WO1989006538A1

WO1989006538A1 - Process for producing a therapeutically active ingredient, in particular for cicatrization or for treatment in geriatry, and a therapeutic preparation containing said active ingredient

Info

Publication number: WO1989006538A1
Application number: PCT/AT1989/000003
Authority: WO
Inventors: Gerhard Hantich; Johanna Krenauer; Volker Eisenreich; Ernst Hesse
Original assignee: Gebro Broschek Kg Pharmazeutische Fabrik
Priority date: 1988-01-13
Filing date: 1989-01-13
Publication date: 1989-07-27
Also published as: EP0396597A1; AT392003B; JPH03505200A; ATA6188A

Abstract

In a process for producing a therapeutically active ingredient, in particular for cicatrization or treatment of cerebrovascular insufficiency, defibrinated blood of young bovines or calves is subjected to enzymatic papain hydrolysis at a pH of 6.5 to 7.4. The reaction is stopped with cold water and ultrafiltration up to 10 000 D is carried out. The filtrate is concentrated, mixed with alcohol, allowed to stand for at least 24 h and then filtered. The preparations obtained possess appreciably improved cicatrizing and geriatric activities.

Description

Process for the production of a therapeutically active agent, particularly for wound healing or for treatment in geriatrics, and a therapeutic preparation containing such an agent

The invention relates to a method for producing a therapeutically active substance, particularly for healing or for treatment in geriatrics, in which defined blood from warm-blooded mammals, in particular from young cattle or calves, undergoes enzymatic hydrolysis with papain subjected, filtered, the permeate concentrated and treated with alcohol, preferably ethanol.

Numerous methods have been known for many years in order to extract preparations with wound healing, cell respiratory or metabolism and growth-promoting properties from blood or blood fractions or from organ ho ogenisateπ of mammals or humans. In many cases, the exact composition of these preparations was largely unclear, but the effects of these had proven to be beneficial for the treatment of poorly healing wounds, such as burns, leg ulcers and the like, or for improving cerebral blood flow. A common feature of the manufacturing process of this

Preparations usually consist of defibrinated, often also hemolyzed, blood from usually young slaughter animals, especially calves, young cattle or horses, in the usual way to free cell substances, minerals and / or protein and to concentrate the extracts as a whole or fractions thereof and to formulate as active ingredient preparations. In the known processes, deproteinization or deproteinization takes place by heating or centrifuging or filtering, or else by the action of acids or alcohols and subsequent centrifugation and / or filtration of the precipitated proteins. A deproteinization by heating and subsequent filtration with the aid of a membrane separation process is described in EP-A 95 170, the continuous membrane multi-stage ultrafiltration using membranes with a molecular exclusion limit of more than about 8000 daltons being used as part of the membrane separation process - Wise separation of the inorganic salts is carried out by electrodialysis with membranes with a permeability of up to about 300 daltons. EP-A 140 134 describes a similar process for the production of a biologically active extract from organs of mammals and from cell cultures, in which the starting material is comminuted with digestion of the cells, rapidly heated to a temperature of 70 to 90 °, cooled, centrifuged, from the solution obtained by ultrafiltration, the substances with a molecular weight of more than 10,000 D are removed and the remaining solution is removed by electrodialysis, the salt ions. Thymus, spleen, liver and other organs, as well as, are examples of the starting material for this * method

10 mammalian blood.

From AT-PS 274 240 it is known to use hemolyzed blood cells from the blood of young mammals, if necessary after complete drying or partial concentration to a higher dry matter content

, _c . Idπeπaustauscherverfahren, absorption chromatography,

High-voltage electrophoresis, high-voltage dialysis, gel filtration, thin-layer chromatography or counter-current distribution can be separated into fractions, of which those with a UV maximum at 245 to 246 mμ are used to isolate the active substance. The optional before

_"N of Fraktionstrenπung made concentration or drying can occur for example by ultrafiltration or by spray drying.

Finally, it is also known from EP-A 38 511 that there are biologically active substances that can be used for wound healing

Organs or body fluids from mammals are obtained in order

₂₅ r Glykosphiπgolipide is of the following formula:

30 NH I • CH ₃ - (CH ₂ ) _g -CH = CH-CH ₂ -C = 0 where R is an oligosaccharide which has 5 sugar groups. Hiebei this active component is by homogenization of the tissue or Kör¬ perflüssigkeit of the mammal with an aqueous o, 2% -igeπ NaCl solution, ± _xιgieren ^• 35 Zentι or filtration this Homogeπisats, Ultrafiltratioπ the obtained filtrate, concentration of the permeate, binding of organic materials from the concentrated permeate on activated carbon, desorption, 1 Purification of the same obtained by multiple chromatographing and elution of these organic substances.

