CA1094453A - TREATMENT OF TUMORS AND NEW DERIVATIVES OF .beta.-1,3- GLUCAN - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

It has been found that water-insoluble, thermogelable .beta.-1,3-glucan produced by microorganisms, lower polymers obtainable upon partial hydrolysis of the glucan or, carboxy-methylated derivatives of said glucan or of said lower polymers are useful for inhibiting growth of tumors in warm-blooded animals. The inhibitory activity against various tumors of these polysaccharides is very strong and significant whenever administered to warm-blooded animals from the early to very late stage of tumor progression or even when administered prior to plantation of tumors. The carboxymethylated derivatives above-mentioned are novel water-soluble compounds which can be pro-duced by carboxymethylation of said glucan and partial hydrolyzate thereof.

Description

- ~0~4~3 1 This is a divisional application of patent application serial number 291,230 filed on November 18, 1977.
This invention relates to a preventive and curative treatment of tumors and a new derivative of ~-1,3-glucan usable for the treatment.
For years there have been searches for substances which, with parenteral or oral administration, will cause the inhibition of tumor growth and the prolongation of the survival time of tumor-bearing warm-blooded animals without serious side effects.
It has now been found by the present inventors that a water-insoluble, thermogelable ~-1,3-glucan produced by micro-organisms belonging to the genus Alcaligenes and the genus A~robacterium, partially hydrolyzed lower polymers thereof, or carboxymethylated derivatives of said glucan or of said lower polymers have a strong antitumor activity against the growth of tumors such as sarcoma, carcinoma or leukemia in warm-blooded animals without undesirable side effect.
The principal object of the present invention is, therefore, to provide a treatment for warm-blooded animals affected or may be affected by neoplasias with the ~-1,3-glucan, the lower polymers, or their carboxymeth~lated derivatives. And another o~ject is to provide an antitumor agent containing the ~-1,3-glucan, the lower polymers, or the carboxymethylated derivatives as active ingredients. A further object is to provide novel carboxymethylated derivatives of either the ~-1,3-glucan or the lower polymers obtainable by partial hydrolysis of the ~-1,3-glucan.
It is already known from Japanese Patent Publications 30 Nos. 32673/1973 and 32674/1973 and British Patent No.1,352,938 that certain strains of microorganisms belonging to the genus '~

`' 109~453 1 Alcaligenes and the genus Agrobacterium produce a water-insoluble, thermogelable ~-1,3-glucan (hereinafter referred to briefly as TAK-N). However, no pharmaceutical usage of the glucan has been known at all.
The extensive studies made by us on TAK-N, lower polymers obtainable by partial hydrolysis thereof (hereinafter referred to as TAK-D) and carboxymethylated derivatives of TAK-N or of said lower polymers (hereinafter referred to as CMTAK) led to the finding that these substances possess a strong antitumor activity giving a markedly high tumor inhibition ratio and a complete regression of tumors. The finding was followed by further studies which have resulted in the completion of this invention.
This invention is directed to a method of inhibiting the growth of a tumor in a warm-blooded animal which comprises administering to said animal to inhibit the growth of said tumor a sufficient amount of a member of the group consisting of a ~-1,3-glucan which is water-insoluble and thermogelable in the presence of water at an average degree of polymerization of 170, a lower polymer obtainable upon partial hydrolysis of said glucan and a carboxymethylated derivative of said glucan or said lower polymer.
And another part of this invention is directed to an antitumor agent containing said glucan, said low polymer or said carboxymethylated derivative.
TAR-N which is employable in accordance with this invention is such that, as described in detail in the afore-mentioned patent literatures,it is produced by the cultivation of certain microorganisms such as Agrobacterium radiobacter (IF0 13127, ATCC6~661. Agrobacterium radiobacter U-l9(IF0 13126, ATCC 21679, FERM P-1166) and Alcaligenes faecalis var. myxogenes . ~

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-` 109~53 1 NTK-u (IFO-13140, ATCC 21680, FERM-P 1168). This ~-1,3-glucan contains ~-1,3-bonds as its glycosidic linkages and is water-insoluble, most species of which have average degrees of poly-merization (hereinafter abbreviated as DP) not less than about 70 and a characteristic property such that they are gelable at DP 170 when heated, for example at about 60C, in the presence of water.
The DP of TAK-N is variable according to the method of preparation. Thus, as determined by the method of Manners _ al 10Carbohydrate Research 17, 109 (1971), it is from about 70 to 1,000 and, in many cases, from 100 to 600. And, while these variants of glucan species are generally thermogelable in the presence of water, they lose gelability as their DP values are lowered. For example, the glucan with DP of 113 is not gelable but that with 170 is gelable. We have discovered that all of TAK-N, TAK-D and CMTAK have a potent antitumor activity.
It is particularly surprising that even some species of TAK-D which may not be called polysaccharides because o~
their low degree of polymerization retain and display such an antitumor activity.
TAK-D is produced by partial hydrolysis of TAK-N. Among the methods of hydrolysis available for this purpose are such known methods as acid hydrolysis, alkaline hydrolysis and enzymatic hydrolysis with ~-1,3-glucanase.
TAK-D can be separated from the reaction mixture by various procedures that are commonly practised for the puri-fication or fractionation of polysaccharides and oligosaccharides, such as precipitation under acidic conditions, precipitation by the addition of ethanol, and gel filtration. By such procedures, various lower polymers each having the desired DP value can be obtained separately.

