KR20030067752A - Novel mixtures of microbial enzymes - Google Patents

Abstract

The present invention relates to a novel mixture of microbial enzymes comprising concentrated Rhizopus delemar lipase, Aspergillus melleus protease and Aspergillus oryzae amylase. . The invention also relates to a pharmaceutical formulation comprising a mixture of said microbial enzymes. The novel pharmaceutical preparations are particularly suitable for the treatment and / or prophylaxis of indigestion, in particular for the treatment and / or prophylaxis of indigestion due to pancreatic deficiency in mammals and humans.

Description

New Mixture of Microbial Enzymes {NOVEL MIXTURES OF MICROBIAL ENZYMES}

Mammalian and human indigestion is usually based on deficiency of digestive enzymes, in particular on deficiency of endogenous lipases, but also on deficiency of proteases and / or amylases. The cause of such digestive enzyme deficiency is often due to pancreatic insufficiency (pancreatic insufficiency), the organ that produces the most important endogenous digestive enzymes. If the pancreatic deficiency is pathological, it may be congenital or acquired. Acquired chronic pancreatic deficiency may be due to, for example, alcoholism. Congenital pancreatic deficiency may be due to, for example, a congenital cystic fibrosis disease. The consequences of digestive enzyme deficiency can be serious symptoms of malnutrition and malnutrition, which involve increased susceptibility to secondary disease.

In the treatment of endogenous digestive enzyme deficiency, replacement by similarly acting exogenous digestive enzymes or digestive enzyme mixtures has proven effective. Nowadays, pharmaceutical preparations (hereinafter 'preparations') comprising pig pancreatin (hereafter pancreatin) are most frequently used for this purpose. Such digestive enzyme mixtures obtained from the pancreas of pigs are virtually ideal for humans for enzyme replacement therapy due to the large similarity of the enzymes and their accompanying substances with the contents of human pancreatic fluid. Because some of the components of the pancreatin--for example, pancreatic lipase and pancreatic amylase--are sensitive to acidic pH values below pH 5, for protection against acid-induced degeneration in the stomach, pancreatin preparations for oral administration are It should be coated with an enteric protective layer. Such a protective layer preserves acid-sensitive pancreatic constituents from irreversible breakage and releases their contents in the upper part of the small intestine only after passing through the stomach. The upper part of the small intestine usually has a higher, harmless pH value of about pH 5.5 to pH 8. At the same time, the small intestine top, eg, duodenum, is largely the point where most of the food components degraded by the enzyme are reabsorbed into the body.

Because pancreatin is a natural product, very significant technical expenditures are required to provide uniform quality and advanced forms. In addition, finding suitable raw materials for processing into pancreatic fluid is also unstable.

Thus, attempts have already been made in various cases to make available digestive enzyme mixtures that are similarly well matched with pancreatic oxime and have improved properties as compared to pancreatin for endogenous digestive enzyme replacement.

To be suitable for the replacement of digestive enzymes in humans, all replacement enzymes have numerous requirements (e.g. Peschke, "Active Components and Galenic Aspects of Enzyme Preparations"): Pancreatic Enzymes in Health and Disease, PGLankisch Edit, Springer Verlag Berlin, Heidelberg 1991, pp. 55-64; cited below as "Peschke". Thus, these replacement enzymes must be particularly stable against pepsin and other endogenous proteases such as pancreatic proteases. Alternative enzymes must maintain their activity even in the presence of endogenous bile salts.

Substitution of underproduced endogenous lipases, such as endogenous lipases underproduced by disease, is generally considered to represent the most important component of human digestive enzyme replacement therapy. However, it has been known for a relatively long time that the simultaneous replacement of underproduced proteases and amylases has additional beneficial effects on the patients (eg, Peschke, p. 55; WO 96/38170, p. 6). Reference). Thus, pharmaceutical preparations for the treatment and / or prophylaxis of indigestion in mammals and humans should mainly replace not only the lipolytic activity of the body but also the proteolytic and starch activity. It is important here that the different replacement enzymes (lipases, proteases, amylases) contained in the pharmaceutical preparations can each exhibit their activity to a sufficient degree at the intended site of action (usually the upper part of the small intestine). Under physiological conditions, especially in the human stomach during or immediately after food intake, higher pH values, for example, usually pH 4-5, than in empty stomachs (approximately pH 1-2), and also the small intestine Since the upper physiological pH values are usually between 5.5 and 8, these digestive enzymes with good pH stability and pH activity in the range of about pH 4-8 are considered suitable for human digestive enzyme replacement.

