DE2660052A1 - Pure anticoagulant heparin prodn. - using salt lakes obtd. from mucus-free animal intestines by sprinkling with salt - Google Patents

Abstract

Use of intestinal salt lakes obtd. by sprinkling animal intestines (freed from intestinal mucus) with common salt for the prodn. of pure heparin is new. Heparin is used as an anticoagulant. Process enables practically colourless heparin to be produced on a technical scale. The salt lake is a storage-stable, almost odourless material, which does not suffer deterioration in quality after prolonged storage.

Description

Process for the preparation of heparin

The present invention relates to a method for obtaining Heparin from brine, which is obtained when processing animal heat (intestinal brine ).

Nit heparin has long been a naturally occurring medicine Anticoagulants are available that are still used in modern medicine as well before is indispensable and is becoming more and more widely used.

In recent years a number of procedures have become known that all aim to remove the active ingredient heparin from heparin-containinga in an increasingly gentle way animal tissue such as the lungs, liver and, more recently, mainly from intestinal mucus (ucosa) from pigs, cattle and sheep (British patent? 54 885, German patent specification 1,228,241, US patent 3,058,884, German patent specification 1 253 868). Such raw materials have the disadvantage because of their perishability a limited shelf life, which are prolonged by freezing can be, which, however, in addition to the additional technical effort, also leads to considerable increases in costs leads. Both the operating staff and residents are involved in the processing from the surrounding area not infrequently because of the unpleasant smell associated with it faced unreasonable problems.

In the German patent 1 253 868, the recovery of heparin summarized for some common procedures. Then you bring that animal tissues in the initial stage in contact with a hot aqueous saline solution, to release the heparin from the cells. The heparin content of the resulting Medium is extremely low.

It has now been found that in the slaughterhouses and intestinal mucilage Accruing brine contains heparin This brine is produced when animal intestines, which have previously been freed from the intestinal mucus for preservation and drainage sprinkled with solid table salt.

It is therefore extremely surprising with regard to DBP 1 253 868 to denote that the brine produced by the mere salting of animal intestines contains large amounts of heparin. It was also not to be expected that this heparin occurs almost completely free of chemically related by-products, so that brine, previously known only as an environmentally harmful waste product, a new source of raw materials for which is already a very scarce heparin due to raw material shortages. By the Work-up to raw heparin can be particularly good if there is a substantial absence of impurities are carried out gently and the previous status is no longer required complex isolation methods known in the art (see US Pat. No. 3,451,996 and British Patent 1,221,784). Finally, one arrives at heparin qualities, the heparin preparations available on the market are nowhere near reached will. The according to the invention used according to known work-up processes Raw heparins isolated from intestinal brine as a raw material are already in such purity suggest that the subsequent purification to heparins with far over 200 USP units / mg leads. Possess the heparin preparations commercially available up to now usually heparin grades of 150 USP units / mg, in a few cases also 155 USP units / mg. A USP unit / mg is understood to be the specific one Activity arising from the U.S.P. assay, with which the inhibition of lump formation measured from preserved sheep plasma. 1 USP unit is approximately 1.1 international units (UJ). Since the activity values of the Heparin preparations remain unsatisfactory despite numerous process improvements USP, for example, stipulates that heparin preparations (from intestinal mucus) must be at least 140 Must contain USP units. Only in the laboratory has L.W. Kananagh and L.B. Jaques [Arzneimittelforschung (Drug Res.) 24, No. 12, 1942 (197) 1 Heparin to be isolated by repeated crystallization as a barium salt with a maximum of 175 USP-U / mg.

By using intestinal brine as a raw material according to the invention succeeded in isolating heparin on an industrial scale in a purity that it exists as an almost colorless substance. All available from other raw materials to this day Raw heparins, on the other hand, first have to be decolorized by a complex and lossy process (U.S. Patent 3,179,566).

Brine is also a storable, almost odorless raw material, which does not suffer any loss of quality due to long storage. This eliminates chemical ones and other preservation methods.

The invention thus relates to a method for isolating particularly pure heparin from saline solutions of animal tissue using methods known per se, characterized in that maB, intestinal brine used as the starting material, the obtained by sprinkling the intestines freed from the intestinal mucus with solid table salt has been.

The following examples illustrate the invention.