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109~4~3 1 Ther term T~K-D used throughout this specification means any and all types of lower polymers which are obtainable by , partial hydrolysis of TAK-N.
As aforesaid, DP of TAK-N as obtained from cultures of the aforementioned microorganisms are broadly dispersed. And since the degree of polymerization of the ~-1,3-glucan produced in the course of the fermentation process tends to decrease as the cultivation time is prolonged, it is even possible to produce TAK-N with DP lower than the above-mentioned range.
On the other hand, since TAK-D is produced by the hydro-lysis of T~K-N, its DP is naturally lower than the starting material TAK-N. But, generally speaking, TAK-N having degrees of polymerization corresponding to those of a large majority of TAK-D species can be produced by the cultivation of said micro-organisms. And so long as TAK-N and TAK-D have the same DP, no substantial difference in antitumor activity and thermogelability has been found between them.
The manner of production of TAK-D from TAK-N will now be described by way of the results of our experimental production runs.
Experiment 1 (hydrolysis with sulfuric acid) In 6 liters of 4 N-sulfuric acid was suspended 60 g of TAK-N (DP : 540 as measured by the method of Manners et al.
Unless otherwise specified hereinafter, all DP values indicated were measured by this method~. The hydrolysis reaction was carried out at 60C. After 30 minutes and after one hour, a 2 liter portion of the reaction mixture was taken. These portions of the reaction mixture were designated Sl and S2, respectively. The remainder of the reaction mixture was further incubated at 6QC for an additional one hour to obtain another sample S3. From Sl, S2 and S3, hydrolysis products were prepared , ~ :

-` 105~44~3 1 by the following procedure. First, each sample was centri-fuged and the precipitate was collected. Each precipl-tate was then washed with 1.6 liters of distilled water and centrifuged.
This procedure was repeated again and the precipitate was suspended in 1.2 liters of distilled water. The suspension was neutralized with an 8 N-solution of sodium hydroxide and lyophilized. The above procedure provided 22 g, 20 g and 19 g of powders, from Sl, S2 and S3, respectively. Then 7 g portions of these powders were respectively suspended in 700 ml of distilled water and the pH was adjusted to 12.5 with an 8 N

solution of sodium hydroxide to yield Sl', S2' and S3', respectively.
Ethanol was added to each solution to give a final con-centration of 60 % and, after the resulting precipitate was removed by centrifugation, ethanol was further added to the supernatant to give a concentration of 70 %. Then, the solution was neutralized with dilute hydrochloric acid. The resulting precipitate was collected by centrifugation, washed 4 times with 250 ml portions of distilled water and, then, lyophilized.
In this manner, 1.8 g and 1.5 g of white powders (S-I

and S-II~ were obtained from Sl' and S2', respectively. To S3' was added a sufficient amount of ethanol to give a final concentration of 70 % and the mixture was centrifuged. The precipitate was resuspended in about 500 ml of distilled water and, after the addition of ethanol to give a concentration of 70 %, the suspension was neutralized with a small amount of hydrochloric acid. The resulting precipitate was collected by centrifugation, washed 4 times with 250 ml portions of distilled water and lyophilized, which provided 5.6 g of white powder (S-III).

~0944~;3 1 The physioehemical properties of S-I, S-II and S-III are shown in Table l.
TABLE l S-I S-II S-III
_*

Elemental analysis(%) C40.1340.65 40.94 H6.74 6.77 6.76 Purity(%)** 93.1 91.1 93.8 Glucose content(%)*** C0.1 ~0.1 ~0.1 [~]25 in 0.1 N NaOH 25.6 19.5 18.8 (C=1.16)(C=1.10) (C=0.82) * Measured by the method of Manners et al ** Calculated from glucose content determined by the phenol-sulfuric acid method *** Measured by gas chromatography Experiment 2 (hydrolysis with formie acid) (I) In 150 ml of 85 % formie aeid was dissolved 6 g of TAK-N
(DP : 540) and the hydrolysis reaction was earried out at 88C
for 20 minutes. After eooling, the reaction mixture was con- -centrated to dryness and the eoneentrate was suspended in water.
The suspension was brought to pH 12.5 with a 5 N-solution of sodium hydroxide to obtain a elear solution. The solution was brought to pH 7.0 with 5 N-hydroehlorie acid and the resulting preeipitate was eolleeted by eentrifugation. The preeipitate was washed well with distilled water and lyophilized. By the above proeedure was obtained 5.4 g of white powder (F-I).
(II) Twelve grams of the same TAK-N as used in (I) was hydrolyzed in 300 ml of 90 % formic acid at 95C for 20 minutes and the reaction mixture was treated in essentially the same manner as -` 10~453 1 in (I) to obtain 10.3 g of white powder (F-II).
(III) Twelve grams of the same TAK-N as used in (I) was hydrolysed in 300 ml of 90 ~ formic acid at 95C for 40 minutes and the reaction mixture was treated in essentially the same manner as in (I) to obtain 5.4 g of white powder. The powder was dissolved in 0.05 N-aqueous solution of sodium hydroxide to give a final concentration of 1.0 % and ethanol was further added to the solution to give a concentration of 57.5 %. The resulting precipitate was collected by centrifugation, suspended in dis-tilled water and neutralized with dilute hydrochloric acid. Thesuspension was centrifuged and the precipitate was washed well with 70 ~ aqueous ethanol and lyophilized to obtain 2.2 g of white powder (F-V).
To the supernatant obtained upon recovery of the above ethanolic precipitate was added ethanol to give a concentration of 70 % and the mixture was neutralized with hydrochloric acid.
The resulting precipitate was collected by centrifugation, washed with 70 % aqueous ethanol and lyophilized. By the above procedure was obtained 2.3 g of white powder ~F-III). The supernatant remaining after recovery of the first precipitate ~5.4 g) from the hydrolysate mixture contained a substantial amount of partial hydrolysates in a solubilized state. Therefore, this supernatant solution was concentrated and centrifuged to separate the generated precipitate. The precipitate was lyophilized to recover 2.9 g of white powder (F-VII~. The resulting supernatant solution was further fractionated by gel filtration chromatography on a column of Sephadex* G-25 (solvent: 0.1 M
ammonium bicarbonate~ followed by lyophilization of combined fractions. By this procedure was obtained 0.7 g of white powder ; 30 (F-VIII~-*Trade Mark 1t)~44S3 1 (IV~ The same TAK-N (18 g) as that used in (I) was hydrolyzed in 450 ml of 90 % formic acid at 95C for 40 minutes and, to this reaction mixture, essentially the same fractionation pro-cedure as in (III) was applied, such as concentration, pre-cipitation with ethanol, gel filtration, etc. sy the above procedure, four fractions of white powder (F-IV, 2.3 ~, F-VI 2.8 g, etc.) were obtained.
The physiochemical properties of these powders - F-I, F-II, F-IIX, F-IV, F-V, F-VI, F-VII and F-VIII - are shown in Table 2. When six samples having lower DP (F-III, IV, V, VI, VII, VIII) were each dissolved in a 0.02 N-solution of sodium hydroxide and, chromatographed respectively on the same Sephadex****
G-200 column equilibrated with the same solution as above, they were eluted in the order of decreasing DP (from F-III to F-VIII), each sample giving a symmetrical peak, which indicated that each sample had a normal distribution in regard to degree of polymerization.

