Hydrogen sulfide production: + (2) Nitrate reduction: (3) Butyric acid production: + 5 (4) Indole production: + (5) Urease: (6) Catalase: (7) Starch hydrolysis : (8) Oxygen behavior: Anaerobic 10 (9) Ammonia evolution: + (10) Carbon dioxide evolution: +

(11) Boundary range for growth: pH 5 - 8.5, temperature 30 ~ 45 ° C

(12) Development of gas from saccharides: L-arabinose (-) 15 D-xylose (-) D-glucose (-) D-mannose (-) D-fructose (-) D-'galal'actose · (- 20 malt sugar (-) sucrose (-) trehalose (-) sorbitol (-) mannitol (-) 25 inositol (-) glycerol (-) starch (-)

The present TF-100 compounds or fractions thereof (designated TF-110, TF-120, TF-130 (TF-1316, TF-132a, TF-132b, TF-133a, TF-133b, TF-1323, TF-136), TF-140 and TF-150) are prepared e.g. in the following way:

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5

Bacterial culture of the Fusobacterium genus that can form TF substances TF-031 (FERM 5077; ATCC 31647)

Culture or its In supernatant fluid |

organic up- J

solvent —I-- - Stand

Solvent, ______.

fraction * - s ^ arerin9 |

precipitate

Phosphate buffer ζ- (sodium chloride) or water

Insoluble L- Seoarerina Fraction Separation Drying h- Va ^ c ^ soluble ^^ y fraction-- -1- -IT- TF-100 - - _ „- * .-- \ - ^ Dialysis or

Outer solution * '^ ultrafiltration j Drying t ------ Internal solution

J

TF "110 ^

Do not adsorb ion exchange - fraction fraction treatment

Phosphate Buffer - (Sodium Chloride)

Eluate - ~ 1 1 ir_ Drying Concentration No. r-Tii1 TF-120 Desalting T -.....- p Drying

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6% ™ J soluble * 2 I I-vrp-100 fraction ^^ - *

Formation of ** ΓΤ. ! ,. ,, * l-— Barium ions everything barium ___

Separation -? Solution In Barium Salt i____! - In Removal! of barium in _

Separation -? Solid "· 1 ------ -

Resolution

Dialysis or ultrafiltration i

Concentration ί-- Drying

V

TF-140

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7

Water-soluble fraction SSrdm -H 1 - ^ - 110 l. _ Y _ / - Treatment

Soluble fraction e_ with quaternary --- ammonium salt

Quaternary - artmonium cat complex

Solution containing - sodium chloride - ^

Treatment for _ dissociation _______1_

Vaskévæske

Soluble fraction

Hydrophilic organic solvent

Soluble fraction ζ- Separation

Precipitation Drying

V

TF-150

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8

The above-described method of preparation can be elucidated as follows: (1) Cultivation_of_bacteria

The cultivation of said bacteria belonging to the genus Fusobacterium is carried out by the usual method of growing anaerobic bacteria. That is, adjusting to a pH of 6 - 8.5, preferably 7.2 - 8.2, a culture substrate containing a nitrogen source such as calf brain heart extracts, various peptones 10 or the like; a vitamin source such as yeast extract or the like; an inorganic salt such as sodium chloride or the like; a carbon source such as glucose, lactose or the like; a reducing agent such as L-cystine, sodium sulfite, thioglycollate or the like, and the bacteria are seeded on the culture substrate, then cultured stationarily under anaerobic conditions at 35-42 ° C, preferably 36-38 ° C. for 1-5 days, preferably 24-72 hours. It is particularly desirable to use the culture substrate described in Table 1 (hereinafter referred to as the TF culture substrate). However, it is not always necessary to use the brain-heart infusion, which has a calf-brain-heart extract, as a carbon source, and it can be replaced by a heart-infusion, which is a calf-heart extract, beef extract, fish extract, corn mold liquid or the like. Among the various peptones, proteose peptone and phyton peptone are not always necessary, and peptic peptone may be replaced by polypeptone.

When agar is not used, it is advantageous to perform a culture with agitation.

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9 TABLE 1

The constituents of the culture substrate (g / l) TF-a TF-b TF-c

Trypticase Peptone 17 17 17 5 Phyton Peptone 3 3 1.5

Proteoseptone 10 5 5

Brain-Heart Infusion 35 17.5

Core infusion 25 Yeast extract 333 10 Sodium chloride 7.5 7.5 7.5

Glucose 666

Lactose 555 L-cystine 0.25 0.5 0.5

Sodium sulphite 0.1 0.1 0.1 Thioglycollate 0.5 0.5 0.5

Agar 0 or 0.7 0 or 0.7 0 or 0.7 (2) Isolation of supernatant liquid from the culture ________________

The organisms are removed from the culture 20 prepared above to obtain the supernatant liquid. To remove the organisms, a conventional method, e.g. centrifugation or a filtration method using a filter aid, such as "Hyflo Super Cel", is particularly preferred for centrifugation, taking into account the workflow, the number of organisms desired to be removed, and the yield of supernatant liquid. The organisms are preferably removed in this treatment step, but they can be removed in a later step (3).

(3) Insulation_of_the_carcinostatic_ compound_TF-100_ 30 A hydrophilic organic solvent is added to the supernatant liquid or culture prepared above

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10 and the precipitate formed is collected. At this point, the supernatant liquid or culture is adjusted, preferably to pH 2-7. The hydrophilic organic solvent comprises e.g. alcohols such as ethanol or methanol and ketones as acetone. Alcohols and especially ethanol lead to the best results. The hydrophilic organic solvent may suitably be added such that its concentration is 30-70%, preferably 50-70% by volume. After addition of the hydrophilic organic solvent, the resulting mixture is allowed to stand at a low temperature, preferably approx. 5 ° C, for several hours up to several days to complete the formation of the precipitate.

The precipitate thus formed is separated by conventional methods such as decantation, centrifugation or filtration.

Then the precipitated water is added in an amount of 10-15 times the amount of the precipitate isolated above [the water can be replaced by a phosphate buffer or a sodium chloride-containing phosphate buffer when it is intended to prepare TF-120 or 130 (1316, 132a, 132b, 133a, 133b, 1323 or 136), as discussed below], and the water-insoluble components thus formed are removed by conventional methods such as centrifugation or filtration, and the water-soluble fraction is isolated. When the culture itself is applied, the organisms are removed by the treatments mentioned above. The water-soluble fraction thus prepared is dried by lyophilization or similarly to form the carcinostatic compound TF-100.

The TF-100 thus produced has the properties shown in Table 2.

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11 (4) Isolation_of_the_carcinostatic_connection_TF; 110_

The water-soluble fraction prepared in section (3) described above is subjected to dialysis or ultrafiltration; Alternatively, TF-100 can be dissolved in water in an amount of 10-5 times the amount of TF-100 and the solution thus prepared is subjected to dialysis or ultrafiltration. Low molecular weight constituents such as amino acids and inorganic materials are removed by the treatment mentioned above. The internal solution prepared by dialysis or ultrafiltration is collected and then dried to form the carcinostatic compound TF-110.

The TF-110 thus produced has the properties shown in Table 2.

15 (5) I§Qlering_of_the_carcingstatic_connection_TF; 120_

The water-soluble fraction prepared in the above-mentioned treatment step (3), or if necessary the internal solution obtained by subjecting the water-soluble fraction to dialysis or ultrafiltration (the 20 internal solution prepared in the above-mentioned treatment step (4)) or a solution of powdered TF-110 in a small amount of water is treated with an ion exchanger. Preferably, as an ion exchanger, a weakly basic ion exchanger or a weakly basic ion exchanger with molecular sieve properties is used, and one can suitably use e.g. diethyl aminoethyl "Sephadex" ® A-50 (registered trademark of Pharmacia Co., Ltd.). A column packed with ion exchanger is equilibrated with e.g. a phosphate buffer having a pH of approx. 8, after which the above-described solution to be treated is passed through the column and the eluted solution is collected, then dried by lyophilization or the like; alternatively, the same buffer as described above and the solution to be treated

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12 lesf is placed in a container containing an ion exchanger and the contents are stirred and then filtered, after which the filtrate is dried; thereby obtaining a carcinostatic compound TF-120 having the properties described below.

