DK163063B - APPLICATION OF TARM SALT LAYER FOR THE PREPARATION OF HIGH PURPOSE HEPARIN - Google Patents

Description

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DK 163063 B

The present invention relates to the use of intestinal brine to produce high purity heparin.

Within the medicine, a naturally occurring anticoagulant has long been available in the form of heparin, which is also indispensable in modern medicine as before and is increasingly used.

In recent years, a number of methods have become known, all of which aim to isolate the active substance heparin in an increasingly gentle manner from heparin-containing animal tissues such as lung, liver and, more recently, primarily from intestinal mucosa (Hucosa). in pigs, cattle and sheep (GB-PS 754,885, DE-PS 1,228,241, US-PS 3,058,884, DE-PS 1,253,868). Such raw materials, because of their slight perishability, have the disadvantage of having a limited storage time which can be extended by grinding, which, however, in addition to further technical effort also leads to significant cost increases. During the work-up, both the operating staff and residents in the adjacent surroundings are not infrequently faced with unacceptable problems due to the associated odor genes.

In DE-PS 1,253,868, the recovery of heparin is summarized for some conventional methods. According to these, the animal tissue is brought into contact with a hot aqueous saline solution at the initial stage in order to release the heparin from the cells. The amount of heparin in the resulting medium is extremely low.

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It has now been found that the brine found in slaughterhouses and intestinal slimming establishments contains heparin.

This brine is formed when, at room temperature, animal intestines previously liberated from intestinal mucus are placed in boiling salt solutions with molar saturation amount of boiling salt for preservation and dewatering.

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It should therefore be noted, with reference to DE-PS 1,253,868, that the brine formed by the introduction of animal intestines into boiling salt solution contains large amounts of heparin. It was not to be expected that this heparin was almost completely free of chemically related by-products, so that brine, hitherto known only as an environmentally polluting waste product, represents a novel source of raw material for the already very scarce heparin. Due to the far-off absence of contaminants, the preparation for crude heparin can be made particularly lenient, and the circumstantial isolation methods known in the prior art thus lapse (see US Pat. Nos. 3,451,996 and GB-PS 1,221,784). Finally, heparin qualities are obtained which are far from being obtained in the heparin preparations available on the market. The intestinal brine used as raw material isolated raw heparins according to the known working methods according to the invention already exists in such purity that the subsequent purification leads to heparins with well over 200 USP units / mg. The commercially available heparin preparations usually have 20 heparin grades of 150 USP units / mg, in a few cases also 155 USP units / mg. By a USP unit / mg is understood the specific activity achieved by the U.S.P. test with which the inhibition of the formation of small chunks of conserved sheep plasma is measured. A USP unit equals approx. 1.1 international 25 needle units (IU). Since, despite numerous process improvements, the activity values of commercially available heparin preparations are still unsatisfactory, for example, USP prescribes that heparin preparations (from intestinal mucus) should contain at least 140 USP units. Only in the laboratory so far have L. L. Kananagh and L. B. Jaques [Arzneimittelforschung (Drug Res.) 24, No. 12, 1942 (1974)] succeeded in isolating heparin by repeated crystallization as a barium salt with a maximum of 175 USP-E / mg. 1

For the preservation and dewatering of animal intestines, saline solutions are suitable, the contents of which are equal to the molar saturation of boiling salt. Under molar saturation amount understand

DK 163063 B

3, the molar amount contained in a concentrated aqueous solution at room temperature of dissolved boiling salt in a slaughterhouse. (A concentrated aqueous saline solution is about 26.4%).

5, as a rule, the slimmed animal gut is treated for 2 hours to 3 days with boiling salt solutions as set forth in the claim. For the most part, however, the treatment of the intestines is completed after 3-24 hours. After removing the preserved and dehydrated animal intestines from the saline solution, an intestinal brine is obtained which has a surprisingly high content of heparin.

In using the invention according to the invention of such intestinal salts as raw material, it is also technically possible to isolate heparin in a purity so that it is almost available as a colorless material. In contrast, all raw heparins obtained hitherto from other raw materials must first be decolorized by cumbersome and loss-making methods (US-PS 3,179,566).

Intestinal brine is another storage suitable, almost odorless 20 raw material, which during prolonged storage is not subject to quality loss. This eliminates chemical and other preservation methods.

The invention thus relates to the use of intestinal brine which is 2 S

been obtained by immersion of intestinal mucus-liberated animals in boiling salt solutions with molar saturation amount of boiling salt at room temperature in a slaughterhouse, to produce high purity heparin.

Example 1 600 liters of brine obtained in the manner specified in the claim were added to 600 liters of methanol in a 1500 liter stirrer at room temperature with stirring over half an hour. After stirring for the same time, the mixture was allowed to stand for approx. 1 hour.

