CH639277A5 - Antitumour agent containing a beta-1,3-glucan - Google Patents

Abstract

beta -1,3-Glucans of the formula: <IMAGE> in which R is hydrogen or -CH2COOH and the degree of polymerisation is between 2 and 1000, are antitumour agents.

Description

The invention relates to an anti-tumor agent containing an effective amount of a β-1,3-glucan which can be used for the preventive and curative treatment of tumors.

Research has been carried out for years with a view to finding a substance capable of causing, by parenteral or oral administration, the inhibition of the growth of tumors and the prolongation of the survival time of warm-blooded animals suffering from tumors, with no serious side effects.

It has been found that a water-insoluble thermogellable β-1,3-glucan produced by microorganisms belonging to the genus Alca-ligenes and to the genus Agrobacterium, partially hydrolysed lower polymers of this glucan, or carboxymethylated derivatives said glucan or said lower polymers exhibit a strong antitumor activity against the growth of tumors such as sarcoma, carcinoma or leukemia in warm-blooded animals,

without harmful side effects.

The object of the invention is to provide an antiumoral agent containing as active ingredients ß-1,3-glucan, its lower polymers or its carboxymethylated derivatives.

It is already known, from the Japanese patents published under Nos. 32673/1973 and 32674/1973, and from the English patent No. 1352938, that certain strains of microorganisms of the genus Alcaligenes and of the genus Agrobacterium produce a ß-l, 3 -glucane thermogellable insoluble in water (designated hereinafter, abbreviated, by TAK-N). However, no pharmaceutical use of glucan has been reported.

The extensive studies carried out by the inventors on TAK-N, its lower polymers obtained by partial hydrolysis (hereinafter designated by TAK-D) and the carboxymethylated derivatives of TAK-N or of said lower polymers (hereinafter designated by CMTAK) led to the discovery that these substances have strong anti-tumor activity, resulting in a remarkably high rate of tumor inhibition, and a complete regression of these tumors.

The TAK-N which can be used is that which, as described in detail in the aforementioned patents, is produced by the culture of certain microorganisms such as the Agrobacterium radiobacter (IFO 13127,

ATCC 6466), Agrobacterium radiobacter U-19 (IFO 13126,

ATCC 21679, FERM P-l 166) and Y Alcaligenes faecalis var. myxoge-nes NTK-u (IFO 13140, ATCC 21680, FERM P-1168). This ß-1,3-glucan contains ß-1,3 bonds, as glucoside bonds,

and is insoluble in water, most species having average degrees of polymerization (hereinafter referred to as DP) which are not less than about 70, and a characteristic property that these products are gellable at DP 170 by heating, for example to around 60 ° C, in the presence of water.

The DP of TAK-N varies depending on the preparation process. 35 Determined according to the method of Manners et al. (“Carbohydrate Research” 17.109 (1971), it is approximately 70 to 1000 and, in some cases, 100 to 600. In addition, while these variants of types of glu-canes are generally thermogellable in the presence of water , they lose their gelling power when their DP decreases. For example, the glucan of a DP of 113 is not gellable, while that of DP 170 is gellable. It has been discovered that all TAK-N, TAK- D and CMTAK had potent anti-tumor activity.

It is particularly surprising that even certain types of 45 TAK-D, which cannot be called Polysaccharides due to their low degree of polymerization, retain and manifest such anti-tumor activity.

TAK-D is produced by partial hydrolysis of TAK-N. Among the hydrolysis methods which can be used for this purpose, mention is made of so known methods such as acid hydrolysis, alkaline hydrolysis and enzymatic hydrolysis with ß-1,3-glucanase.

TAK-D can be separated from the reaction mixture according to various techniques commonly used for the purification or fractionation of Polysaccharides and oligosaccharides, such as precipitation in an acid medium, precipitation by addition of ethanol and gel filtration. By such techniques, various lower polymers each having the desired DP can be obtained separately.

The term TAK-D used in the present description means any and all types of lower polymers which are obtained by partial hydrolysis of TAK-N.

As already mentioned, the DPs of TAK-N as obtained from the cultures of the aforementioned microorganisms are widely dispersed. Because the degree of polymerization of ß-1,3-glucan obtained during a fermentation process tends to decrease when the culture time is extended, it is even possible to obtain a TAK-N of DP lower than values of the aforementioned range.

Furthermore, because TAK-D is obtained by hydrolysis of TAK-N, its DP is naturally lower than that of TAK-N

3

639,277

of departure. However, in general, TAK-N with degrees of polymerization corresponding to those of a large number of types of TAK-D can be obtained by culture of said microorganisms. As long as TAK-N and TAK-D had the same DP, no appreciable difference was found between them in their antitumor activity and in their gelling power.

The method of producing TAK-D from TAK-N will now be described using the results of the experimental production trials.

Test I (hydrolysis with sulfuric acid):

In 6 l of 4N sulfuric acid, 60 g of TAK-N are suspended (DP 540, measured according to the method of Manners et al. Unless otherwise indicated, all the DPs indicated below are measured according to this method.) The hydrolysis reaction is carried out at 60 ° C. After 30 min and 1 h, a fraction of 21 is withdrawn from the reaction mixture. These fractions of the reaction mixture are designated respectively by S, and S2. The rest of the reaction mixture is further incubated at 60 ° C for an additional 1 h to obtain another sample S3. From Sj, S2 and S3, the hydrolysis products are prepared as follows. First, each sample is centrifuged and the precipitate is collected. Each precipitate is then washed with 1.6 l of distilled water and centrifuged. This technique is repeated, and the precipitate is suspended in 1.2 l of distilled water. The suspension is neutralized with an 8N solution of sodium hydroxide and lyophilized. The precipitated technique makes it possible to obtain, from Si, S2 and S3, respectively 22, 20 and 19 g of powder. Portions of 7 g of these powders are each suspended in 700 ml of distilled water, and the pH is adjusted to 12.5 with an 8N solution of sodium hydroxide, so as to obtain, respectively, S /, S2 'and S3'.