TF-120 can also be prepared by desalting the eluted solution or the filtrate, e.g. by dialysis using cellophane tubes or the like, using "Sephadex1® G-25 (registered trademark of Pharmacia Co., Ltd.) or by ultrafiltration, and subsequent drying of the solution.

The TF-120 thus prepared has the properties shown in Table 2.

(6) Isolation_of_the_carcinostatic_connection_TF-130

A column containing the adsorbed constituents from which TF-120 has been removed in the treatment step (5) described above is treated with a phosphate buffer, preferably a phosphate buffer having a sodium chloride concentration of 0.1-0.6 M and a pH of approx. 8 (hereafter, the phosphate buffer having a sodium chloride concentration of 0.1-0.6 M will be referred to as a 0.1-0.6 M sodium chloride phosphate buffer); or alternatively subjecting the water-soluble fraction prepared in the above-mentioned treatment step (3) to dialysis or ultrafiltration, if necessary, and then treatment with an ion exchanger using a 0.1-0.6 M sodium chloride phosphate buffer; thereby forming a fraction which is not eluted with a phosphate buffer without sodium chloride, but eluted with a 0.1-0.6 M sodium chloride-phosphate buffer.

This method of treatment can be performed either with a system of columns or in portions. The thus obtained desired fraction eluted with the sodium chloride-phosphate buffer is dried by lyophilization or similarly

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13 to form a carcinostatic compound TF-130. The eluate containing the desired fraction eluted with the above-mentioned sodium chloride phosphate buffer may be desalted e.g. by dialysis using a cello-5 fan tube, using a molecular sieve such as Sepadex G-25 or by ultrafiltration and drying.

The term "TF-130" used herein denotes collectively the desired eluted fractions prepared by treating the eluate obtained in the above-mentioned treatment step (3), or optionally the internal solution component obtained by subjecting the water-soluble fraction to dialysis. or ultrafiltration, with an ion exchanger, which is not eluted with a phosphate buffer without sodium chloride, but eluted with a 0.1 - 0.6 M sodium chloride phosphate buffer.

In particular, the TF-130 comprises the following fractions.

(i) A fraction which has not been eluted with a phosphate buffer without sodium chloride, but which has been eluted with a 0.6 M sodium chloride phosphate buffer in the above-mentioned ion exchange treatment [this fraction is referred to as TF-1316].

(ii) A fraction which has not been eluted with a 0.1 M sodium chloride phosphate buffer, but which has been eluted with a 0.2 M sodium chloride phosphate buffer by the above-mentioned ion exchange treatment [this fraction is referred to as TF-132a and b; TF-132a is prepared by washing an ion exchanger containing the above-mentioned adsorbed component with a 0.1 M sodium chloride phosphate buffer, treating the ion exchanger with a 0.2 M sodium chloride phosphate buffer, and subsequent collection of the fraction eluted with a 0.2 M sodium chloride phosphate buffer? and wherein TF-132b is obtained by treating the water-soluble fraction prepared in the above-mentioned treatment step (3) or the component of the internal solution (TF-39 110) prepared in the above-mentioned treatment step (4) adjusted to a appropriate molarity, cf. (iii-2)

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(Iii) A fraction which has not been eluted with a 0.2 M sodium chloride phosphate buffer, but which has been eluted with a 0.3 M sodium chloride phosphate buffer in the above-mentioned ion exchange treatment [this fraction is referred to as such as TF-133a and b; the letters a and b respectively having the same meaning as defined in the case TF-132a and b].

(iv) A fraction which has not been eluted with a 0.1 M sodium chloride phosphate buffer, but which has been eluted with a 0.3 M sodium chloride phosphate buffer in the above-mentioned ion exchange treatment [this fraction termed TF 1323].

(v) A fraction that has not been eluted with a 0.5 M sodium chloride phosphate buffer, but which has been eluted with a 0.6 M sodium chloride phosphate buffer in the above-mentioned ion exchange treatment [this fraction is referred to as TF 136].

Since these compounds TF-1316, 132a, 132b, 133a, 133b, 1323 and 136 have common properties in connection with the following conditions, the compounds having these common properties are referred to as TF-130. That is, the TF-130 prepared in the manner described above has the following characteristics: (a) Gray-white to light brown powder.

(B) It inhibits the spread of Ehrlich ascites tumor in mice, Ehrlich solid tumors and Sarcoma-180 carcinoma, and it possesses immunostimulatory activity.

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

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(D) It has no clear melting point, it begins to decompose at about 110 ° C and decomposes significantly above 200 ° C.

(e) Its infrared absorption spectrum recorded using the KBr tablet method exhibits absorption bands 5 in the range 3600 - 3200, 2960 - 2930, 1670 - 1640, 1550, 1440 - 1380, 1240, 1140 - 1000 and 820 cm -1.

(f) The ultraviolet absorption spectrum of a solution thereof in water exhibits a strong absorption at the absorption edge and it exhibits an absorption peak 10 in the vicinity of 256-280 nm.

(g) It gives positive result in Molisch's reaction, in the phenol-sulfuric acid reaction, in the anthron-sulfuric acid reaction, in the indole-hydrochloric acid reaction and in the Lowry-Folin reaction, and it gives a negative result in the ninhydrin reaction. .

(h) Elemental analysis values: C 27.5 - 39.8% H 3.2 - 5.7% N 2.8 - 6.3% 20 (i) Its saccharide content measured by the phenolic sulfuric acid method is about 19 , 0 - 64.0% by weight (expressed as glucose), and its protein content measured by Lowry-Folin1's method is in the range of 6.0 - 28% by weight (expressed as calf serum albumin).

The ion exchange treatment mentioned above will be described in more detail below. As ion exchangers used in the subsequent treatments, a weak basic ion exchanger or a weak basic ion exchanger with molecular sieve properties is preferred, appropriately use di-

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16 ethylaminoethyl-nSephadex® A-50, as used in the treatment step (5) described above, and the subsequent method can be carried out in batch treatment.

(i) EnO., 6 M sodium chloride phosphate buffer, preferably having a pH of approx. 8 is poured through a column with an ion exchanger containing the adsorbed component and through which the solution to be treated is passed in the above-mentioned treatment step (5) and the eluted fraction is collected, concentrated if desired and desalted by dialysis, ultrafiltration or the like, and then dried by lyophilization or the like to form a carcinostatic compound TF-1316.

The TF-1316 thus produced has the properties shown in Table 2.

(Ii) -1) A 0.1 M sodium chloride phosphate buffer, which has a pH value of approx. 8, is poured through a column with an ion exchanger containing the adsorbed component and through which the solution to be treated is passed in the above-mentioned treatment step (5) to leach the column, after which a column is poured through the column. 0.2 M sodium chloride phosphate buffer, preferably having a pH of approx. 8, and the eluted fraction is collected, concentrated if desired and desalted by dialysis, ultrafiltration or the like, and dried by lyophilization 25 or the like to form a carcinostatic compound TF-132a.

The TF-132a thus prepared has the properties shown in Table 2.

(ii) -2) Methods for collecting the fraction that has not been eluted with 0.1 M sodium chloride phosphate buffer, the eagles have been eluted with 0.2 M sodium chloride phosphate buffer in the ion exchange treatment,

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17 is prepared as follows using the water-soluble fraction prepared by the above-mentioned treatment step (3) or by the internal solution prepared in the above-mentioned treatment step (4) to form TF-132b. That is, the column packed with ion exchanger is equilibrated with a 0.2-0.3 M sodium chloride phosphate buffer, preferably having a pH of about 8, followed by the 0.2 to 0 mentioned above. 3 M sodium chloride phosphate buffer with the water-soluble fraction prepared in the above-mentioned treatment step (3) or the internal solution component prepared 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 0.2 - 0.3 M sodium chloride phosphate buffer mentioned above, and the eluted solution and the washings are combined. The ion-exchange treatment can be carried out on several occasions. A solution prepared by diluting the aforementioned eluted solution with a phosphate buffer such that the sodium chloride concentration is 0.1 M is then passed through a column in which the ion exchanger has been equilibrated with 0.1 M sodium chloride buffer. preferably having a pH of about 8 and the fraction eluted with the above-mentioned 0.1 25 M sodium chloride phosphate buffer is removed and the 0.2 M sodium chloride phosphate buffer mentioned above is passed through the column to formation of an eluate. In the above-mentioned concrete example of ion exchange treatment, a fraction that has not been adsorbed on an equilibrium ion exchanger is collected with the aforementioned 0.2 M sodium chloride phosphate buffer, it is adsorbed on an ion exchanger equilibrated with the above 0.1 M sodium chloride-phosphate buffer, washed with the above-mentioned 0.1 M sodium chloride-phosphate buffer, and then eluted with the above-mentioned 0.2 M sodium chloride-phosphate buffer to form an eluate. One can also use a method by which one inversely collects

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18, the fraction that has been adsorbed on an ion exchanger which is equilibrated with the above-mentioned 0.1 M sodium chloride phosphate buffer, and then performs a method for separating this fraction from a fraction adsorbed on a ion exchanger equilibrated with the above 0.2 M sodium chloride phosphate buffer and a fraction which has been eluted with the above 0.2 M sodium chloride phosphate buffer is collected.