DP Elemental Purity Glucose[a]D5 analysis(~) (%)** content in 0.1 N NaOH
F-I 299 40.73 6.46 92.3< 0.03 30.6(C=0.35) F-II113 41.71 6.91 98.4< 0.03 22.3(C=0.57) F-III50 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-VII16 42.46 6.99 91.8< 0.03 0.4(C=1.06) F-VIII7 39.84 6.37 94.1< 0.03 0.0(C=0.57) *, ** and ***: see the footnote to Table 1.
****Trade ~ark - 8 -, ~

--`` 109~4~3 1 We have also discovered that the carboxymethylation oE
TAK-N and TAK-D yields new derivatives (CMTAK) in which hydroxyl groups of these glucans are more or less carboxymethylated.
Some aspects of this invention are predicated upon the above finding. Thus, the invention is also directed to ~-l,3-glucan derivatives of the following general formula and their salts.

2 2 ~H2R

~ ~ H ~ 0 ~ ~ ` (I) OR~ ~ ~ J ~1, OR

(wherein at least one of R's is -CH2COOH, with the remainder, if any, being H; n is zero or an i~tegral number; or its salt.
C~qTAK can be obtained by carboxymethylating TAK-N or TAK-D in a known manner, for example by reacting TAK-N or T~K-D
with monochloroacetic acid in the presence of alkali. In addition to this method, any other conventional methods that are used for the carboxymethylation of carbohydrates may be - employed.
To recover CMTAK from the reaction mixture, it is possible to employ conventional methods that are commonly used for the purificatlon of carbohydrates, such as precipitation by the addition of an organic solvent. In addition, such other procedures as ion exchange chromatography, gel filtration, etc.
; may likewise be utilized according to the content of carboxymethyl groups in, or the solubility in water of, CMTAK.

While the CMTAK thus obtained contains carboxymethyl ; 30 groups in its molecule, the content of carboxymethyl groups varies widely depending upon the conditions of the carboxymethyla-_ 9 ~

. . .

- 10944~3 I tion reaction. As determined by titration, the content of carboxymethyl groups of the CMTAK obtained by a normal carboxy-methylation reaction procedure is generally less than 3 carboxy-methyl groups per glucose residue in the CMTAK molecule. CMTAK, as it is suitable for the p~poses of this invention, includes all substances which are obtainable by the carboxymethylation of TAK-N or TAK-D and the molecules of which contain detectable carboxymethyl groups, irrespective of the degree of carboxy-methylation.
As will be apparent from the general formula (I) given hereinbefore as its free acid, CMTAK can react with various bases to form the corresponding salts, such as the sodium, potassium, calcium, aluminum, magnesium and amine salts. In the context of this invention, CMTAK includes not only its free acid but also its salts, particularly those with low toxicity.
Table 3 shows the physical properties of some re-presentative species of CMTAK which we prepared from TAK-N and TAK-D by the procedure described hereinbefore. The determinations of such physical properties were also made after each sample was previously dried under reduced pressure and over phosphorus pentoxide at 60~C for 10 hours.