In parallel with this development, there are a number of processes in which the defibrinated and, if appropriate, hemolyzed blood is hydrolyzed, prior to each further treatment, in order to produce the biologically active substance. is subjected to digestion with a proteolytic enzyme. Protein breakdown products that are then incorporated into the final supplements appear to promote and improve the effectiveness of the products obtained.

ECf According to US Pat. No. 2,912,359, defibrinated and hemolyzed blood with proteolytic enzymes is pre-digested with warming, dialyzed for desalination after cooling, acidified and the resulting precipitate, which is neutralized with lye, is obtained as an active ingredient.

According to DE-PS 1 076 888, defibrinated blood of young slaughter

1-5 animals are broken down by fermentation and / or subjected to a dialysis with water or alcohols after a fractional deprotection with aliphatic alcohols or acids to remove the higher molecular weight components. If necessary, the dialysate is freed from the organic solvents and gently concentrated.

20 ⁾ DE-OS 2 349 186 proposes to add a physiologically usable acid addition salt of heptaminol- (6-amino-2-methylheptan-2-ol) to the preparation obtained according to DE-PS 1 076 888 above to potentiate the effect of the preparation.

A method of the type described at the outset is also known

25 (DE-0S 1 949 195), in which the enzymatic digestion is carried out at a pH of 5 to 5.5, followed by partial deproteinization by heating to about 80 ° C. and subsequent filtration. Since it is not possible to achieve complete de-proteinization, a second de-proteinization stage is used, which is thermal in

SO ⁾ presence of Papain is conducted. This process is complicated and lengthy and difficult to carry out. There is a relatively high tendency towards turbidity in the end product if the preparations are stored in liquid form, the yields are not too favorable in relation to the blood used and the effectiveness of the active substance itself appears to be

S5 is not given everywhere in the desired manner.

The object of the invention is to avoid these disadvantages of the known methods, to eliminate the tendency to turbidity and to increase the yield and the effectiveness of the active ingredient with improved procedural economy. According to the invention, it is proposed that papain hydrolysis be carried out on u-hemolyzed blood with activated papain at a pH of 6.5 to 7.4, that the reaction is then stopped by cooling, that up to 10,000 D for deproteinization and removal of papain is ultrafiltered, and then the essentially protein-free permeate is concentrated to 1/25 to 1/35, based on the blood used, and left to cool for at least 24 hours, mixed with alcohol, and ^{■ is} finally filtered off.

In contrast to the last-described known methods a- enzymatic hydrolysis is carried out in non-hemolyzed blood and not applied ^{'ärmebehandlung} as long as protein in the solution. Rather, the method according to the invention results in heating only when the concentration is narrowed down and, if appropriate, also during sterilization of the end product, but there is no change in the product in either case. While in the known method a further removal of undesired substances is carried out in a complex manner by means of downstream column separation, this is not necessary in the method according to the invention, which may be a reason for the different effectiveness. While in the known process the end product has a maximum of the molecular weight distribution in the range of about 350 to 400, in the process according to the invention the molecular weight maximum is considerably lower than 300. The molecular weight distribution of the end product of the process according to the invention therefore differs significantly from that of known products. This may also be a reason for the different effectiveness. But another reason for this is also likely to be that ^'ysiertes the ER findungεgemäßeπ method as a starting point essentially unhämol blood is used, which contains so the red corpuscles Blutkör¬ still intact. A slight hemolysis, which occurs, for example, when the blood freezes, is irrelevant to the method according to the invention and has no influence on the end product, as studies have shown. The protein protein which is carried out in the process according to the invention by means of ultrafiltration and in which papain is also removed, since its molecular weight is greater than 20,000, also removes the hemoglobin (molecular weight greater than 50,000). 1 ", so that at the time of the alcohol addition there is no more protein in the solution, since protein molecules generally have a molecular weight of more than 12,000. It is therefore reasonable to assume that in the process according to the invention the alcohol precipitation for removal In contrast to this, the product obtained in the process according to the invention remains completely free of turbidity. The simplified process procedure is also advantageous since a hemolysis step is saved. This is to be understood in this way that in the course of the enzymatic hydrolysis with papain

I-CD hemolysis takes place, so that the two steps of hydrolysis and hemolysis take place in one. A later addition of water to facilitate ultrafiltration or to thermally stop the reaction no longer results in significant hemolysis.

In addition, an end product is achieved, which in comparison with

• C5 known preparations has an increased wound healing effect. In addition, a comparatively improved oxygen utilization is achieved. In particular, cerebral blood flow and peripheral arterial blood flow are increased in comparison to known preparations. Furthermore, the method according to the invention provides surprisingly high

ZLX booties based on the amount of blood used and is extremely economical in terms of process technology.

Another important advantage of the method according to the invention is that it is possible to standardize the clear-filtered mixture to the desired concentration, preferably by

25ό dilution.

