IL28649A - Glycopeptides and their preparation from animal organs - Google Patents

Description

28649/2 New glycopeptides and their preparation from animal organs PREPHAR PRQSPECflON DE RECHERCHSS PHARMACISUTIQXIES S.A. - 2 - 28649/2 The existence in animal organs of glycoproteins is known. These are macromolecules whose nature is intermediate between those of proteins and polysaccharides, and their structure can be conceived, by a rough approximation, as resulting from the combination of one or more molecules of a polysaccharide with one or more molecules of a protein. This combination is due to covalent linkages and the structure is thus not an adduct or a complex but rather a single defined molecule. In some types of glycoproteins the protein moiety predominates, in others the polysaccharide moiety. It is mainly the latter type which is usually referred to as glycopeptides . Some substances of this class have a high biological importance, e.g. those that characterize the blood groups.

This invention provides a new glycopeptide or group of glycopeptides, which is or are characterized by the following composition (in per cent by weight of the dry matter): total nitrogen - 5-6 to 6.3; protein -13 to 18; amino acids - 13 to 18; hexosamine - 40 to 50; neuraminic acid derivative - 4 to 6; hexose - 27 to 31; and further characterized, in the purified state, by the substantial absence of sulfur and phosphorus and of uronic acids.

These new glycopeptides have been found to possess valuable pharmacological properties, mainly for the treatment of inflammatory diseases, and especially of ulcers.

The new glycopeptides behave as homogeneous substances in electrophoresis, paper chromatography and the - 3 - 28649/2 like analytical processes. They are precipitated by copper sulfate from an alkaline medium (biuret reagent) in a quantitative yield. The composition indicated above characterizes them as glycopeptides, and the presence of hexosamines and absence of uronic acids, sulfur-containing groupings of all kinds and of phosphorus imparts to their pol saccharic moiety a characteristic nature.

Riarmacologieal tests on laboratory animals have shoTim,¾n doses of 12, t 5 or 50 mg kg the new glycopeptides inhibit local edfena upon administration of carragenine and aerotonine, the diffusion of Blue Evans in the intradermal pomphi by serotonine, dextrane and bradiquinine, as well as the growth of cotton-pellets granuloma in the rat. At a dose of 100 mg/kg, the substance being tested reduces the percentage of rats affected and the gravity of pylorus-ligation ulcer according to Shay, and of fast ulcer. Moreover, it can promote the recovery of the function of inflamed limbs (arthritis caused by AgHO^).

The invention also provides processes for preparing the new glycopeptides from animal organs, especially from those of the digestive system of svrine, in particular the (hereinafter "the source material") gastric mucosa and the duodenum* The process according to the invention comprises the following main operations: of the source material a. hydrolysis/in a mildly acidic or alkaline aqueous medium (followed by mild acidulation if the hydrolysis is carried out in an alkaline medium) ; b. removal of precipitated solid ballast substances; - 4 - 28649/2 c. addition of acetone to the liquid, and recovery of the precipitate thereby produced; d. enzymatic proteolysis of the precipitate (c), and removal of solid ballast matter; e. recovery of the glycopeptide from the liquid pro- teol sate and, if desired, purification thereof* It is an important feature of the process of the invention that the enzymatic proteolysis does not appear to a ffect the protein or peptide moiety of the glyco-peptides, but does destroy the protein present as a ballast, i.e. not as a part of the glycopeptide molecule. IJ is behaviour is surprising in view of previous statements in tie literature, which, however, refers to other types types of compounds.

For the hydroly ic treatment, the organ is preferably minced and slurried in water. Before or during the hydrolysis the aqueous slurry is preferably heated, e.g. at 50° to 100°C, preferably for 10 to 45 minutes. The heating operation can be carried out and controlled most easily at or near boiling temperature, i.e. in the vicinity of 100°C. She pH of the aqueous medium in the hydrol zing operation may be within the range from 1 to 10, it is adjusted by suitable additions of acids or alkaliesf.

A pH above 10 must be avoided since at such a degree f alkalinity the material is quickly degraded. For the same reasons, at a high pH (above 9) the heating temperature - 5 - 28649/2 and time must be carefully controlled.

