FI65073B - FOERFARANDE FOER FRAMSTAELLNING AV HEPARIN - Google Patents

Description

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Federal Republic of Germany-Förbundsrepubliken Tyskland (DE) P 20009 ^ 3.0 Verified-Styrkt (71) Schering Aktiengesellschaft, Berlin / Bergkamen, DE; Mullerstrasse 170-178, 1 Berlin 65, Federal Republic of Germany-Förbundsrepubliken Tyskland (DE) (72) Hans-Jörg Vidic, Berlin, Federal Republic of Germany-Förbundsrepubliken lyskland (DE) (7 * 0 Oy Kolster Ab (5 * 0 Method for the production of heparin - For the purposes of this Regulation,

The invention relates to a method to obtain a heparin salt solution that is formed during work-casings (intestinal salt solution).

Heparin has long been available to medicine as a naturally occurring anticoagulant, which is also necessary for modern medicine and is increasingly used.

In recent years, there has become known a number of methods, which all have the aim to isolate the active ingredient in heparin are increasingly saving manner heparin-containing animal tissues such as the lung, liver and later time especially intestine of pigs, cattle and sheep, in the cytoplasm (mucosa) in British Patent No. 754 885, DE Patent Publication 1,228,241, U.S. Patent 3,058,884, DE Patent 1,253,868). Such raw materials are perishable, which limits their storage, which, however, can be improved by freezing. However, this will lead to a significant increase in costs. Furthermore, the treatment often causes difficult problems for both the factory workers and the residents of the surrounding area due to the odor caused by the treatment. DE-A-1 253 868 describes the recovery of heparin in connection with some conventional methods. According to this, the animal tissue is initially contacted with hot aqueous saline to dissolve the heparin from the cells. The amount of heparin in the resulting medium is very small.

It was now found that the saline solution formed during the cleaning of mucus in slaughterhouses contains heparin. This salt solution was formed when the casings, which has in advance been removed by the intestinal mucus, is set to the preservation of water and removal of the saline.

In view of DE-A-1 253 868, it can be considered surprising that the saline solution formed when keeping the intestines of animals in saline contains larger amounts of heparin. Nor was it expected that this heparin was almost completely free of chemically related by-products, so saline, hitherto known only as an environmentally hazardous waste product, is a new source of raw material for already very little heparin due to lack of raw materials. Due to the low content of impurities, further processing to crude heparin can be performed with particular savings, thus avoiding the cumbersome isolation methods known in the art (see U.S. Patent 3,451,996 and GB 1,221,784). In addition, grades of heparin are obtained which have not yet been found among the heparin preparations available on the market. Further processing of the known methods used in accordance with the invention, the isolated intestinal salt solution used for soaking raakahepariinin the purity is already such that the final purification of heparin results in products containing more than 200 USP units / mg.

65073 3 Heparin preparations marketed to date generally contain 150 USP units / mg, and in some rare cases 155 USP units / mg. In this case, one USP unit / mg refers to the specific activity that results from U.S.P. an assay to measure the formation of clots in preserved sheep plasma. 1 USP unit corresponds to about 1.1 International Units (UI). Because the activity values of commercial heparin preparations are still unsatisfactory despite numerous improvements in the method, the USP, for example, dictates that heparin preparations (derived from intestinal mucus) must contain at least 140 USP units. L.W. Kananagh and L.B. Jaques / Arzneimittel-Forschung (Drug Res.) 24, No. 12, 1942 (1974) _7 has so far only been able to isolate heparin up to 175 USP / mg active in the laboratory by crystallization several times as the barium salt.

Saline solutions with a salt salt content of 3 molar to a saturating arrow are particularly suitable for preserving the intestines and removing water from animals. By impregnation molar amount is meant the molar amount contained in the saturated aqueous saline solution at the slaughterhouse temperature. (Saturated aqueous NaCl is about 26.4%).

Generally, the intestines of animals from which the mucus has been removed are treated with saline for 2 hours to 3 days. In most cases, however, the treatment of the intestines of the animals is completed after only 3-24 hours. After the preserved intestines of the animals, which have been completely dehydrated, are removed from the saline solution, a saline solution containing a surprising amount of heparin is obtained.

By using such intestinal saline solutions as a raw material in accordance with the invention, it was also possible to isolate heparin as pure according to the technical standard so that it appeared as an almost colorless substance. In contrast, of all the crude heparins obtained so far from other raw materials, it is first necessary to decolorize by cumbersome and disadvantageous methods (U.S. Patent No. 3,179,566).

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In addition, the saline solution for intestinal processing is a storable, almost odorless raw material, the quality of which does not suffer during storage of a single piece. In this case, chemical and other preservation methods are eliminated.

The invention thus relates to a process for recovering heparin from a saline solution obtained from the treatment of animal tissue, which is diluted with water in a ratio of saline to water of 1: 3 to 1: 5, and from which heparin is separated in a manner known per se. which is obtained by soaking the slaughterhouses processing at ambient temperature for cooking salt solution with a concentration between 3 M and a saturated animal intestines, from which the intestinal mucus has been removed.

Example 1 600 L of saline was added to one 500-liter mixer-evaporator at room temperature for half an hour with stirring in 600 liters of methanol. The mixture was stirred further for half an hour, after which the mixture was allowed to stand for about 1 hour.