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-_ 1 ~ - 1094453 1 Figs. 1 and 2 show the I.R~ spectra in potassium bromide of CMTAK No. 2 and No. 11 in Table 3, respectively. Numerals at the topside, the left side and the bottom side of each of the figures indica-te wavelength (~), transmittance (%) and wave number (cm~l), respectively.
The characteristic absorptions were found at 1440-1400 and 891 cm 1 The TAK-N, TAK-D and CMTAK obtained as above, ranging from 2 to about 1,000, preferably from 15 to 800, more preferably from 40 to 600 in DP, possess a significant inhibitory activity against various kinds of tumors in warm-blooded animals including domestic animals, poultry, dogs, cats, rabbits, rats, mice, etc., and particularly against solid tumors which are known to be less sensitive to treatments with generally known cytotoxic antitumor drugs.
For instance, the growth of sarcoma 180, Ehrlich carcinoma, SN-36 leukemia, CCM adenocarcinoma, or NTF reticulum cell sarcoma each transplated subcutaneously into mice was markedly inhibited when TAK-N, T~K-D or CMTAK was administered intraperitoneally, lntraveneously or subcutaneously, before, after or at the time of transplantation, once or repeatedly at dose levels of about 1 to 1,000 mg/kg per administration per day.
The toxicities of TAK-N, TAK-D and CMTA~ are extremely low. For example, no toxic effect was observed both in mice and rats at a dose of 3,000 mg/kg by intraperitoneal route or 10,000 mg/kg by oral route. Thus, these polysaccharides can be safely administered to warm-blooded animals repeatedly.
The administration may be accomplished by general procedures which are normally applied in the management of cancers. Thus intra-tumor, subcutaneous, intramuscular, or ~0~44~
1 intravenous injections, if necessary, oral and rectal administrations, or in external applications, coating, instillation and other methods of administration are feasible.
According to the present invention, TAK-N, TAK-D or CMTAK is administered in a sufficient amount to a warm-blooded animal to inhibit growth of its tumor.
The dosage and procedure of the administration of TAK-N, TAK-D or CMTAK to the warm-blooded animals affected by neoplasias may vary according to the kind of agents ~TAK-N, TAK-D, CMTAK), animals and patients, tumorous symptoms, administration forms and other factors. Generally, the sufficient amount of dosage per administration is about 0.02 to 2,000 mg/kg body weight, preferably 0.2 to 2,000 mg/kg body weight, the preferred upper limit being about 500 mg/kg body weight. In many cases, about 200 mg~kg is the most desirable upper limit.
The administration may be conducted, for example, one to six times per day for consecutive or intermittent days.
The-form of in]ection is varied in each case but can be realized in a per se conventional manner, that is to say, TAK-N, TAK-D or CMTAK is dissolved or suspended in an aqueous liquid medium directly or with a conventional solubilizer such as alcohols (e.g. ethanol, propyleneglycol~, non-ionic surface active agents, physiological saline and isotonic solutions.
In the case of CMTAK, the sample is easily dissolved in distilled water or physiological saline.
For oral preparation, any member or pharmaceutical forms can be used, for example, syrups, elixirs, suspensions or the compound can be processed into wafers, pills, tablets supposi-tories, gel and the like. In a preferred embodiment, the oral dosage form consists of a tablet containing between about 10 and 1 1,000 mg o the above active ingredient per tablet. Such tablets can be coated in the usual fashion, preferably using a readily soluble coating material, for example, sugar, etc.
The above amount can also be incorporated into gelatin capsules which promptly dissolve upon introduction into the stomach.
In any event, the usual flavoring and coloring agents can be used without effect on the active ingredient so incorporated.
Tablets of this type are prepared in the usual fashion by compounding the active ingredient with a binding agent, e.g., starch, sugar and the like, granulating the mixture and, after adding the necessary fillers, flavoring agents, lubricants, etc., the mixture is slugged and passed through a 30-mesh screen.
The thoroughly blended mixture is then compressed into tablets of desired hardness with the usual punch, preferably to make bisected tablets for easier administration.
TAK-N, TAK-D and CMTAK may be administered in combi-nation with other antitumor agents. Combination with other anti-tumor agents leading to the enhanced immunological activity of the tumor-bearing animals is also desirable.
The invention will be further described by way of examples. Some of these examples show the process of production of TAK-D from TAK-N, the process of production of CMTAK, the physical properties of TAK-N, TAK-D and CMTAK, and some of the injectable forms of these agents. Other examples demonstrate the antitumor activities of TAK-N, TAK-D and CMTAK against mouse tumors by way of models.
As demonstrated above, the treatment of tumor-bearing animals with the compounds produces drastic regression of various kinds of tumors such as sarcoma, carcinoma, reticulum cell sarcoma and leukemia. All of these types of tumors in rodents ~L0~ ~453 1 have been known to be reliable models for tumors in other warm-blooded animals.
It should, o~ course, be understood that these examples are by no means limitative of this invention.
~ ost of the following experiments on antitumor activity of TAK-N, TAK-D, and CMTAK were tested by approximately the same methods as those described for antitumor activity of lentinan, a branched glucan from an edible mushroom, by Chihara, G. et al. (See Nature 222, 687-688, 1969, and Cancer Research 30, 2776-2781, 1970).
Mouse tumors used in the experiments such as sarcoma 180 (S180), Ehrlich carcinoma (Ehrlich~, NTF reticulum cell sarcoma (NTF), CCM adenocarcinoma ~CCM) and leukemia SN-36(SN-36) were maintained in ICR-JCL mice, 5 to 7 weeks old, each in an ascites form. A suitable number of these tumor cells was subcutaneously transplanted into ICR-JCL mice, and the tumor nodes which had grown up were excised for weighing on the 35th day after the tumor transplantation.
A TAK-N or TAK-D, was suspended or dissolved in distilled water or in physiological saline, and CMTAK was dissolved in distilled water or in physiological saline in adequate concentrations and at around neutral pH values. Each preparation was administered intraperitoneally, subcutaneously or intra~eneously to mice (0.2 ml/20g body weight) daily for 10 days starting at 24 hours afer the tumor transplantation, daily for 5 days before tumor transplantation, or only once during the 7th day before and the 21st day after the tumor implantation.

Tumor Suppressive Effect of TAK-N (DP 540): Single Injection Four millions of S 180 cells were subcutaneously trans~

~ 15 --- 109 ~S3 1 planted into the leEt in~uinal region oE ICR-JCL female mice,

4 to 5 weeks old. The tumor nodes were excised on the 35th day after the transplantation, and weighed. The average tumor weight of a treated group ¢T) consisting of 5 mice was compared with that of control group (C) consisting of 10 mice, and the tumor inhibition ratio (% Inhibition: (C-T)/C x 100) was calculated. The number of tumor-free animals (complete regression) on the 35th day was also counted.

TAK-N (DP 540) suspended in physiological saline (10 mg/ml) was administered intraperitoneally (0.2 ml/20 g body weight) once to each animal at the dose level of 100 mg/kg on the 7th, 3rd or 1st day before, or the 1st, 2nd, 3rd, 5th, 7th, lOth, 14th, or 21st day after the tumor transplantation, or on the day of tumor transplantation.
As shown in Table 4, TAK-N at the dose level of 100 mg/kg inhibited the growth of S 180 significantly when the test sample was administered only once during the 7th day before and the 21st day after the tumor transplantation.