The hydrolysis dissolves the desired glycopeptides. Acidic hydrolysis leaves undesired ballast matter, mainly proteins and sulfur-containing mucopolysaccharides, undissolved. Alkaline hydrolysis dissolves these ballast substances as well, and in this case they have to be precipitated by acidulation of the reaction mixture after the termination of the hydrolyzing operation, preferably with a weak acid such as acetic acid.

In either case the precipitate is removed, .e.g by filtration or centrifugation and the liquid hydrolysate is further processed.

Optionally the liquid hydrolysate may be subjected to a further purifying operation prior to being further processed in that some more protein ballast is precipitated, e.g. with trichloroacetic acid, and this precipitate is again removed.

The liquid reaction mixture produced by the enzymatic proteolytic treatment will as a rule have to be freed from solid ballast matter. For the recovery of glycopeptides from the clarified proteolysate the latter is preferably concentrated by evaporation, and from the concentrate the glycopeptides can be precipitated with acetone. The precipitate, after drying is a white to hazel powder.

This can be further purified, mainly by the removal of salts, so that a product of constant analytical - 6 - 28649/2 composition is obtained, The purification may be carried out, for example, by dialysis which allows the impurities by to pass through the membrane, or/ion exchange by means of exchange ion resins of the acidic type or mixed type, which removes the strongly ionic impurities. In either case the yields of purified glycopeptide are from 65 to 75$ by weight of the crude product. The purified product is a colourless, inodorous and amorphous powder whose phosphorus and sulfur contents are practically negligible.

The invention is illustrated by the following Examples to which it is not limited.

EXAMPia.-l .2 Kg of deep-frozen, fat-free duodenum of swine were minced in a mincer provided with a perforated plate with 1.1 mm hole diameter. The minced material was suspended in 10 litres of water, the pH of the mixture was adjusted to 9*2 with 3H sodium hydroxide, and the mixture was boiled with stirring for 15 minutes while its pH was maintained at 9·2 by repeated additions of 3N sodium hydroxide. When the heating was interrupted, the pH of the mixture was adjusted to 4.7 by adding about 600 ml of 80$ acetic acid, then the mixture was allowed to cool to ?0°C and, after the addition of 10 liters of acetone and 1 kg of a filtration aid (celite, celatom or the like) , filtered by being poured through a cloth. The filtrate, amounting to 22 liters, was admixed with 55 liters acetone. The precipitate thereby produced - 7 - 28649/2 was collected by decantation, washed with acetone, dried tinder vacuum, and ground. Its weight was then approximately 3 kg.

This powder was dispersed in 9 liters of water, the pH of the solution was adjusted to 5.7 with 21ί hydrochloric acid, 20 grs of sodium chloride and 20 grs of papain were added, then the mixture was incubated with stirring at 60°C for 24 hours* At the end of the papain treatment, 50 grs of a filtration aid were added and the mixture was filtered through a cloth. The filtrate was concentrated under reduced pressure (50 mm.Hg.) to a volume of 3 liters, and then admixed with 2.7 liters of acetone. The precipitate thereby formed was collected by decantation, washed with a cetone and dried under vacuum. This a3 glyco crude/peptide, amounting to approximate^ly 66 grs. It had the following characteristics: P = 3. 3 S = 0.21%; H = 5.91 ; Acetyl groups: 7.12%; Viscosity (on 1% solution) at 20 C = 1.2699 cps; pH (1% solution) = 6.9.

EXAMPLE 2 3.8 Kg of deep-frozen gastric mucosa of swine were minced in a mincer provided with a perforated plate with 1.1 mm hole diameter. The minced material was suspended in 7.5 liters of water and boiled with stirring for 15 minutes, at a pH of 8.6, whic as maintained by repeated additions of 2N sodium hydroxide. Then the mixture was admixed first with 250 ml of 80% acetic acid, then with so much trichloracetic acid (about 160 grs) - 8 - 28649/2 as to lower the pH to 1.8. The precipitate thereby formed was removed by centrifugation and the clear liquid was admixed with four times its volume of acetone. The precipitate thereby produced was collected by filtration, washed with acetone, dried under vacuum, and ground.