In the method according to the invention, defibrinated blood from slaughter animals, in particular from calves or young cattle (Ms maximum 3 years) is preferably used as the starting material. This blood is pre-digested with an activated papain solution. Hiebei becomes a preparatory

3Ö-- commercial papain suspended in distilled water and freed of any solids by centrifugation. Commercial papain has an approximate molecular weight of 25,000, but it also contains low molecular weight substances with a molecular weight of less than 10,000. In order to avoid that these low molecular weight substances from the

3S Papaiπ get into the final active ingredient concentrate, the aqueous suspension is ultrafiltered. The concentrate is then mixed with cysteine solution, which activates the papain and uses it as an incubation solution. is applied.

The blood, which may have been stored in frozen form and in this case is thawed beforehand, is heated to 35-42 ° C in the incubation kettle, the pH is adjusted to 6.5-7.4 with 1N HC1 and preferably approximately with the incubation solution Incubated for 14 to 18 h. The completely incubated blood is mixed with fresh aqua dest.ad inject at a temperature of 0-10 ° C., whereby the enzymatic hydrolysis or pre-digestion is interrupted and the mass is diluted for the ultrafiltration. Before the ultrafiltration, the ultrafilter is usually prefiltered to protect it, in order to separate particles with a size of - 50 μm.

This is followed by ultrafiltration, during which samples are taken at regular intervals to check the filter tightness by means of trichloroacetic acid precipitation. The results must be negative. Ultrafiltration removes practically all of the protein from the solution. This could be confirmed by determining the molecular weight by means of SDS gel electrophoresis. From ultrafiltration, it is advisable to continue working under aseptic conditions. The permeate obtained is in the as soon as possible

Vacuum evaporator at 60 - 80 ° C to 1/25 - 1/33 of the original Blutvolumeπs concentrated. Precipitation occurs again, which is filtered off by a filter. However, this strong narrowing largely reduces the likelihood of reprecipitation. The - filtered concentrate is then mixed with about 2 1/2 times the volume of pre-cooled 96% ethanol and left in the cold, preferably at: cold room temperature (4-6 ° C). Instead of ethanol, methanol, propanol or butanol can also be used. After a standing time of at least 24 h, preferably about 48 h, X is filtered clear, which can be done with the help of a black band filter. The alcohol added for the precipitation is then distilled off in a Rotavapor. This usually leads to precipitation again, which is clear-filtered using black tape and glass fiber pre-filters.

The strong concentration before the alcohol precipitation makes it possible to carry out only a dry weight adjustment to about 200 mg / ml by dilution with the appropriate amount of fresh water for injection purposes. ^• * ^{■■ •} The finished active ingredient ^concentrate is filtered through a sterile filter and again through an ultrafilter and then into sterile

Bottled. In general, sterile filtration for germ removal is always required when the alcohol is withdrawn

5. becomes. The clear filtration is carried out to remove the precipitates.

By ultrafiltration before derÄikoholfälluπg ^■ that uneconomically large amounts of alcohol are used is avoided. The manipulation of smaller quantities is always easier and therefore cheaper.

The alcohol precipitation turns the solution into higher peptides

L-0- ^' separated, which could cause precipitation or turbidity in the end product through agglomerate formation. A product is obtained which is extremely stable in the liquid state and shows no reprecipitation in the pharmaceutical end product, even if it is autoclaved at higher temperatures, for example about 120 ° C.

-C5 The concentration is reduced to at least 1/25 - 1/33 of the original blood volume so that the product no longer has to be concentrated after the alcohol precipitation. After alcohol precipitation, only the alcohol is evaporated and the mass is diluted to the appropriate dry weight.

2 * © Although a molecular weight determination using SDS gel electrophoresis turns out to be negative for molecules with a molecular weight of more than 10,000, it is advantageous to carry out a second ultrafiltration at the end in order to obtain a reliably clean, sterile and pyrogen-free end product. Losses regarding

2 ^* 5 ^* "of the yield do not occur here.

It has been found that, without the interposition of an alcohol precipitate, prolonged storage of the active ingredient or the dilute ampoule solution leads to clouding and precipitation by peptide agglomerations, the formation of which occurs by shaking the solution

SCI ^* at 50 ° C or by storing the solution in the refrigerator at 5 ° C can be reduced to a few days. If the ultrafiltration is only carried out after the alcohol precipitation, the positive effect on the shelf life can no longer be determined without clouding the active ingredient, despite the concentration mentioned above. One more way

S ^* ****** ^* to determine the tendency to precipitate consists in autoclaving the finished product at 121 ° C. Here, without performing alcohol precipitation, turbidity occurs after a few days to weeks. ^{■ • **} A further advantage of the method is that trafiltratioπ by the corresponding pre-treatment with papain and the VcraTteiweißung by ultra- surprisingly, a product is obtained, which in the

Compared to the preparations on the market, much higher

5 yields of active substance with equal amounts of blood.