Example 1 600 liters of brine were added to a 1500 liter agitator Room temperature with stirring within half an hour with 600 liters of methanol offset. It was stirred for the same time, after which the mixture was about an hour stopped.

The cloudy supernatant was then decanted into a second vessel and left the fancy salt cake behind.

The suspension obtained now remained for two days.

It was then decanted again, the supernatant discarded and the precipitate isolated by centrifugation.

After drying in vacuo, 1.68 kg of a product were obtained which 11 USP admissions / mg. It still consists of approx. 60% table salt.

Example 2 Similar to British patent 754 885, 3 liters of brine acidified to pH 3.1-3.2 with acetic acid while stirring (approx. 50ml). After some Hours were decanted and the remainder centrifuged. The precipitate was discarded, the combined solutions were neutralized with sodium hydroxide solution.

The same volume of methanol was then slowly added with stirring. After 45 minutes, the precipitated common salt was decanted. This suspension left standing for several hours, decanted the supernatant and centrifuged the rest. The precipitate was dried in vacuo.

3.76 g at 23 USP units / mg were obtained.

Example 3 3 liters of brine were diluted with 14 liters of water. then 50 g of kieselguhr and a solution of 7 g of bezethonium chloride (= Hyamin 1622, Rohm & Haas) added in 100 ml of water. After a few hours it was decanted and the rest sucked off. After washing with water, the precipitate was still moist three times using the method from German Patent 1,228,241 with 200 each ml of 2 molar saline solution extracted. The combined extracts were 2 parts by volume Methanol precipitated.

The isolated and dried precipitate weighed 560 mg and possessed a Activity of 163 USP units / mg.

Example 4 3 liters of brine were made up with acetic acid while stirring pH 3.1 - 3.2 brought. After several hours it was decanted and the remainder centrifuged. The combined solutions were diluted to 15.5 liters with water and 40 g of kieselguhr and 7 g of benzethonium chloride (Hyamin 1622) are added to 100 ml of water.

After a few hours, the precipitate was isolated as in Example 3 and further processed.

Finally, 796 mg with 105 USP units / mg were obtained.

Example 5 3 liters of brine were diluted with 13 liters of water (cf. German patent specification 1 156 938). Then was with acetic acid (approx. 50 ml) to pH 3.2 acidified. After a few hours, a precipitate had settled, which after was centrifuged off after decanting the clear supernatant. After drying 16.6 g of raw heparin were obtained at 5.5 USP units / mg.

Example 6 10 liters of brine were added at 50 liters. Water diluted. These Solution was filtered through an ion exchange column with a diameter of 26 mm and a length of 380 mm (approx. 3 liters / hour). It became a medium base Macroporous anion exchange resin used in the Cl form (Lewatit CA 9249). Other anion exchange resins can also be used, e.g.

the Dowex I-X-1 described in German Patent 1 253 868.

The resin was then removed from the column, twice with 500 ml 0.9 molar saline solution and washed with 400 ml 2 molar saline solution Eluted for 5 hours at 40 ° C with stirring. This solution was made with 1.5 parts by volume Methanol precipitated. The isolated and dried precipitate weighed 1.5 g and possessed 152 USP units / mg.

Example 7 An aliquot of the raw heparin from Example 1 accordingly 425,000 USP-E or 38.65 g, was three times with 200 ml two molar Extracted saline solution, stirring at 60 ° C for one hour each time. the combined extracts were diluted with water until the solution had about 0.9 mol / l chloride ions contained. There were now 200 ml of an anion exchanger (Lewatit CA 9249) in the Chloride form was added and the mixture was stirred for one hour. It was then suctioned off and washed with approx. 200 ml 0.9 molar saline solution. The ion exchanger was then stirred for five hours with two molar saline solution at 40 ° C. and thereafter Aspirated and washed with two molar saline solution. The elution solution was combined with the washing solution and precipitated with 1.5 times the volume of methanol. Of the Precipitate was centrifuged off, first with approx. 50 ml water-methanol (1 + 1.5 Parts by volume), then washed with approx. 50 ml of methanol and dried.

Han received 1.91 g Na heparinate at 202 USP units per mg.

Claims (1)

Pat ent claims method for isolating pure heparin from saline solutions of animal tissue according to methods known per se, characterized in that that one uses intestinal brine as the starting material, which is obtained by sprinkling with vom Intestinal mucus freed animal intestines with solid table salt has been obtained.