The eluate prepared in the manner described above can then, if desired, be concentrated and desalted by dialysis, ultrafiltration or the like, and then dried by lyophilization or the like to form a carcinostatic compound TF-132b.

The TF-132b thus prepared has the properties shown in Table 2.

(iii) -l) The internal solution component prepared in the above-mentioned treatment step (4) is passed through a column with an ion exchanger equilibrated with a phosphate buffer preferably having a pH value of about 8, and through this column a 0.2 M sodium chloride phosphate buffer, preferably having a pH of about 8, is passed to leach the column and then through this column a 0.3 M sodium 25 chloride-phosphate buffer, preferably having a pH of about 8, or alternatively, the aforementioned 0.3 M sodium chloride phosphate buffer may be passed through the column which has been treated in the above-described treatment steps (ii) - 1) and which still contains the remaining adsorbed 30 component. The eluted fraction is collected, concentrated if desired and desalted by dialysis, ultrafiltration or the like, and then dried by lyophilization or the like to form a carcinostatic compound TF-133a.

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19

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

(iii) -2) The fraction which has not been eluted with a 0.2 M sodium chloride phosphate buffer but eluted with a 0.3 M sodium chloride phosphate buffer in the ion exchange treatment is collected as follows analogous to that used in the above processing step (ii) -2) to form TF-133b. That is, a column packed with ion exchanger is equilibrated with a 0.3 M sodium chloride phosphate buffer, preferably having a pH of about 8, and then passing through a column the 0 mentioned above. 3 M sodium chloride phosphate buffer with the water-soluble fraction formed in the above-mentioned treatment step (3), or the internal solution component obtained by subjecting the water-soluble fraction to dialysis or ultrafiltration and the eluted solution record lesson. If necessary, the column is washed with the above-mentioned 0.3 M sodium chloride phosphate buffer, and the eluted solution and the washings are combined. The ion-exchange treatment can be carried out on several occasions. A solution prepared by diluting the above-mentioned eluted solution with a phosphate buffer such that the sodium chloride concentration is 0.2 M is then passed through a column in which the ion exchanger has been equilibrated with a 0.2 M sodium chloride. Phosphate buffer, preferably having a pH of about 8 and the fraction eluted with the above-mentioned 0.2 M sodium chloride phosphate buffer, is removed and the 0.3 M sodium chloride phosphate buffer mentioned above is passed through the column to form an eluate. In the above-mentioned concrete example of ion exchange treatment, a fraction which has not been absorbed on an ion exchanger equilibrated with the 0.3 M sodium chloride phosphate buffer mentioned above is adsorbed on an ion exchanger brought in equilibrium with the 0.2 M sodium chloride phosphate mentioned above.

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20 buffer washed with the above-mentioned 0.2 M sodium chloride phosphate buffer and then slurried with the above-mentioned 0.3 M sodium chloride phosphate buffer to give an eluate. One can also use a method by which, conversely, a fraction which has been adsorbed on an ion exchanger equilibrated with the above-mentioned 0.2 M sodium chloride phosphate buffer is collected, and then the fraction is separated from a fraction. which has been adsorbed on an ion exchanger equilibrated with the above-mentioned 0.3 M sodium chloride-phosphate buffer, and a fraction which has been eluted with the above-mentioned 0.3 M sodium chloride-phosphate buffer is collected. ies.

The eluate obtained in the manner described above is then subjected to concentration as desired and desalted by dialysis, ultrafiltration or the like and then dried by lyophilization or the like to form the desired carcinostatic compound TF-I33b.

The TF-133b thus prepared has the properties shown in Table 2.

(iv) The water-soluble fraction prepared in the above-described treatment step (3) or the internal solution component prepared in the above-mentioned treatment step (4) is placed in a 0.3 M sodium chloride phosphate buffer, preferably having a pH of approx. 8, and the buffer thus prepared is passed through an ion exchange column which has previously been equilibrated with a 0.3 M sodium chloride phosphate buffer, preferably having a pH of approx. 8. The eluted solution is adjusted by adding phosphate buffer, then

For example, the sodium chloride concentration is 0.1 M and is then passed through an ion exchange column which has previously been equilibrated with a 0.1 M sodium chloride phosphate buffer, preferably having a pH of approx. 8, and 35 are then passed through this column referred to above 0.3 M

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21 sodium chloride phosphate buffer, after which the eluted fraction is collected, concentrated if desired and desalted by dialysis, ultrafiltration or the like, and dried by lyophilization or the like to form a carcinostatic compound TF-1323.

The TF-1323 thus prepared has the properties shown in Table 2.

(v) The internal solution component prepared in the above-mentioned treatment step (4) is passed through an ion exchange column which is equilibrated with a phosphate buffer, preferably having a pH of approx. 8, and a 0.5 M sodium chloride phosphate buffer, preferably having a pH of approx. 8 is passed through the column to wash it out, and through this column is passed a 0.6 M sodium chloride phosphate buffer, which preferably has a pH of approx. 8, or alternatively, the above-mentioned 0.5 M sodium chloride phosphate buffer is passed through the column which has been treated in the above-mentioned treatment step (ii) -2) # and still contains residual absorbent component, after which the above-mentioned 0, 6 M sodium chloride phosphate buffer is passed through this column.

The eluted fraction is collected, concentrated if desired, and desalted by dialysis, ultrafiltration or the like, and then dried by lyophilization or the like to form a carcinostatic compound TF-136.

The TF-136 thus produced has the properties shown in Table 2.

(7) Isolation_of_the_carcinostatic_ compound_TF-140 Barium ions are added to the water-soluble 22

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fraction prepared in the above-mentioned treatment step (3) or to the solution obtained by dissolving TF-100 in water to form a barium salt, after which the precipitate is isolated and subjected to a barium removal treatment and the water-soluble fraction is isolated and then dialyzed or ultraviltered to form TF-140. In more detail, an aqueous barium hydroxide solution, preferably a 0.1 - 0.5 M aqueous barium hydroxide solution, is added to the water-soluble fraction prepared in the above-mentioned treatment step (3) or to an aqueous solution prepared by dissolving TF -100 in water in an amount of approx. 10-15 times the amount of TF-100 and the pH of the mixture thus prepared is preferably adjusted to 10-13 to form a barium salt. A precipitate is precipitated by the addition of a barium hydroxide solution, and preferably the amount of barium ion added is adjusted such that the total barium concentration in the final volume of the mixture is between 0.005 and 20 0.1 M. The mixture thus prepared is filtered and a treatment is carried out to separation of barium from the barium salt formed. The barium is preferably removed by adding the barium salt to sodium sulfate, preferably a 5-15% aqueous sodium sulfate solution, and stirring the mixture thus prepared, filtering to obtain a solution. The precipitate which has been separated and removed is washed again with the above mentioned solution. The solution thus prepared is subjected to dialysis, ultrafiltration or the like to remove the excess sodium sulfate or low molecular weight compounds and the solution thus prepared is dried by lyophilization or the like to form a carcinostatic compound TF-140

The TF-140 thus produced has the characteristics shown in Table 2.