As already mentioned, the molecular weight distribution of the product produced in accordance with the invention differs significantly from that of products produced in a known manner. This was determined by comparative tests, the following products being used as comparison products:

Comparative Product I: A commercially available, protein and pyrogen-free product which is obtained from the blood of calves ready for slaughter. Comparative Product II: Another, commercially available product, 15 produced essentially by the process according to DE-PS 1076888. Comparative Product III: A product produced by the process according to DE-OS 1949 195. Method: Zσ column: TSK 2000 SW , 0.75 x 60 cm.

Guard column: Merck Hiber 100 Diol, 0.4 x 25 cm Sample volume: 20 ul

Pump: LKB 2150 HPLC pump with Rheodyne injector Flow: 0.5 ml / min 255 Pbratome er: LKB 2138 Uvicord S, 280 nm Recorder: Servogor S, 30 cm / h Mblekulargewichtsstandard:

Substance molecular weight retention time (min)

Tyrosine 181.19 49.6

Sft∑ Insulin Chain a 2.660 41.8

Ovalbumiπ 45,000 33.5

The results are shown in FIG. 1 in comparison to the measurement results of the product (IV) according to the invention. The diagrams show the full absorption AFS (absorptioπ fill scale) as a function of the retention time RT.

The comparative products I and II each show only a single main maximum with a retention time of 47.8 and 48.2 minutes, respectively. This corresponds to an approximate molecular weight of 350-400. 1 * same product I has one shoulder each with a retention time of

49.3 min and 56.1 min (corresponds to - _* ✓ MG 200 or MG <100), the comparison product II, however, only one shoulder at RT 56.2 (~ MG <100). The total substances detectable in the experiment are shown in a range from MG <100 to MG-v 2,500. Comparative product III, on the other hand, shows two main maxima, one at RT = 48.4 min (MG ~ 300), the second at RT = 52.4 min (MG <150), and furthermore three shoulders in the lower MG range (<100) .

The preparation (IV) according to the invention differs significantly from UCC from the substances mentioned hitherto. It has three distinct maxi a, the first maximum being in a lower molecular weight range than in the previously mentioned preparations, namely approximately at MG 200. This is probably achieved by the additional ethanol precipitation. The other two maxima are 115 at MG <150 and MG <100. In addition, the preparation according to the invention shows no shoulders in the low-molecular range, but two in the higher-molecular range at MG 500 and MG 300.

The mass maxima appearing to the left of the maxima in the curves for the comparative preparations I and II are due to the preservatives and therefore do not belong to the respective preparation.

The exact measurement results are summarized in Table 1 below:

Table 1: £ .. Maxima 255 retention time approximate molecular weight

(min)

SEGO comparative preparation m 48.4 52.4 300 150 e preparation IV 49.6 52.0 56.2 200 150 100 2nd shoulder

Approximate molecular weight (min)

Comparative preparation I covering preparation II comparative preparation m preparation IV according to the invention

^: 3.blekula__ weight of the total substances detectable in the experiment

Retention time approximate molecular weight

(min)

Comparative preparation I 43.0 - 58.5 2500- <100 Dressing preparation H 40.4 - 59.2 2700 - <100

Comparative preparation in 39.8 - 67.2 3000 - <100 eα ± preparation IV 39.5 - 63.5 3000 - <100 according to the invention

The product produced according to the invention was also tested in various test and examination series for its effectiveness in therapeutic terms. The exact test conditions will be given later.

When examining the effect of the preparation on the incorporation of ³ H-thymidine in DNA from human fibroblasts and liver cells, it was found that the preparation has a significantly stronger (5 to 10-fold) effect on the growth of both fibroblasts and compared to known products of liver cells. For this purpose, the regenerating effect of the preparation produced according to the invention on human foreskin fibroblasts was examined, which were damaged by the withdrawal of fetal calf serum. As a result, it was found that the preparation according to the invention, while increasing the DNA synthesis, clearly has a regenerating effect on damaged human fibroblast cells and that the concentration ranges and the inhibition range are similar to known products. For these examinations, human foreskin fibroblasts were mixed in the 8th 5 ^' to 10th passage with variable amounts of the product according to the invention or the known comparative preparation I and the ³ H-thymidine incorporation in DNA was measured. In order to stress the cells, after plotting, they were first allowed to grow to 1/2 to 2/3 confluence and then in artificial medium without the addition of fetal calf serum. an essential medium component, incubated for 6 to 9 hours. The medium was then changed to one which contained only small amounts of fetal calf serum and increasing amounts of the preparation according to the invention and ³ H-labeled thymidine or of the comparative preparation I. 5- This approach was incubated for 12 to 24 hours and the radioactivity vity in the DNA fraction determined after appropriate processing of the cells.

