DE1069329B - Process for the production of an injectable permanent proteolytic enzyme preparation - Google Patents

Description

Method of making an injectable resistant proteolytic Enzyme preparations The invention relates to the stabilization of proteolytic Enzymes.

More recently, trypsin has been used in more acute cases Thrombophlebitis and phlebothrombosis recommended. For parenteral administration however, difficulties arose from trypsin preparations.

Trypsin is relatively unstable in aqueous solution and as a result solutions must be freshly prepared immediately before use. It was further found that intramuscular administration of trypsin in aqueous solution, aqueous isotonic solution or oily suspension often causes considerable pain at the injection site.

It has now been found that trypsin preparations, the aqueous gelatin contain, are significantly more stable and also those mentioned, at the injection site largely reduce the pain that occurs. There is still no binding one Theory for explaining this fact; but one can assume that the pain reduction is due to the fact that the trypsin digests the gelatin rather than the tissue, which is injured during the injection, and thereby the pain factor is considerable belittles. It is also believed that the presence of polypeptides leads to a Shifting the balance between effective and ineffective trypsin, and that an improved durability is achieved thereby. The latter is extreme important because the effectiveness of trypsin is known to decrease rapidly in aqueous media.

It has also been found that proteolytic enzymes in general be stabilized by such a combination with aqueous gelatin. This here The term "proteolytic enzyme" used includes the trypsin itself, as well as the related proteolytic enzymes like chymotrypsin, pepsin and bromelin as well also streptokinase. Trypsin is preferably used as the proteolytic enzyme or chymotrysin, but especially trypsin.

The gelatin used in the practice of the invention should be sterile, be free of fever pathogens and without antigenic effects. This gelatin is common and known (e.g. Knox 250 A or Pharmagel A). It is usually made from purified Beef bone or pig skin collagen is made and is commercially available as drier Solid available. The gelatin can be used in aqueous solution and should be partially hydrolyzed (e.g. by heating in dilute acid). So you can get an aqueous solution of partially hydrolyzed gelatin (polypeptide hydrolyzate) produce by adding 22 percent by weight gelatin (e.g.

Knox 250 A or Pharmagel A) in warm 0.001 N hydrochloric acid solution, the 0.01 volume percent propyl para-oxybenzoate and 0.09 volume percent methyl para-oxybenzoate contains, dissolves the mixture for 2 to 5 hours at 100 "C or 1 to 3 hours heated at 1200 C in an autoclave, then filtered and with distilled Water containing the para-oxybenzoic acid ester in the concentrations given above contains, diluted to the desired concentration.

The pE value is adjusted to a value of with additional hydrochloric acid 3.2 to 4.8, preferably from about 3.6, set. Generally a 50 /, ige aqueous solution can be used. But you can also use more or less concentrated solutions use. The gelatin solution can also be prepared in such a way that Dissolves 10 percent by weight of gelatin in warm 0.01 N hydrochloric acid solution, which is 0.01 percent by volume propyl para-oxybenzoate and 0.09 volume percent methyl para-oxybenzoate contains. The solution obtained in this way is heated to 80 to 100 ° C. and held for 1 hour long at this temperature. It is then filtered and the filtrate obtained adjusted to a pH of 3.2 to 4.8 with hydrochloric acid. Then you dilute the filtrate with water, the 0.01% para-oxybenzoic acid propyl ester and 0.09% contains methyl para-oxybenzoate, to a 50% strength solution.

When preparing the gelatin solution, you can use the above used hydrochloric acid also other suitable, non-reactive strong acids, such as phosphoric or lactic acid. However, it is important that the pE value the finished gelatin solution 3.2 to 4.8, preferably about 3.6, amounts to. The para-oxybenzoic acid esters are used as protective agents, and on Instead of the methyl and propyl esters mentioned, you can also use other parabens or other means of protection such as B. phenol, a cresol, p-chlorocresol or thimerosal use.