m · *

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(8) Isolation of the Carcinostatic Compound_TF-150 The water-soluble fraction formed in the above-mentioned treatment step (3) or the internal solution prepared in the above-mentioned treatment step (4) is treated with a quaternary ammonium salt and the resulting The precipitate is isolated and then subjected to a dissociation treatment using a solution containing sodium chloride, to which is added a soluble hydrophilic organic solvent to the soluble fraction and the precipitate thus formed is isolated and dried to form TF-150. As quaternary ammonium salt which can be used in this step, any complexing agent with a polysaccharide is commonly used, and particularly preferred examples thereof include cetylpyridinium chloride, cetyltrimethylammonium bromide, etc. Treatment with the quaternary ammonium salt is usually carried out in the near , such as a borate buffer or the like. A more detailed description of the above-mentioned treatment with a quaternary ammonium salt and dissociation treatment using a solution containing sodium chloride is as follows. One can, e.g. adding drop by drop an aqueous solution of a quaternary ammonium salt to the water-soluble fraction prepared in the above-mentioned treatment step (3) or to the internal solution prepared in the above-mentioned treatment step (4), the pH is slightly alkaline and the mixture thus prepared is stirred at 0 ° C - 50 ° C for 10 minutes to several hours, after which the precipitated precipitate is isolated. The precipitate is washed several times with a buffer, preferably a 0.1% borate buffer containing 0.1 M sodium chloride and a small amount of quaternary ammonium salt, and then dissociated with a buffer, preferably a 0.1% borate buffer containing 0%. 5 M sodium chloride and a small amount of quaternary ammonium salt to form a soluble fraction, upon which the soluble fraction

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24 is preferably adjusted to pH 2-6. Then, to the soluble fraction, a hydrophilic organic solvent, preferably an alcohol, is added such that its final concentration is 50-90%, preferably 70-90% by volume, and the mixture thus prepared is allowed. to stand at 0 ° - 30 ° C for several hours up to 90 hours, after which the precipitated precipitate is collected to obtain a carcinostatic compound TF-150.

The TF-150 thus produced has the properties shown in Table 2.

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DK 151639B

32 Table 2 used for the "Sephadex'® G-50 marked" & ", a column of 50 mmø x 600 mm and distilled water as the eluent; for the" Sephadex'® G-200 (registered trademark from the company Pharmacia Co., Ltd. marked "SS", a column of 44 mm x 500 mm was used for compounds TF-110, 120, 132a, 133a, 136 and 140, as well as a column of 21 mmo x 400 mm for compounds TF-1316, 132b, 133b, 1323 and 150, and as eluent a 0.1 M phosphate buffer with a pH of 7 used for all of these and to obtain the high liquid chromatogram performance (HPLC) labeled "SSS" was used a 7.9 mm x 600 mm x 2 column with TSK-GEL G300SW (trademark of Toyo Soda Co., Ltd.) and the elution was carried out at room temperature with a throughput. flow rate of 0.8 ml / min. using 0.1 M phosphate buffer having a pH of 7 as the eluent.

The pharmacological properties of the subject carcinostatic compounds TF-100, TF-110, TF-120, TF-1316, 20 TF-132a, TF-132b, TF ~ 133a, TF-133b, TF-1323, TF-140 and TF-150 are as follows: (I) Impact of the TF compounds on cell viability-excavation_mith_hen'Ryn_tir_colony formationj _______

Based on the method discussed by Kim. J. H. et al.

[Cancer Res. 2i5, 698 (1965)] HeLa S-3 cells grown in Eagle's MEM were added 10% calf serum for 3-5 days at 37 ° C, CC CC incubator, after which the culture was taken out, and for the cells adhering to the glass. , a PBS (-) solution (an aqueous solution containing 8.0 g / l sodium chloride, 0.2 g / l potassium chloride, 1.15 g / l sodium phosphate, monobasic, and 0.2 g / l was added potassium phosphate, dibasic) containing 0.01% by weight ethylenediaminetetraacetic acid and 0.1% by weight trypsin. The cells were removed

DK 151639B

33 from the glass surface of the culture flask, and they were divided into single cells by pipetting, whereupon the number of cells was counted under a microscope. The cell suspension was diluted with Eagle's MEM supplemented with 20% calf serum so that it could contain 200 cells per ml. ml. After 1 ml of cell suspension was seeded on Petri dishes and grown in a CO 2 incubator at 37 ° C for 24 hours, to the cell suspension was added the supernatant liquid prepared in Example 1 (1) so that concentration 10 16 to 1/128. And to each of the 1 ml aliquots of cell suspension grown at 37 ° C for 24 hours, each of the compounds to be tested was added and the HeLa cells were grown in each case for 7 days.

After culture, the culture substrate was removed, after which the culture dishes were washed with Hank's solution, and the cells were fixed with 70% by weight ethanol and then washed with 100% ethanol. After the ethanol was removed, the cells were stained with a Gimsa staining solution, on which the colonies were counted and cell viability calculated.

The results are as shown in Tables 3 and 4. The cell viability in the tables was calculated from the following equation.

The cell viability of each substance tested is given the average value for five repeated experiments.

The number of colonies in the case of a treated group

Cell viability = The number of colonies in up to x 100.

felled an untreated group

DK 151639 B

34 TABLE 3

Effect of supernatant liquid on cell viability Cell viability (%)

Dilution - (ml / ml). 48 hours 72 hours 96 hours 1/128 79.6 66.4 80.4 1/64 30.0 7.9 5.8 1/32 0 0 0 1/16 000 Control 100 100 100

DK 151639 B

35 TABLE 4 Effect of TF Substances on Cell Viability

Compound Concentration Cell viability (µg / ml) (%) 5 10 99, 4 TF-100 50 97.4 100 95.9 10 94.5 TF-110 50 88.9 10 100 87.3 10 98.8 TF-120 50 91.2 100 83.3 10 96.7 15 TF-132a 50 97.4 100 95.9 10 96.9 TF-133a 50 94.5 100 88.8 20 10 95.9 TF-136 50 96, 4 100 95.4 10 98.5 50 98.6 25 TF-140 100 98.6 200 96.8 500 93.4

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36

As can be seen from this experiment, the present TF compounds possess very low cell killing effect on HeLa cells as compared to the supernatant fluid.

5 (II) Immunostimulatory Activity In each group, four mice of the strain ICR were used. Each of the compounds to be tested was dissolved in 0.2 ml of physiological saline and then administered intraperitoneally to the mice. Twenty-four hours after administration 10, 0.2 ml of a carbon suspension was prepared by mixing 1 ml of Pelican ink No. 17 Black (manufactured by Giinther-Wagner Co., Ltd.) and 2 ml of physiological saline solution containing 3% by weight gelatin and this suspension. was injected into the tail of the mouse, whereupon 1, 5, 10 15 and 15 minutes after injection 0.02 ml of blood was collected from the eye cavity using a hematocrit capillary tube lined with heparin which was then diluted and hemolysed with 1.6 ml of an aqueous 0.1% by weight sodium carbonate solution. This suspension was subjected to color measurement at 675 nm and the phagocytosis index, the so-called K value, was determined from the equation prepared by Halpern et al.

0.2 ml of a physiological saline solution was administered to the mice in the control group.

log Cn - log C

K = -j ---

t - tQ

wherein Cq = content of carbon powder in the blood at time t, and C = content of carbon powder in blood at time t.

The results obtained are shown in Tables 5-7.

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37 TABLE 5

Dosage

Compound (mg / kg) Average K values TF-100 10 0.0851 - 0.0145 50 0.0605 - 0.0302 TF-110 5 0.0901 - 0.0213 20 0.1025 - 0.0025 TF -120 5 0.0718 - 0.0152 20 0.0559 - 0.0197 TF-1316 5 0.0832 - 0.0193 20 0.1001 + - 0.0246 TF-132a 5 0.1102 - 0.0203 20 0.0809 - 0.0296 TF-133a 5 0.0851 - 0.0057 20 0.0769 - 0.0174 TF-136 5 0.0959 - 0.0087 20 0.1027 - 0.0154

Control - 0.0300 - 0.0024 TABLE 6

Dosage

Compound (mg / kg) Average K values TF-132b 5 0.1190 - 0.0051 20 0.1073 - 0.0031 TF-133b 5 0.0550 - 0.0020 20 0.1099 - 0.0075 TF -150 5 0.1399 - 0.0011 20 0.0577 - 0.0098 TF-1323 5 0.1400 - 0.0075 20 0.0685 - 0.0126

Control - 0.0286 + 0.0035

Table 7

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38

Compound Dose Average K values _ (mq / kq) _ T "140 1 0.0891 ± 0.0156 5 0.0946 ± 0.0139 20 0.0815 * 0.0200

Control - 0.0379 * 0.0048

As can be seen from Tables 5-7, the groups to which the TF compounds in question had been administered showed activation of the reticuloendothelial macrophages in comparison with the control group, and normal mouse immunity of cells was increased.

