CH638818A5 - Use of intestinal brine for producing heparin. - Google Patents

Description

638818

2nd

PATENT CLAIM Use of intestinal brine, which has been obtained by placing intestinal mucus freed from intestinal mucus in saline solutions, for the production of pure heparin.

The invention relates to the use of intestinal brine, which is obtained by placing animal intestines freed from intestinal mucus in saline, for the production of pure heparin.

With heparin, medicine has long been a naturally occurring anticoagulant, which is still indispensable in modern medicine and is being used more and more.

In recent years, a number of processes have become known, all of which aim to use heparin more and more gently from animal tissue containing heparin, such as the lungs, liver, and more recently from intestinal mucus (pigs, cattle and Isolate sheep (GB-PS 754885, DE-PS 1228241,

U.S. Patent 3,058,884, DE Patent 1253,868). Because of their perishability, such raw materials have the disadvantage of a limited shelf life, which can be extended by freezing, but this also leads to considerable cost increases in addition to additional technical outlay. During the refurbishment, both the operating staff and residents from the surrounding area are often faced with unreasonable problems due to the associated odor nuisance.

DE-PS 1253 868 summarizes the production of heparin for some common processes. Then the animal tissue is brought into contact with a hot aqueous salt solution in the initial stage in order to remove the heparin from the cells. The heparin portion of the medium is extremely low.

It has now been found that brine obtained in the slaughterhouses and intestinal mucilages contains heparin. This brine usually arises when animal intestines that have previously been freed from intestinal mucus are placed in saline solutions for preservation and drainage.

With regard to DE-PS 1253 868, it is therefore extremely surprising that the brine formed when animal intestines are placed in saline contains large amounts of heparin. It was also not to be expected that this heparin would be almost completely free of chemically related by-products, so that brine, previously only known as an environmentally harmful waste product, represents a new source of raw material for heparin, which is already very scarce due to raw material shortages. Due to the largely lack of impurities, the processing to crude heparin can be carried out particularly gently, and the elaborate isolation methods known from the prior art are thus eliminated (see US Pat. No. 3,451,996 and GB Pat. No. 122,1784). Ultimately, heparin qualities are achieved that are far from being achieved in the heparin preparations available on the market. The raw heparin isolated as raw material according to known processing methods from intestinal brine used as raw material are already in such purity that the subsequent purification leads to heparins with well over 200 USP units / mg. The heparin preparations commercially available so far generally have heparin qualities of 150 USP units / mg, in a few cases also 155 USP units / mg. A USP unit / mg is the specific activity that results from the U.S.P. assay with which the inhibition of the

Lump formation from preserved sheep plasma is measured. 1 USP unit corresponds to approximately 1.1 international units (UI). Since the activity values of the commercially available heparin preparations are still unsatisfactory despite numerous process improvements, z. B. USP stipulates that heparin preparations (from intestinal mucus) must contain at least 140 USP units. Only in the laboratory has L.W. Kananagh and L.B. Jaques [Drug Research (Drug Res.) 24, No. 12, 1942 (1974)] to isolate heparin by repeated crystallization as a barium salt with a maximum of 175 USP-E / mg.

For the preservation and drainage of animal intestines, saline solutions are particularly suitable, the saline content of which is 0.5 m to the saturation molar amount. Those saline solutions whose saline content is 3 m to the saturation molar amount are particularly preferred. Saturation molar amount is the molar amount of a concentrated aqueous solution of dissolved common salt at room temperature in a slaughterhouse. (A concentrated aqueous saline solution is approx. 26.4%.)

A concentrated NaCl solution is created e.g. also by sprinkling the dripping animal casings with table salt.

As a rule, the degummed animal intestines are treated with saline solutions for 2 hours to 3 days. In most cases, however, the treatment of the animal intestines ends after 3-24 hours. After removing the preserved and drained animal intestines from the saline solution, an intestinal brine is obtained which has a surprisingly high heparin content.

By using such intestinal brine as raw material according to the invention, it was possible to isolate heparin on a technical scale in a purity that it is present as an almost colorless substance. On the other hand, all crude heparin available from other raw materials to date only have to be decolorized by complex and lossy processes (US Pat. No. 3,179,566).

In addition, intestinal brine is a storable, almost odorless raw material that does not lose any quality due to long storage. This eliminates the need for chemical and other conservation methods.

The invention thus relates to the use of intestinal brine, which has been obtained by placing animal intestines freed from intestinal mucus in saline solutions, for the production of pure heparin. Pure heparin can be isolated from this intestinal brine by known methods.

example 1

600 liters of brine were mixed in a 1500 liter stirrer at room temperature with stirring within half an hour with 600 liters of methanol. The same time was stirred, after which the mixture remained for about an hour.