Preparations which further contain microbial lipases in addition to mammalian pancreatic extracts are already known from European patent application EP A 0 387 945. However, because it still contains the contents of animal pancreatin, such preparations cannot always be manufactured in simple laboratory processes as they are always standardized to achieve a certain quality and desired amount.

International patent application WO 96/38170 includes, in particular, acid-stable Aspergillus niger amylase and optionally acid-stable Rhizopus javanicus lipase, which is to be used as a digestive aid. Formulations that can be described are described. However, there is no specific presentation in this document of the replacement of endogenous proteolytic activity. Instead, only reference is given to the fact that it is possible to replace all other human pancreatic components with pig pancreatin except for lipase and amylase. This indicates that the formulations described in WO 96/38170 are not intended or unsuitable for the total replacement of endogenous digestive enzymes.

Furthermore, in a 1995 paper by S. Schcheler of the University of Erlangen-Nurnberg, entitled "Multi-component Formulation with Optimal Digestibility by Highly Active Aspergillus oryzae Enzyme," a commercially available enzyme The combination of Phosphorus Rhizopus oryzae lipase, Aspergillus oryzae protease and Aspergillus oryzae amylase has been mainly investigated from a pharmaceutical point of view. However, for example, the lipases used therein do not have satisfactory stability to endogenous pancreatic proteases.

From the above features, it is clear that pharmaceutical preparations intended for the total replacement of endogenous digestive enzymes in mammals and humans must contain alternative enzymes or mixtures of replacement enzymes that carefully match the endogenous conditions.

Accordingly, it is an object of the present invention to replace endogenous lipolysis, proteolysis, and starch enzymatic activity containing an improved digestive enzyme mixture and containing such a mixture, and due to the high specific activity of the replacement enzyme contained therein, To provide a pharmaceutical formulation for the treatment and / or prophylaxis of indigestion in mammals and humans in which the dosage is relatively low. At the same time, the replacement enzymes (lipases, proteases, amylases) contained in the digestive enzyme mixture, as well as individually and in mixtures with each other, meet all the requirements for the digestive enzymes intended for the treatment of humans as well as possible. You have to. For example, replacement enzymes should have good pH stability and pH activity within the predominant pH range at their physiological point of action. In addition, replacement enzymes should be readily compatible with endogenous actives such as, for example, bile salts or endogenous proteases such as pepsin or pancreatic proteases. Another object of the present invention consists in selecting alternative enzymes which can be obtained in a desired amount with a certain quality by a manufacturing process that is simple in terms of process and product quality.

The present invention relates to novel enzyme mixtures comprising certain combinations of microbial lipases, proteases and amylases. The invention also relates to pharmaceutical preparations comprising mixtures of these microbial enzymes. These new pharmaceutical agents are particularly well suited for the treatment and / or prevention of indigestion in mammals and humans, and in particular for the treatment and / or prevention of indigestion based on chronic exocrine pancreatic insufficiency. Is very suitable.

1 is a pH profile of the microbial lipase "Lipase D Amano 2000",

2 is a pH profile of the microbial protease "Prozyme 6",

3 is a pH profile of the microbial amylase "Amylase A1".

The object of the present invention is achieved by providing a novel mixture of microbial enzymes comprising:

a) concentrated lipase from Rhizopus delemar,

b) neutral protease of Aspergillus melleus, and

c) amylase of Aspergillus oryzae.