Fig. 2 of the accompanying drawing shows the results in diagram form, where the measurement curve for a product according to the invention is shown with full lines, that for the known comparative preparation I is shown with dashed lines. On the abscissa the preparation additions are indicated ^• in microliters, on the ordinate the radioactive count. 5 The determination of the regenerating effect on human hepatoma cells, which had also been damaged by the withdrawal of fetal calf serum, also showed an extremely favorable effect with an increase in DNA synthesis. The examination methodology corresponded to that of the fibroblast

ICC investigation, however, the increase was only measured compared to untreated controls. 3 shows the measurement results for the preparation according to the invention.

The investigation of the increase in cellular respiration in liver cells, which after homogenization is relatively low or relatively high

15 Eigenatmuπg have shown that the new preparation can produce a stronger activation than the known comparative preparation. In the area of maximum respiratory increase, both preparations work to a comparable extent. The measurement was carried out using the known Warburg method (described in GB-PS 824 375). In the following day

Belle ²⁰⁾ the measurement results for three different approaches to liver ho ogeπaten are given, whereby a comparison with both the known processes same preparation was carried out I, as well as to measurements without ^'preparation addition.

Table 2:

25

SEGD

heights (eg partial activity of damaged fibroblasts or increase in cellular, mitochondrial oxygen consumption). These cell-activating effects can, for example, be regarded as desirable in the context of wound healing. On the other hand, the activation of neoplastic cells would be undesirable. Investigations showed that, in contrast to the known comparative preparation I, the preparation according to the invention reduced oxygen. 1 - need of tumor cells (mouse ascites tumor cells) not activated. This was checked in the following experiment, in which the oxygen activity of mouse ascitic tumor cells was examined after addition of the preparation according to the invention or of the comparison preparation I: 5 Measurement of the oxygen consumption: by means of the "Clark" electrode suspension buffer: Na ₂ HP0 ₄ 0.04 Mol / 1, NaCl 0.08 mol / 1, gCl ₂ 0.005 mol / 1, pH 7.4 (3 ml per chamber) ^* Ascitic tumor cells: according to H0RNYKIEWICS and SATKE-EICHLER (drug research 6, 1956) 0.1 and 0 .05 ml per chamber; Preparation according to the invention: injection solution, Q._ 40 mg; dry weight / ml (0.2 ml / chamber)

Comparative preparation I: ampoules, commercial preparation (0.2 ml / chamber)

The results for the preparation (IV) according to the invention and for the comparison preparation (I) are shown graphically in FIG. An average of six determinations was determined for preparation IV according to the invention, and a mean of four determinations for comparative preparation I. It can be seen that, in contrast to the comparative preparation I, the preparation (IV) according to the invention not only does not increase the oxygen activity (cellular respiration) of mouse-ascite tumor cells, but even causes a decrease in the oxygen activity. It is particularly surprising that when the preparations according to the invention are used in geriatrics in patients with cerebral vascular insufficiency, treatment over four weeks shows a clear improvement in the flicker frequency analysis, furthermore in a psychological score determined by an evaluation score

255 total pressure and the geriatric assessment scheme according to Gottfries and: Gö LCries. The new preparation is therefore also excellently suited for the symptomatic treatment of psychopathological side effects - cerebral vascular insufficiency.

Furthermore, investigations were carried out using the WARBURG method,

30) to find out to what extent the preparation produced according to the invention actually causes an increase in the 0 -, - metabolism. The preparation produced according to the invention was compared with two commercially available products (comparison preparation I and II). A pronounced increase in breathing in the rat liver homogenate occurred in all three preparations

35., depending on the amount and / or the state of the active cell components, the results for a known preparation and the preparation produced according to the invention are similar until for the invented 1 preparation prepared in accordance with the invention was significantly better, but was reduced by up to 20% in the other known preparation.

It was found that the preparation produced by the process according to the invention compared to the first known preparation

5 4 times and in comparison to the second known preparation

1.4 times the yield of active ingredient could be obtained with the same amount of blood used.

Finally, a measurement of the cerebral blood flow or peripheral arterial blood flow (at the femoral artery) was carried out in -Q-baboons that were exposed to an artificial lack of oxygen. Such a measurement represents a recognized model for the evaluation of medications which influence blood flow to the brain or arterial blood flow (especially in the legs) (Meyer JS, Ishikawa S., Lee TK, J. Neurosurg. 21, 524 ( 1964)). * 5 A comparative investigation shows that the preparation according to the invention (IV) (4 ml / kg body weight intravenously as an injection solution) compared to the known comparative preparation I (4 ml / kg body weight intravenously as an injection solution) both the cerebral and promotes peripheral blood flow in the baboon much more. 0 ^'The measurement results are shown for the cerebral blood flow in Figure 5, the peripheral arterial blood flow in Fig.6.

Other cardiovascular parameters (blood pressure, heart rate, vascular resistance) did not change or changed only slightly, so that the preparation produced according to the invention has a specific pharmacological effect on the blood circulation areas mentioned.

The concentrate produced according to the invention can be processed for use in therapy according to methods known per se for injection solutions, tablets, dragees, wound ointments or gels. Therapeutic preparations according to the invention, which can be wound healing or geriatric preparations in particular, contain a concentrate, optionally after it has been dried, which was produced by the previously described method.