The 47 grs of powder thus obtained were slurried in water and subjected to proteolysis at pH of 5·7» as described in Example 1. The crude product amounted to 20 grs. and contained 4$ of phosphorous and 6»2 of nitrogen.

EXAMPLE 3 1 Kg of deep-frozen, fat-free duodenum of swine was minced in a mincer provided with a perforated plate of 1.1 mm hole diameter. The minced material was suspended in 1 liter of water, brought to pH 4 with 80% acetic acid, and boiled with stirring.

The mixture was kept boiling for 15 minutes, then ms permitted to cool and centrifugated. The liquid was admixed first with 10 grs of sodium acetate, then wit four times its volume of acetone. The precipitate thus formed was separated by decantation, washed with acetone, dried under vacuum and ground. 11 Grs of powder were thus obtained which were subjected to the proteolytic treatment with papain at pH 5·7 as described in Example 1.

The crude product amounted to 5.2 grs and contained 2.5≠ of and 6.03$ of ».

BXAMPLE 4 1 Kg of deep-frozen, fat-free duodenum of swine - 9 - 28649/2 was minced in a mincer provided with a perforated plate of 1.1 mm hole diameter, The minced material was suspended in 1 liter of water acidulated to pH 1.7 with trichloroacetic acid, and the mixture was heated to 50°C. for minutes with stirring, then allowed to cool and centri-fugated. The filtrate was admixed with 15 grs of sodium acetate, then with four times its volume of acetone.

The precipitate thus formed was separated by decantation, washed with acetone, dried under vacuum and ground. The 9 grs of powder so obtained were subjected to the proteolytic treatment with papain at pH to 5.7, as described in Exaaple 1.

The crude product obtained by this procedure amounted to 4.8 grs and contained 2,6% of P and 6.1 of K.

EXAMPLE 5 100 Grs of the crude glycopeptide obtained by any of the processes described in Examples 1 to 4 were suspended in 1 liter of water. The suspension was freed by centrifugation from undissolved matter and the pH of 8 · the clear liquid was brought to 7.β with 2K sodium hydroxide. This solution was subjected to dialysis with deionized water at 20°C through a 0.03 mm thick cellulose membrane for 48 hours. At the end of this treatment, the volume of the solution had risen?2 liters. The solution was concentrated back to the original volume of 1 liter by evaporation under reduced pressure (50 mm.Hg.). The concentrate was admixed with 5 grs of sQdium chloride, - 10 - 28649/2 then with 1200 ml of acetone or 2500 ml of methanol. The precipitate thereby formed was collected by decantation, washed with the solvent used for the precipitation, dried under vacuum and ground. Yield, 69 gra.

The product had the following composition and properties: P * none*, 3 = 0..25 = 5.8196; CH^CO = 9.75 ; viscosity (in 15¾ solution) at 20°C = 1.6409 cps; Ha = 0.71 pH (1 solution) = 6.3» hexosamines = 39.2 ; F uronic acids = none; proteins = 16.3 (folin) ; hexoses = 9. neua inic acid derivatives 4.55 .

EXAMPLE 6 100 grs of the crude glycopeptide obtained by any of processes described in Examples 1 to 4 were suspended in 1 liter of water* The suspension was freed by centri-fugation from undissolved matter and the clear liquid was treated with 150 ml of "Amberlite IR 120" resin (acidic form, 26-50 U.S.A. mesh) at room temperature for half an hour. The resin was then washed with 100 ml of distilled water and the wash liquors were combined with the main solution. The solution was admixed with 5 grs of sodium chloride, then with 1320 ml of acetone. The precipitate thereby formed was collected by decantation and washed 3 times with acetone, then redissolved in 75 ml of water. The pH of the solution was brought to 6.2 with NaOH 2N.

Then the solution was admixed with 5 grs of sodium acetate and with 2.5 times its volume of acetone, whereby a precipitate was obtained which was collected by - 11 - 28649/2 decantation, washed with acetone, dried under vacuum and ground. Yield 67 grs.

In operation on an industrial scale, the ion exchange operation^preferably performed toy percolation of the solution through an ion exchanger column, which does not substantially modify the process as auch.