Ethanol is added to each solution, to have a final concentration of 60%, and, after removing the precipitate formed by centrifugation, ethanol is added to the supernatant, so as to have a concentration of 70%. The solution is then neutralized with dilute hydrochloric acid. The precipitate formed is collected by centrifugation, washed 4 times with 250 ml portions of distilled water, then lyophilized.

1.8 and 1.5 g of white powders (S-I and S-II) are obtained in this way, from S / and S2 ′, respectively. Sufficient ethanol is added to S3 'to obtain a final concentration of 70%, and the mixture is centrifuged. The precipitate is resuspended in approximately 500 ml of distilled water and, after addition of ethanol to have a concentration of 70%, the suspension is neutralized with a small amount of hydrochloric acid. The precipitate formed is collected by centrifugation, washed 4 times with 250 ml portions of distilled water and lyophilized, which gives 5.6 g of a white powder (S-III).

The physicochemical properties of S-I, S-II and S-III are indicated in Table I.

Table I

IF

S-II

S-III

DP *

125

82

68

Elementary analysis (%)

C 40.13

C 40.65

C 40.94

H 6.74

H 6.77

H 6.76

Purity (%) **

93.1

91.1

93.8

Glucose content (%) ***

<0.1

<0.1

<0.1

[a] | J in 0.1N NaOH

25.6

19.5

18.8

(C = 1.16)

(C = 1.10)

(C = 0.82)

* Measured according to the method of Manners et al.

** Calculated from the glucose content determined by the phenolsulfuric acid method.

*** Measured by gas chromatography.

Test 2 (hydrolysis with fornic acid):

I) In 150 ml of 85% formic acid, 6 g of TAK-N (DP 540) are dissolved and the hydrolysis reaction is carried out at 88 ° C. for 20 min. After cooling, the reaction mixture is concentrated to dryness, and the concentrate is suspended in water. The suspension is brought to pH 12.5 with a 5N sodium hydroxide solution, so as to obtain a clear solution. This solution is brought to pH 7 with 5N hydrochloric acid, and the precipitate formed is collected by centrifugation. The precipitate is washed well with distilled water and lyophilized. 5.4 g of a white powder (F-I) are obtained by this process.

II) 12 g of the same TAK-N as that used in I are hydrolyzed in 300 ml of 90% formic acid for 20 min, at 95 ° C., and the reaction mixture is treated almost in the same way as in I, which provides 10.3 g of a white powder (F-II).

III) 12 g of the same TAK-N as that used in I are hydrolyzed in 300 ml of 90% formic acid, at 95 ° C, for 40 min, and the reaction mixture is treated almost in the same way as in I, which provides 5.4 g of a white powder. This powder is dissolved in a 0.05N aqueous solution of sodium hydroxide to obtain a final concentration of 1.0%, and ethanol is added to the solution, so as to have a concentration of 57, 5%. The precipitate formed is collected by centrifugation, suspended in distilled water and neutralized with dilute hydrochloric acid. The suspension is centrifuged and the precipitate is washed well with 70% aqueous ethanol and lyophilized, which provides 2.2 g of a white powder (F-V).

To the supernatant obtained after recovery of the ethanolic precipitate mentioned above, ethanol is added to have a concentration of 70% and the mixture is neutralized with hydrochloric acid. The precipitate formed is combined by centrifugation, washed with 70% aqueous ethanol and lyophilized. By the above process, 2.3 g of a white powder (F-III) are obtained. The supernatant remaining after recovery of the first precipitate (5.4 g) from the mixture of hydrolyzates contains a significant amount of partial hydrolysates in the solubilized state. Consequently, this supernatant solution is concentrated and centrifuged to separate the precipitate formed. The precipitate is lyophilized to obtain 2.9 g of a white powder (F-VII). The supernatant solution is then fractionated by gel filtration and column chromatography on Sephadex G-25 (solvent: 0.1M ammonium bicarbonate), followed by lyophilization of the combined fractions. 0.7 g of a white powder (F-VIII) is obtained by this process.

IV) The same TAK-N (18 g) as that used in I is hydrolyzed in 450 ml of 90% formic acid, at 95 ° C., for 40 min, and almost the same technique is applied to this reaction mixture. fractionation as in III, such as concentration, ethanol precipitation, gel filtration, etc. This technique gives four fractions of white powders (F-IV, 2.3 g; F-VI, 2.8 g, etc.).

The physico-chemical properties of these powders (F-I, F-II,

F-III, F-IV, F-V, F-VI, F-VII and F-VIII) are indicated in Table II. When six samples with weaker DP (F-III to F-VIII) are each dissolved in a 0.02N solution of sodium hydroxide, and chromatographed respectively on the same Sephadex G-200 column equilibrated with the same solution as above, they are eluted in decreasing order of PD (from F-III to F-VIII), each sample giving a symmetrical peak, which indicates that each sample has a normal distribution as regards the degree of polymerization.

(Table at the top of the next page)

It has been discovered that the carboxymethylation of TAK-N and TAK-D provides new derivatives (CMTAK), in which the hydroxyl groups of these glucans are more or less carboxymethylated.

