Using enzymes prepared from infusoria as digestion-promoting medicinal agents

FIELD: medicine, pharmacology, biochemistry, enzymes.

SUBSTANCE: invention relates to an enzyme-containing medicinal agent wherein enzymes are chosen from a group consisting of hydrolases, lipases, amylases, glycosidases, phospholipases, phosphodiesterases, phosphatases prepared from infusoria. Also, invention relates to a method for using indicated enzymes in improving digestion or in treatment of digestion disorders. Invention enhances resistance to stomach acid media and preparing from safety microorganism.

EFFECT: valuable medicinal properties of enzymes.

7 cl, 2 tbl, 7 dwg, 2 ex

The invention relates to a medicinal product for the treatment of digestive disorders, containing enzymes of the infusoria.

In General medical and therapeutic practices digestive disorders play an increasingly important role. These digestive disorders in many cases are the result of more or less pronounced lack of the so-called pancreatic enzymes. These enzymes in a healthy condition in the pancreas synthesized by specialized cells, the so-called almosnino cells, and secreted by exocytosis through the salivary glands and the main excretory duct (Ductus pancreatics) in the duodenum. The daily number of pancreatic secretion is approximately 2 liters Along with digest fats by lipase in pancreatic secretion are also enzymes for digesting proteins (trypsin, chymotrypsin, and carboxypeptidase) and carbohydrates (α-amylase). Thus the secretion of pancreatic enzymes precisely regulated by endogenous regulatory mechanisms by which hormones, such as gastrin, secretin, pancreozymin. This regulatory mechanism may be broken due to a variety of reasons, so there comes a throttling secretion of pancreatic enzymes or complete regression of the exocrine functions of the pancreas. This leads to the fact that dietary CA is Itza in the small intestine is not digested and there is indigestion. It is also called exocrine insufficiency of the pancreas, the digestive tract can be caused by various reasons. Along with medication due to dyspepsia, chronic atrophic gastritis and chronic, often provoked by alcohol consumption, pancreatitis, etiological factors of pancreatic insufficiency are promptly caused the violation (for example, resection of the stomach Billroth I and II, vagotomy, pancreatectomy) and cystic fibrosis. In each case of chronic digestive disorders have important socio-medical and economic importance, as patients due to symptoms are often limited healthy or not healthy and have a shorter life expectancy.

Pancreatic digestive disorders cause patients many ailments as diarrhea, massive chair, feeling content, difficulties in the epigastric region, the decrease in body weight and other.

Regardless of the reasons and manifestations of pancreatic maldigestion or failure of the pancreas to correct disorders of the digestive always necessary replacement therapy with the help of enzymes. This means that from the outside you need to enter the missing EN zymes is s, preferably the lipase, protease and amylase and other enzymes. During therapy, the enzyme is administered to the patient orally, often in the middle of the meal, and through the stomach they fall into the small intestine, where they perform the digestion of food gruel (chyme) and thus fulfil the function of a missing inherent in the body of the pancreas. Thus it is necessary to use products that contain a sufficient quantity of enzymes. In addition, the enzymes must be resistant to acid gastric juice, have a small particle size and completely bioavailable in the digestive tract.

For the treatment of digestive disorders, which are based on the absence of pancreatic enzymes, in particular the main enzyme lipase and protease, in the market there are many products containing enzymes. They are based partly on the enzymes from the pancreas of pigs, such as drugs Combizym^®, The Festal^®, Pankreon^®, Not available^®, Panzytrat^®, Meteozym^®or Enzym-Lefax N^®or , as Citrapepsin^®on the gastric enzymes. Partly, however, the drugs also contain enzymes from extracts of fungi, such as Combizym^®and Festal^®. Next, describes the use of enzymes from fish or other marine stomach is s (French patent 1015566), as well as the composition of the enzymes of the gastrointestinal tract krill (types of cancers class Euphauslaceae) and capelin (U.S. patent 4695457). Preparations containing enzymes of the pancreas, often produced from waste when cutting fish, or from the pancreas of pigs. The final product of the cooking process is Pancreatin. Pancreatin is a homogenate of cells of the pancreatic tissue (usually pigs). Due to the destruction of many Acinonyx cells it contains, along with pancreatic enzymes many other enzymes and proteins, as well as other high - and low-molecular compounds. The composition of Pancreatin caused by industrial method thereof. To obtain Pancreatin pancreas of pigs after slaughter as quickly as possible subject to freezing, collect and mechanically destroy. For stabilization and activation of enzymes in the homogenate injected with various additives. Then carry out degreasing with organic solvents, such as acetone, removal of fibrous substances, as well as dehydration and drying by lyophilization. For the preparation of certain forms of application can be further herbal processing of obtaining the micro granules, tablets, capsules, pastes, creams, gels, oils or other dosage forms. Often Pancreatin mixed media and buffer substances. Further, for protection against low pH of human gastric juice granulated Pancreatin cover acid-resistant films or lacquers. Both of these last, stage of processing must enforce unstable to acid by the enzymes of the pancreas at the destination, the duodenum (small intestine), its digestive functions.

