DE3019614A1 - Hydrolysed glucan with immunosuppressive activity - for use in treating cancer and arthritis - Google Patents

Abstract

Hydrolysed glucan (I) with beta(1-3) bonds in the main chain and beta(1 6) bonds in the side-chains in which the ratio of the glucose gps in the side-chains to those in the main chain is about 2 to 7. (I) is prepd. by fermentation of Corticiaceae, treating the polysaccharide formed with formic acid and then hydrolysing the prod. The glucan. freeze dried prod. is a white amorphous solid, soluble in water, DMSO and insol. in ethanol, acetone, ethyl acetate, benzene and ether. Its analysis supports the formula (C6H10O5)n in H2O where n is >800. The molecular mess is calc. to be 150,000 to 160,000.

Description

DESCRIPTION

The invention relates to a new hydrolyzed glucan, a method preparations suitable for its manufacture and for pharmaceutical purposes which contain this hydrolyzed glucan. The glucan of the present invention is a valuable one immunomodulating agent for the treatment of cancer and rheumatoid arthritis.

It is well known that fungi of the Basidiomycetes class are polysaccharides that have activity against cancers and other tumors. These However, polysaccharides are only slightly soluble in water and even if it is it is possible to prepare an aqueous solution from such polysaccharides, gelatinized this solution easily. As a result, it is very difficult to conduct pharmacological experiments carry out with these polysaccharides and encounter even greater difficulties when trying to use these polysaccharides therapeutically.

Rheumatoid arthritis is an incurable disease and although numerous Medicines have been studied to cure the disease, attempts were made not successful. Known treatments, such as treatment with steroids, steroid-free anti-inflammatory agents, gold-containing drugs, etc. Symptomatic treatments in all cases, but not causal treatments.

Japanese Patent 828,248 discloses a method of manufacturing of a polysaccharide with anti-tumor activity in which a glucan-producing Microorganism of the genus Corticium or of the genus Hypnochus is bred and then a glucan is separated from the culture broth, which in the Main chain has ß- (1- + 3) -bonds and in the side chains has p- (1-> 6) -bonds. However, as explained above, although the glucan formed has antitumor activity, However, it is difficult to apply in practice because of its aqueous solution tends to gel.

According to the invention it has now surprisingly been found that by Treatment of glucans of this type with formic acid and subsequent hydrolysis of the product formed is a glucan with significantly improved solubility in water can be produced and that, moreover, the glucan thus obtained in unexpected Way shows a very much improved anti-tumor activity.

The invention thus relates to a glucan that has 3- (1 p 3) bonds in the main chain and p (1 - + 6) bonds in the side chains, the Ratio of glucose units in the side chains to glucose units in the main chain is about 2: 7, which is characterized by the following properties: (a) In In the form of the freeze-dried product, it is a white, amorphous solid; (b) it is soluble in water, dimethyl sulfoxide and in dimethylformamide and is insoluble in ethanol, acetone, ethyl acetate, benzene and diethyl ether; (c) its elemental analysis corresponds essentially to the values for a polysaccharide of the hexose type with bound water can be calculated using the following formula: (C6H1005) n-nH20 s in the n is a number calculated from property (e) greater than 800; (d) its infrared spectrum (KBr powder) corresponds to the spectrum shown in the attached drawing and (e) in the ultracentrifugal analytical distribution curve only a single peak observed and its calculated from the sedimentation constant Molecular weight is 150,000 to 160,000.

This glucan can be obtained by treating a polysaccharide, that is produced by a fungus of the Corticiaceae family, with formic acid and Hydrolysis of the product so treated.

The invention also relates to a method for production of the hydrolyzed glucan according to the invention by treating a polysaccharide, formed by a fungus of the Corticiaceae family with formic acid and subsequent hydrolysis of the product so treated.

The invention also relates to a medicament which hydrolyzed glucan according to the invention in admixture with a pharmaceutically suitable one Contains carrier or thinning agent.

That used as the starting material for the process according to the invention Polysaccharide is a metabolic product of a funug of the Corticiaceae family the Basidiomycetes, for example a fungus of the genus Corticium or Hypnochus. This starting polysaccharide can be produced by using the chosen fungus is grown as described in more detail in Japanese Patent Publication No. 828,248 is.

The hydrolyzed glucan according to the invention is preferably produced adding 70 to 90 percent by weight aqueous formic acid to the starting polysaccharide is added and the mixture obtained is preferably heated with stirring. The temperature to which the reaction mixture is heated is not particularly critical, although a temperature of 800 to 1000C is preferred. The one for that reaction required time depends on the reaction conditions, especially the temperature, away; however, the reaction will normally proceed within a period of 5 minutes up to 1 hour completely.