Mixtures of microbial enzymes according to the invention can be included in conventional pharmaceutical formulations with conventional auxiliaries and / or carriers. These pharmaceutical preparations exclusively comprise a mixture of microbial enzymes of certain fungi according to the invention as active substances and are suitable for the total replacement of endogenous digestive enzymes in mammals and humans. What the individual enzymes (lipases, proteases, amylases) contained in the mixture of microbial enzymes according to the invention have in common is within the physiological and pathological pH range of the digestive tract (approximately pH 4-8), especially during or immediately after food intake. Under conditions prevailing, it has good pH stability and good pH activity. In addition, the pharmaceutical formulations are differentiated by good efficacy and good suitability.

Concentrated lipases of Rhizopus delemar have an inactivity of at least 1,800,000 FIP units / g (internationally standardized enzymatic active units, which are set to conform to the specifications of the "International Pharmaceutical Federation" (Belgium)). Strain Rhizopus delemar is considered a subspecies of strain Rhizopus oryzae. Lipases of the strain Rizopus delemar mold are also known per se and can be obtained, for example, using known processes from the corresponding cultures of the fungus. Methods of fermenting fungi and isolating enzyme products formed therefrom are described, for example, in professional biotechnology books (eg, H. Diekmann, H.Metz, "Grundlagen und Praxis der Biotechnologie). ", Gustav Fischer Verlag Stuttgart, New York 1991) or through a professional scientific publication known to those skilled in the art. Lipases thus separated can be enriched or concentrated, for example, by means of known methods to remove the substances accompanying them and until the specific activity required according to the invention is obtained. Preferably, it is also known as "Lipase D Amano 2000" ("Lipase D2") by Amano Pharmaceuticals of Japan, which is a lipase (EC No. 3.1.1.3) of Rhizopus delemar. ) Can be used. This lipase-like natural pancreatic lipase-has 1,3 position specificity with respect to fatty acid glycerides. The inactivation is between about 1,800,000 and about 2,250,000 FIP units / g, depending on the charge. "Lipase D Amano 2000" is differentiated due to its high stability against pancreatic proteases. Therefore, in the laboratory test after 2 hours of acting with pancreatic pancreatic protease in the pH range of 6-8, the lipolytic activity of "Lipase D Amano 2000" maintained 55% of the initial activity. In laboratory tests after at least 120 minutes in a pH range of 37 ° C., pH 4-8, the pH stability of “Lipase D Amano 2000®” was at least 70% of the initial activity.

For example, the pH profile of concentrated lipase of Rhizopus delemar is suitable as a characteristic determinant of the lipase. Thus the pH profile of "Lipase D Amano 2000" is determined as inactive as a function of pH value. According to a variation of the FIP method to determine the activity of microbial lipases, the inactivity at individual pH values was measured. In addition, the pH profile in the presence of various concentrations of bile salts was also determined.

a) Manufacture of olive oil emulsion

44g gum arabic,

115g olive oil and

400 ml water was homogenized in an electric mixer for 15 minutes.

b) preparation of bile extracts of different concentrations;

Bile-free: 120ml water

0.5mmol / l bile: 120ml water + 200mg bile extract (FIP standard)

5mmol / l bile: 120ml water + 2g bile extract

10mmol / l bile: 120ml water + 4g bile extract

c) substrate emulsion preparation

480 ml olive oil emulsion (see above),

160 ml aqueous calcium chloride solution (28.3 g CaCl ₂ · 2H ₂ O / l water) and

120 ml bile extract of desired concentration was mixed (see above).

d) preparation of enzyme solution

50 mg of "Lipase D Amano 2000" (inertness determined at 2,230,000 FIP units / g) was dissolved in 100 ml of 1% aqueous sodium chloride solution. 1 ml of this stock solution was taken and diluted with ultrapure water to 200 ml. In each of the following determinations, 1 ml of diluted stock solution (equivalent to 5.575 FIP units / g) was used.