In the following, more precise manufacturing information and the description of the examination methods and their results for the preparation according to the invention are given.

Papai pretreatment and activation: Pretreatment of the papain is advisable in order to separate insoluble substances and to 1. To avoid that low molecular weight substances (MW - 10,000) from the Pa¬ paiπ get into the active ingredient concentrate.

200 g papayotin (from Extract-Chemie / activity: 80 U / mg) are added to 11 a.d. suspended and then centrifuged (centrifuge: Sorvell 5 RC-3, rotor: HG-4L: 3,000 rpm. 20 min).

Prefiltration for ultrafiltration (the solution should be free of particles> 10 μm) via: black filter or round filter 595, glass fiber pre-filter (from Sartorius), 8 μm membrane filter (from Sartorius) Ultrafiltration: device Pellicon cassette system from M- llipαre N> EG: 10,000 0- sample volume after centrifugation and prefiltration - ^* 0.77 1 concentrate volume: 0.38 1 flow rate: 60 ml / min

The concentrate is diluted with cysteine solution (3 g cysteine at o, 62 1 ad) to ^'11 diluted = activated Papaiπlösuπg (= incubation) 5- to erfindungsge äßeπ preparation of the novel active ingredients will now proceed as follows: raw materials.

100 1 blood of calves or young cattle defibrinated by stirring (up to ax. 3 years old) 0 11 activated papain solution HCL 1 N ethanol 96% aqua dest. ad. injection incubation: The thawed blood is warmed to 37 ° C. in the incubation vessel, the pH is adjusted to 7.0 with 1 N HC1. The :: incubation solution is then added and the mixture is allowed to incubate for 14 to 18 h. The incubated blood is mixed with fresh a.d. Diluted 1: 1 and prefiltered for the subsequent ultrafiltration.

Prefiltration: particle size - 50 μm; device: Seitz plate filter 0: for 7 filter layers Seitz Supra 300.

Ultrafiltration: Device: Millipore Ultrafilter, filter area: 4.6 m ² , NMG

10,000

Output volume: - ~ 2001; flow rate: ~ 0.71 / min.

The concentrate is diluted at - ^ 60 1 with 30 1 and filtered - 5: again down to - * -> - 20 - 25 1 retentate. Total filtration volume: 205 ^~ - 2101. After 30, 100 and 2001 permeate, samples are taken to check the filter tightness using TCA precipitation (must be negative!). 1 Concentration in the evaporator: The ultrafiltrate is concentrated as quickly as possible in the evaporator at 60 - 80 ° C to 3 - 4 1 final volume under vacuum.

1 1 centered (concentration on 25 ~ 33 of ^the original

Blood volume). This leads to strong precipitations, which exceed one

5 ^" black band filter are filtered off. One concentrates so high to the

To reduce the likelihood of reprecipitation.

Ethanol precipitation: The filtered concentrate is now 2.5 times

Amount (10 1) of pre-cooled ethanol 96 vol .-% stirred in in the cold

(Cold room temperature: 4 - 6 ° C). After a standing time of 48

1 * 0 ^' hours is clear filtered.

Filters used: black band filter, glass fiber pre-filter (Sartorius). The ethanol added for the precipitation is distilled off in a Rotavapor. This usually leads to precipitation again. In this case, the clear filtration is repeated using black belt and glass fiber pre-filters.

T5 Due to the severe constriction before the ethanol precipitation, it is now only necessary to adjust the dry weight to 200 mg / ml by dilution with the appropriate amount of fresh water for injection purposes.

The finished active ingredient concentrate is passed through a sterile filter (0.2 μm membrane filter, from Sartorius) and again through an ultrafilter (NMGG:

2D_ 10,000) filtered and then filled into sterile bottles. This active ingredient concentrate is the basis for the production of ampoules, infusion solutions, tablets, dragées, capsules, ointments, gels etc. Preferred galenical forms are powders or granules which are filled into hard or soft gelatin capsules.

25 Example 1:

10 1 cattle blood are defibrinated at the slaughterhouse by stirring. The fibrin can be filtered off through ^'a sieve. The blood is frozen in small portions (4 - 5 1) and thawed again shortly before processing. For incubation, the pH is adjusted with HC1