Injectable preparations can be prepared in such a way that crystalline trypsin, e.g. B. in distilled water or preferably in a small one Dissolves amount of 0.01 N hydrochloric acid and then an aqueous, partially hydrolyzed, sterile, fever-free, non-antigenic gelatin solution incorporates the for example as described above. Lyophilized trypsin, by dissolving the trypsin in water or 0.01 N hydrochloric acid, allowing it to freeze and Freeze-drying is produced, you can easily directly in a 5% aqueous, partially hydrolyzed, sterile, fever pathogen-free, non-antigenic gelatin solution dissolve to form a composition suitable for injecting. The received The composition is adjusted to a pE value by further addition of acid or alkali of about 4, and it can be used to maintain this pE value the addition of acid or alkali can be buffered. For setting the pE value hydrochloric acid or sodium hydroxide are generally used. But you can too other acids, e.g. B. phosphoric or lactic acid and other alkalis, e.g. B. Potassium Hydroxide, use. The composition is in an acidic pE value range from 3.2 to 4.8, preferably held at 3.6. Protectants such as propyl para-oxybenzoate (about 0.01 to 0.02 percent by weight) and methyl para-oxybenzoate (about 0.09 up to 0.18 percent by weight) can also be added.

According to another embodiment of the invention, trypsin and prepare gelatin ingredients separately and both immediately before use unite. Here the trypsin can be used as such. Preferably there However, enough unreactive, strong acid is added to the trypsin (e.g.

Q.01 n-hydrochloric acid) to produce a solution with a pE value of about 2 to 3 manufacture. The solution obtained is then introduced into by freeze-drying Easily soluble product ready for mixing with the gelatin solution. The gelatin component becomes in the manner described above (i.e. by dissolving the partially hydrolyzed gelatine, polypeptide hydrolyzate -), in dilute acid, Adding protective agents, heating, filtering, diluting and then adjusting the p made to 3.2 to 4.8.

According to a further embodiment of the invention, a lyophilized Combination of trypsin and gelatin for mixing immediately before use Prepare with an aqueous solvent.

This can be done by getting a solution of trypsin freeze-dried in gelatin, a protective agent (e.g. B. 0.1 01o para-oxybenzoic acid ester) or acid (z. B. hydrochloric acid) can, but it can also be isotonic saline solution. It is definitely important that the finished composition made by mixing the trypsin gelatin component is formed with the aqueous solvent, a pH of 3.2 to before use 4.8, preferably 3.6.

Administration by injection is preferably intramuscular using 21/2 to 5.0 mg of trypsin or other proteolytic Enzyme (where 1/2 or 1.0 ccm of the composition has a concentration of 5 mg trypsin / ccm carried out 1 to 4 times or more per day, as required.

The invention will now be explained in more detail by means of examples. All Unless otherwise stated, parts given therein relate to parts by weight.

Example 1 To a 5% aqueous solution of non-antigenic, fever pathogen-free, partially hydrolyzed gelatin (Knox 250 A), which can be obtained by heating for 3 hours the gelatine in 0.001 N hydrochloric acid at 100 ° C, are about 5 mg of crystalline trypsin are given per ccm of solution. Add to the mixture obtained then about 0.09 01o para-oxybenzoic acid methyl ester and 0.01 ° lo para-oxybenzoic acid propyl ester. After the mixture has been uniformly stirred, make the p, value by adding hydrochloric acid or sodium hydroxide to a value of 3.6 a. You can of course also achieve a pE value of 3.2, 3.5 or 4.5, by changing the amount of acid or alkali accordingly. The trypsin concentration can be changed by adding the amount to the gelatin solution Enzyme varies. The product adjusted to a pE value of 3.2 to 4.8 can used as such for injections of the usual type.

Example 2 a) Gelatin Solvent Gelatin (Knox 250 A) 3000 g propyl paraben .................. 6.0 g methyl paraben .................. 54.0 g concentrated Hydrochloric acid (36.5 to 38%) ................ 90 cc of distilled water, for total amount of the solution of ................. 60 liters. Bring 551 of water to the boil and admits propyl paraben and methyl paraben. Then you add the hydrochloric acid to the aqueous solution and mixes until a uniform mixture is achieved. The gelatin is then added and the mixture obtained is heated to 80 to 100 ° C. and held at this temperature for 1 hour.

Then the value is brought to 3.5 with additional hydrochloric acid and dilute the solution to the appropriate volume with distilled water.