Along with the usual preparation of Pancreatin in the final product, you can increase the content of lipase by stepwise extraction with chloroform, butanol and acetone. Just as in the case of Pancreatin, the composition of enzymes derived from krill species Euphausia suberba and guts capelin (called in German Lodden) (see application U.S. And 4695457). Also in this case, first of all get the homogenate of the tissue, it is then purified by centrifugation, extraction with chloroform, drying and chromatography steps. In each case, the aim of the process is to achieve a high content of pancreatic enzymes like lipase, protease and α-amylase. Further, the composition of the enzymes for the treatment of digestive disorders should be resistant to acid gastric juice. In addition, in the digestive tract, especially in the duodenum, the enzyme should preferably b is constructed to be liberated and to exert its biological action. Avoid allergies composition of enzymes as possible should not contain inactive proteins, i.e. must have a high degree of purity. In addition, pharmacoeconomic point of view the cooking process should be inexpensive.

In the case of Pancreatin usually do not carry any further purification chromatographic methods, so that the target enzymes are not in purified or in a homogeneous form. The drawback of such protein mixtures from cell homogenates is that they contain plenty of proteins from intracellular compartments (cytosol, cell nucleus, membranes, organelles) porcine pancreatic cells. They do not have any or any desired enzyme activity and thus significantly reduce the amount of biologically active substances on the input form application. The same thing happens if you get concentrated cell homogenates from krill or gastrointestinal tract capelin.

Another disadvantage of the preparation of enzymes and compositions of the enzymes of the waste when cutting fish (pancreas, gastro-intestinal tract of fish and krill is periodic method of obtaining. Usually organs (pancreas) at different stages of crushed and homogenized. The homogenate was then degreased and dried. These studies shall obtain enzymes cannot be carried out continuously, as always work with a substantial proportion of solid substances, which because of the stages of extraction and centrifugation cannot be processed further continuously. To obtain enzymes, however, the optimal continuous or partially continuous production processes, so that the output per unit time per unit volume in the case of such processes above and, thus, lower costs. For continuous receipt of enzymes, however, it is necessary that they be extracellular in dissolved form and is released only by grinding or homogenization of the cells.

For continuous receipt of enzymes therefore, the process of preparation of Pancreatin unsuitable.

Another disadvantage of enzymes from the pancreas is unstable to acid. Pancreatic enzymes are neutral or alkaline hydrolases. This means that, firstly, they show the maximum of its activity at pH values from 7 to 8 and in acidic conditions, they have greatly reduced activity. Secondly, the low pH values inactivate their catalytic function through reversible or irreversible denaturation. For this reason, derived from pancreatic enzymes (Pancreatin) with the help of special methods of encapsulating or adding buffer substances need to be protected from low value is the pH of acid gastric juice during the passage through the stomach. Such methods also increase the cost of medicines, based on the action of Pancreatin.

In addition, a negative factor in the use of pancreatic enzymes for specific groups of patients with impaired digestion is the origin of Pancreatin. Usually use the pancreas of pigs, which is unacceptable for patients of Jewish and Muslim religion because of religious precepts.

Finally, the enzymes from the pancreas of pigs cannot be used in patients with dyspepsia who have allergies to proteins pigs. Pigs in addition consider natural "reservoir" of pathogenic human influenza viruses, so it is impossible to exclude contamination of Pancreatin such viruses.

The invention therefore is to obtain enzymes, respectively, of the compositions of enzymes medicines that:

1) are located in the extracellular region and can be received and cleared without homogenization (destruction) of tissues or cells from free from cell supernatant;

2) can be obtained continuously in biotechnological process from a safe, unmodified by genetic engineering of microorganisms;

3) are acidic hydrolases, therefore, have a Maxim is active at acidic pH and are more resistant to acid, than the enzymes from the pancreas;

4) do not originate from tissues or organs of pigs.

The problem is solved thanks to a medicinal product that contains enzymes selected from the group consisting of hydrolases, lipases, proteases, amylases, field of glycosidase inhibition, phospholipases, phosphodiesterase, phosphatases, which are obtained from infusoria.

The object of the invention is particularly medicines that are used to promote digestion and to treat digestive disorders.

Proposed according to the invention medicines preferably contain enzymes used for digestion contained in food macromolecules like proteins, nucleic acids and carbohydrates, as well as other components of food, such as fats or phospholipids in the gastrointestinal tract of humans or animals.