Now the cloudy supernatant was decanted into a second vessel and the failed salt cake was left behind. The suspension obtained now remained for two days. The mixture was then decanted again, the supernatant discarded and the precipitate isolated by centrifugation. After drying in vacuo, 1.44 kg of a product was obtained which has approximately 10 USP units / mg. It still consists of about 45% table salt. An aliquot of the crude heparin obtained in this way, corresponding to approximately 425,000 USP-E or 42.5 g, was extracted three times with 200 ml of two-molar sodium chloride solution, stirring in each case at 60 ° C. The combined extracts were diluted with water until the solution contained about 0.9 mol / 1 chloride ion. 200 ml of an anion exchanger (Lewatit CA 9249) in the chloride form were then added and the mixture was stirred for one hour. Then s

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was suctioned off and washed with about 200 ml of 0.9 molar saline. The ion exchanger was then stirred for five hours with two-molar sodium chloride solution at 40 ° C. and then suctioned off and washed with two-molar sodium chloride solution. 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 about 50 ml of water-methanol (1 + 1.5 parts by volume), then with about 50 ml of methanol and dried.

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

15.5 g of a sodium heparin containing about 200 USP units per mg obtained in the manner described above were dissolved in 200 ml of two-molar saline. The solution was sucked through a suction filter and the residue was washed first with 30 ml of two-molar sodium chloride solution, then with about 250 ml of completely deionized water. The combined solutions were then brought to a chloride molarity of 0.9 with deionized water. The cleaning with one liter of Lewatit CA 9249 was then repeated as above.

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

Example 2

Similar to British patent 754 885, 3 liters of brine were acidified to pH 3.1-3.2 (approx. 50 ml) with acetic acid. After a few hours it was decanted and the rest centrifuged. The precipitate was discarded, the combined solutions were neutralized with sodium hydroxide solution.

The same volume of methanol was then slowly added while stirring. After 45 minutes, the precipitated table salt was decanted. This suspension was left to stand for several hours, the supernatant decanted and the rest centrifuged. The precipitate was dried in vacuo. 3.43 g with 21 USP units / mg were obtained. An aliquot of the crude heparin obtained in this way, corresponding to approximately 425,000 USP-E or 20.2 g, was cleaned as in Example 1. 10.65 g of sodium heparin was obtained, with an activity of 262 USP units per mg.

Example 3

3 liters of brine were diluted with 12 liters of water to a chloride molarity of approximately 0.85 m. Then 50 g of diatomaceous earth and a solution of 6 g of bezethonium chloride (= Hyamin 1622, Rohm & Haas) in 100 ml of water were added. After a few hours, it was decanted and the rest was suctioned off. After washing with water, the still wet precipitate was removed three times according to the procedure

638818

German Patent 1228 241 extracted with 200 ml of 2 molar saline. 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

30 liters of brine were diluted with 120 liters of water to a chloride molarity of approximately 0.85 m (cf. DE-PS 1 156 938). The mixture was then acidified to pH 3.2 with acetic acid (approx. 50 ml). After a few hours, a precipitate had settled, which was centrifuged off after decanting the clear supernatant. After drying, 111 g of crude heparin with 6.5 USP units / mg were obtained. An aliquot of the crude heparin obtained in this way, corresponding to approximately 425,000 USP units or 65.5 g, was first purified analogously to Example 1. After the first purification stage using ion exchangers, 196 g of Na heparinate with 198 USP-E per mg were obtained.

15 g of a sodium heparinate obtained in the manner described above with about 200 USP units per mg were dissolved in 200 ml of two-molar calcium chloride solution. The solution was sucked through a suction filter and the residue was washed first with 30 ml of two normal calcium chloride solutions, then with about 250 ml of completely deionized water. The combined solutions were then brought to a chloride molarity of 0.9 with deionized water. Now one liter of Lewatit CA 9249 was used for stirring. Elution and washing were carried out with two molar calcium chloride solution instead of saline.

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

Example 5

10 liters of brine were diluted with 40 liters of water to a chloride molarity of approximately 0.85 m. This solution was pumped through a filter over an ion exchange column with a diameter of 26 mm and a length of 380 mm (approx. 3 liters / hour). A medium-based macroporous anion exchange resin in the Cl form was used (Lewatit CA 9249). Other anion exchange resins can also be used, e.g. the Dowex 1-X-1 described in German Patent 1,253,868.

The resin was then removed from the column, washed twice with 500 ml of 0.9 molar sodium chloride solution and eluted with 400 ml of 2 molar sodium chloride solution 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 had 149 USP units / mg.

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