The excess formic acid is then preferably removed by evaporation removed under reduced pressure. Then water is added to the residue to hydrolyze. The hydrolysis is preferably carried out by heating the mixture reflux until deformylation is complete. The formed gelatinous Material can then be removed by centrifugation and becomes the mother liquor an organic solvent was added to precipitate a precipitate, which then is won. The type of solvent used in this stage is subject no particular limitation provided that it is the hydrolyzed polysaccharide does not solve. Preferably an alcohol such as methanol or ethanol is used. The resulting precipitate is then dissolved or dispersed in water and the solution or dispersion is freeze-dried, with the desired hydrolyzed polysaccharide is obtained in the form of a white, amorphous solid.

This amorphous solid is in water, dimethyl sulfoxide and in dimethylformamide soluble and is insoluble in ethanol, acetone, ethyl acetate, benzene and diethyl ether. It is also heat-resistant.

The data of his elemental analysis correspond to the data for a Polysaccharide of the hexoside type containing bound water of the formula (CgH1005),. LiH20 can be calculated.

The infrared spectrum of the product (in potassium bromide powder) is in Fig. 1 shown.

The connection has a specific rotation 20 7 = -15 ° + O (c = 0.1, Dimethyl sulfoxide).

D In the Anaaltytic ultracentrifugal distribution (ultra-centrifugal spectrum) of the product, only a single peak was observed and the sedimentation constant became a molecular weight in the region of 150,000 to 160,000 obtained, from which the value n in the formula given above becomes 833 to 889 could be calculated.

The saccharides making up the hydrolyzed glucan according to the invention can be identified in the following ways. The hydrolyzed glucan is in dissolved in aqueous sulfuric acid and then for 120 minutes at 1000C in a further hydrolyzed closed tube. Then the product is made with barium hydroxide neutralized and subjected to paper chromatography. By developing the paper chromatogram with an aqueous-ammoniacal silver nitrate solution, only a single stain is left observed. The product characterized by this single spot can pass through Comparison with repetition of the experiment using a standard sample identified as glucose. The types of binding of the glucose units that make the hydrolyzed glucans of the invention can be determined by the Glucah is completely methylated using conventional methods, the methylated Glucan is hydrolyzed to form methylated glucose and the methylated glucose reduced and the reduction product is acetylated. Is by gas chromatography the mixture thus formed as acetates of 2,3, -4, 6-tetra-0-methylglycitol, 2,4, 6-tri-0-methylglycitol and 2,4-di-0-methylglycitol in a molar ratio of about 1: 2.5: 1 identified. This result confirms that the glucan of the present invention was about two Side chains (each from a single glucose unit) bound with (1> 6) bonds to 7 glucose units each, with (1 o 3) bonds, in the main glucan chain.

By examining the decomposition products produced with the help of Endo-p-1,3-glucanase and exo-β-1,3-glucanase were obtained, it was also confirmed that all glucose bonds templates in the p configuration.

The hydrolyzed glucan according to the invention is more soluble in water and is less subject to gelation in aqueous media Solution, than the polysaccharide from which it was made.

In addition, its immunomodulatory activity (e.g. the Anti-cancer activity and the inhibitory effect against arthritis) very strong and of lower Accompanied toxicity. In particular, the strong arthritis inhibition activity and the low toxicity were in view of the corresponding properties or

Activities of polysaccharides produced directly by fungi of the class Basidiomycetes were generated not to be expected. The hydrolyzed according to the invention Glucan is therefore valuable in the treatment of diseases caused by disorders of the Immune system, e.g. rheumatoid arthritis.

The biological activity and toxicity of the hydrolyzed according to the invention Glucans are shown by the tests below.

(1) Anti-neoplastic activity These trials were for 7 weeks old male mice of the ICR strain. 2 x 106 cancer cells from sarcoma 180 were grafted onto the axillary skin of each mouse, 6 and 7 days after grafting, respectively a sample of the hydrolyzed glucan according to the invention (prepared according to the manner described in Example 1 below) in sterilized physiological Saline solution administered by injection into the abdominal cavity.

The concentration of the hydrolyzed glucan in the saline solution was 0.1% (weight / volume). 25 days after grafting, the diameter of the Tumor measured. The experiment was performed using a control group of mice repeated to which no hydrolyzed glucan was administered. The tumor inhibition ratio (%)) was calculated using the following formula: d d x x 100 do where d the mean tumor diameter (mm) of the Control group and d is the mean tumor diameter of the treated group. The results are listed in Table 1 below, showing the effects of varying Amounts of the hydrolyzed glucan according to the invention administered per day to the Shows tumor inhibition ratio.