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

", Dose Day of Average Tumor No. of complete Samp1 injection tumor inhibi- regression/No.
m(g/)g ratlot%) ----- 7.06 ---- 0/10 TAK-N 100 x 1 - 7* 2.27 67.8 3/5 (DP540 100 x 1-3 1.64 76.8 3/5 100 x 1 -1 0 100 5/5 ~100 x 1 0** 0.56 92.1 3/5 ~ 6.94 ---- 0/10 TAK-N 100 x 11*** 1.19 82.9 4/5 100 x 12 0.59 91.5 4/5 (DP540 100 x 1 3 0 100 5/5 100 x 1 5 1.25 82.0 3~5 100 x 1 7 0 100 5/5 ----- 5.08 ---- 0/10 TAK - N (100 x 1 7 0 100 5/5 ~100 x 1 10 0.01 99.8 4/5 (DP540 100 x 1 14 0.01 99.8 4/5 2~100 x 1 21 :1.71 66.3 1/5 _ ----- 5.36 ---- 0/5 TAK - N r20 x 1 7 3.72 30.6 1/5 ~ 60 x 1 7 0.12 97.8 3/5 (DP540)lloo x 1 7 , * TAK-N was administered on the 7th day before the tumor transplantation ** TAK-N was administered on the 1st day after the tumor transplantation *** TAK-N was administered on the day of tumox transplantation - 17 - -., ~ 1094453 1 These results indicate that l'AK-N ~DP 540) is effective in suppressing the tumor growth at all stages of tumor proyression in warm-blooded animals even by a single administration.

Tumor Suppressive Effect of TAK-N (DP 540) and Autoclave-curdled TAK-N (DP 540):

. . _ .
Six millions of S 180 cells were subcutaneously trans-planted into the right inguinal region of ICR-JCL mice with body weights of about 23 g. The tumor nodes were excised on the 35th day after the transplantation, and weighed. The tumor inhibition ratio in a treated group was calculated as in Example 1.
TAK-N (DP 540) and autoclave-curdled TAK-N (DP 540) were used. The latter was obtained by heating a 2 % suspension of TAK-N (DP 540) at 120 for 25 minutes followed by drying and powdering the formed gel. The samples were suspended in dis-tilled water at desired concentrations, and each suspension was administered intraperitoneally to a group of 5 mice daily for 10 days starting at 24 hours after the tumor transplantation.
As shown in Table 5, TAK-N ~DP 540) showed a pronounced effect on the growth of tumors at the dose level from 1 to 50 mg/kg, the effect being particularly significant at the dose level from 5 to 25 mg/kg.
The autoclaved TAK-N (DP 540)was also effective in tumor growth suppression at the dose level of 10 mg/kg. There was no observed toxicity.

lo~ ^C~3 .
Dose Average Tumor No. of complete Sample mg/~g tumor inhibition regression/No.
(ip) weight ratio (%) of treated mice - 6.00 ----- 0/6 1 x 10 4.80 20.0 0/6 3 x 10 3.58 40.3 1/6

5 x 10 0.02 99.7 5/6 7 x 10 0.73 87.8 5/6 10 x 10 0.01 99.8 5/6 TAK-N
15 x 10 0.10 98.3 4/5 (DP 540) 20 x 10 0 100 5/5 25 x 10 0.42 93.0 3/6 50 x 10 1.19 80.2 1/6 TAK-N ----- 3.67 ----- 0/6 (DP 540) 10 x 10 0.29 92.1 5/6 * Autoclave-treated These results indicate that TAK-N is useful in tumor retardation at relatively low dose levels and that the sample can be sterilized by autoclaving without loss of its activity.

Tumor Inhibitory Activity of Various Kinds _f TAK-N and TAK-D
.
From 24 hours after the transplantation of S 180 as described in Example 2, various kinds of TAK-N and TAK-D suspended in distilled water were administered in-traperitoneally at the dose level of 10 mg/kg to a group of 5 mice daily for 10 days. The tumor nodes were excised on the 35th day after the tumor transplantation, and weighed. The inhibition ratios were cal-culated as in Example 1.

- ~0~44S3 1 As shown in Table 6, all samples used exhibited an inhibitory activity against the tumor growth. Especially, those samples having DP over 50 showed a strong antitumor activity with many complete regression.

Dose Average Tumor No. of complete Sample mg/kg tumor inhibition regression/No.
(ip) weight(g) ratio(~) of treated mice ~ 5.46 ---- 0/6 (DP 565) lOxlO 0.07 98.7 5/6 (DP 540) " 0.20 96.3 3/5 TAK-N (DP 380) 0.46 91.6 4/6 (DP 125) " 0 100 6/6 5.46 ---- 0/6 S-II
(DP 82) lOxlO 0 100 6/6 S-III
(DP 68) " 0.03 99.5 5/6 TAK-D F-III
(DP 50) " 0 100 6/6 F-V
(DP 39) " 2.03 62.8 1/6 F-VI
(DP 24) " 3.60 34.1 0/6 F-VII
(DP 16) ~ 1.54 71.8 1/6 .-These results indicate that both TAK-N and TAK-D

are effective in tumor growth regression irrespective of their DP values.

Tumor Suppressive Effects of CMTAK and Autoclave-treated CMTAK

The antitumor activity of three kinds of CMTAK prepared from TAK-N (CMTAK Nos. 1, 4, 5) and of autoclaved CMTAK

~', .

10~ 4~;3 1 was assayed as described in Example 2. Samples were dissolved in distilled water for injection, and the pH was adjusted to around 7Ø In some experiments one of these samples (CMTAK No. 1) was sterilized by autoclaving (120C, 25 minutes) before further administration.
As shown in Table 7, all CMTAK samples at the dose level of 10 mg/kg inhibited significantly the tumor growth.
These growth inhibition by CMTAK was observed at dose levels from 1 to 40 mg/kg.
TAB~E 7 Dose Average Tumor No. of complete Sample mg/kg. tumor inhibition regression/No.
~ip) weight(g) ratio(%) of treated mice _--- 2.87 CMTAK No. 1lOx9 0 100 7/7 No. 4 lOx9 0.28 90.2 4/7 No. 5 lOx9 0.01 99.7 6/7 ---- 3.65 ---- 0/6 ~lxlO 0.77 78.9 1/6 Autoclaved CMTAK No. 13xlO 0.03 99.2 5/6 5xlO 0.23 93.7 4/6 ____ 2.17 ~~~~ 0!6 5xlO 0 100 6/6 I lOxlO 0.07 96.8 5/6 CMTAK No. 1~ 0.08 96.3 5/6 20xlO 0.21 90.3 3/6 l40xlO 0.18 91.7 1/6 These results indicate that CMTAK can be sterilized without loss of its antitumor activity and that they are useful in an injectable form for cancer treatment.