19 ml of samples were taken from each of the substrate emulsions having a constant bile salt concentration and maintained at 37 ° C. Next, for different substrate emulsion samples, pH values were set to 3, 4, 5, 6, 7 and 8 by adding 0.1M NaOH or 1M HCl. Then 1 ml of the enzyme solution was added to each of the samples of the prepared substrate emulsion (note: in principle, to determine the optimal titration rate, the appropriate amount of lipase ideally contained in the enzyme solution is known, In the manner of dilution series). Once addition was complete, pH stat titration with 0.1 M NaOH was performed for 10 minutes. Next, end-point titration was performed to pH 9 in 30 seconds to completely dissociate the released fatty acids. The total consumption of 0.1 M NaOH required was converted to lipase active unit E: 1 lipase active unit E corresponds to a consumption of 1 μmol per minute. The determined lipase active units can be converted to units of E / mg by reference to the amount of dry enzyme g used each time. To prepare the pH profile, the E / mg units and each bile salt concentrations irradiated for each pH value irradiated are shown in Table 1, and the values shown are shown on the graph of FIG.

[Table 1] "Lipase D Amano 2000" activity according to pH and bile salt concentration

(Unit: E / mg)

pH No bile Bile 0.5mmol / l Bile 5mmol / l Bile 10mmol / l 3 608 416 500 300 4 936 972 1076 480 5 1432 1342 960 1076 6 1048 1570 1382 1220 7 1024 2092 1832 988 8 308 732 1104 64 9 0 0 200 0

From this pH profile at a FIP standard bile salt concentration of 0.5 mmol / l, the optimal pH for "Lipase D Amano 2000" can be set at about pH 7, as the maximum value of lipase activity.

The neutral protease of Aspergillus melleus has an activity of at least 7,500 FIP units / g. The optimum pH is between pH 6 and pH 8. Neutral proteases of the strain Aspergillus meleus fungus are also known per se and can be obtained, for example, using known processes from the corresponding cultures of the fungus. Methods of fermenting fungi and isolating enzyme products formed therefrom are described, for example, in professional biotechnology books (eg, H. Diekmann, H.Metz, "Grundlagen und Praxis der Biotechnologie). ", Gustav Fischer Verlag Stuttgart, New York 1991) or through a professional scientific publication known to those skilled in the art. The proteases thus separated can be enriched or concentrated, for example, by known methods to remove the substances accompanying them and until the specific activity required according to the invention is obtained.

Preferably, "Prozyme 6" (sometimes referred to as "alkaline proteinase") from Amano Pharmaceuticals, Japan, which is a neutral protease from Aspergillus melleus (EC No. 3.4.21.63) Can be used. This microbial protease hydrolyzes 1,4-α-D-glucoside linkages of polysaccharides containing at least three 1,4-α-D-glucose units and has an inactivity of about 7,800 FIP units / g. In laboratory tests after at least 120 minutes in a pH range of 37 ° C., pH 5-8, the pH stability of “Prozyme 6®” was at least 60% of the initial activity.

For example, the pH profile of the neutral protease of Aspergillus melleus is suitable as a characteristic determinant of the protease. Thus the pH profile of the "Prozyme 6" protease is determined as inactive as a function of pH value.

To this end, various substrate solutions were prepared in accordance with the instructions of the FIP method for measuring the activity of pancreatic proteases. By modifying the FIP instructions, 4% hemoglobin solution was used as substrate solution instead of casein. In addition, using the FIP instructions, for different substrate solutions, 1 M NaOH or 1 M HCl was added in corresponding amounts to set different pH values to 2, 3, 4, 5, 6, 7 and 8, respectively. . "Prozyme 6" samples were added to the substrate solutions.

Next, the protease activities of the "Prozyme 6" samples in the substrate solutions with different pH values were measured according to the FIP instructions. Enzyme activities found in the individual samples were normalized to the maximum value (as 100%) found in this series of measurements. The measured values of the pH profile of "Prozyme 6" are shown in Table 2 and shown on the graph of FIG. As a result, "Prozyme 6" is optimally effective in the physiological pH range.

[Table 2] "Prozyme 6" relative activity (%) according to pH

pH % 2 One 3 0 4 63 5 67 6 77 7 77 8 100

Note) Standard activity at pH 7.5: 7812 FIP unit / g,

Protease determination using hemoglobin as substrate according to a modified FIP.

From this pH profile, the optimal pH of "Prozyme 6" may be set to about pH 8, as the maximum value of protease activity.