5

10

15

20

25

30

35

40

45

50

55

60

65

639 277 4

Table II

DP *

Analysis

elementary (%)

C H

Purity (%) **

Glucose content (%) ***

[apD5 in 0.1N NaOH

F-I

299

40.73 6.46

92.3

<0.03

30.6 (C = 0.35)

F-II

113

41.71 6.91

98.4

<0.03

22.3 (C = 0.57)

F-III

50

41.98 6.77

93.5

<0.03

12.1 (C = 1.06)

F-IV

44

42.35 6.50

90.8

<0.03

10.9 (C = 2.24)

F-V

39

41.44 6.71

93.3

<0.03

4.0 (C = 1.62)

F-VI

24

42.62 6.99

92.5

<0.03

-1.5 (C = 1.07)

F-VII

16

42.46 6.99

91.8

<0.03

0.4 (C = 1.06)

F-VIII

7

39.84 6.37

94.1

<0.03

0.0 (C = 0.57)

*, ** and ***: see the note at the bottom of Table I.

The invention relates to ß-1,3-glucan derivatives having the following general formula, and their salts.

^ N

ch2or ch2or h, gold

(I)

\

(Formula in which at least one of the Rs is - CH2COOH, the rest, if any, being H; n is an integer between 2 and 1000.)

CMTAK can be obtained by carboxymethylation of TAK-N or TAK-D, in a known manner, for example by reacting TAK-N or TAK-D with monochloroacetic acid in the presence of an alkali. In addition to this method, any other conventional process used for the carboxymethylation of carbohydrates can be used.

To recover CMTAK from the reaction mixture, it is possible to use conventional methods which are commonly used for the purification of carbohydrates, such as precipitation by addition of an organic solvent. In addition, other techniques such as ion exchange chromatography, gel filtration, etc. may be used depending on the content of carboxymethyl groups in CMTAK, or depending on the water solubility of CMTAK.

While the CMTAK thus obtained contains carboxymethyl groups in its molecule, the content of the carboxymethyl groups varies widely depending on the conditions of the carboxymethylation reaction. As determined by titration, the content of

/

n carboxymethyl groups of CMTAK obtained by normal carboxymethylation reaction is generally less than three carboxymethyl groups per glucose residue in the CMTAK molecule. CMTAK, as a product suitable for the purposes of the invention, contains all the substances which can be obtained by carboxymethylation of TAK-N or TAK-D, the molecules of which contain detectable carboxymethyl groups, independently of the degree of carboxymethylation.

As emerges from the general formula I indicated above, CMTAK, in the form of its free acid, is capable of reacting with various bases to form the corresponding salts, such as 45 sodium, potassium, calcium, d aluminum, magnesium and amines. In the context of the invention, CMTAK includes not only its free acid, but also its salts, in particular those of low toxicity.

Table III illustrates the physical properties of some so-typical CMTAK species prepared from TAK-N and TAK-D, according to the techniques previously indicated. The determinations of these physical properties were carried out after each sample had been previously dried under reduced pressure and on phosphorus pentoxide at 60 ° C, for 10 h.

Table III

CMTAK N °

Example No. *

Starting product

Content***

-ch2cooh

Elementary analysis (%)

Viscosity **** (cPo)

[a] jf in 0.1N NaOH

Type

DP **

VS

H

N / A

1

13

TAK-N

540

0.54

40.97

5.57

2.7

2.57

6.0 (C = 0.77)

2

14

TAK-N

540

0.75

38.51

5.45

5.7

2.71

10.1 (C = 0.76)

3

15

TAK-N

540

1.07

37.89

4.77

10.0

2.60

3.7 (C = 0.79)

4

16

TAK-N

255

0.30

40.82

6.05

2.7

2.10

15.1 (C = 0.80)

5

17

TAK-N

255

0.36

39.82

6.06

3.9

2.27

10.0 (C = 0.74)

6

18

TAK-D

299

0.59

38.84

5.71

4.8

2.25

14.6 (C = 0.82)

5

Table III (continued)

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* Numbers of the examples in which the CMTAK is produced.

** Determined by the method of Manners et al.

*** Number of carboxymethyl groups per glucose residue.

**** Determined using a viscometer with coaxial rotation, under the following conditions: solvent: 0.1N solution of sodium hydroxide;

concentration: 0.2%; temperature: 30 ° C;

shear speed: 1046.7 s-1.

CMTAK

No.

Example No. *

Starting product

Content *** -CH2COOH

Elementary analysis (%)

Viscosity **** (cPo)

[a] ff in 0.1N NaOH

Type

DP **

VS

H

N / A

7

18

TAK-D

299

1.15

37.93

4.86

9.0

2.29

1.5 (C = 0.81)

8

19

TAK-D

113

0.51

40.22

5.74

4.7

1.96

12.2 (C = 0.71)

9

20

TAK-D

113

1.22

37.03

4.78

9.9

1.49

3.1 (C = 0.78)

10

19

TAK-D

68

0.36

40.46

5.96

3.5

1.68

10.0 (C = 0.74)

11

20

TAK-D

68

0.45

39.41

5.93

4.6

1.83

9.5 (C = 0.78)

Figs. 1 and 2 illustrate the IR spectra in potassium bromide of CMTAK, respectively Nos 2 and 11 in Table III. The numbers at the top, left and bottom of each figure indicate respectively the wavelengths (| x), the transmission coefficients or transmittances (%) and the wave numbers (cm "1).

The characteristic absorptions were found at 1440-1400 and 891 cm "1.

The TAK-N, TAK-D and CMTAK obtained as above, with DP ranging from 2 to approximately 1000, preferably from 15 to 800, and more advantageously from 40 to 600, exhibit a remarkable inhibitory activity against different kinds of tumors in warm-blooded animals including domestic animals, poultry, dogs, cats, rabbits, rats, mice, etc., and especially against serious tumors known to be less susceptible to treatment with generally known cytotoxic anti-tumor drugs.

For example, the growth of sarcoma 180, Ehr-Iich carcinoma, SN-36 leukemia, CCM adenocarcinoma or NTF reticular cell sarcoma, each tumor being transplanted subcutaneously in mice, was significantly inhibited by 40 administering TAK-N, TAK-D or CMTAK intraperitoneally, intravenously or subcutaneously, before, after or at the time of transplantation, in one or more doses doses of approximately 1 to 1000 mg / kg / d.