Specialist it is clear that according to the invention to facilitate digestion or for the treatment of digestive disorders can also use the enzymes of the infusoria, which are not used so far, lipases, proteases or amylases, as well as other enzymes due to catalytic cleavage of components of food can contribute to digestion in the gastrointestinal tract.

According to one variant of the invention, f is renty have optimal pH value of from 4 to 6.

Proposed according to the invention medicines preferably contain enzymes that originate from ciliates of the genus Tetrahymena, Colpidium, Paramecium.

In the proposed according to the invention medicinal products preferably contain pharmaceutically acceptable auxiliary substances and carriers.

Proposed according to the invention medicines used in particular in the form of tablets, microgranules, oil, juice, gels, suppositories, capsules, pills.

The object of the invention is also the use of enzymes from ciliates selected from the group consisting of hydrolases, lipases, proteases, amylases, field of glycosidase inhibition, phospholipases, phosphodiesterase and/or phosphatases, to obtain drugs for the treatment of digestive disorders, especially drug induced dyspepsia, chronic atopic gastritis, chronic pancreatitis, acute pancreatitis, promptly due to indigestion or caused by cystic fibrosis disorders of digestion.

The enzymes that produce the simplest group of ciliates and which are used in proposed according to the invention medicines, especially suitable for the treatment of digestive disorders. In addition, enzymes and compositions of the enzymes of the infusoria, which are contained in the offer with the according to the invention medicines as well as receive them, do not possess the above disadvantages of pancreatic enzymes or enzymes of capelin or krill.

The enzymes of the ciliates come into their culture medium. Table 1 presents, for example, the enzymatic activity of various enzymes in the culture medium of the ciliates. Listed in table 1 enzyme activity was determined by the usual described in the literature methods. To determine the activity in the case of lipase used azocasein test (Muricane, 1986). To determine the activity in the case of lipase and amylase was carried out tests in accordance with methods FIP for fungal amylase and microbial lipase (Demeester and other " Pharmaceutical Enzymes, resp. Ed. A. Lauwers, S. Scharpe, Marcel Dekker, new York, 1997, S. 372-382). To determine the activity in the case of acid phosphatase and β-hexosaminidase used p-nitrophenylphosphate, respectively, p-nitro-phenyl-N-acetyl-β-D-glucosaminyl substrate, which is described Kiy and others (1996). The activity of phospholipase A₁were determined using radiometric enzyme test (Hartmann and others, 2000).

Insects that secrete enzymes into the culture medium, it is possible at lower cost to ferment when using low-cost fermentation medium at a high density of cells and continuously. While enzymes can be filtered free of cells from the fermenter through the perfusion module (filter) and thus continuously removed from the fermentation medium. The fermentation process can be maintained for longer periods of time due to the constant supply of low-nutrient environments (components: the environment on the basis of skim milk and yeast extract).

Example 1

The figure 1 presents the kinetics of secretion of ciliates within the 14-day period of fermentation. In the case of continuous perfusion fermentation module when this pic is ouput as follows.

The bioreactor system is based on a managed processor fermenter with a capacity of 2 liters (patch BIOSTAT MD, Braun Diessel Biotech, Melsungen, Germany) with a digital DCU-block adjustment and pump unit. Collection of free cell supernatants was performed by perfusion through the module, as stirrers used blade impeller. When this is used the concentration of silicone oil 1 ml/L. the Number of revolutions of the stirrer to protect against cell damage was limited to 800 rpm./minutes the Concentration of dissolved oxygen was maintained constantly at 60% with cascade. It was chosen in the first place, the rate of aeration as the first adjustable parameter and, in the second place, the number of revolutions of the agitator as the next parameter regulation. Measurement of oxygen concentration was performed using amperometrical About₂electrode (Ingold Messtechnik, Steinbach). The temperature in the fermenter was maintained at 30^aboutWith through the use of the vessel with double walls and thermostat, regulating pH 7 during the continuous phase of the fermentation was carried out via DCU-adjustable pump for correcting tools with 4 M acetic acid. Continuous phase fermentation in the fermenter was introduced Wednesday on the basis of skim milk and consumed that contains the enzymes of the medium were removed from the fermentation process through the use of the pump, which ran through the conductive probe, the working volume of 2 l was able to be kept constant. Free cell supernatant was collected without particles through the perfusion module with the pore size of the membrane of about 0.3 microns. Perfusion module was located on the holder polypropylene capillary S6/2 (ENKA, Wuppertal) with outer, respectively, an inner diameter of 2, respectively 1 mm and a length of 2.8 m Exchange medium volume per unit time was determined as the rate of perfusion. The flow medium on the basis of skim milk could regulate the number of revolutions of the sleeve of the pump in such a way that set the rate of perfusion of one volume of the fermenter (2 litres) a day. Before autoclaving, the fermenter was made to complete the foam and downloaded 1.8 l environment on the basis of skim milk without glucose. After autoclaving through the inlet fitting of the pump filed 200 ml of 10%glucose solution. Connect the flask with the environment and the flask collection was carried out through the high speed clutch in a sterile chamber. In the sterile chamber of the seed culture was poured into the Erlenmeyer flask 500 ml through the drain at the bottom and through the hose and separate nozzles for starters filed pump bioreactor.