(Ill) Carcinostatic activity (a) Antitumor activity against Ehrlich ascites tumor

Ehrlich ascites tumor cells were injected intraperitoneally into mice of the strain ICR (female mice, 6 weeks old) 10 at a rate of 1 x 10 mouse. Then, each of the compounds to be tested was dissolved in 0.2 ml of physiological saline and then administered intraperitoneally to the mice once daily for 7 days from the first day after injection of tumor cells, or 15 times cm day on the first day and from the third to the seventh day for a total of six days. For the test compounds listed in Table 9, each of these was administered to the mice once a day on the first and from the third to the seventh day 20 altogether for a total of six days after injection with tumor cells. 0.2 ml of physiological saline solution was administered in the same manner as described above.

The results obtained are shown in Tables 8 and 9.

Number of survival days of mice in a group, 25 m / p _ to which the compound was administered 'Number of survival days of mice in the control x 100% group

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41

Tables 8 and 9 show that the group that had been administered each of the TP compounds in question lived longer than the control group.

(B) Anti-tumor activity against Ehrlich solid tumor (1) Ehrlich tumor cells were transplanted subcucantly into the armpit of mice of the strain ICR (female mice, 6 weeks old) in a C.

amount of 4 x 10 mouse. Then, each of the compounds to be tested was administered to the mice 10 times a day for 7 days from the first day after transplantation of the tumor cells, or once a day on the first day and from the third to the seventh day for a total number of six days. 0.2 ml of physiological saline solution was administered in the same manner as described above. The weight of the tumors was determined on the 11th and 14th day after the transplantation of the tumor cells. The weight of the tumors was determined by measuring the largest diameter (a) mm and the smallest diameter (b) mm by means of a caliper with calculation based on the following equation: 20 Weight of the tumor = - ^ b · (mg)

The results obtained are shown in Table 10.

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DK 151639 B

43 (2) Ehrlich tumor cells were transplanted subcutaneously into the armpit of mice of the ICR strain (female mice, 6 weeks old) in an amount of 4 x 10 6 cells per cell. mouse. Then, each of the compounds to be tested was administered to the mice, which were divided into groups for intravenous, intraperitoneal, subcutaneous and intramuscular doses of 10 mg / kg or 20 mg / kg once daily, respectively. on the first day and from the third to the seventh day for a total of six days after transplantation of the tumor-10 cells. 0.2 ml of physiological saline solution was given in the same manner as shown above. The weight of the tumors was determined on the thirteenth day after the transplantation of the tumor cells. The results obtained are shown in Table 11.

TABLE 11

Connect- Dose Average T / C

Method of administration (mg / kg) tumor weight (g) (%) TF-132a Intravenous 10 3.83 47 8.11 100 20 TF-133a Intravenous 10 1.81 26 20 1.84 26

Intraperitoneal 10 2.82 40 20 1.82 26

Subcutaneous 10 3, .57. 51 25 20 2.86 41

Intramuscular 10 4.65 67 20 3.22 46 6.99 100

As shown in Tables 10 and 11, an effect of growth inhibition of tumors is observed in the groups to which each of the TF compounds in question had been administered.

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4 ·; (c) Antitumor activity against Sarcctna -180 carcinoma

Sarcoma-180 carcinoma was transplanted intraperitoneally to mice of the ICR strain (female mice, 6 weeks old) in an amount of 1 x 10 mouse. Then, each of the compounds to be tested was administered intraperitoneally to the mice at a dose of 1 mg / kg, 5 mg / kg or 10 mg / kg once a day from the first day to the seventh day, all for a total of seven days after the transplantation of the tumor cells, or once a day on the first day and from 10 the third to the seventh day for a total of six days.

To the control group, 0.2 ml of physiological saline solution was given in the same manner as described above.

The results obtained are shown in Table 12.

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DK 151639 B

46

As can be seen in Table 12, the groups to which the TF compounds were administered exhibited antitumor activity in comparison with the control group.

(d) Anti-tumor activity against B-16 melanoma 5 (1) B-16 melanoma tumor cells were transplanted subcutaneously into the armpit of mice of the BDF- (male mouse, 7 weeks old) strain.

- X

old) in an amount of 1 x 10 mouse. Then, the compound TF-133a was administered intraperitoneally to the mice at a dose of 5 rng / kg or 10 mg / kg once daily on 10 the first day and from the third day to the seventh day, a total of six days after the transplantation of the tumor cells. . In the same manner as described, 0.2 ml of physiological saline was administered to the control group and 20 mg / kg of the compound 5-FU (FU = fluorouracil) to the group to receive 5-FU.

The results obtained are shown in Table 13.

TABLE 13

Combine Dose (mg / kg x admi-Average number of nitrations); tumor weight (g) T / C (%) 20 TF-133a 5x6 4.07 70 10 x 6 2.52 43 5-FU 20 x 6 4, 21 72

Control - 5.84 100

As shown in Table 13, the groups to which the compound TF-133a was administered exhibited an apparent growth inhibitory effect on tumors compared to the control group, and each of the groups given 5 mg / kg and 10 mg / ml. of the compound TF-133a, had equal and significant growth inhibitory activity against tumors in comparison with the group to which the compound 5-FU was administered.

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(2) The transplantation of B-16 melanoma tumor cells and administration of the compound TF-132a, TF-133a, 5-FU and a physiological saline solution was performed analogously to that described above (1). On the 21st day after transplant 5 of the tumor cells, the mice were dissected and metastases of the melanoma tumor cells in the lungs were observed.

The results obtained are shown in Table 14.

TABLE 14

percent

Combine Dose (mg / kg x number of Average metastasis-10 administration-number of metastases inhibition TF-132a 5x6 16.0 48 10 x 6 9.86 69 TF-133a 5x6 9.3 70 10 x 6 6.7 78 15 5-FU 20 x 6 24.8 20

Control - 31.0 0

As shown in Table 14, metastases of B-16 melanoma tumor cells were significantly inhibited in the groups to which compounds TF-132a and TF-133a were administered, in comparison with the group to which compound 5-FU had been filed.

(IV) Acute toxicity

The LDr-n for mice was found to be as shown in Table 15. oU

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48 TABLE 15

Compound Method of administration LD, -q (mg / kg) TF-100 Intraperitoneal 500 TF-110 Intravenous> 250 Intraperitoneal> 1000 TF-120 Intravenous> 250

Intraperitoneal> 1000 TF-132a 'Intravenous 300

Intraperitoneal> 500 10 TF-133a Intravenous 300

Intraperitoneal> 500 TF-136a Intravenous 300

Intraperitoneal> 500 TF-132b Intravenous,> 100 15 TF-133b Intravenous> 200 TF-140 Intravenous> 100 TF "150 Intravenous> 200 TF-1323 Intravenous> 200 TF-1316 Intravenous> 200 20 As is clear from the above described Pharmacological studies, the present TF compounds are useful as carcinostatic agents and can be expected to be active against various cancers, as well as to exhibit remarkable activity against particular solid cancers. very good effects, although TF-130 and especially 132-a, 132b, 133a, 133b and 1323 are preferred for various reasons.

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49

The dosage of the present TF compounds is suitably selected depending on the patient's condition, although it is generally desirable to administer them in a dosage to an adult of 0.01 to 50 mg / kg once a day or several times over. day, preferably subcutaneously, intramuscularly or intravenously or by injection into the affected body part.