The toxicities of TAK-N, TAK-D and CMTAK are extremely low. For example, no toxic effect is observed, both in mice and in rats, at a dose of 3000 mg / kg intraperitoneally, or 10000 mg / kg orally. Thus, these Polysaccharides can be administered, safely and repeatedly, to warm-blooded animals.

The administration can be carried out according to the general techniques usually applied for the treatment of cancers. Are thus applicable intratumoral, subcutaneous, intramuscular or intravenous injections, if necessary oral and rectal J5 administrations, or even external applications, whitewashing, instillations and other methods of administration.

According to the invention, TAK-N, TAK-D or CMTAK is administered to a warm-blooded animal in an amount sufficient to inhibit the growth of its tumor. . 60

The dosage and method of administration of TAK-N, TAK-D or CMTAK to warm-blooded animals with neoplasia may vary depending on the type of agent (TAK-N, TAK-D or CMTAK), animals and patients, tumor symptoms, administration forms and other factors. In general, the sufficient dosage amount per administration is about 0.02 to 2000 mg / kg bodyweight, preferably 0.2 to 2000 mg / kg bodyweight, the preferred upper limit being about 500 mg / kg bodyweight. In many cases, the most advantageous upper limit is about 200 mg / kg.

The administration can be carried out, for example, one to six times a day, for several consecutive days or intermittently.

TAK-N, TAK-D and CMTAK can be administered in combination with other anti-tumor agents. Combination with such agents causing increased immunological activity in animals with tumors is also advantageous.

The invention will now be described with reference to the examples below. Some of these examples illustrate the technique for producing TAK-D from TAK-N, the technique for producing CMTAK, the physical properties of TAK-N, TAK-D and CMTAK and certain injectable forms of these products. Other examples demonstrate the anti-tumor activities of TAK-N, TAK-D and CMTAK against tumors in mice, using standard models.

As indicated above, the treatment of animals suffering from tumors with the compounds produces an energetic regression of the various types of tumors, such as sarcomas, carcinomas, reticular cell sarcomas and leukemias. All of these types of tumors in rodents have been shown to be exact classic types of tumors in other warm-blooded animals.

These examples obviously do not have any limiting character of the invention.

Most of the following tests on the anti-tumor activity of TAK-N, TAK-D and CMTAK are evaluated using approximately the same methods as those described for the anti-tumor activity of lentinan, branched glucan from an edible fungus, according to Chihara , G. et al. (see “Nature”, 222, 687-688,1969, and “Cancer Research” 30.2776-2781.1970).

The tumors of the mice used in these tests, such as sarcoma 180 (S 180), carcinoma of (Ehrlich) and sarcoma with reticular cells NTF (NTF), adenocarcinoma CCM (CCM) and leukemia SN-36 (SN-36), are maintained in mice of the ICR-JCL type, from 5 to 7 weeks, each in ascites forms. An appropriate component of these tumor cells is transplanted subcutaneously into ICR-JCL mice, and the tumor nodules which have developed are excised for weighing on the 35th day after transplantation of the tumor.

A TAK-N or TAK-D was suspended or dissolved in distilled water or in physiological solution, and CMTAK was dissolved in distilled water or in physiological saline, at appropriate concentrations and at pH close to neutral. Each preparation was administered intraperitoneally, subcutaneously or intravenously to mice (0.2 ml / 20 g bodyweight), daily for 10 d, starting 24 h after tumor transplant, daily

639,277

6

ment for 5 d before transplanting the tumor, or only once between the 7th d before and the 21st d after the implantation of the tumor.

Example 1:

TAK-N (DP 540) tumor suppression effect: single injection

Four million S 180 cells are transplanted subcutaneously into the left inguinal region of female ICR-JCL mice, 4 to 5 weeks old. The nodes of the tumor are excised on the 35th day after transplantation and weighed. The average weight of a tumor in a treated group (T) comprising 5 mice was compared with that of a control group (C) in 10 mice, and the inhibition rate of the tumor was calculated ( % inhibition = (C — T) / C x 100). The number of tumor-free animals (complete regression) was also counted in the 35th d.

TAK-N (DP 540), suspended in physiological saline solution (10 mg / ml), was administered intraperitoneally (0.2 ml / 20 g bodyweight) once to each animal, at the dose of 100 mg / kg, on the 7th, 3rd or 1st before, or on the 1st, 2nd, 3rd, 5th, 7th, 10th, 14th 5 or 21st after the transplantation of the tumor, or on the day of transplantation of the tumor.

As seen in Table IV, TAK-N at the dose of 100 mg / kg markedly inhibits the growth of S 180, when the test sample is administered once between the 7th before and the 21st after 10 tumor transplant.

In addition, with single administration of TAK-N (DP 540) on the 7th day after tumor transplantation at doses of 20, 60 and 100 mg / kg, marked suppression of tumor growth was observed on both higher doses, 60 and 100 mg / kg.

Table IV

Tumor inhibition rate (%)

Number of

Sample

Dose mg / kg (i.p.)

Injection day

Average tumor weight (g)

complete regressions / number of mice treated

7.06

-

0/10

100x1

-7 *

2.27

67.8

3/5

TAK-N

100x1

-3

1.64

76.8

3/5

(DP 540)

100x1

-1

0

100

5/5

100x1

0 **

0.56

92.1

3/5

6.94

0/10

riooxi

1.19

82.9

4/5

TAK-N (DP 540)

100x1 100x1 100x1

2

3 5

0.59 0

1.25

91.5 100 82.0

4/5 5/5 3/5

[lOOxl

7

0

100

5/5

5.08

-

0/10

100x1

7

0

100

5/5

TAK-N

100x1

10

0.01

99.8

4/5

(DP 540)

100x1

14

0.01

99.8

4/5

100x1

21

1.71

66.3

1/5

5.36

0/5

TAK-N (DP 540)

20x1 60x1 .100x1

7 7 7

3.72 0.12 0.03

30.6 97.8 99.4

1/5 3/5 4/5

* TAK-N was administered on the 7th before the tumor transplant.

