Antiproteinases drug

 

(57) Abstract:

The invention relates to medicine, namely to treatment antiproteinase drugs, and can be used for correction of conditions associated with increased proteolytic activity in the tissues, blood and other biological fluids. The invention allows to create antiproteinases the drug on the basis of protein proteinase inhibitor with polyvalent antiproteinase activity against pancreatic serine and leukocyte proteinases, the ability to provide prolonged therapeutic effect and does not suppress the activity of serine proteases kallickrein - kinin and coagulation of blood systems. The essence of the invention lies in the fact that as a main component antiproteinases drug use ovomucoid of protein duck eggs. table 2.

The invention relates to medicine, namely to treatment antiproteinase drugs, and can be used for correction of conditions associated with increased proteolytic activity in the tissues, blood and other biological fluids.

Many serious diseases are accompanied by activation of processes of proteolysis. A key role in their tapasam (trypsin, chymotrypsin, elastase). In this regard, one of the most effective ways of correction of pathological conditions associated with hyperactively proteolytic enzymes, is a selective suppression of their activity.

Known used for this purpose, preparations, obtained from the pancreas of cattle -"the Drug", "Contrical", "Aprotinin", "Tsalal", "Gordox and others [1] However, showing high affinity for trypsin and chymotrypsin, these drugs are ineffective in relation to the other, the most aggressive in the pathology of the body, the serine proteases leukocyte and pancreatic elastase, the leukocyte cathepsin G. in addition, therapeutic effect of antifermentny products obtained from the pancreas, due to their low molecular weight and rapid excretion from the body briefly.

The closest (prototype) to declare to the technical essence and the achieved effect is antiproteinases drug (Contrical) containing protein proteinase inhibitor, sodium chloride and water for injection [2] as a proteinase inhibitor, this drug contains the basic pancreatic trypsin inhibitor in the following ratio to the of jacci To 1.0 l

The basic pancreatic trypsin inhibitor has a high affinity to the activated pancreatic enzymes trypsin and chymotrypsin, and therefore the most effective in the pathology of this organ.

The disadvantages of the known antiproteinases of the drug are low activity against other serine proteinases, especially leukocyte (elastase, cathepsin G), a high rate of excretion from the body (half-life when administered intravenously is 90 minutes), and the ability to inhibit the activity of serine proteases kallickrein-kinin and coagulation of blood systems, which could cause regional disturbance of microcirculation.

The objective of the invention is the creation of antiproteinases of the drug on the basis of protein proteinase inhibitor with polyvalent antiproteinase activity against pancreatic serine and leukocyte proteinases, the ability to provide prolonged therapeutic effect and does not suppress the activity of serine proteases kallickrein-kinin and coagulation of blood systems.

This is achieved by the fact that antiproteinases preparation containing protein inhibitor of Proteus the duck eggs in the following ratio of components, g/l:

Ovomucoid of protein duck eggs 0.5 to 50.0 Sodium chloride 8,5-10,0 Water for injection To 1.0 l

Ovomucoid of protein duck eggs receive according to the method described [3]

The composition is prepared as follows.

P R I m e R 1. 10.0 g obtained under aseptic conditions, purified by ultrafiltration and freeze-dried ovomucoid of protein duck eggs are dissolved in water for injection, add 9.0 g pyrogen-free sodium chloride solution and bring the solution volume to 1.0 l with water for injection. The solution is filtered through a sterilizing filter with a pore diameter of 0.22 μm. Antiproteinases the product is ready to use.

P R I m e R s 2-5. Antiproteinases the product prepared according to example 1, using 0.5 g of ovomucoid and 8.5 g of sodium chloride; 3.0 g of ovomucoid and 10.0 g of sodium chloride; 25,0 g ovomucoid and 9.0 g natra chloride; 50.0 g of ovomucoid and 9.0 g of sodium chloride, respectively.

Comparative analysis with the prototype shows that the proposed facility has the chemical nature of the protein proteinase inhibitor, that is, the proposed solution meets the criterion of "novelty". It also meets the criterion of "inventive step" as among the famous antiproteinuric drugs no substance, obladaushiy) and leukocyte (elastase and cathepsin G) proteinases, as well as the ability to provide prolonged therapeutic effect.

P R I m e R 6. In experiments on 92 Wistar rats of both sexes weighing 200,0 g study therapeutic effect antiproteinases of the drug on the background of acute destructive pancreatitis. Modeling inflammation of the pancreas is carried out by introducing canned medical bile in General biliopancreatic duct by canulli it through the duodenum blunt needle (0.4 mm in diameter. The volume of injected bile 0.1 ml per 100 g body weight. Animals are divided into control (48 individuals) and two experimental series: to study therapeutic effectiveness of the proposed object (22 individuals) and the comparison drug Kontrikala (22 individuals). Compare the drugs are administered intravenously once 2 hours after modeling of acute pancreatitis in the dose of 10 mg per kg of body weight. In the control series no treatment is not carried out.

In the development of the disease carry out biochemical analysis of blood plasma, assess the mortality of animals, study the morpho-structural features of the pathologic process in the pancreas.