The present invention is further explained below with reference to Examples and the accompanying drawings, in which: Figure 1 shows a photomicrograph showing the appearance of Fusobacterium nucleatum TF-031 used in the method of the present invention; Figure 2 shows an infrared absorption spectrum of the carcinostatic substance TF-100; Figure 3 shows an ultraviolet absorption spectrum of this substance; Figure 4 shows an elution pattern recorded when this substance was subjected to gel filtration using "Sephadex" ® G-50; 5 shows a high performance liquid chromatography (HPLC); 6 shows an infrared absorption spectrum of the carcinostatic substance TF-110; FIG. 7 shows an ultraviolet absorption spectrum for this substance; FIG. Figure 8 shows an elution pattern recorded when this substance was subjected to gel filtration using "Sephadex" ® G-20 0 0,

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50 FIG. Figure 9 shows the HPLC chromatogram of this substance; Figure 10 shows an infrared absorption spectrum of the carcinostatic substance TF-120; 11 shows an ultraviolet absorption spectrum of this substance; FIG. 12 shows an elution pattern recorded when this substance was subjected to gel filtration using "Sephadex" ® G-200; Figure 13 shows an HPLC chromatogram of this substance; Figure 14 shows an infrared absorption spectrum of the carcinostatic compound TF-1316; Figure 15 shows an ultraviolet absorption spectrum of this compound; 16 shows an elution pattern recorded when this compound was subjected to gel filtration using "Sephadex" ® G-200; 17 shows an HPLC chromatogram of this compound; FIG. Fig. 18 shows an infrared absorption spectrum of the carcinostatic compound TF-132a; 19 shows an ultraviolet absorption spectrum of this compound; FIG. 20 shows an elution pattern recorded when this compound was subjected to gel filtration using "Sephadex" ® G-200; 21 shows an HPLC chromatogram of this compound,

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51 FIG. 22 shows an infrared absorption spectrum of the carcinostatic compound TF-132b; 23 shows an ultraviolet absorption spectrum of this compound; Fig. 24 shows an elution pattern recorded when this compound was subjected to gel filtration using "Sephadex" G-200; Fig. 25 shows an HPLC chromatogram of this compound; Fig. 26 shows an infrared absorption spectrum of the carlo cinostatic compound TF-133a. Fig. 27 shows an ultraviolet absorption spectrum of this compound; Fig. 28 shows an elution pattern recorded when this compound was subjected to "gel filtration" using "Sephadex® G-200; Fig. 29 shows an HPLC chromatogram of this compound; Figure 30 shows an infrared absorption spectrum of the carcinostatic compound TF-133b; 31 shows an ultraviolet absorption spectrum of this compound; FIG. Fig. 32 shows an elution pattern recorded when this compound was subjected to gel filtration using Sephadex1® G-200, Fig. 33 shows an HPLC chromatogram of this compound, Fig. 34 shows an infrared absorption spectrum of the carcinostatic compound TF-1323,

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52 FIG. 35 shows an ultraviolet absorption spectrum of this compound; FIG. Fig. 36 shows an elution pattern recorded when this compound was subjected to gel filtration using Sephadex® G-200; Fig. 37 shows an HPLC chromatogram of this compound; Fig. 38 shows an infrared absorption spectrum of the carcinostatic compound. TF-136, Fig. 39 shows an ultraviolet absorption spectrum of this compound, Fig. 40 shows an elution pattern recorded when this compound was subjected to gel filtration using "Sephadex® G-200," 41 shows an HPLC chromatogram of this compound; FIG. 42 shows an infrared absorption spectrum of the carcinostatic compound TF-140; 43 shows an ultraviolet absorption spectrum of this compound; Figure 44 shows an elution pattern recorded when this compound was subjected to gel filtration using Sephadex® G-200, Figure 45 shows an HPLC chromatogram of this compound, Figure 46 shows an infrared absorption spectrum of the carcinostatic fabric TF-150,

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53 FIG. 47 shows an ultraviolet absorption spectrum of this substance; FIG. 48 shows an elution pattern recorded when this substance was subjected to gel filtration using "Sephadex" G 200, and Fig. 49 shows an HPLC chromatogram of this substance.

EXAMPLE · 1 (1) In each of 15 two-liter culture flasks each equipped with screw caps were placed 2 liters of culture medium containing 34 g of trypticase peptone, 6 g of phyton peptone, 2 g of proteoseptone, 70 g of heart-heart infusion. , 6 g yeast extract, 15 g sodium chloride, 12 g glucose, 10 g lactose, 0.5 g L-cystine, 0.2 g sodium sulfite, 1.0 g sodium thioglycolate and 1.4 g agar, and the culture substrate was adjusted to a pH of 7. The culture substrate was sterilized under 1.2 atmospheric pressure at 120 ° C for 15 minutes, heated in a boiling water bath for 20 minutes and then immediately water-cooled, followed by grafting a starter culture solution of Fuso-bacterium nucleatum TF-031, which had previously been prepared by growing in a culture medium of the same composition as above, into the culture medium described above under sterile conditions in an amount of 100 ml per ml. culture flask, and stationarily grown in an incubator at 37 ° C for 48 hours.

At the end of cultivation, the culture was centrifuged to remove organisms at 4,000 rpm. at 5 ° C for 20 minutes. The amount of supernatant liquid produced was approx. 27 liters.

(2) To the supernatant liquid prepared under (1) was added 40 liters of ethanol with stirring at 5 ° C and the mixture thus prepared allowed to stand in a

DK 151639 B

54 rooms at low temperature until the amorphous precipitate had completely stabilized. The mixture was then centrifuged at 6,000 rpm. at 5 ° C for 15 minutes, and the precipitate was collected, washed with ethanol and then dried under reduced pressure to form ca. 60 g of a crude powdered product.

(3) 20 g of the crude product prepared in (2) was dissolved in 120 ml of water and the water-insoluble materials formed at this time were removed by centrifugation at 7,500 rpm. . for 10 minutes, after which the water-soluble fraction thus prepared was combined with the washing liquids prepared by washing the water-insoluble material twice with 60 ml portions of water, and the solution thus prepared was dried under reduced pressure to form 14 g greyish white light brown powder of the substance TF-100.

EXAMPLE 2

The solution described in Example (1- (3) prepared by combining the water-soluble fraction liberated from water-insoluble materials with the wash water obtained by washing the water-insoluble material) was subjected to ultrafiltration under using a hollow fiber ultrafiltration system (membrane no .: HI-1; manufactured by Asahi Kasei Kogyo Kabushi-25 ki Kaisha) and the internal solution was lyophilized to form 10 g of greyish-white-brown powder of the fabric TF-110 is required.

EXAMPLE 3

10 g of the powder prepared in Example 2 was dissolved in a small amount of water and the solution thus prepared was charged to a column packed with diethylaminoethyl "Sephadex" A-50, equilibrated with a 0.025 M

DK 151639B

55 phosphate buffer of pH 8 and the eluted solution was collected. 2.5 liters of the same phosphate buffer as mentioned above was further poured through the column and the thus prepared eluted solution was combined with the previously collected eluted solution, after which the resulting solution was desalted using a hollow fiber ultrafiltration system (membrane #:> HI-1), concentrated under reduced pressure and finally subjected to lyophilization to give 470 10 mg of greyish-white-brown powder of the substance TF-120.

EXAMPLE 4 2 g of the powder prepared in Example 1- (2) was dissolved in 24 ml of water and the water-insoluble material was removed by centrifugation at 7,500 rpm.

15 for 10 minutes. The water-soluble fraction was dialyzed overnight against distilled water at 5 ° C using a cellophane tube, and the internal solution was concentrated under reduced pressure. The concentrated solution was then placed on a column packed with 150 ml of 20 diethylaminoethyl "Sephadex" ® A-50 which had previously been equilibrated with a 0.025 M phosphate buffer of pH 8 and the column was washed by pouring 600 of a 0.025 M phosphate buffer of pH 8, to which a 0.025 M phosphate buffer containing a sodium chloride concentration of 0.6 M and having a pH of 8 was passed through the column to form an eluate. The eluate was concentrated under reduced pressure to ca. 100 ml volume, dialyzed against distilled water at 5 ° C, again concentrated under reduced pressure, desalted using a column of "Sephadex, Aiy G-25, and subjected to lyophilization to give 470 mg of a gray-white-light brown compound TF-1316.

