DD136572B1 - METHOD FOR THE PRODUCTION OF A THERAPEUTICALLY ACTIVE MUCOPOLYSACCARID POLYESTERIC ACID ESTER FROM BOVINE TRACHEA - Google Patents

Description

Title of the invention

Process for the preparation of a therapeutically effective mucopolysaccharide polysulphuric acid ester on bovine trachea

Application area of the invention

The invention relates to a process for the heratilation of a therapeutically active mucopolysaccharide polychloric acid adea from bovine trachea, which is suitable as a pharmaceutical preparation for the treatment of joint diseases and is particularly injectable intra-articularly.

Characteristic of the known technical solutions

It is known that Mukopolyaaccharidpolyschwefölsäureester with blood anticoagulant and / or lipolytic (antilipämiacher) effect can be obtained by sulfation of animal or vegetable polysaccharides (Pharm · Praxis 12. 281 (1969)).

Although the corresponding methods often contain technically complicated purification stages, such as enzymatic degradation steps with papain, trypsin, hyaluronidase or bacterial proteases, the previously known preparations are generally due to their high heparinoid effect or their other composition for the treatment of joint diseases in terms of effect and tolerability unsuitable, which is especially evident in intra-articular injection,

Bs are already known processes for the isolation of acid mucopolysaccharides (DT-OS 1.492.017, DT-OS 1.467.810, BE 843.095, JA 031353, JA 076160, JA 123444), which, however, are not suitable for intra-articular injection in joint diseases.

According to DT-OS 1.492.017, acidic polysaccharides are obtained from bone or cartilage tissue by extraction with an aqueous weakly alkaline solution of ethylenediaminetetraacetic acid, treatment of the extracts. pH 5 with a cation exchange resin IR 50 and an enzymatic degradation of the insoluble residues by means of papain and trypsin and subsequent precipitation of the acidic polysaccharides with quaternary ammonium compounds.

Furthermore, duodenal heparinoid with antilipemic effect was purified by fractional precipitation with quaternary ammonium salts or zinc chloride according to DT-OS No. 1,467,810. Chondroitin sulfates were obtained by treatment of cartilage with pepsin and papain (BE 843.095), a culture solution of Serratia or Vas. subt. (JA 031.353), by treatment of animal skin with lipase, pronase and Dowex-1 chromatography (JA 076.160) or by purification by chromatography on a coarse-pored, strongly basic anion exchanger (JA 123.444).

Also according to the Japanese patents Hr. 7000 156-R (Derwent 04390 R), Hr. 7001 445 (Derwent 06504 R), Hr. 7004 734 (Derwent 14519 R), Hr. 7314 078 (Derwent 25548 U) obtained low molecular weight Chondroitinpolyaulfate (MW 400-5000), by an approximately 10-hour treatment of chondroitin sulfate (MW 400-4000) with chloroalfaic acid in pyridine or formamide with subsequent alkalization, steam distillation or treatment with a cation exchange resin or obtained by depolymerization of Chondroitinpolyaulfat by means of chondroitinase and fractional precipitation with organic solvents, although have a high antilipemic and thus an anti-arteriosclerotic effect, a suitability for the treatment of joint diseases is not known.

Object of the invention

The object of the invention is to obtain a mucopolysaccharide polysulfuric acid ester which can be injected directly into the joint capsule as a therapeutic agent in degenerative joint diseases.

Explanation of the essence of the invention

The invention has for its object to develop a process for the preparation of a highly purified Mukopolyeaccharidpolyschwefelsäureesters that is free of incompatible Polyaaccharid- and Prcteinanteilen.

According to the invention, it has been found that a mucopolysaccharide fraction is isolated by alcohol fractionation of a crude Mukopolysaceharidgemisches obtained in a known manner from bovine trachea by alkaline extraction, preferably by precipitation of an i'orfraktion for the production of a therapeutically effective and joint-compatible preparation for the removal of interfering high molecular weight polysaccharide and protein components pH 1-2 and 20 ⁰ C with an ethanol concentration of 30-34 VoI.%, Which is discarded and subsequent precipitation of the desired mucopolysaccharide fraction with an ethanol concentration of 70-74 VoI ,%, and this for the separation of low molecular weight fractions after partial hydrolysis and sulfation in the form of a 5% aqueous solution of pH 9-1O at 20 ⁰ C dialyzed via Hohlfaaern or membranes with an exclusion limit of 5000 MW, wherein the partial hydrolysis with hydrochloric acid and the sulfation using chlorosulfonic acid in the form amide is carried out in a known manner, and cleaned with a strongly acidic cation exchanger and decolorized with hydrogen peroxide.

The mucopolysaccharide polysulfuric acid ester (MPS) obtained has a sulfur content of 11-13.5 %, a nitrogen content of 1.7-2.2 % _f, a uronic acid content of 20-26 % and an anticoagulant activity of less than 15 ΙΕ / mg compared to standard heparin.