Then, the cloudy supernatant was decanted into another container and the formed salt cakes were allowed to

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4 bake. The obtained suspension was allowed to stand for 2 days. Then it was decanted again, the supernatant was discarded and the precipitate was isolated by centrifugation. After drying in vacuo, 1.44 kg of a product having 5 10 USP units / mg. It still exists for approx. 45% of boiling salt. An aliquot of the crude paper thus obtained corresponding to approx. 425,000 USP-E or 42.5 g were extracted three times, each time with 200 ml of 2 M brine, stirring for one hour at 60 ° C. The combined extracts 10 were diluted with water until the solution contained ca. 0.9 mol / l chloride ions. 200 ml of an anion exchanger ("Lewatit® CA 9249") was now added in the chloride form and stirred for one hour. Then it was extracted and washed with ca. 200 ml 0.9 molar boiling salt solution. The ion exchanger was then stirred for 5 hours with 2 molar saline at 40 ° C and then suctioned off and washed with 2 molar saline. The elution solution was combined with the washing solution and precipitated with 1.5 times the volume of methanol. The precipitate was centrifuged off, first washed with ca. 50 ml of water-methanol (1 + 1.5 20 parts by volume) and then with approx. 50 ml of methanol, and dried.

198 g of Na heparinate were obtained with 195 USP units per ml. mg.

15.5 g of a Na-heparinate obtained in the manner described above with about 200 USP units per mg was dissolved in 200 ml of 2 molar saline. The solution was sucked through a filtration apparatus and the residue was washed first with 30 ml of 2 molar saline solution and then with ca. 250 ml of completely desalinated water. The combined solutions were then adjusted to a chloride molarity of 0.9 with completely desalinated water. Then, as above, the purification with one liter of "Lewatit® CA 9249" was repeated.

The combined elution and washing solution was further processed as indicated above. After washing and drying the precipitated material, 11.2 g of sodium heparinate was obtained, which had an activity of 251 USP units per liter. mg.

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Example 2 In accordance with British Patent Specification No. 754,885, 3 liters of brine were obtained in the manner specified in the claim, with stirring, acidified with acetic acid to pH 3.1-3.2 (about 50 ml).

After a few hours, decant and centrifuge the remainder. The precipitated material was discarded and the combined solutions were neutralized with baking soda.

With stirring, the same volume of methanol was slowly added.

After 45 minutes, decanted brine was decanted. This suspension was allowed to stand for several hours, decanted the supernatant and centrifuged the residue. The precipitated material was dried in vacuo. 3.43 g of 21 USP units / mg were obtained. An aliquot of the crude heparin thus obtained, corresponding to approx. 425,000 USP-E or 20.2 g were purified analogously to that of Example 1. 10.65 g of sodium heparinate was obtained with an activity of 262 USP units per mg.

EXAMPLE 3 20 3 L brine obtained in the manner specified in the claim was diluted with 12 L of water to a chloride molarity of approx. 0.85 M. Then 50 g of diatomaceous earth and a solution of 6 g of benzethonium chloride ("Hyamin 1622", Rohm & Haas) were added in 100 ml of water. After 25 hours, decant and extract the remainder. After washing with water, the still moist precipitate was extracted three times in accordance with the procedure of German Patent No. 1,228,241, each time with 200 ml of 2 molar saline solution. The combined extracts were precipitated with 2 parts by volume of methanol. The isolated and dried precipitate weighed 465 mg and had an activity of 155 USP units / mg.

EXAMPLE 4 35 liters of brine obtained in the manner specified in the claim

diluted with 120 L of water to a chloride molarity of approx. 0.85 M

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6 (see DE-PS 1,156,938). Then, it was acidified with acetic acid (about 50 ml) to pH 3.2. After a few hours, a precipitate had settled, which precipitate was centrifuged after removing the clear supernatant. After drying, 111 g of crude heparin was obtained with 6.5 USP units / mg. An aliquot of the thus obtained crude heparin corresponding to 425,000 USP units or 65.6 g was initially purified. After the first purification step over ion exchange, 196 g of Na heparinate was obtained with 198 USP-E per liter. mg.

10 g of a sodium heparin obtained in the manner described above with approx. 200 USP units per mg was dissolved in 200 ml of 2 normal calcium chloride solution. The solution was aspirated through a filtration apparatus and the residue was first washed with 30 ml of 2 normal calcium chloride solution and then with ca. 250 ml of completely desalinated water. The combined solutions were then adjusted to a chloride molarity of 0.9 with completely desalinated water. Then, as above, a liter of "Lewatit® CA 9249" was stirred. The elution and post-washing were carried out with 2 normal calcium chloride solution instead of boiling brine.

The combined elution and washing solution was further processed as indicated above. After washing and drying the precipitated material, 10.7 g of calcium heparinate was obtained, which had an activity of 260 USP units per liter. mg.

Example 5 30 10 l of brine obtained in the manner specified in the claim was diluted to a chloride molarity of approx. 0.85 with 40 liters of water. This solution was pumped through a filter through a 26 mm diameter ion exchange column and a length of 380 mm (about 3 liters / hour). A medium-base, macroporous an-32 ion exchange resin was used in the CT form ("Lewatit® CA 9249"). Other ion exchange resins can also be used, such as the "Dowex® 1-X-1" described in German Patent No. 1,253,868.

Claims (1)

Finally, the resin was removed from the column, washed with 500 ml of 0.9 molar saline and eluted with 400 ml of 2 molar saline for 5 hours at 40 ° C with stirring. This solution was precipitated with 1.5 parts by volume of methanol. The isolated and dried precipitate weighed 1.2 g and contained 149 USP units / mg. Claims. 10 ____________________ The use of intestinal brine obtained by immersion of intestinal mucus-liberated animals in boiling salt solutions with molar saturation amount of boiling salt at room temperature in a slaughterhouse, to produce high purity heparin. 20 25 1 35