The amylase of Aspergillus oryzae according to the invention (EC No. 3.21.1.1) is α-amylase and has an activity of at least 40,000 FIP units / g (measured at pH 5.8). The optimum pH is in the range between pH 4 and pH 6.5. Amylases of the fungus strain Aspergillus oryza are also known per se and can be obtained, for example, using known processes from the corresponding cultures of the fungus. Methods of fermenting fungi and isolating enzyme products formed therefrom are described, for example, in professional biotechnology books (eg, H. Diekmann, H.Metz, "Grundlagen und Praxis der Biotechnologie). ", Gustav Fischer Verlag Stuttgart, New York 1991) or through a professional scientific publication known to those skilled in the art. The amylases thus separated can be enriched or concentrated, if desired, by the known methods to remove the substances accompanying them and until the specific activity required according to the invention is obtained. Preferably, "Amylase A1" from Amano Pharmaceuticals, Japan, an amylase from Aspergillus melleus, and from Extrakt-Chemie, Germany, from amylase from Aspergillus melleus "Amylase EC" may be used. "Amylase A1" is preferred.

This microbial amylase "Amylase A1" has an inactivity of about 52,000 FIP units / g (measured at pH 5.8). In laboratory tests after at least 120 minutes in the pH range of 37 ° C., pH 5-8, the pH stability of “Amylase A1®” was at least 85% of the initial activity. In another laboratory test, for pancreatic pancreatic protease (measured in the range of pH 6-8), for "Prozyme 6" (measured in the range of pH 4-8), and for pepsin, Amylase A1® has been confirmed to have good stability.

For example, the pH profile of amylase of Aspergillus oryzae is suitable as a characteristic determinant of the amylase. Thus the pH profile of "Amylase A1" is determined as inactive as a function of pH value.

Various substrate solutions were prepared in accordance with the instructions of the FIP method for measuring the activity of microbial amylase. By varying the FIP instructions, for different substrate solutions, a corresponding amount of 5M NaOH or 5M HCl was added in advance to the acetate buffer used according to the FIP method, and the different pH values were respectively 3.25, 4, 5 , 6, 6.8 and 7.4. "Amylase A1" samples were added to the substrate solutions.

Next, according to the FIP specification, the amylase activities of the "Amylase A1" samples were measured in the substrate solutions having different pH values. Enzyme activities found in the individual samples were normalized to the maximum value (as 100%) found in this series of measurements. The measured values of the pH profile of "Amylase A1®" are shown in Table 3 and shown on the graph of FIG.

Table 3 "Amylase A1" relative activity (%) according to pH

pH % 3.25 33 4 60 5 100 5.8 87 5 80 6.8 33 7.4 10

Note) Standard activity at pH 5.8: 51870 FIP unit / g,

From this pH profile, the optimal pH of “Amylase A1®” can be set at about pH 5, as the maximum value of amylase activity.

The microbial amylase "Amylase EC" has an inactivity of about 42,500 FIP units / g (measured at pH 5.8). In addition, small amounts of β-amylase can be detected. The optimum pH (measured according to the assay for "Amylase A1" above) is about pH 5. In laboratory tests after at least 120 minutes in the pH range of 37 ° C., pH 6-8, the pH stability of “Amylase EC®” was at least 80% of the initial activity. In another laboratory test, for pancreatic pancreatic protease (measured in the range of pH 6-8), for "Prozyme 6" (measured in the range of pH 4-8), and for pepsin, Amylase EC® has confirmed good stability.

Pharmaceutical preparations according to the invention can be, for example, oral administration of solids, such as powders, pellets or microspheres, which can be poured into capsules or sachets or compressed into tablets, if desired You can choose the form. Liquid pharmaceutical preparations such as suspensions or solutions may also be contemplated. In this case, the separate enzymes lipase, protease and amylase may be present together or spatially separated from one another. If the individual enzymes are not spatially separated from one another, a drying process and / or storage is preferred. In addition, the pharmaceutical preparation may further include conventional auxiliaries and / or carriers. Suitable auxiliaries and / or carriers are, for example, microcrystalline cellulose, polyethylene glycols (eg PEG 4000) or alternatively lower alcohols (especially straight chain or ground phase C such as 2-propanol). ₁ to C ₄ alcohols), and also water.