• 333 ^{) set} to 7.0, 20 g of pretreated activated papain were added and the mixture was left to stand at 37 ° C. for 15 hours with stirring. The pretreatment of papain is used to clean commercially available papain (∑rBPapayotin, from Extract-Chemie, 80,000 U / g). 20 g of commercially available paper are suspended in 200 ml of ad and then centrifuged. The supernatant is prefiltered for the subsequent ultrafiltration. The solution should be free of particles> 10 μm. In ultrafiltration, the mixture is filtered to a concentration of 50 ml and then to 1 Activation of the Papaiπs with Cysteiπ solution (300 mg Cysteiπ in 151 ml ad) diluted to a total of 200 ml. The ultrafiltrate is discarded. The incubated blood is stirred in 10 l of pre-cooled distilled water. The diluted blood is prefiltered for ultratiltration through a plate filter system for 7 filter layers and then filtered through an ultrafilter with a molecular weight limit of 10,000 daltons and immediately concentrated to 360 ml. The substances which have precipitated out due to the concentration are separated off by filtration through a black band filter. The protein-free filtrate is stirred into 900 ml of 10% pre-cooled ethanol 96% and left in the refrigerator at 7 ° C. The protein-free test is carried out in a rapid test with trichloroacetic acid. After standing for at least 48 hours, the precipitates are filtered off and the alcohol is removed in a Rotavapor. 15 The product obtained in this way is diluted with sterile, pyrogen-free water to a dry substance content of 200 mg / ml and filled aseptically into sterile bottles. Analysis: pH 6.1

20 density (g / ml.) 1.0760

Chloride * ^' 2.56

Sodium * 1.94

Potassium * 0.16

Yield * 10.9

25 Osmolality (mosm / kg) 3,110

Activity (Warburg) ** + 32.1%

SDS electrophoresis: free of substances with MW> 10,000

* g from 1 kg of blood

** Difference against reference standard like this:

Example 2 50 l of calf blood are defibrinated at the slaughterhouse by stirring. The fibri flakes are filtered off through a sieve. The blood is processed immediately as in Example 1. -.-.- Aπalyseπdateπ: pH 6.0

Density (g / ml) 1.0840 1 chloride * 2.28

Sodium * 1.60

Potassium * 0.14

Yield * 8.3

5 Osmolality (mosm / kg) 3,725

Activity (Warburg) ** 35.8%

SDS electrophoresis: free of substances with MG> 10,000 * g from 1 kg blood

** Difference against reference standard ID. Example 3:

1011 cattle blood are defibrinated at the slaughterhouse by stirring. The fibrin flakes are filtered off through a sieve. The pH is then adjusted to 7.1 with HC1, incubated with 20 g of activated papain pretreated according to Example 1 for 14 hours at 37 ° C. and the iπ-E5-incubated blood with dist. Diluted water 1: 1. To remove all particles> 10 μm, pre-filter through a plate filter and then take place. an ultrafiltration with a separation limit of 10,000 daltons. The protein-free ultrafiltrate (checked by means of precipitation with trichloroacetic acid) is in a rotary evaporator to about 350 ml ZΩ. concentrated, mixed with 900 ml of ethanol and stored in the refrigerator for 48 hours. The precipitated substances are separated off by filtration and the alcohol is distilled off in a vacuum evaporator. This concentrate is sterile filtered to 400 ml and filled aseptically into ampoules. 25 Analysis data: pH value 6.1

Density (g / ml) 1.0912

Chloride * 1.68

Sodium * 1.19

333 ⁾ Potassium * 0.11

Yield 6.0

Osmolality (mosm / kg) 3,635

Activity (Warburg) ** 28.3%

SDS electrophoresis: free of substances with MG> 10,000 35 * g from 1 kg blood

** Difference against reference standard 18th

1 Example 4:

10 '1 defibrinated fresh bovine blood is adjusted to pH 6.5 with HC1, incubated with 20 g of activated papain pretreated according to Example 1 at room temperature for 18 hours and then filtered undiluted ultra-5. The protein-free ultrafiltrate is concentrated to 300 ml in a vacuum evaporator, mixed with 0.75 l of ethanol and then stored in the refrigerator for 24 hours. The precipitate is separated off by centrifugation or filtration and the alcohol is evaporated off in a rotary evaporator. The concentrate is adjusted with sterile, pyrogen-free water at 0 ^'200 mg dry matter / ml and dispensed into sterile bottles. Example 5: The concentrate produced according to Examples 1 to 4 is injected with Aqua ad. diluted to 40 mg / ml, the pH checked again or adjusted to 6.9 ^" and filtered again for depyrogegenization with an ultra-5 filter with a separation limit at 10,000 MW. This injection solution is poured into 2 ml under aseptic conditions , 5 ml or 10 ml ampoules.

Example 6: The concentrate produced according to Examples 1 to 4 is injected with Aqua ad 0. adjusted to a dry matter content of 50-70 mg / ml and lyophilized. Tablets are made by 500.0 glyophilisier. Concentrate 1,987.5 g of methyl cellulose, 12.5 g of Mg stearate 25. Are mixed and compressed to 250 mg tablets. Since the lyophilized concentrate is extremely hygroscopic, the tableting must take place under dry: room air (<20% relative humidity). The cores are coated with a saliva-resistant, white film by 30U g spray solution consisting of: 30: 12.0 g Eudragit E 100 for 750 g cores

1.5 g of polyethylene glycol 6,000 91.8 g of opaspray white, K 1-7000 (50% Festetϊ_faπte_, Fa.θDlαrcoπ) 97.5 g of isopropanol 97.5 g of methylene chloride 35-: are sprayed on.