30 g of a sterile, washed filter aid are then added (Hi-Flo) and filter the solution through Whatman # 52 paper.

The solution is then filled into ampoules and in quantities of 5.8 ccm sterilize them for 1/2 hour at 100 ° C. b) Lyophilized Trypsin Crystallines Trypsin .............. 125 g 0.01 N hydrochloric acid, for the total amount of the solution of ................ 5000 cc. The trypsin is dissolved in the hydrochloric acid and washed through Whatman No. 52 paper filtered. It is then filtered through a Selas 02 filter into a sterile one Container.

1 ccm is placed in each ampoule under aseptic conditions filled, followed by lyophilization.

For the purpose of the injection, 5 ccm of gelatine solvent a) mixed with 25 mg of lyophilized trypsin b).

Example 3 a) aqueous solvent propyl para-oxybenzoate ... 6.0 g of methyl para-oxybenzoate .... 54.0 g of distilled water, for total amount the solution of ................ 60 liters The para-oxybenzoic acid esters are dissolved in the distilled water, and the resulting solution is through a medium sintered glass filter filtered, then in quantities of 5.8 ccm in Ampoules filled and sterilized for 20 minutes at 120 "C. b) Trypsin gelatin, lyophilized A gelatin solution is prepared as described in Example 2, only that the gelatin concentration is increased to 25%.

Then prepare a trypsin solution as described in Example 2, and filter it through a bacterial filter (Selas 02) into a sterile container.

Under sterile conditions, 5 l of the above-described are sterile Gelatin solution is added to the trypsin solution and the p-value is adjusted to 3.6.

2.0 cc of the solution obtained, the 25 mg of trypsin and 0.5 g of gelatin are filled into ampoules and the contents lyophilized in the usual manner.

For injection purposes, 5 cc of the aqueous solvent a) are added the lyophilized trypsin gelatin mixed.

The compositions of the invention are particularly useful for alleviating inflammatory and edematous manifestations, especially thrombophlebitis and Phlebothrombosis, can be used. You can also help treat others Inflammatory diseases can be applied. This is how they turned out to be in the treatment from iritis and other eye infections as well as diabetic and other leg ulcers as useful. If used intramuscularly, a deep injection is preferred. The syringes used for this purpose are preferably those with no. 20 needles.

The injection is usually given slowly into the gluteal muscle. the Compositions can also be used in the same way to dilute mucous secretions used in the upper airways, such as those found in bronchial asthma will.

With regard to the durability test, a solution of 3 mglccm crystalline trypsin in 50/0 but Gelatin with a pE value of 4.1 according to the usual Hemoglobin digestion processes (especially that of Northrop, Kunitz, and Herriot, »Crystalline Enzymes, Columbia University Press, 1948, p.107). The test results showed that after 2 weeks at room temperature there was no change in trypsin potency had occurred. Studies have also been carried out on a method that essentially that of Fabinyi-Szebehelz, Brit. J. Pharmacol, Vol. 8, [1953] p. 30, corresponds to also using the above-mentioned solution, and the obtained Data showed that the effectiveness of trypsin in gelatin was similar to that of trypsin in Ö1 (Parenzyme) was comparable.

PATENT CLAIM 1. Process for the manufacture of an injectable, stable proteolytic enzyme preparation, characterized in that one partially hydrolyzed, sterile, fever pathogen-free, non-antigenic gelatin and incorporates a proteolytic enzyme into an aqueous medium and adjusts the pE value so that that the aqueous solution obtained has a value of 3.2 to 4.8.

Claims (1)

2. The method according to claim, characterized in that the enzyme as such or an aqueous solution of the enzyme with an aqueous solution of the gelatin united. 3. The method according to claim, characterized in that the enzyme adding in lyophilized form to an aqueous solution of the gelatin. 4. The method according to claim 1, characterized in that one is a Mixture of the enzyme and gelatin in lyophilized form to the aqueous medium gives. 5. The method according to any one of claims 1 to 4, characterized in that that the proteolytic enzyme is trypsin. ~~~~~ Publications considered: Swiss patent specification No. 90 689.