The microbial replacement enzymes according to the invention are differentiated due to their good stability over a wide pH range and can therefore be used directly in the preparation of pharmaceutical preparations for oral administration, without further treatment such as film coating. To this end, individual replacement enzymes (lipases, proteases, amylases) can be pelletized together or spatially separated from one another. If desired, individual replacement enzymes may be filmcoated with a suitable known enteric layer. If not all of the replacement enzymes will be enteric coated, it is appropriate to pelletize the individual types of replacement enzymes separately and to film coat each pellet separately. In particular, the proteases and / or lipases are suitable for enteric film coating of each of them individually after pelletizing. If desired, all three enzymes present in the enzyme mixture may be provided together by one enteric film coating, or two enzymes may be provided by one enteric film coating, and the other enzyme may not be film coated.

The high inactivity of the replacement enzymes used according to the invention makes it possible to achieve high efficiencies even with relatively low dosages. For example, in one embodiment, the pharmaceutical agent may be in the form of a capsule for oral administration of size 0. Lipases of about 10,000-50,000 FIP units, amylases of 8,000 FIP units, and proteases of 200 FIP units may be present in such dosage forms. Alternative enzymes are preferably present with lipase: amylase: protease in a ratio of 50-500 FIP units: 40-120 FIP units: 1 FIP units, respectively.

The suitability of the pharmaceutical preparations for the treatment and / or prophylaxis of indigestion in mammals and humans according to the invention can be exemplified by the in-vitro test model for measuring fat digestion presented below.

One. Example of Fat Digestion in the Pig Feed Test

In a pig feed test that also contained other food components, the effect of microbial enzyme mixtures usable in accordance with the present invention on fat metabolism was investigated. The aqueous calcium chloride solution added serves to precipitate the released fatty acid as calcium soap.

A) Preparation of pig feed test

The components are as follows:

64.8g "Altromin 9021" commercial feed (about 2-3% fat content, consisting almost of milled wheat, manufactured by Altromin, Germany),

3.85 g "Sojamin®" protein mixture (manufactured by Lukas Meyer, Germany)

24.5 g Arabian gum (manufactured by Merck, Germany), and

26.7 g soya oil (main fat component; average molecular weight 932 g / mol, manufactured by Roth, Germany).

The components were mixed with 265 ml of ultrapure water and then homogenized with a home mixer for 15 minutes. Ultrapure water was added to the resultant homogenate so that the volume became 450 ml.

B) Preparation of Bile Extract Solution

1.35 g bile extract (FIP standard; lipase activating material) was dissolved in 50 ml of ultrapure water.

C) Preparation of Enzyme Solution

1. Lipase Solution

63.1 mg of "Lipase D Amano 2000" (inactive at pH 7 of 1,888,137 FIP units / g, manufactured by Amano Pharmaceuticals, Japan) was dissolved in 10 ml of ultrapure water. 250 μl of this stock solution was used for the next measurement.

2. Protease Solution

319 mg of "Prozyme 6" (inactive at pH 7.5, 7,812 FIP units / g, manufactured by Amano Pharmaceuticals, Japan) was dissolved in 10 ml of ultrapure water. 250 µl of this stock solution was used for the next measurement.

3. Amylase Solution

595 mg of "Amylase EC" (13,466 FIP units / g with specific activity at pH 5.8, manufactured by Extrakt-Chemie, Germany) was dissolved in 10 ml of ultrapure water. 1,000 μl of this stock solution was used for the next measurement.

D) Preparation of Measuring Solution

Successively with 2 ml of the bile extract solution, the three enzyme solutions C1) to C3) were added to 15.5 ml of the pig feed test formula, and ultrapure water was added so that the volume of the mixture was 29 ml.

E) Run Measurement

The prepared solution was stored at a constant temperature of 37 ℃, the end point titration with 1M NaOH was adjusted to pH 7. Immediately after adding the three enzyme solutions, the pH fixation titration was started and proceeded for 20 minutes, and the consumption of 1M NaOH was recorded every 10 seconds. While the titration was in progress, 1 ml of 4 M calcium chloride solution was manually added in steps of 50 µl to obtain a maximum reaction rate.