** TAK-N was administered on the 1st after the tumor transplant.

*** TAK-N was administered on the day of the tumor transplant.

These results indicate that TAK-N (DP 540) is effective in suppressing tumor growth, at all stages of tumor progression, in warm-blooded animals, even by single administration.

Example 2:

Effect of tumor suppression by TAK-N (DP 540) and TAK-N flocculated in autoclave (DP 540)

Six million S 180 cells are transplanted subcutaneously into the right inguinal region of ICR-JCL mice of a live weight of approximately 23 g. Tumor nodes are excised on the 35th after transplantation and weighed. The tumor inhibition rate in a treated group was calculated as in Example 1.

TAK-N (DP 540) and TAK-N (DP 540) were used. The latter was obtained by heating a 2% suspension of TAK-N

(DP 540) at 120 ° C for 25 min, then drying and spraying

55 the gel formed. The samples were suspended in distilled water at the desired concentrations, and each suspension was administered intraperitoneally to a group of 5 mice, daily for 10 d, starting 24 h after transplantation of the tumor.

60

As seen in Table V, TAK-N (DP 540) has a pronounced effect on tumor growth at doses of 1 to 50 mg / kg, this effect being particularly important at doses of 5 to 25 mg / kg.

65

TAK-N (DP 540) has also been shown to be effective in suppressing tumor growth at doses of 10 mg / kg. No toxicity was observed.

7

Table V

639,277

Tumor inhibition rate (%)

Number of

Dose

Average weight regressions

Sample mg / kg of complete /

(i.p.)

tumor (g)

number of

treated mice

-

6.00

-

0/6

"1x10

4.80

20.0

0/6

3x 10

3.58

40.3

1/6

5x10

0.02

99.7

5/6

TAK-N

7x10

0.73

87.8

5/6

(DP 540)

10x10 15x10

0.01 0.10

99.8 98.3

5/6 4/5

20x10

0

100

5/5

25x10

0.42

93.0

3/6

-50x10

1.19

80.2

1/6

Gel*

-

3.67

-

0/6

TAK-N

10x10

(DP 540)

0.29

92.1

5/6

* Autoclaved.

These results show that TAK-N can be used in slowing the growth of tumors, at relatively low doses, and that the sample can be sterilized by autoclave treatment, without losing its activity.

Example 3:

Inhibitory activity of tumors of various types of TAK-N and TAK-D

From 24 hours after the transplantation of S 180, as described in Example 2, various types of TAK-N and TAK-D suspended in distilled water were administered, intraperitoneally, to a dose of 10 mg / kg, to a group of 5 mice daily for 10 d. The tumor nodules were excised on the 35th after transplantation of the tumor and weighed. The inhibition rates were calculated as in Example 1.

As seen in Table VI, all of the samples used show inhibitory activity against tumor growth. In particular, samples of PD greater than 50 show strong antitumor activity with complete regression.

Table VI

Tumor inhibition rate (%)

Number of

Dose

Average weight regressions

Sample mg / kg of complete /

(i.p.)

tumor (g)

number of mice treated

-

5.46

-

0/6

(DP 565)

10x10

0.07

98.7

5/6

TAK-N

(Dp380)

10x10 10x10

0.20 0.46

96.3 91.6

5/5 4/6

(DP 125)

10x10

0

100

6/6

5.46

0/6

S-II

(DP 82)

10x10

0

100

6/6

S-III (DP 68)

10x10

0.03

99.5

5/6

F-III TAK-D (DP 50)

10x10

0

100

6/6

F-V (DP 39)

10x10

2.03

62.8

1/6

F-VI (DP 24)

10x10

3.60

34.1

0/6

F-VII (DP 16)

10x10

1.54

71.8

1/6

These results show that TAK-N and TAK-D are both effective in regressing tumor growth, regardless of the values of their PD.

639,277

8

Example 4:

Effects of tumor suppression by CMTAK and autoclaved CMTAK

The anti-tumor activity of three types of CMTAK prepared from TAK-N (CMTAK No. * 1, 4, 5) and autoclaved CMTAK was tested as described in Example 2. The samples were dissolved in distilled water for injection, and the pH

has been adjusted to approximately 7.0. In certain tests, one of these samples (CMTAK No. 1) was sterilized by autoclave treatment (120 ° C., 25 min) before any other administration.

As seen in Table VII, all of the CMTAK samples, at doses of 10 mg / kg, significantly inhibited tumor growth. Growth inhibition by CMTAK has been observed at doses of 1 to 40 mg / kg.

Table VII

Dose

Sample mg / kg (i.p.)

Average tumor weight (g)

Tumor inhibition rate (%)

Number of complete regressions / number of mice treated

CMTAK N ° 1 10x9 CMTAK N "4 10x9 CMTAK N" 5 10x9

2.87 0

0.28 0.01

100 90.2 99.7

0/7 7/7 4/7 6/7

CMTAK N ° 1 autoclaved

1x10 3x10 5x10

3.65 0.77 0.03 0.23

78.9 99.2 93.7

0/6 1/6 5/6 4/6

CMTAK N “1 autoclaved

-

2.17

C 5x10

0

10x10

0.07

15x10

0.08

20 x 10

0.21

140x10

OO

o

100 96.8 96.3 90.3 91.7

0/6 6/6 5/6 5/6 3/6 1/6

These results show that CMTAK can be sterilized without losing its anti-tumor activity and be used in an injectable form for the treatment of cancer.