DK 151639 E

EXAMPLE 5

The solution described in Examples 1- (3) prepared by combining the water-soluble fraction liberated from water-insoluble materials and the wash water obtained by washing the water-insoluble material was dialyzed overnight against distilled water at 5 ° C using a cellophane tube, and the internal solution was concentrated under reduced pressure. The concentrated solution was then added to a column packed with 50 g of 10 diethylaminoethyl-nSephadex® A-50 which had previously been equilibrated with a 0.025 M phosphate buffer having a pH of 8 and 2 liters of a 0.025 M phosphate buffer with pH 8 and 2.5 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.1 M 15 and a pH of 8 were poured through the column after which 2.5 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.2 M and pH 8 was poured through the column to elute the active compound adsorbed on the column and the thus prepared eluted solution was desalted using an ultrasonic filtration system of the type with hollow fibers (Menbran No .: HI-1) and then subjected to lyophilization to give 600 g of greyish-white-brown powder of the compound TF-132a.

EXAMPLE 6 44.2 g of the powder prepared in Example 1- (2) was dissolved in 1.2 L of 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and pH of 8, and the suspension Insoluble material was removed by filtration using "Hyflo Super Cel" and then the filtrate thus prepared was added to a 33 cm diameter column packed with 500 ml of diaminoethyl "Sephadex" A-50 which had previously been placed in equilibrium with a 0.025 M phosphate buffer with a sodium chloride concentration.

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57 tration of 0.3 M and pH of 8 and the eluted solution was collected. The column was washed with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and pH of 8, and the washings were combined with the above-mentioned eluted solution to give a solution of 1.67 liters. This solution was then again placed on an 8 cm diameter column packed with 400 ml of diethylamino-ethyl "Sephadex" A-50, previously equilibrated with a 0.025 M phosphate buffer with a sodium chloride concentration of 0, 3 M and pH of 8 to give 1.68 liters of an eluted solution. The column was washed with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and pH of 8 to give 1.64 liters of wash liquid.

The eluted solution and the washings were combined and a 0.025 M phosphate buffer of pH 8 was added to them to give 4.98 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.2 M and pH 8. The buffer solution thus formed was added to a column of 8 cm diameter packed with 500 ml of diethylaminoethyl "Sepha-20 dex'® A-50 which had previously been equilibrated with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.2 M and pH of 8, and the column was washed with 2 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.2 M and pH of 8. To 7 liters of a solution prepared by combining the washings with the eluted solution was added a 0.025 M phosphate buffer with pH of 8 to form 14 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.1 M and pH of 8. The thus prepared eluted solution 30 was added to an 8 cm diameter column packed with 500 ml diethylaminoethyl "Sephadex ' A-50 previously equilibrated with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.1 M and pH of 8 and the eluted solution removed. The column was washed with 35 2 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.1 M and pH of 8, then 2 liters

DK 151639B

58 of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.2 M and pH of 8 was poured through the column and the eluted solution was collected. The eluted solution was concentrated and desalted using an ultrafiltration system (filter used: Toyo Ultravilter UK-10), and further desalted using a column of "Sephadex" G-25, decolorized with activated charcoal and then subjected to lyophilization to give 880 mg of greyish-white-brown powder of compound TF-132b.

EXAMPLE 7 10 g of the powder prepared in Example 2 was dissolved in a small amount of water and the solution thus prepared was added to a column packed with diethylaminoethyl-15 "Sephadex® A-50 which had previously been equilibrated with A 0.025 M phosphate buffer with pH of 8. Then 5 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.2 M and pH of 8 were poured through the column, then 2.5 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and pH of 8 was poured through the column to elute the active material adsorbed in the column, and the eluted solution thus prepared was concentrated under reduced pressure. The reduced solution was desalted using a cellophane tube. completely desalted using Sephadex® G-25 and then subjected to lyophilization to give 600 mg of greyish-white-brown powder of the compound TF-133a.

EXAMPLE 8 44.2 g of the powder prepared in Example 1- (2) was dissolved in 1.2 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and pH of 8, and the insoluble material in suspension. was removed

DK 151639 B

59 by filtration using "Hyflo Super Cel", after which the filtrate produced was added to a 33 cm diameter column packed with 500 ml of diethylaminoethyl "Sephadex1® A-50 which had previously been equilibrated 5 with a 0.035 M phosphate buffer with a sodium chloride concentration of 0.3 M and pH of 8 was collected and the column was washed with a column of 0.025 M phosphate buffer with a sodium chloride concentration of 0 ',' 3 M and pH of 8, and the washings were combined with the above-mentioned eluted solution to give 1.67 liters of a solution The solution was again added to an 8 cm diameter column packed with 40 ml of diethylaminoethyl "Sepha-dex" ® 2-50, which had previously been equilibrated with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8 to give 1.68 liters of the eluted solution. The column was washed with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and pH of 8 to form Read of 1.64 liters of wash liquid. The eluted solution and washings were combined and a 0.025 M 20 phosphate buffer with pH of 8 was added thereto to give 4.98 liters of a 0.025 M phosphate buffer having a sodium chloride concentration of 0.2 M and pH of 8. solution was added to a column of 8 cm in diameter packed with 500 ml of diethylaminoethyl "Se-25 phadex" A-50 which had previously been equilibrated with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.2 M and pH of 8, and the column was washed with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.2 M and a pH of 8, then 2 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and a pH of 8 were poured 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 "Se-phadex" G -25 column which was decolorized with activated charcoal and then subjected to lyophilization to give 590 mg of off-white-brown-brown powder of compound TF-133b.

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EXAMPLE 9 (1) 20 g of the crude powdered product prepared in Example 1- (2) was dissolved in 120 ml of water and the water-insoluble material formed at this time was removed by centrifugation at 7,500 rpm. ./mine. for 10 minutes, after which the water-soluble fraction thus prepared and the wash water formed by washing the water-insoluble material twice with 3 ml portions of water were combined and then subjected to ultra-filtration using an ultrafiltration system hollow fiber type (membrane no .: HI-1) and the internal solution was subjected to lyophilization to give 10 g of a greyish-light brown powder.

(2) 25 g of the powder prepared by the above treatment (1) was dissolved in 1 liter of 0.025 M phosphate buffer with 0.3 M sodium chloride concentration of pH M of 8 and the solution thus prepared was added to a column of 33 cm in diameter packed with 500 ml of diethylaminoethyl "Sephadex" A-50 which had previously been equilibrated with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and pH of 8. similarly washed with a 0.025 M phosphate buffer having a sodium chloride concentration of 0.3 M and pH of 8, and the eluted solution and washings were combined to give 1.6 liters of a mixture. an 8 cm diameter column packed with 400 ml of diethylaminoethyl "Sephadex" A-50, which had been similarly equilibrated with a 0.025 M phosphate buffer, has a sodium chloride concentration of 0.3 M and a pH of 8 to form of 1.65 liters of the eluted solution. This eluted solution was combined with 1.7 liters of the wash water prepared by washing the column with a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and pH of 8, and the mixture thus prepared was diluted with. a 0.025 M phosphate buffer with

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61 pH of 8 to form 10.35 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.1 M and pH of 8.

The buffer solution thus prepared was then added to a column of 8 cm diameter packed with 500 ml of diethylaminoethyl "Sephadex" A-50 which had previously been equilibrated with a 0.025 M phosphate buffer with a sodium chloride concentration of 0 , 1 M and pH of 8, and the column was washed with 2 liters of a 0.025 M phosphate buffer with a 10 sodium chloride concentration of 0.1 M and pH of 8, then 2 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.3 M and the pH of 8 was poured through the column and the eluted solutions were collected The eluted solution thus prepared was desalted and concentrated using an ultrafiltration system (filter used: Tovo Ultrafilter UK-10) and further desalted on Sephadex® G-25, decolorized with activated charcoal, and then subjected to lyophilization to give 1.65 g of grayish-white-brown powder of compound TF-1323.

EXAMPLE 10 10 g of the powder prepared in Example 2 was dissolved in a small amount of water and the solution thus prepared was added to a column packed with diethylamino-rg).