Example 7: 1.25 1 of the concentrate prepared according to Examples 1 to 4 are made on 1 kg of granules consisting of 98% methyl cellulose and 2% gelatin, very long • sprayed on. The granules obtained in this way are subsequently dried at 60 ° C. in a drying cabinet with circulating air for at least 18 hours. After reaching a residual moisture of max. 0.5%, the granules are mixed with 0.5% Mg stearate and compressed to 250 mg tablets. & The tablets are coated as in Example 6. Example 8: For the production of a wound gel with a dry substance content of the active ingredient concentrate of 8 mg / g gel prepared according to Examples 1 to 4, 1-0 1.75.0 g methyl cellulose 4,000 cps are used

2.210 g of propylene glycol

3. 90.0 g polyethylene glycol

4. 120.0 ml active ingredient concentrate

5. 4.5 g of p-OH benzoic acid methyl ester E5 6. 0.9 g of p-OH benzoic acid propyl ester

7. 2,496.6 g aqua dest. processed into a gel in such a way that a dispersion is prepared from 1st, 2nd and 3rd and this is incorporated into 1,800 ml of hot aqueous phase (in which 5th and 6th are dissolved). With the remaining H-, 0 that will. 2 ^Q > active ingredient concentrate diluted, sterile filtered and the gel stirred at 50 ° C cooling temperature.

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Claims

Patent claims:

1. A process for the preparation of a therapeutically active substance, in particular for wound healing or for treatment in geriatrics, in which defibrinated blood from warm-blooded mammals, in particular from young cattle or calves, is subjected to enzymatic hydrolysis with papain , the permeate is concentrated and treated with alcohol, preferably ethanol, characterized in that the papain hydrolysis is carried out on unhemolyzed blood with activated papaiπ at a pH of 6.5 to 7.4, and then the reaction is stopped by cooling, ultrafiltration is carried out to detect and remove papain up to 10,000 D, and then the essentially protein-free permeate is concentrated to 1/25 ^{^} to 1/35, based on the blood used, with alcohol is left to stand chilled for at least 24 hours and finally clarified.

2. The method according to claim 1, characterized in that the Klar¬ filtered mixture to a predetermined concentration, e.g. 200 mg / ml, is diluted with distilled water.

3. The method according to claim 1 or 2, characterized in that the alcohol is removed from the clear filtered mixture, preferably by evaporation.

4. The method according to any one of claims 1 to 3, characterized gekennzeich¬ net, that the reaction is stopped by dilution with cold distilled water, preferably at a temperature of 0 to 5 ° C.

5. The method according to claim 4, characterized in that the thinning is carried out to the extent of about 1: 1.

6. The method according to any one of claims 1 to 5, characterized gekennzeich¬ net that a pre-filtration for the separation of particles with a size of more than 50 microns is carried out before the Ultrafiltratioπ.

7. The method according to any one of claims 1 to 6, characterized gekennzeich¬ net that the concentration of the permeate is carried out in a vacuum evaporator at a temperature of 60 to 80 ° C.

8. The method according to any one of claims 1 to 7, characterized in that a further filtration is carried out before the alcohol addition.

9. The method according to any one of claims 1 to 8, characterized in that the alcohol addition to the extent of about 65 to 80 Vol .-%, preferably about 70 vol .-% of the resulting mixture takes place.

10. The method according to any one of claims 1 to 9, characterized in that the mixture is left to stand at a temperature of 4 to 6 ° C.

11. The method according to any one of claims 1 to 10, characterized gekenn¬ characterized in that for papain hydrolysis, the papain is freed by ultrafiltration of substances with a molecular weight of less than 10,000 and activated with cysteine.

12. The method according to any one of claims 1 to 11, characterized gekeπn- ^* 0 records that the papain hydrolysis at a pH of about 7 and one

Temperature of about 37 ° C for 14 to 18 hours.

13. The method according to any one of claims 1 to 12, characterized gekenn¬ characterized in that the standing time after the alcohol precipitation is 24 to 55 hours, preferably about 48 hours. * 5

14. The method according to any one of claims 1 to 13, characterized gekenn¬ characterized in that the clear-filtered mixture after dilution is again sterile and / or ultrafiltered.

15. Active ingredient, in particular for wound healing or for the treatment of geriatrics, produced by the method according to one of claims 1 0 to 14.

16. A process for the preparation of therapeutic preparations, in particular for wound healing or for geriatrics, characterized in that concentrates prepared according to one of claims 1 to 14 are processed into injection solutions, tablets, dragées or wound ointments or gels.

17. Therapeutic preparation, in particular wound healing agent or geriatric preparation, containing a concentrate produced according to one of claims 1 to 14.

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