Example 5:

Effects of tumor suppression by TAK-N, TAK-D and CMTAK using various methods of administration

_ TAK-N (DP 540), TAK-D (F-III, DP 50), TAK-D (F-VII, DP 16) and CMTAK N ° 1 were administered at doses of 5 mg / kg per intravenously, subcutaneously or intraperitoneally, during

10 consecutive d, to ICR-JCL mice which had previously undergone a transplant with 4 x 10 6 cells, 24 h before the first injection. The tumor growth inhibition rates were calculated as in Example 1.

As seen in Table VIII, significant inhibition of tumor growth was observed in each case. By intravenous administration, TAK-N, CMTAK and even F-VII (DP 16) almost completely inhibit tumor growth, the inhibition rate being greater than 85%, with complete regression of 45 the tumor on 4 or 5 of the 5 mice treated.

40

Table VIII

Tumor inhibition rate (%)

Number of

Sample

Dose mg / kg

Method of administration

Average tumor weight (g)

complete regressions / number of mice treated

-

7.30

-

0/10

CMTAK No 1

'5x10 5x10

i.v. s.c.

0.84 3.77

87.5 48.4

4/5 0/5

5x10

i.p.

0.91

88.5

2/5

-

7.30

-

0/10

TAK-N (DP 540)

5x10 5x10 5x10

i.v. s.c. i.p.

0

2.72 0.17

100 63 98

5/5 3/5 4/5

TAK-D

5x10

i.v.

3.02

59

2/5

F-III

5x10

s.c.

0.91

88

3/5

(DP 50)

5x10

i.p.

2.30

68

3/5

639,277

9

Vili painting (continued)

Sample

Dose mg / kg

Method of administration

Average tumor weight (g)

Tumor inhibition rate (%)

Number of complete regressions / number of mice treated

TAK-D

5x 10

i.v.

1.06

85

4/5

F-VII

5x 10

s.c.

2.67

63

3/5

(DP 16)

5x10

i.p.

2.34

68

2/5

These results show that TAK-N, CMTAK and TAK-D are effective in suppressing tumors by any route of administration.

Example 6:

Effects of tumor suppression by TAK-N, TAK-D 20

and CMTAK administered before tumor transplant: pretreatment

TAK-N and TAK-D of different degrees of polymerization and CMTAK No. 1 were administered intraperitoneally to ICR-JCL mice of live weight of approximately 23 g, at doses of 3 to 80 mg / kg , daily for 5 consecutive d. TAK-N and TAK-D were suspended, and CMTAK dissolved, in distilled water. In the case of CMTAK, the final pH was adjusted to about 7.0. Then, on the 1st or 3rd after the last administration, 6 x 10 6 S 180 cells were transplanted subcutaneously into the right inguinal region of each animal. On the 35th after transplantation, the tumor nodes were excised and weighed. The tumor inhibition rates were calculated as in Example 1.

As seen in Table IX, a marked inhibition of tumor growth is observed for each sample. In particular, TAK-N (DP 540), CMTAK and TAK-D with DP greater than 50 exhibit a strong inhibitory activity. CMTAK was effective in delaying tumors at low doses of around 3 mg / kg.

Table IX

Tumor inhibition rate (%)

Number of

Sample

Dose mg / kg (i.p.)

Injection day

Average tumor weight (g)

complete regressions / number of

treated mice

-

5.06

0/5

TAK-N (DP 540)

20x5

—5 to - 1

0.37

92.7

4/6

TAK-N (DP 125)

20x5

- 5 to - 1

0.98

80.6

2/6

-

10.10

-

0/6

S-II (DP 82)

20x5

- 5 to —1

0.20

98.0

4/5

S-III

(DP 68)

20x5

- 5 to - 1

0.35

96.5

5/6

F-III (DP 50)

20x5

- 5 to - 1

1.30

87.1

3/5

F-V (DP 39)

20x5

- 5 to - 1

5.52

45.3

0/6

F-VI (DP 24)

20x5

- 5 to —1

4.18

58.6

0/6

F-VII (DP 16)

20x5

—5 to —1

5.84

42.2

0/6

-

6.43

-

0/6

'3x5

—5 to - 1

1.44

77.6

3/5

CMTAK

5x5

- 5 to - 1

3.55

44.8

1/6

# 1

10x5

- 5 to -1

0.19

97.1

3/6

s20x 5

- 5 to -1

0.05

99.2

4/6

-

5.03

0/5

CMTAK 1

40x5

- 5 to - 1

0.11

97.8

4/6

No 1 I

80x5

—5 to - 1

0.15

97.0

4/6

639,277

10

Table IX (continued)

Tumor inhibition rate (%)

Number of

Sample

Dose mg / kg (i.p.)

Injection day

Average tumor weight (g)

complete regressions / number of mice treated

-

7.45

-

0/5

TAK-N (DP 540)

20x5

—7 to —3

0.18

97.6

3/5

TAK-D

S-III

20x5

CO

1

-cd r-1

0.75

89.9

4/5

(DP 68)

These results show that the above-mentioned polysaccharides are useful as prophylactic agents in the treatment of cancer.

Example 7:

TAK-N and CMTAK repressive activity of tumors against various types of tumors

Ehrlich's carcinoma, CCM adenocarcinoma, NTF reticular cell sarcoma and SN-36 leukemia are used to examine the activity of TAK-N and CMTAK for the delay of various types of tumors.

We transplanted into ICR-JCL mice, with an average weight of

23 g, subcutaneously, 3.1 x 10® Ehrlich cells,

1.1 x 107 TLC cells, 4.5 x 10® NTF cells or 7.2 x 104 SN-36 cells, and these mice were administered intraperitoneally with TAK-N (DP 540) suspended in distilled water, or CMTAK N ° 1 dissolved in distilled water (pH 7.0) and autoclaved, at doses of 10 to 50 mg / kg, daily for 10 d, in starting 24 hours after transplant. The tumors were excised and weighed after transplantation, and the tumor inhibition rates in the treated groups were calculated as in Example 1.