, ~

10~4453 1 EX~MPLE 5 Tumor Suppressive Effects of TAK-N, TAK-D and C~lTAK by various Routes of Administration TAK-N (DP 540), TAK-D (F-III: DP 50), TAK-D tF-VII:
DP 16), and CMTAK No. 1 were administered at the dose level of 5 mg/kg intravenously, subcutaneously or intraperitoneally for 10 consecutive days to ICR-JCL mice which were previously transplanted with 4 x 106 cells at 24 hours before the first injection. The inhibition ratio of tumor growth were calculated as in Example 1.
As shown in Table 8, in each case, the significant inhibition of tumor growth was observed. By intravenous adminis-tration, TAK-N, CMTAK, and even F-VII (DP 16) inhibited the tumor growth almost completely, the inhibition ratio being over 85 `% with the complete tumor regression in 4 or 5 out of 5 mice treated.

Dose Route of Average Tumor No. of com-Sample(mg/kg) administra- tumor inhibi- plete re-tion weight(g~ tion gression/No.
ratio(%) of treated mice ----- 7.30 ----- 0/10 CMTAKJ 5 x 13 iv 0.8487.5 4/5 No. 1~ 5 x 10 sc 3.7748.4 0/5 ~5 x 10 ip 0.9188.5 2/5 ------ 7.30 ----- 0/10 TAK-N~5 x 10 iv 0 100 5/5 (~P 540)~ 5 x 10 sc 2.72 63 3/5 ~5 x 10 ip 0.17 98 4/5 TAK-D~5 x 10 iv 3.02 59 2/5 F-III~ 5 x 10 sc 0.91 88 3/5 (DP 50)~5 x 10 ip 2~3a 68 3~5 30 TAK-D(5 x 10 iv 1.06 85 4/5 (DP 16)~ 5 x 10 sc 2.67 63 3/5 5 x 10 ip 2.34 68 2/5 , . - 22 -`` 10~44~i3 .

1 These results indicate that TAK-N, CMTAK and TAK-D
are e~fective in tumor suppression by any route of administration.

Tumor Su ressive Effects of TAK-N, TAK-D and CM~AK Administered P P ~
Before Tumor Transplantation : Pre-treatment TAK-N, TAK-D having different average degrees of po~y-merization and CMTAK No. 1 were administered intraperitoneally to ICR-JCL mice with body weights of about 23 g at dose level from 3 mg/kg to 80 mg/kg daily for 5 consecutive days. TAK-N
and TAK-D were suspencled, and CMTAK was dissolved, in distilled water. In the case of CMTAK the final pH was adjusted to around 7Ø Then, on the 1st or 3rd day after the last administration, 6 x 106 cells of S180 were transplanted sub-cutaneously into the right inguinal region of each animal. On the 35th day after the transplantation, the tumor nodes were excised and weighed. Tumor inhibition ratios were calculated as in Example 1. -As shown in Table 9, a marked tumor growth inhibition was observed by each sample. Especially TAK-N (DP 540), CMTAK and TAK-D having DP over 50 exhibited a~strong inhibitory activity.
ClMTAK was effective for tumor retardation at dose level as low as 3 mg/kg.

. .

. _ . ... _ .
Dose Day of Average Tumor No. of complete Sample mg/kg injection tumor inhibition regression/No.
(ip) _ _ weight(g) ratio(~) 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 ~--V
(DP 39)20x5 -5 to -1 5.52 45.3 0/6 F-VI
(DP 24)20xS -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 ~ 5x5 -5 to -1 3.55 44.8 1/6 CMTAK No.l lOx5 -5 to -1 0.19 97.1 3/6 20 20x5 -5 to -1 0.05 99.2 4/6 . .
5.03 ----- 0/5 CMTAK ~o 1~4X5 -5 to -1 0.11 97.8 4j6 ~80x5 -5 to -1 0.15 97.0 4/6 -... . . ~
7.45 ----- 0/5 TAK-N
(DP 540) 20x5 -7 to -3 0.18 97.6 3/5 TAK-D
S-III 20x5 -7 to -3 0.75 89.9 4/5 (DP 683 These results indicate that the polysaccharides mentioned above are useful as prophylactics in cancer treatment.

10~ ~453 Tumor suppressive Activity o~ TAK-N and CMTAK against Various Kinds of Tumors Ehrlich carcinoma, CCM adenocarcinoma, NTF reticulwm cell sarcoma and SN-36 leukemia were used to examine the usefulness of TAK-N and CMTAK for the retardation of various kinds of tumors.
ICR-JCL mice with an average body weight of 23 g were transplanted subcutaneously with 3.1 x 106 Ehrlich cells, 1.1 x 107 CCM cells, 4.5 x 106 NTF cells or 7.2 x 104 SN-36 cells and were administered intraperitoneally with TAK-N (DP 540) suspended in distilled water or CMTAK No. 1 dissolved in distilled water (pH 7.0) and autoclaved, at dose levels from 10 to 50 mg/kg daily for 10 days starting at 24 hours after the transplantation. The tumors were excised and weighed 35 days after the transplantation and the inhibition ratios of tumors in the treated groups were calculated as in Example 1.
As shown in Table 10, in each case, a significant ~0 inhibition of tumor growth was observed.