25 Ethyl Sephadex, * A-50, previously equilibrated with a 0.025 M phosphate buffer with pH of 8. Then 6 liters of a 0.025 M phosphate buffer with a sodium chloride concentration of 0.5 M and pH of 8 poured through the column, after which 2.5 liters of a 0.025 M phosphate 30 buffer with a sodium chloride concentration of 0.6 M and pH

of 8 was poured through the column to elute the active ingredient adsorbed on the column and the resulting solution solution was desalted using a

DK 151639E

62 hollow fiber ultrafiltration system (membrane no .: HI-1), concentrated under reduced pressure and then subjected to lyophilization to give 130 mg of greyish-white-brown-brown powder of compound TF-136.

EXAMPLE 11 15 g of the crude powder product prepared in Example 1- (2) was dissolved in 200 ml of water and the water-insoluble materials were removed by centrifugation at 7,500 rpm. for 10 minutes, after which the water-soluble fraction so prepared was adjusted to pH 10 by the dropwise addition of 0.2 M aqueous barium hydroxide solution. A white precipitate was precipitated by the addition of the aqueous barium hydroxide solution.

Barium ions were added such that the total concentration of barium ions in the final volume was 0.01 M, then the solution was stirred for 30 minutes and the precipitate was collected by filtration. The precipitate thus obtained of a barium salt was suspended in 10 ml of an aqueous 10% by weight containing 20% sodium sulphate solution, kept sufficiently stirred and then filtered and the filtrate collected. The precipitate was again suspended, stirred and then filtered in the same manner as described above, initially with 10 ml of 10 wt% aqueous sodium sulfate solution and then twice with 5 ml portions of this solution, and each filtrate was collected. Finally, the remaining precipitate was washed twice with 10 ml portions of water and then filtered to collect the wash water. The filtrate mentioned above was collected and the washings were combined, dialyzed against distilled water, concentrated under reduced pressure and then subjected to lyophilization to give 0.45 g of crude white-light brown powder of the compound TF-140.

DK 151639B

EXAMPLE 12 32 g of the powder prepared in Example 1- (2) was dissolved in 450 ml of water and the insoluble material was removed by filtration using "Hyflo Super Cel" and then the filtrate thus formed was dialyzed. overnight to tap water using a cellophane tube. To the dialyzed solution was added drop by drop 80 ml of a 20% by weight cetylpyridinium chloride solution in water with stirring, while further 200 ml of a 10% by weight borate buffer (pH 9.0) was added and the mixture thus prepared was stirred. at room temperature for 30 minutes, after which the precipitate was collected by filtration. The precipitate was suspended in 150 ml of a 0.1% by weight borate buffer (pH 9.0) containing 0.1 M sodium chloride and 0.2% by weight cetylpyridinium chloride and the suspension thus prepared was kept stirring for 30 minutes. The precipitate was then separated from the suspension by filtration and resuspended in 150 ml of borate buffer (pH 9.0) of the above composition, and the suspension thus prepared was kept under stirring for 30 minutes, after which the precipitate was collected by filtration. The precipitate thus collected was suspended in 150 ml of a 0.1% by weight borate buffer (pH 9.0) containing 0.5 M sodium chloride and 0.2%. cetylpyridinium chloride, and the suspension thus prepared was kept under stirring and dissociated for 30 minutes. The precipitate was then separated from the suspension by filtration to form a soluble fraction. The precipitate separated by filtration was further subjected to the same dissociation treatment as described above to form a soluble fraction. The two soluble fractions were

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64 and the soluble fraction thus obtained was adjusted to pH 3-4 with 10% hydrochloric acid, after which ethanol was added so that its concentration was eventually 80% by volume. The 5 solution thus prepared was allowed to stand overnight at 5 ° C and the precipitate formed was collected by filtration to give 5.6 g of greyish-white-brown powder of the compound TF-150.

Claims (17)

A process for preparing TF-100 or fractions thereof with carcinostatic and immunostimulatory effects, characterized in that Fusobacterium nucleatum TF-031 (FERM 5077; ATCC 31647) is grown in a nutrient substrate containing nitrogen sources, carbon sources, vitamin sources, reducing agents. and inorganic salts with or without agar under anaerobic conditions, adding a hydrophilic organic solvent to the culture or supernatant liquid, isolating the formed precipitate 10 and then isolating the water-soluble fraction from the precipitate and, if desired, subjecting subsequent treatment 1, 2, 3 or 4, 1. dialyzing or ultrafiltrating the water-soluble fraction, then isolating the desired fraction from the internal solution, 2. treating the water-soluble fraction or internal solution obtained under 1 with ion exchange, and the adsorbed fraction or the non-adsorbed fraction is isolated, 3. the water-soluble fraction is added barium ions are isolated, the barium salt obtained is isolated, barium is removed from the salt and the water-soluble fraction is then dialyzed or ultrafiltered and the desired fraction is isolated from its internal solution, or 4. a quaternary ammonium salt is added to the water-soluble straight fraction or internal solution obtained below 1, the resulting precipitate is isolated to treat for dissociation and the desired fraction is then isolated from the treated liquid. DK 151639E Process according to claim 1, characterized in that the isolated water-soluble fraction from the precipitate is dried. Process according to claim 1, characterized in that the fraction adsorbed by an ion exchanger is eluted with a 0.6 M sodium chloride-phosphate buffer and the eluted fraction is isolated. Process according to claim 1, characterized in that, by ion exchange, a fraction which is adsorbed by treatment with a 0.1.M sodium chloride-phosphate buffer is isolated but eluted with a 0.2M sodium chloride-phosphate buffer. Process according to claim 1, characterized in that, by ion exchange, a fraction adsorbed by treatment with a 0.2 M sodium chloride phosphate buffer is isolated but eluted with a 0.3 M sodium chloride phosphate buffer. Process according to claim 1, characterized in that, by ion exchange, a fraction adsorbed by treatment with a 0.1 M sodium chloride-phosphate buffer is isolated but eluted with a 0.3 M sodium chloride-phosphate buffer. is. Process according to claim 1, characterized in that, by ion exchange, a fraction which is adsorbed by treatment with 0.5 M sodium chloride-phosphate buffer is isolated but eluted with a 0.6 M sodium chloride-phosphate buffer. Process according to claim 1, characterized in that the water-soluble fraction of the precipitate is subjected to ion-exchange treatment in a 0.2 - 0.3 M sodium chloride-phosphate buffer; the eluate is then adjusted so that its sodium chloride concentration is 0.1 M and then treated with an ion exchanger which is equilibrated with a 0.1 M sodium chloride phosphate buffer to adsorb thereto; and that a fraction is then eluted with a 0.2 or 0.3 M sodium chloride phosphate buffer and isolated. Process according to claim 1, characterized in that the water-soluble fraction of the precipitate is subjected to ion-exchange treatment in a 0.3 M sodium chloride-phosphate buffer; that the eluate is then adjusted to 0.2 M and the solution then treated with an ion exchanger which is equilibrated with a 0.2 M sodium chloride-phosphate buffer for adsorption thereon, and that a fraction is then eluted with 0, 3 M sodium chloride phosphate buffer and isolated. Process according to any one of claims 1-9, characterized in that the hydrophilic organic solvent added to the culture or to its supernatant liquid is an alcohol. Process according to any one of claims 1-9, characterized in that the hydrophilic organic solvent added to the culture or its supernatant liquid is used in a solvent concentration 25 of 30-70, preferably 50-70% by volume. Process according to any one of claims 1-9, characterized in that the ion exchanger used is a weakly basic ion exchanger or a weakly basic ion exchanger with molecular sieve properties. Process according to any one of claims 3-9, characterized in that the pH of the sodium-containing chloride-containing phosphate buffer used to treat the adsorbed fraction is approx. 8th Process according to any one of claims 3-9, characterized in that the fraction treated with ion exchange is concentrated, desalted and dried. Process according to claim 1, characterized in that the quaternary ammonium salt used is cetylpyridinium chloride. Process according to claim 1, characterized in that the treatment with the quaternary ammonium salt is carried out in the presence of a borate buffer. Process according to claim 1, characterized in that the sodium chloride-containing solution is a borate-buffer containing sodium chloride and a quaternary ammonium salt.