After injection of 0.2 ml of a 50 mg MPS / ml solution into the right knee joint of the rabbit hindlimbs (3 injections per week), no inflammatory reaction of the joint capsule occurred after

With 10 intra-articular injections of 50 mg / 1 ml each in humans, the preparation was found to be well-tolerated and effective in the treatment of degenerative arthritis of the knee.

embodiments

10 kg of fresh bovine tracheas were comminuted in a meat grinder and 3 times with 35 1 of acetone at room temperature (20 ⁰ C) stirred for 3 hours, was subsequently being respectively filtered through a coarse screen. The coarse-fiber trachea powder was dried in the air. Yield: 2.150 kg

2 kg dry Tracheapulver were digested with 600 g of caustic soda and 15 1 of water for 20 hours at 15 ⁰ C with occasional stirring. It was then adjusted to pH 1 with 3.9 l of 18% strength hydrochloric acid and centrifuged. The clear centrifugate (16 1) was mixed with 48 1 of ethanol. After a 15-hour life at -4 ⁰ C, the precipitate was removed by centrifugation, washed with alcohol and acetone and dried in a vacuum desiccator. Yield: 203.4 g of crude mucopolysaccharide mixture

200 g of crude mucopolysaccharide mixture were dissolved in 1800 ml of water. After adjusting the solution with hydrochloric acid to pH 1 was made up to 2000 ml with water. By adding 1000 ml of 96% ethanol at 20 ⁰ C, a pre-fraction was precipitated, which was spun after a lifetime at +4 ⁰ C overnight and discarded. From the centrifugate at 4 ⁰ C, a Mukopolysaccharidpraction has been precipitated by the addition of another 5000 ml of ethanol, which has been obtained by centrifugation and washing with ethanol and acetone. Yield: 118.2 g mucopolysaccharide fraction

A 10% solution of fractionated 10og of mucopolysaccharides in 0.1 IT hydrochloric acid was left for 3 hours at 80 ⁰ C. The precipitation of the partially degraded mucopolysaccharide fraction was carried out with 5000 ml of ethanol. The precipitate was recovered by centrifugation and washed out with alcohol and acetone

 Yield: 76.1 g

75 g of the partially degraded mucopolysaccharide fraction were dissolved in 750 ml of formamide and while stirring and cooling in the sulfonation flask within 1 hour with 112.5 ml of chlorosulfonic acid dropwise, the temperature was maintained at 10-15 ⁰ C. Then it was stirred for 4 hours at room temperature (20 ⁰ C). By pouring the solution into 6 L of methanol, the sulfated mucopolysaccharide was precipitated. The precipitate was filtered off with suction and dissolved in 1500 ml of water. The pH was adjusted to 10 with sodium hydroxide solution and, for the purpose of dialysis, the solution was pumped through a hollow fiber cartridge (exclusion limit MW 5000), which was flushed through in the outer jacket with running tap water and finally with distilled water. After concentrating the dialyzed mucopolysaccharide solution by ultrafiltration through the same hollow fiber cartridge to 695 ml, the sulfated MPS was precipitated with 3500 ml of ethanol with addition of sodium acetate. Yield: 75.8 g

A solution ^{ѵо в} 75 g of the sulfated MPS Group in 750 ml of distilled water was 750 ml of a strongly acidic cation exchanger based on a styrene-Divinylbenzolcopolymerieates filtered with nuclear pendant sulfonic acid groups (H ⁺ form). After adjusting the filtrate with 4 N sodium hydroxide solution to pH 7.0, 75 ml of 30%

Waaaeratoffperoxid added and the Anaatz 60 min at 70 ⁰ C belaaaen, with 0.1 N NaOH after 10 min nachgeatellt every 10 min. After cooling, daa Muko poly saccharide was precipitated by pouring the slurry into 5000 ml ethanol (addition of sodium acetate to inhibit flocculation). The precipitate was centrifuged, washed with acetone and dried in vacuo. Yield: 71.5 g Mukopolyaaccharidpolyachwefelaäureeater-Na-SaIz

Sulfur content: 11.32 % Uronic acid: 23.0% Sodium: 11.3% Nitrogen: 1.9 %

Claims (1)

invention claim Process for the preparation of a therapeutically effective mucus polysaccharide polysulphuric acid residue from bovine trachea, characterized in that a mucopolysaccharide fraction is isolated by fractionation with a lower aliphatic alcohol of a crude mucopolysaccharide mixture obtained from bovine trachea by alkaline extraction in a known manner, preferably by precipitation of a prefraction at pH 1-2 and 20 ⁰ C with an ethanol concentration of 30-34 vol.%, which is discarded, and subsequent precipitation of the desired fraction mucopolysaccharide with an ethanol concentration of 70-74 vol.%, after this carried out in a known manner in the form of partial hydrolysis and sulfation filtered a 5% aqueous solution of ρΉ 9-10 at 4-25 ° C for dialysis over HohlJfasern or membranes with an exclusion limit of 5000 MW and cleaned with a strongly acidic cation exchange resin and decolorized with hydrogen peroxide ·