~0~44~;3 . _ _ _ _ _ Tumor Sample Dose Average Tumor No. of complete mg/kg tumor inhibition regression/No.
_ _ _ (ip) weight(g)ratio(%) of treated mice --- 3.54 --- 0/5 TAK-N ~lOxlO 2.37 33.1 0/5 Ehrlich(DP 540)~20xlO 0.50 85.9 , 2/6 carcinoma 40xlO 0.93 73 7 0~5 _____________________________ _____________ ____________ --- 1.93 --- 1/6 CMTAK J20xlo 0.80 58.5 3/6 No. 1 ~40xlO 0.60 68.9 2/6 TAK-N --- 3.42 --- 0/6 CCM adeno- (DP 540)20xlO 0.79 76.9 2/6 __________________________________________ ____________ carcinoma CMT~K 3.42 -__ 1/6 No. 1 20xlO 1.03 69.9 1/6 --- 0.91 --- 2/6 Leukemia TAK-N flOxlo 0.18 80.2 4/5 SN-36(DP 540)J25xlO 0.19 79.1 3/5 ~50xlO 0.23 74.7 2/6 .. _ _ . ~ . ...
NTFTAK-N --- 7.64 --- 0/6 reticulum (DP 540)~10xlO 0.38 95.0 2/6 sarcoma~OxlO 2.21 71.1 0/6 __ .. . ...... . _ ~ _ . .

The results indicate that TAK-N and CMTAK have an antitumor activity against several types of tumors, i.e.
sarcoma, carcinoma, adenocarcinoma, leukemia and so forth.

The four preparations of CMTAK (CMTAK No. 2, 6, 9 and 11) were each dissolved in physiological saline and, according to the procedure of Example 4, their effects of S180 tumor were examined. All samples, at the daily dose level of 5 mg/kg per ~, -10~4~53 1 administration for 10 days starting at 2~ hours after the tumor transplantation, significantly suppressed the tumor growth.

EXAMPLE_9 TAK-N (replaceable by TAK-D or CMT~K)150 mg Lactose 48 mg Magnesium stearate 2 mg Total200 mg The above amount makes up one capsule.
TAK-N (TAK-D or CMTAK) and lactose are mixed in the above-indicated proportions, tableted and pulverized. Then, magnesium stearate is added. The mixture is dispensed into capsules.

.. .. . _ _ TAK-N (replaceable by TAK-D or CMTAK 400 mg Lactose 95 mg HPC-L (hydroxypropyl cellulose) 5 mg Total 500 mg The above amount makes up a single dose.
The above three ingredients are mixed together in the indicated proportions and with the addition of a small amount of water, the mixture is kneaded in a kneader, granulated, dried, regranulated, size-selected and packaged in doses indicated above.

. .
(a) One gram of TAK-D (DP 16 ) is dissolved in 1,000 ml of distilled water for injection (or physiological saline). The solution is ~iltered and the filtrate is distributed into ampoules in 500 ml portions. After sealing, the ampoules are hea~-sterilized in a routine manner.

~0~9 ~453 (b) Two gram of CMTAK (the product of Example 14 is dissolvedin lO0 ml of distilled water for injection (or physiological saline) and the solution is filtered. The filtrate is dis-tributed into ampoules in 20 ml portions and, after sealing by fusion, the ampoules are heat-sterilized in a routine manner.

TAK-N (replaceable by TAK-D or CMTAK) 160 mg Sorbitol 200 mg Sodium carboxymethylcellulose 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 make a total volume of 4 ml. ~When CMTAK is employed, the solution is neutralized, if necessary, with N/10 - sodium hydroxide) In 80 ml of isopropyl alcohol was suspended 3 g of TAK-N (DP 5~0) and the suspension was stirred at room temperature for 30 minutes. Then, 8 ml of a 30~ solution of sodium hydroxide was slowly added with stirring over a period of about 60 minutes.
The mixture was further stirred vigorously at room temperature for about 90 minutes for the purpose of preventing the formation of a gel. Then, 3.6 g of monochloroacetic acid was added, and the mixture was stirred at 60 - 70C for 5 hours to allow the carboxymethylation to proceed. The product was recovered by filtration and thoroughly washed with a mixture of methanol and acetic acid (7 : 3, v/v). The precipitate was collected by filtration, washed well with 80 ~ aqueous methanol, methanol and 1094~3 1 acetone in the order mentioned and dried under reduced pressure.
By the above procedure was obtained 2.9 g of CMTAK. Car~oxymethyl content (the number of carboxymethyl group per glucose residue;
the same definition also applies hereinafter): 0.54.

EX~PLE 14 In 40 ml of isopropyl alcohol was suspended 1.5 g of TAK-N (DP 540) and the suspension was stirred at ro3m temperature for 30 minutes. Then, under stirring, 2 ml of a 30 % solution of sodium hydroxide was added in four installments, i.e. 0.5 ml each at intervals of 15 minutes. The mixture was further stirred at room temperature for 90 minutes. Then, 0.9 y of monochloroacetic acid was added in three installments, i.e. 0.3 g each at intervals of 10 minutes. The carboxymethylation was thus conducted at 50C with stirring for 150 minutes. The product was collected by centrifugation, dissolved in 50 ml of water and neutralized with acetic acid. To this neutral solution was added 120 ml of methanol and the resulting precipitate was collected by centrifugation. The precipitate was washed with a mixture of 300 ml of 80 % aqueous methanol and 100 ml of :20 '' ethanol and, then, with a mixture of 300 ml of 80 % aqueous methanol and 200 ml of ether. The product was finally lyophili2ed to recover 1.7 g of CMTAK. Carboxymethyl content :
0.75.

. .
In 40 ml of isopropyl alcohol was suspended 1.5 g of TAK-N (DP 540) and the suspension was stirred at room temperature for 30 minutes. Then, 4 ml of a 30% solution of sodium hydroxide was added in 4 installments, 1 ml each at intervals of 15 minutes, followed by stirring at room temperature for 90 minutes. Thereafter, 1.8 g of monochloroacetic acid was added 109~453 1 in 3 installments, i.e. 0.6 g each at intervals of 10 minutes.
The mixture was stirred at 50C for 150 minutes, whereby the carboxymethylation was allowed to proceed. The praduct was collected by centrifugation, dissolved in 40 ml of water and neutralized with acetic acid. To this neutral solution was added 90 ml of methanol and the resulting precipitate was collected by centrifugation. The precipitate was washed well with a mixture of 200 ml of 80 % aqueous methanol and 100 ml of ethanol and, then, with a mixture of 200 ml of 80 % aqueous ethanol and 200 ml of ether. It was then lyophilized to recover 2.0 g of CMTAK. Carboxymethyl content: 1.07.