The cloudy solution on top was decanted into another vessel, leaving a precipitated salt cake. The resulting suspension was now allowed to stand for two days. It was then decanted again, the supernatant was discarded and the precipitate was isolated by centrifugation. The precipitate was dried in vacuo to give 1.44 kg of product containing about 10 USP units / mg. The product still consists of about 45% common salt. An aliquot of the crude biparine thus obtained, corresponding to about 425,000 USP units, or 42.5 g, was extracted three times with 200 ml each of 2 M saline, stirring for 1 hour at 60 ° C each time. The combined extracts were diluted with water until the solution contained about 0.9 mol / L chloride ions. 200 ml of anion exchanger (Lewatit CA 9 249) in chloride form were now added and stirred for 1 hour. Finally, it was suction filtered and washed with about 200 ml of 0.9 M saline. The ion exchanger was then stirred for 5 hours with 2-M brine at 40 ° C and then suction filtered and washed with 2-M brine. The elution solution was combined with the washing solution and precipitated with 1.5 times the volume of methanol. The precipitate was centrifuged, washed first with about 50 ml of water-methanol (1 + 1.5 vol), then with about 50 ml of methanol and dried.

198 g of Na-heparinate containing 195 USP units / mg were obtained. 15.5 g of sodium heparinate obtained as described above, containing about 200 USP units / mg, was dissolved in 200 ml of 2-M saline solution. The solution was filtered off with suction and the residue was washed first with 30 ml of 2-M brine, then with about 200 ml of completely desalted water. The chloride molarity of the combined solutions was adjusted to 0.9 with completely salt-free water. Purification was then performed as described above with 1 liter of Lewatit CA 9 249.

The combined elution and washings were further treated as described above. After washing and drying the precipitate, 11.2 g of sodium heparinate having an activity of 251 USP units / mg were obtained.

Example 2 As described in GB 754 885, 3 liters of brine was adjusted to pH 3.1-3.2 with acetic acid (about 50 ml). After a few hours, the residue was decanted and the residue was centrifuged. The precipitate was discarded, the combined solutions were neutralized with NaOH solution.

The same volume of methanol was now slowly added with stirring. After 45 minutes, the suspension on top of the precipitated saline was decanted. The suspension was allowed to stand for several hours, the supernatant was decanted and the residue was centrifuged. The precipitate was dried in vacuo. 3.43 g of product containing 21 USP units / mg were obtained. An aliquot of the crude heparin thus obtained, corresponding to about 425,000 USP units, i.e. 20.2 g, was purified as described in Example 1. 10.65 g of sodium heparinate with an activity of 262 USP units / mg were obtained.

65073 e

Example 3 3 liters of brine was diluted with 12 liters of water until a chloride molarity of 0.85-m was reached. Then 50 g of diatomaceous earth and a solution of 6 g of benzethonium chloride ("Hyamine 1 622", Rohm & Haas) in 100 ml of water were added. After a few hours, the mixture was decanated and the residue was filtered off with suction. The precipitate was washed with water and extracted wet three more times by the method of DE-A-1,227,241 in each case with 200 ml of 2-M saline solution. The combined extracts were precipitated with 2 volumes 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 until a chloride molarity of about 0.85 m was reached (cf. DE patent publication 1,156,938). The solution was acidified with acetic acid (about 50 ml) to pH 3.2. After a few hours, a precipitate had precipitated, the solution was decanted and the precipitate was centrifuged. After drying, 111 g of crude heparin containing 6.5 USP units / mg were obtained. An aliquot of the crude heparin thus obtained, corresponding to about 425,000 USP units or 65.5 g, was first purified as in Example 1. After the first ion exchange purification step, 196 g of Naheeparinate containing 198 USP units / mg were obtained.

15 g of sodium heparinate obtained as described above, containing about 200 USP units / mg, was dissolved in 200 ml of 2N calcium chloride solution. The solution was filtered off with suction and the residue was washed first with 30 ml of 2N calcium chloride solution, then with about 200 ml of completely salt-free water. The chloride molarity of the combined solutions was now adjusted to 0.9 with completely desalinated water. The above mixture was now mixed with Lewatit CA 9 249 (1 liter). Elution and washing were performed with 2N calcium chloride solution instead of brine.

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The combined elution and washings were further treated as described above. The precipitate was washed and dried to give 10.7 g of calcium heparinate having an activity of 260 USP units / mg.

Example 5 10 liters of brine was diluted with 40 liters of water until a chloride molarity of 0.85-m was reached. This solution was pumped through a filter through an ion exchange column 26 mm in diameter and 380 mm in length (approximately 3 liters / hour). In this case, a medium-strength large-pore anion exchange resin in the Cl form (Lewatit CA 9 249) was used. Other anion exchange resins can also be used, such as Dowex 1-X-1 described in DE-A-1 253 868.

Finally, the resin was removed from the column, washed twice with 500 ml of 0.9 M saline and eluted with 400 ml of 2 M saline for 5 hours at 40 ° C with stirring. This solution was precipitated with 1.5 volumes of methanol. The isolated and dried precipitate weighed 1.2 g and contained 149 USP units / mg.