45 days after grafting, all treated mice became careful studied to determine the number of mice undergoing full regression of the tumor showed. The results of this study are also shown in the table 1, in which the number of mice showing complete regression is shown in Relation to the total number of examined mice in the respective group is given is.

Table 1 Amount of Tumor Inhibition Complete Administered (mg / kg body weight / day ratio (96) regression 0.1 47 1/5 1.0 85 3/4 10.0 100 5/5 (2) Arthritis Inhibitory Activity These experiments were carried out according to the procedure of Winder et al (CV Winder, LA Lembke and MoD. Stephens, Arth. Rheum., 12, 472 (1969)) to measure the ability of compounds to suppress AdJuvans-induced Arthritis carried out by rats. This test is commonly used to test antineoplastic agents. Lewis rats received a subcutaneous injection of the Adjuvant into the sole surface of each right hind paw to cause the disease. A solution of the hydrolyzed glucan of the present invention was injected into the abdominal cavity daily for 15 days after the adjuvant inoculation. The experiments were repeated with a control group of animals to which the hydrolyzed glucan was not administered. On the 18th, 20th, 24th, 28th and 32nd

The day after inoculating the adjuvant, the feet were swollen of the treated rats with the swelling of the feet of the untreated rats compared and using a formula similar to that used to calculate the The formula used was the inhibition ratio for the tumor inhibition ratio each dose of the hydrolyzed glucan is calculated. The results are in the form of the Average values are listed in Table 2 below.

Table 2 Amount administered ID50 inhibition number (mg / kg body weight / day animals ratio 0.1 5 36.8 + 3.6 0.33 1.0 10 59.0 + 3.9 (3) Acute toxicity An acute toxicity test (observation for one week) was carried out by injecting samples of the hydrolyzed glucan into the abdominal cavity of male ddy mice. After the administration of the samples at 100 and 300 mg / kg, respectively, no deaths were observed and the body weight increased normally.

From the results discussed above, it can be seen that the hydrolyzed glucan of the present invention has considerable value as an immunomodulatory Has means. The agent is preferably administered parenterally, for example by subcutaneous injection, intravenous injection or intramuscular injection.

The daily dose varies depending on the disease to be treated, the route of administration and the frequency of administration; generally amounts to However, the daily dose for an adult is 0.5 to 50 mg, for example about 5 mg. This amount can be administered as a single dose or in divided doses.

The hydrolyzed glucan of the present invention can be prepared in a form appropriate for the route of administration chosen, any Of the preparations that are normally used for other immunomodulatory effective means are used. For example, an agent in an ampoule be provided in the amount of a unit dose or it can be in a container for several dose units, preferably together with an antiseptically active Substance, to be provided.

The agent can be in the form of a suspension, a solution or a Emulsion can be used in an oily or aqueous carrier medium customary additives, for example suspension auxiliaries and / or stabilizers, if appropriate and / or dispersants.

According to another embodiment, the active substance can be in the form a powder is present, which before administration in a suitable carrier, for example, is dissolved in sterile, pyrogen-free water.

If the agent according to the invention is provided in the form of a dose unit it preferably contains 0.5 to 10 mg of the active substance per dose unit.

The invention will be further elucidated by the following examples explained. Example 1 shows the production of the inventive hydrolyzed Glucans and Example 2 the preparation of a pharmaceutical agent which Contains hydrolyzed glucan.

Example 1 To 3 g of Corticane (a polysaccharide produced according to the following Preparation example was obtained) 240 ml of a 90 percent (weight / weight) added aqueous formic acid and the mixture was stirred for 20 minutes 95 ° C heated. After evaporation of the formic acid under reduced pressure were 600 ml of water was added to the residue and the resulting aqueous mixture was added heated to reflux. The reaction mixture was then at 10,000 for 15 minutes Centrifuged rpm. 2.4 l were added to the supernatant liquid separated off in this way Ethanol was added, forming a precipitate, which was then centrifuged was won. To the precipitate was added 500 ml of water and then became the aqueous mixture freeze-dried, with 2.0 g of the desired hydrolyzed Glucans were obtained in the form of a white amorphous solid. The product had following properties: Elemental analysis calculated for (C6HlnO,) .nH20: C: 40.00 %; H: 6.71%; Water: 10.00%.

Found: C: 40.22%; H: 6.52%; Water: 9.63%.

Specific rotation: t 20 - 150 (c = 0.1, dimethyl sulfoxide).

Infrared absorption spectrum (KBr) vcm-1: 3400, 1640, 1080, 1040.

Molecular weight (analytical ultra-centrifugal distribution, phosphate buffer, pH 6.5): 159,000.