As seen in Table X, a significant inhibition of tumor growth was observed in each case.

Paintings

Tumor

Sample

Dose mg / kg (i.p.)

Average tumor weight (g)

Tumor inhibition rate (%)

Number of complete regressions / number of mice treated

Ehrlich's carcinoma

TAK-N (DP 540)

10x10 20x10 40x10

3.54 2.37 0.50 0.93

33.1 85.9 73.7

0/5 0/5 2/6 0/5

CMTAK N "1

20x10 40x10

1.93 0.80 0.60

58.5 68.9

1/6 3/6 2/6

Adenocar Cinome CCM

TAK-N (DP 540)

20x10

3.42 0.79

76.9

0/6 2/6

CMTAK No 1

20x10

3.42 1.03

69.9

1/6 1/6

SN-36 leukemia

TAK-N (DP 540)

10x10 25x10 50x10

0.91 0.18 0.19 0.23

80.2 79.1 74.7

2/6 4/5 3/5 2/6

NTF reticular cell sarcoma

TAK-N (DP 540)

10x10 30x10

7.64 0.38 2.21

95.0

71.1

0/6 2/6 0/6

The results show that TAK-N and CMTAK have anti-tumor activity against various types of tumors, namely sarcomas, carcinomas, adenocarcinomas, leukemias, etc.

Example 8:

The four preparations of CMTAK (CMTAK Nos 2, 6, 9 and 11) are each dissolved in physiological saline solution, and

11

639,277

their effects on tumor S 180 are examined, in accordance with the process of Example 4. All the samples, at daily doses of 5 mg / kg administered for 10 d starting 24 hours after transplantation of the tumor, remove remarkably the growth of the tumor.

Example 9:

TAK-N (replaceable with TAK-D or CMTAK) 150 mg

Lactose 48 mg

Magnesium stearate 2 mg

Total: 200 mg

The above quantity forms a capsule.

TAK-N (TAK-D or CMTAK) and lactose are mixed in the indicated proportions, pelletized and sprayed. The magnesium stearate is then added. The mixture is distributed in capsules.

Example 10:

TAK-N (replaceable with TAK-D or CMTAK) 400 mg Lactose 95 mg

HPC-L (hydroxypropylcellulose) 5 mg

Total: 500 mg

The above quantity forms a single dose.

The three ingredients indicated are mixed in the proportions mentioned and, with the addition of a small amount of water, the mixture is passed through the mixer, granulated, dried, re-granulated, sieved and agglomerated according to the doses indicated above.

Example 11:

a) 1 g of TAK-D (DP 16) is dissolved in 1000 ml of distilled water for injection (or of physiological saline solution) and the solution is filtered, the filtrate being distributed in ampoules in 500 ml portions. After sealing, the ampoules are hot sterilized in the usual way.

b) 2 g of CMTAK (product of Example 14) are dissolved in 100 ml of distilled water for injection (or of a physiological saline solution) and the solution is filtered. The filtrate is distributed in ampoules in 20 ml portions and, after sealing, the ampoules are sterilized hot in the usual way.

Example 12:

TAK-N (replaceable with TAK-D or CMTAK) 160 mg

Sorbitol 200 mg

Carboxymethylcellulose, sodium salt 10 mg

Polysorbate 80 3.2 mg

Methyl-p-hydroxybenzoate 4 mg

Propyl-p-hydroxybenzoate 0.4 mg

The above ingredients are mixed in distilled water for injection, to form a total volume of 4 ml. (When CMTAK is used, the solution is neutralized, if necessary, with sodium hydroxide N10.)

Example 13:

In 80 ml of isopropyl alcohol, 3 g of TAK-N (DP 540) are suspended and this suspension is stirred at room temperature for 30 min. 8 ml of a 30% sodium hydroxide solution are then added slowly, with stirring, for approximately 60 minutes. The mixture is continued to be vigorously stirred at room temperature for approximately 90 minutes in order to prevent the formation of a gel. 3.6 g of monochloroacetic acid are then added and the mixture is stirred at 60-70 ° C for 5 h, so that the carboxymethylation takes place. The product is collected by filtration and washed thoroughly with a mixture of methanol and acetic acid (7: 3, v / v). The precipitate is collected by filtration, washed well with 80% aqueous methanol, methanol and acetone, in the order mentioned, and dried under reduced pressure. We obtain,

according to this technique, 2.9 g of CMTAK. Carboxymethyl content (number of carboxymethyl groups per glucose residue, the same definition also applies below): 0.54.

Example 14:

1.5 g of TAK-N (DP 540) are suspended in 40 ml of isopropyl alcohol and the mixture is stirred at room temperature for 30 min. Then added, with stirring, 2 ml of a 30% sodium hydroxide solution, in four fractions, each of 0.5 ml, at 15 min intervals. The mixture is then stirred at room temperature for 90 min. 0.9 g of monochloroacetic acid are added in three fractions, each of 0.3 g, at 10 min intervals. The carboxymethylation is thus carried out at 50 ° C., with stirring for 150 min. The product is collected by centrifugation, dissolved in 50 ml of water and neutralized with acetic acid. To this neutral solution is added 120 ml of methanol, and the precipitate formed is combined by centrifugation. The precipitate is washed with a mixture of 300 ml of 80% aqueous methanol and 100 ml of ethanol, then with a mixture of 300 ml of 80% aqueous methanol and 200 ml of ether. The product is finally lyophilized, so as to obtain 1.7 g of CMTAK.

Carboxymethyl content: 0.75.

Example 15:

1.5 g of TAK-N (DP 540) are suspended in 40 ml of isopropyl alcohol and the suspension is stirred at room temperature for 30 min. Then added 4 ml of a 30% solution of sodium hydroxide, in four fractions of 1 ml each, at 15 min intervals, then stirring is continued at room temperature for 90 min.