The product obtained in this Example had the following composition and properties :· p = 0.08%; S = 0.2$; N = 6.03%; CI^CQ = 9.34 ; Na - 0.43%; pH (1% solution) = 6.2; hexosamines = 39.5%; uronic acids; none; hexoses = 29.5$ viscosity (on 1 solution) at F C = 1.9189 cps; protein (^folin) = 14.05$; neuraminic acid derivatives 5.33 .

EXAMPLE 7 100 grs of crude glycopeptide obtained by any of the processes described in Examples 1 to 4 were suspended in 1 liter of water. The suspension was freed from undissolved matter by centrifugation and the clear liquid was treated with 100 ml of "Amberlite IE 120" resin (acidic form activated with 10% HC1; 26-50 U.S.A. mesh). After 30 minutes the resin was removed by filtration and washed with 100 ml of distilled water; the wash liquors were combined with the filtrate. This solution, in turn, was treated with 100 ml of "Amberlite IBA 410" (basic form, activated with 5% NaOH; 20-50 U.S.S. mesh) for an additional 30 minutes. The resin was removed by filtration and washed with 150 ml of distilled water; the wash liquors were combined ith the filtrate and the pH - 12 - 28649/2 was brought to 6.2 with 2H HaOH. The solution was admixed with 5 grs of sodium acetate and with an equal volume of acetone. The precipitate thereby formed was collected by decantation, washed with acetone, dried under vacuum, and ground. Yield, 70 grs.

The product had the following composition and properties: P = 0.04% S = 0.11$; Ha = 0.27 ; acetyl groups = 9.93$; S = 5.74ίί; hexosamines *= 40 ; uronic P acids = none; protein (jfolin) = 14.5%; hexoses = 31 pH (1 solution) = 6.3; viscosity (on 1% solution), at 20°C cps 1.900 ; neuraminic acid derivatives 5.20%. «■•13 - 28649/2

Claims (10)

1. -iiew- Glycopeptide or group of glycopeptides, which is or are characterized by the following composition (in per cent by weight of the dry matter) : total nitrogen -5.6 to 6.3; protein - 13 to 18; amino acids - 13 to 18; hexosaraine - 40 to 50; neuraminic acid derivative - 4 to 6; hexose - 2? to 31» and further characterized, in the purified state, by the substantial absence of sulfur and phosphorus and of uronic acids.

2. Crude glycopeptides according to Claim 1, substantially as described herein with reference to any of Examples 1 to 4.

3. Purified glycopeptides according to Claim 1, substantially as described herein with reference to any of Examples 5» 6 or 7.

4. A process for the preparation of a glycopeptide according to Claims 1 or 2, comprising the steps of of the source material a. hydrolysis/in a mildly acidic or alkaline aqueous medium (followed by mild acidulation if the hydrolysis is carried out in an alkaline medium) ; b. removal of precipitated solid ballast substances; c. Addition of acetone to the liquid, and recovery of the precipitate thereby produced; d. enzymatic proteolysis of the precipitate (c), and removal of solid ballast matter; e. recovery of the glycopeptide from the liquid pro- teol sate. - 14 - 28649/2

5. A process according to Claim 4» wherein the proteolysis is effected by means of papain.

6. A process a ccording to Claims 4 or 5, wherein before or after the removal of the solid ballast substances ( operation b. ) the liquid hydrolysate is f rther purified by the addition of trichloroacetic acid and removal of the precipitate thereby formed.

7. A process according to any of Claims 4, 5 or 6, wherein the hydrolysis effected at a pH of about 8.5 to about 9.5.

8. A process according to any of Claims 4, 5 or 6, wherein the hydrolysis is effected at a pH of about 1.5 to 4.5.

9. A process according to any of Claims 4 to 8, wherein the proteolysate is concentrated and crude glyco-peptide is precipitated from the concentrate with acetone,

10. A process for the preparation of a purified glycopeptide according to Claim 3, wherein a crude glyco-peptide produced by the process according to any of Claims 4 to 8 is subjected to purification by dialysis or ion exchange· RC/rb