Example 2 5 mg of the hydrolyzed glucan obtained in Example 1 were dissolved in 2 ml of physiological saline and the solution was then converted into Usually sterilized by heating to obtain an injectable solution became.

Preparation of the starting glucan In five 500 ml Sakaguchi flasks were placed In each case 100 ml of a glucose-potato medium (with a content of 2% by weight glucose in a boiled broth containing 200 g / l potatoes) and the contents of the flask was sterilized.

A culture of Corticium vagum -31-9 (FERM No. 302) was then inoculated and the shake culture was carried out at 260C for 8 days. After the termination in the cultivation, 800 ml of distilled water was added to 400 ml of the culture broth and the pH of the mixture was adjusted to 7.4. The mixture was then homogenized in a homogenizer and the solid substance was through Centrifugal separation removed.

To 1 1 of the supernatant liquid 3 1 of ethanol were added and the precipitate formed was collected by centrifugation and distilled Dissolved in water. Then 162 ml of a 0.1 M aqueous solution of cetyltrimethylammonium bromide and 13 ml of a 0.5 M aqueous solution of sodium hydroxide added to adjust the pH adjust the solution to 12.6 so that the polysaccharides were precipitated. 300 ml of a 10% (w / v) aqueous acetic acid was added to the precipitate given to dissolve it, after which 1.2 liters of ethanol were added to the solution. Of the formed precipitate was dissolved in 350 ml of distilled water and the solution was filled into a cellulose tube and against distilled water dialyzed. 2 liters of ethanol were added to 500 ml of the dialysate in order to restore the polysaccharides to fail. The formed precipitate was dissolved in 100 ml of distilled water and then freeze-dried to obtain 2 g of a crude glucan.

13.5 mg of the crude glucan thus obtained was distilled in 27 ml Water and the solution was passed through a Sephadex G "200 (trademark) filled column. The column was then eluted with distilled water and the eluate obtained in 20 ml fractions. The glucan showed a peak in the third Fraction and in fractions 2 through 11 were 75.3% of the total Polysaccharide obtained. Fractions 2-11 were combined and the one obtained Solution was added to 800 ml of ethanol to cause precipitation to occur.

The resulting precipitate was dissolved in distilled water and then freeze-dried, taking 9.2 mg of a glucan known as "Corticane" 0 Corticane is a white neutral substance.

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Claims (7)

Hydrolyzed Polysaccharide PATENT CLAIMS Hydrolyzed glucan with p- (1 ß- (1-> 3) bonds in the main chain and I3- (1 p 6) bonds in the Side chains, indicated that there is a ratio of glucose units in the side chains to glucose units in the main chain of about 2: 7 and the following Has properties: (a) In the form of the freeze-dried product it is a white, amorphous solid; (b) it is soluble in water, dimethyl sulfoxide and in Dimethylformamide and is insoluble in ethanol, acetone, ethyl acetate, benzene and diethyl ether; (c) its elemental analysis corresponds essentially to the values for a polysaccharide of the hexoside type with bound water can be calculated using the following formula: (C6Hi0O5) nnH2Og where n is a number calculated from property (e) greater than 800; (d) be Infrared spectrum (KBr powder) corresponds to that shown in the accompanying drawing Spectrum and (e) in the ultracentrifugal analytical distribution curve is only a single Pe-.w- observed and calculated from the sedimentation constant the molecular weight is 150,000 to 160,000; taking the glucan by treating a polysaccharide produced by a fungus of the Corticiaceae family, with formic acid and hydrolysis of the product so treated. 2. A method for producing a hydrolyzed glucan, thereby It is noted that one is a polysaccharide that is produced by a fungus of the family Corticiaceae is produced, treated with formic acid and the so treated Product then hydrolyzed. 3. The method according to claim 2, characterized in that g e k e n n ze i c h -n e t that you can treat with a 70 to 90 percent by weight watery Performs formic acid. 4. The method according to claim 2 or 3, characterized in that g e k e n n -z e i c h n e t that the polysaccharide with the formic acid at a temperature of 800 to 1000C converts. 5. The method according to any one of claims 2 to 4, characterized g e k e n n notices that the hydrolysis can be achieved by heating a mixture of the treatment product carried out with formic acid and water under reflux until the deformylation is complete is. 6. Medicinal product with immunomodulatory activity, which is an immunomodulator contains in admixture with a pharmaceutically acceptable carrier or diluent, in that it is a hydrolysed immunomodulator Glucan according to claim 1. 7. Medicament according to claim 6, characterized in that g e k e n n -z e i c h n e t that it is in the form of a preparation for parenteral administration is.