Then 1.8 g of monochloroacetic acid are added in three fractions, each 0.6 g, at 10 min intervals. The mixture is stirred at 50 ° C for 150 min, which allows the carboxymethylation to be carried out. The product is collected by centrifugation, dissolved in 40 ml of water and neutralized with acetic acid. To this neutral solution is added 90 ml of methanol and the precipitate formed is collected by centrifugation. The precipitate is washed well with a mixture of 200 ml of 80% aqueous methanol and 100 ml of ethanol, then with a mixture of 200 ml of 80% aqueous ethanol and 200 ml of ether. Then lyophilized to obtain 2.0 g of CMTAK. Carboxymethyl content: 1.07.

Example 16:

3.2 g of TAK-N (DP 255) are suspended in 33 ml of water and 1 g of sodium hydroxide is added with stirring, at room temperature, then 2.4 g of monochloroacetate sodium. The carboxymethylation reaction is thus carried out at room temperature, with constant stirring, for 2 h. Then 1 g of sodium hydroxide and 2.4 g of sodium monochloroacetate are added again, and the reaction is continued at room temperature,

with stirring, for 3 h. Another 1 g of sodium hydroxide and 2.4 g of sodium monochloroacetate are added. The reaction is continued at room temperature, with stirring, for an additional 2 h. 1 l of ethanol is added to the reaction mixture, and the precipitate formed is washed properly with ethanol on a sintered glass filter, until the filtrate ceases to give a red coloration to the phenolphthalein. Then dried at 50 ° C under reduced pressure. The powder formed (3.7 g) is dissolved in 90 ml of water and neutralized with acetic acid, then 210 ml of ethanol are added. The precipitate formed is combined by centrifugation, washed with 80% aqueous ethanol and lyophylized. This method gives 2.6 g of CMTAK. Carboxymethyl content: 0.30.

Example 17:

6.4 g of TAK-N (DP 255) are suspended in 66 ml of water and 4 g of sodium hydroxide are added under ice cooling and stirring, followed by 9.6 g sodium monochloroacetate. The carboxymethylation is carried out under ice cooling and stirring for 2 h. Again 4 g of hydr-

5

10

15

20

25

30

35

40

45

50

55

60

65

639,277

12

sodium oxide and 9.6 g of sodium monochloroacetate, and the mixture is stirred for 3 h while cooling with ice. Then 4 g of sodium hydroxide and 9.6 g of sodium monochloroacetate are added again, and the reaction is continued,

with ice cooling and stirring, for 3 h. 1 l of ethanol is added to the reaction mixture and the precipitate formed is washed well with ethanol on a sintered glass filter, until the filtrate stops giving a red color to the phenolphthalein, then it is dried at 50 ° C. under reduced pressure. The powder formed (8.6 g) is dissolved in 172 ml of water and neutralized with acetic acid. Then, after adding 480 ml of ethanol, the precipitate is combined by centrifugation, washed with 80% aqueous ethanol and lyophilized. According to this method, 5.6 g of CMTAK are obtained. Carboxymethyl content: 0.36.

Example 18:

1.5 g of TAK-D (FI, DP 299) are suspended in 40 ml of isopropyl alcohol and the carboxymethylation is carried out as in Example 14. The product is washed as in Example 14 , to obtain 1.9 g of CMTAK. Carboxymethyl content: 0.59.

The same TAK-D as above is suspended in 40 ml of isopropyl alcohol, and the carboxymethylation is carried out according to the method described in Example 15. The reaction product is washed as in Example 15, which gives 2.2 g of CMTAK. Carboxymethyl content: 1.15.

Example 19:

1.5 g, respectively, of TAK-D (F-II, DP 113) and TAK-D (S-III, DP 68) are suspended in 40 ml of isopropyl alcohol carboxymethylation as in Example 14. The respective reaction products are combined by centrifugation, dissolved in 40 ml of water and neutralized with acetic acid. 90 ml of methanol are added to each of these two neutral solutions, and the resulting precipitate is collected by centrifugation and washed well, firstly with 200 ml of 80% aqueous methanol, then lyophilization is carried out. Two types of CMTAK are obtained by this method.

Yield

Content

(g)

carboxymethyl

CMTAK (obtained from

F-II)

1.4

0.51

CMTAK (obtained from

S-III)

1.4

0.36

Example 20:

1.5 g, respectively, of TAK-D (F-II, DP 113) and TAK-D (S-III, 20 DP 68) are suspended in 40 ml of isopropyl alcohol Carboxymethylation is carried out as in Example 15. The reaction products are each washed as in Example 19, which makes it possible to obtain two types of CMTAK.

Yield

Content

(g)

carboxymethyl

CMTAK (obtained from

F-II)

2.3

1.22

CMTAK (obtained from

S-III)

1.4

0.45

R

1 sheet of drawings

Claims (5)

639,111 1. Antitumor agent containing an effective amount of a 1,3-glucan having the following formula: ch2or h, or in which R is hydrogen or at least one of the R is - CH2COOH, the remainder being H, n is an integer and the average degree of polymerization is between 2 and 1000. 2. Antitumor agent according to claim 1, characterized in that the average degree of polymerization is between 15 and 800. 3. Antitumor agent according to claim 1, characterized in that the average degree of polymerization is between 40 and 600. 4. Antitumor agent according to claim 1, characterized in that the degree of substitution of the carboxymethyl groups is Between 0.30 and 1.22 when the compound contains at least one - CH2COOH defined in group R. 5. Antitumor agent according to claim 1, characterized in that the degree of substitution of the carboxymethyl groups is between 0.45 and 0.75 when the compound comprises at least one 25 - CH2COOH defined in group R.