F) results

Fat (ie fatty acid triglycerides) contained in the pig feed test formula was hydrolyzed to about 67% after a 20 minute reaction time. This corresponds to at least 100% of the catabolism that forms 2-fatty acid monoglycerides, which are physiological hydrolysis products (values above 100% are 2 which form 1- and 3-fatty acid monoglycerides). Due to spontaneous rearrangement and subsequent lipolytic breakdown of fatty acid monoglycerides).

The excellent fat digestion function of the digestive enzyme mixture containing enzymes usable according to the invention can also be exemplified by in vitro testing of olive oil test food.

Particularly good suitability of the pharmaceutical preparations for treating and / or preventing indigestion due to indigestion of mammals and humans, in particular due to pancreatic fluid deficiency, according to the present invention is in vivo in animal models such as pancreatic deficiency It can also be illustrated by using against pigs suffering from:

2. In vivo utility of the enzyme mixture according to the invention for pigs suffering from pancreatic juice deficiency

The test was performed on nine adult female Goettingen miniature pigs (33–40 kg) in the Ellegaard line, all of which had an ileocaecal bypass cannula inserted. The bypass intubation was used to collect chyme from test animals.

Six of these ligated the pancreatic duct (test group). The other three kept the pancreatic duct intact and served as controls for the test results (control). This test was carried out using three different total doses of the enzyme mixture according to the invention. The following enzyme doses were administered:

Dose 1: 111,833 FIP unit / meal "Lipase D Amano 2000"

1,775 FIP unit / meal "Prozyme 6"

89,760 FIP Unit / Meal "Amylase A1"

Dosage 2: 223,665 FIP Unit / Meal "Lipase D Amano 2000"

3,551 FIP unit / meal "Prozyme 6"

179,520 FIP Units / Meal "Amylase A1"

Dose 3: 335,498 FIP unit / meal "Lipase D Amano 2000"

5,326 FIP unit / meal "Prozyme 6"

269,280 FIP unit / meal "Amylase A1"

Per dose, all test animals were 170 g of mini pig domestic feed (Altromin®, consisting of nearly double milled wheat, manufactured by Lukas Meyer), 10 g of protein concentrate (Sojamin 90®, manufactured by Lukas Meyer). ), A dietary diet containing 70 g soybean oil (Roth) and 0.625 g Cr ₂ O ₃ (non-resorbable label, Roth) mixed with 1 liter of water, 250 g twice daily for 22 days It was. In addition, the individual enzymes of the enzyme mixture according to the invention were admixed in an amount corresponding to the feed amount only to the test group animals immediately before feed feed. In addition, a series of tests were performed with five of the test groups, where no enzyme mixture was added to their test feed. The results obtained in this series of tests are given below as "zero values". In each case corresponding to days 20 to 22 of the irradiation period, samples of rice porridge were taken over 12 hours from the bypass intubation of the test group animals and the samples were investigated in terms of crude fat, crude protein and starch content. This salary test and its evaluation were done in a known manner (see PCGregory, R. Tabeling, J. Kamphues, "Biology of Pancreasin Growing Animals"; Developments in Animal and Veterinary Sciences 28 ( 1999) 381-394, Elsevier, Amsterdam; Editors: SG Pierzynowski and R. Zabielski.

In test animals, the apparent precaecal digestibility of crude fat, crude protein, and starch determined in the in vivo test is, in each case, a relative percentage relative to the absolute amount of fat, protein, and starch originally fed. Are given in Table A below. The values given as "when digestion" correspond to "apparent seein digestibility", in that an endogenous component of the irradiated material, for example, an endogenous protein, may also be contained, which means that Is different. WPC digestibility values were obtained from labeling test animals using a labeling method using the equation given below:

Last Alliance Digestion sV

sV (%) = 100-(indicator in salary / indicator in chamois% x

% Nutrients in rice porridge / nutrients in salary x 100)

Table A:

Determination of Slurp Digestion of Crude Fat, Crude Protein, and Starch in Test Group Animals

Last League's Digestive Capacity (%) Crude fat Crude protein Starch Zero value 29.0 ± 9.8 33.7 ± 5.2 63.8 ± 6.7 Test Animal-Dosage 1 43.5 ± 9.9 56.3 ± 4.5 71.9 ± 9.3 Test Animal-Dose 2 52.1 ± 8.3 64.0 ± 3.7 74.2 ± 5.8 Trial Animal-Dose 3 55.3 ± 8.0 68.7 ± 3.3 81.6 ± 3.7 Control animals 97.6 ± 0.02 82.3 ± 1.5 96.9 ± 0.5

All values are given as mean values with standard deviations.

From the results given, it can be clearly seen that by administering the enzyme mixture according to the invention to pigs suffering from pancreatic fluid deficiency, the digestibility of fats, proteins and carbohydrates is dramatically improved and this improvement is dose dependent.

Example 1:

400 g of "Lipase D Amano 2000", 400 g of PEG 4000, and 1,200 g of "Vivapur®" (microcrystalline cellulose) were added in a known manner by adding some 2-propanol and water. A pellet of 0.7-1.4 mm in diameter was produced.

7000 g of "Amylase A1®", 2,000 g of PEG 4000, and 1,000 g of "Vivapur®" (microcrystalline cellulose) were added in a known manner by adding some 2-propanol and water, A pellet of 0.7-1.7 mm in diameter was produced.

1,750 g of "Prozyme 6", 500 g of PEG 4000, and 250 g of "Vivapur®" (microcrystalline cellulose) were added with a little 2-propanol and water in a known manner, to a diameter of 0.7 A pellet of ˜1.7 mm was prepared.

Among the pellets prepared as above, in each case 32 mg lipase pellets, 325 mg amylase pellets, 40 mg protease pellets were poured into one gelatin capsule of size zero. A dosage form with the following activity per capsule was obtained.

Lipase: about 10,000 FIP units

Protease: about 200 FIP units

Amylase: about 8,000 FIP units

Claims (14)

Enzyme mixtures comprising: a) concentrated lipase from Rhizopus delemar, b) neutral protease of Aspergillus melleus, and c) amylase of Aspergillus oryzae. The enzyme mixture of claim 1, wherein the lipase has an activity of at least 1,800,000 FIP units / g. The enzyme mixture of claim 1, wherein the protease has an activity of at least 7,500 FIP units / g. The enzyme mixture according to claim 1, wherein the protease has an optimal pH between pH 6 and pH 8. A pharmaceutical preparation comprising the enzyme mixture according to claim 1, further comprising a conventional adjuvant, carrier or adjuvant and carrier. 6. A pharmaceutical formulation according to claim 5, wherein the form is powder, pellet, microspheres, capsule, pouch, tablet, suspension or solution. The pharmaceutical formulation of claim 5, wherein the at least one enzyme selected from the group consisting of lipase, protease and amylase is in individually pelletized form. The pharmaceutical formulation according to any one of claims 5 to 7, wherein at least one enzyme selected from the group consisting of lipase, protease and amylase is film coated with an enteric layer. 9. A pharmaceutical formulation according to claim 8, wherein the protease, lipase or both are in individually pelletized form and film coated with an enteric layer. 6. The pharmaceutical formulation of claim 5, wherein the ratio of enzymes is lipase: amylase: protease, respectively, 50-500 FIP units: 40-120 FIP units: 1 FIP unit. The pharmaceutical formulation of claim 5 comprising at least 10,000 FIP units of lipase, 8,000 FIP units of amylase and 200 FIP units of protease per dose. Use of the enzyme mixture according to claim 1 for the manufacture of a medicament for the treatment, prophylaxis or treatment of and indigestion in mammals and humans. 13. The use of the enzyme mixture according to claim 1, wherein the indigestion is due to pancreatic deficiency. Use of concentrated lipase of Rhizopus delemar having an inactivity of at least 1,800,000 FIP units / g for the manufacture of a medicament for treating, preventing or treating indigestion in mammals and humans.