Ciliates during the fermentation remain intact, so that the culture medium contains only the extracted infusoria proteins and does not contain any of intracellular components. This fermentation, respectively culture medium using a small number of chromatographic purification stages can achieve a high degree of purity.

Example 2

In the figure 2 is presented, for example carried out using column chromatography stage of purification of phospholipase from the culture medium of the ciliate Tetrahymena. In this case presents the elution profiles of three consecutive stages column chromatography. In particular, figure 2A as a stage 1 shows the elution profile in case chromatography with hydrophobic interaction; figure 2b as a stage 2 shows the elution profile in the case of anion-exchange chromatography and in figure 2C as a stage 3 shows the elution profile in case visokoaktivniy chromatography. For subsequent chromatography used active fractions from the previous chromatography. Thus, the enzyme after the last stage could be cleaned up to almost complete homogeneity (no alien activities, a minor proportion of alien proteins). In accordance with this purification scheme you can also clear other enzymes of ciliates as protease, lipase, amylase or β-hexosaminidase.

Ciliates are up to is not necessarily pathogenic representatives of the th (Balantidium coli), free-living (not parasitic) non-pathogenic microorganisms. For example, for the ciliate Tetrahymena in the literature proved GRAS-status (general recognized as save") (Tledtke, 1994). Besides, is believed to be reliable, that the ciliates do not contain any of endoparasites, which may be transferred to other organisms. In case important for biotechnology species of ciliates, as Tetrahymena or Colpidium, there are no viruses or other endoparasites. Therefore, it is possible to exclude contamination of the composition of enzymes toxic or pyrogenic contamination.

The figure 3 presents the obtaining of the relative enzyme activities of three enzymes from the culture medium of the ciliate Tetrahymena at different pH values (a - phospholipase a₁b - triacylglycerides; - β-hexosaminidase).

Enzymes of ciliates as acid hydrolases have an optimum pH value in the acidic region. The figure 3 presents the enzymatic activity of three enzymes from the culture medium of the ciliate Tetrahymena at different pH values. In particular, presents the relative enzyme activity of phospholipase A₁(figure 3A) and β-hexosaminidase (figure 3b), as well as the absolute enzyme lipase activity (figure 3C).

Hence, it is clear that the optimal pH value for the enzyme of the ciliates is 4.1 to 6.5. The protease of the ciliate has a high enzyme is active even when the pH value, equal 3. In the following table 2 presents the protease activity of ciliates from free from cell supernatant (culture medium) depending on pH values. Enzyme activity at the same time, as already described above, was determined in accordance with the methods FIP for fungal amylase and microbial lipases.

On this basis, the enzymes of the ciliates are more acid resistant than the pancreatic enzymes. Consequently, after passing through the stomach, they have significantly higher activity in the duodenum than the enzymes of the pancreas.

The figure 4 presents the acid protease from the ciliate Tetrahymena compared with Pancreatin in the case of the 10-minute phase impacts typical pH values, which is present in the gastric juice (pH of 1.5). Low pH value mimicked by exposure to high molar concentration of the buffer with a pH value in the acidic region (1 M glycine/HCl, pH 1,5) at a temperature of 37^aboutC.

1. L of the drug remedy to improve digestion or for the treatment of digestive disorders, containing enzymes selected from the group consisting of hydrolases, lipases, proteases, amylases, field of glycosidase inhibition, phospholipases, phosphodiesterase, phosphatases, and obtained from infusoria.

2. The drug according to claim 1, characterized in that the enzymes have an optimum pH value of from 4 to 6.

3. The drug according to any one of claim 1 and/or 2, characterized in that the enzymes come from ciliates of the genus Tetrahymena, Colpidium, Paramecium.

4. The drug according to any one of claims 1 to 3, characterized in that it comprises pharmaceutical excipients and carriers.

5. The drug according to any one of claims 1 to 4, characterized in that it is in the form of tablets, microgranules, oil, juice, gels, suppositories, capsules, pills.

6. The use of enzymes selected from the group consisting of hydrolases, lipases, proteases, amylases, field of glycosidase inhibition, phospholipases, phosphodiesterase and/or phosphatases and obtained from ciliates, for the preparation of a medicine for improving digestion or treatment of digestive disorders.

7. The use according to claim 6, in which digestive disorders caused by medication due to dyspepsia, chronic atrophic gastritis, chronic pancreatitis, acute pancreatitis, quickly caused by indigestion or cystic fibrosis.