.
In 33 ml of water was suspended 3.2 g of TAK-N (DP 255) and, under stirring at room teperature, 1 g of sodium hydroxide was added, followed by addition of 2.4 g of sodium monochloro-acetate. The carboxymethylation reaction was thus conducted at room temperature with constant stirring for 2 hours. There-after, 1 g of sodium hydroxide and 2.4 g of sodium monochloro-acetate were added again and the reaction was further conductedat room temperature with stirring for 3 hours. Then, 1 g of sodium hydroxide and 2.4 g of sodium monochloroacetate were further added. The reaction was continued at room temperature with stirring for an additional 2 hours. To the reaction mixture was added 1 liter of ethanol and the resulting precipitate was washed well with ethanol on a glass filter until the filtrate ceased to give a red color with phenolphthalein. It was then dried at 50C and under reduced pressure. The resulting powder (3.7 g~ was dissolved in 90 ml of water and neutralized with acetic acid, followed by addition of 210 ml of ethanol. The resulting precipitate was collected by centrifugation, washed 109~4S3 with 80 % aqueous ethanol and lyophilized. By the above procedure was obtained 2.6 g of CMTAK. Carboxymethyl content:
0.30.
EX~MPLE 1 7 In 66 ml of water was suspended 6.4 g of TAK-N (DP Z55) and, under ice-cooling and stirring, 4 g of sodium hydroxide and, then, 9.6 g of sodium monochloroacetate were added. The carboxymethylatlon was conducted under ice-cooling and stirring for 2 hours. Then, 4 g of sodium hydroxide and 9.6 g of sodium monochloroacetate were added again and the mixture was stirred under ice-cooling for 3 hours. Thereafter, 4 g of sodium hydroxide and 9.6 g of sodium monochloroacetate were further added and the reaction was continued under ice-cooling and stirring for 3 hours. To the reaction mixture was added 1 liter of ethanol and the resulting precipitate was washed well with ethanol on a glass filter until the filtrate ceased to give a red color with phenolphthalein, followed by drying at 50C
and under reduced pressure. The resulting powder (~.6 g) was dissolved in 172 ml of water and neutralized with acetic acid.
Then, following the addition of 480 ml of ethanol, the pre-cipitate was collected by centrifugation, washed with 80 %
aqueous ethanol and lyophilized. By the above procedure was obtained 5.6 g of CMTAK. Carboxymethyl content: 0.36.
EXAMPLE_18 In 40 ml of isop~opyl alcohol was suspended 1.5 g of TAK-D (F-I, DP 299) and the carboxymethylation was conducted as in Example 14. The product was washed as in Example 14 to obtain 1.9 g of CMTAK. Carboxymethyl content: 0.59.
The same TAK-D as above was suspended in 40 ml of isopropyl 10~ ;3 1 alcohol and, by the same procedure as that described in Example 15, the carboxymethylation was carried out. The reaction product was washed as in Example 15 to recover 2.2 g of CMTAK.
Carboxymethyl content: 1.15.

. .
In 40 ml of isopropyl alcohol was suspended 1.5 g of each of TAK-D (F-II, DP 113) and TAK-D (S-III, DP 68), respectively. And, as in Example 14, the carboxymethylation was carried out. The respective reaction products were collected by centrifugation, dissolved in 40 ml of water and neutralized with acetic acid. To each of these two neutral solutions was added 90 ml of methanol and the resulting precipitate was collected by centrifugation and washed well first with 200 ml of 80 % aqueous methanol and then with 200 ml of aqueous ethanol, followed by lyophilizing. By the above procedure were obtained two species of CMTAK.

Carboxymethyl Yield content CMTAK (obtained from F-II 1.4 g 0.51 " ~ " from S-III) 1.4 g 0.36 In 40 ml of isopropyl alcohol was suspended 1.5 g of each of TAK-D (F-II, DP 113~ and TAK-D (S-III, DP 68), res-pectively and, as in Example 15, the carboxymethylation was carried out. The reaction products were washed respectively as in Example 19 to recover two species of CMTAK.

Carboxymethyl Yield content 30CMTAK (obtained from F-II) 2.3 g 1.22 " ( " from S-III) 1.4 g 0.45 .

Claims (4)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. An antitumor agent which contains a member selected from the group consisting of .beta.-1,3-glucan being water-insoluble and thermogelable in the presence of water at an average degree of polymerization of 170, a lower polymer obtainable upon partial hydrolysis of said glucan, and a carboxymethylated derivative of said glucan and of said lower polymer and a suitable vehicle.

2. An antitumor agent as claimed in Claim 1 wherein the average degree of polymerization of said .beta.-1,3-glucan and said lower polymer lies be-tween 5 and 1,000 as determined by the method of Manners et al described in Carbohydrate Research 17, 109(1971) and said carboxymethylated derivative is obtainable from the above-mentioned glucan and lower polymer.

3. An antitumor agent as claimed in Claim 1 wherein the average degree of polymerization of said .beta.-1,3-glucan and said lower polymer lies between 15 and 800 as determined by the method of Manners et al described in Carbohydrate Research 17, 109 (1971) and said carboxymethylated dexivative is obtainable from the above-mentioned glucan and lower polymer.

4. An antitumor agent as claimed in Claim 1 wherein the average degree of polymerization of said .beta.-1,3-glucan and said lower polymer lies between 40 and 600 as determined by the method of Manners et al described in Carbohydrate Research 17, 109 (1971) and said carboxymethylated derivative is obtainable from the above-mentioned glucan and lower polymer.