When the selected model of pancreatic necrosis in tissue pagelog is with hemorrhages in necrotic tissues, fat necrosis and suppuration. There is a sharp increase in the proteolytic activity of blood plasma of animals, reaching a maximum after 3-4 h after the start of the experiment, when repeated, even more pronounced rise after 1 day. It should be noted a clear correlation between biochemical and histological studies on the height of haemorrhagic component of necrosis and the emergence of stefanakos title amidase activity (BAPNA) blood plasma is the highest (table. 1). The progression of the pathological process in this series of animals causes the death of some of them: mortality after 24 and 48 h after the start of the experiment account for 45.8% (22 individuals) and 77.1 percent (37 individuals), respectively.

Histological examination of the tissues of the pancreas in a series of animals with intravenous claimed antiproteinases of the drug show that already after 1 h in the pancreas regressing phenomena interstitial edema, hemorheological disorders, degenerative and necrobiotic changes acinar cells. In the same period, significant, more than 30 times compared to the same period in animals from the control series, and more than 15 times the source data, snizeni Intego drug its inhibitory effect on amidase blood system is stored, moreover, the level of proteolytic activity in these time intervals does not exceed the initial value.

On histological data through 22 hours after the introduction of the proposed facility shows no signs of progression of necrosis and hemorheological disorders in systems of macro - and microvasculature. Around foci of inflammation there is a clear inflammatory demarcation. Reduction of interstitial edema, absence of dystrophic changes in acinar cells remaining viable segments leads to the restoration of exposee of imagenow in the lumen of the ducts, thus preventing parapet them in the interstitium and the flow in the blood and lymphatic vessels. Treatment of the claimed object slows the progression of dystrophic and necrobiotic changes in acinar cells and contributes, thereby reducing lesions. Under the influence of the treatment not only stops the spread of hemorheological disorders, but recovered after lysis or recanalization of blood clots, blood in the initially damaged arteries and veins. Most of the animals in this series survives. Mortality after 24 h is 13.6% (3 individuals), through 48-22,7% (5 individuals) CE is the group of the comparison drug Kontrikala. Compared with rats treated with the claimed subject, after 1 h after infusion Kontrikala observed a greater level of total proteolytic activity of blood plasma (PL.1). However, the introduction of the comparison drug significantly (p<0,05) decrease amidase activity relative to the comparable period in control. Histological differences between a power series in this time interval does not occur. However, after 4 h after injection Kontrikala proteolytic activity of the plasma reaches the corresponding values in the control series, more than 2 times higher than that in animals with the introduction of the proposed object. Histologically, in this period there is a picture of the exacerbation of the pathological process in the pancreas with elements of repeated hemorrhages, aggravation of degenerative and necrobiotic changes in acinar cells, the increase in areas with hemorheological disorders. Further through 22 h after the correction of pancreatic necrosis limited Kontrikala level of proteolytic activity is even higher, statistically significantly different not only from the original values, but also on indicators to compare the experimental group (table.1). However, the parenchyma of the pancreas suggests, introduction Kontrikala also contributes to the inhibition of development of pathological process in the body, although the severity of this treatment effect is significantly lower in comparison with the claimed subject, and the ability to inhibit the active forms of serine proteases short term, is seen only within 1-2 h after infusion. Injections of the proposed facility on the contrary contribute to the stable retention of the level of total amidase activity in the plasma of animals with experimental pancreatic necrosis within the boundaries of the physiological norm almost for 22 h after injection, thereby ensuring the mobilization of compensatory-adaptive proliferative and reparative mechanisms. Mortality in the treatment Contrical after 24 h is 22.7% (5 individuals), 48 54,5% (12 individuals), respectively.

The results obtained indicate a high therapeutic antiproteinase activity claimed antiproteinases of the drug, its ability long-term (prolonged) to inhibit the active forms of serine proteases that can be attributed to features of the chemical structure of part of the preparation of ovomucoid of protein duck eggs.

P R I m e R 7. To determine antiproteinuric-Hcl buffer, pH 8.0, containing 0.02 M l2to the concentration of ovomucoid 0,h-9mol 1 ml, 0.5 ml of the resulting solution (the content of ovomucoid 0,h-9mol) are added to 0.5 ml of trypsin solution in the same buffer (the contents of trypsin 0,h-9mol) and incubated for 5 min at 37aboutC. To determine the residual activity of the enzyme to the incubation mixture is added 3.5 ml of a solution of p-nitroanilide N-benzoyl-D,L-arginine (0,h-3M) in the same buffer. The reaction is stopped after 10 minutes by addition of 0.5 ml of 30% aqueous solution of acetic acid and measured the density of the solution at 410 nm. A control experiment is carried out according to a similar method in the absence of ovomucoid. The residual enzyme activity calculated by dividing the optical density of the sample on the optical density of the control and expressed in percent.

The measurement results are shown in table.2.

P R I m e R s 8-12. The experiment is carried out according to example 7, using drugs, containing 0.5 g/l of ovomucoid and 8.5 g/l sodium chloride; 3.0 g/l of ovomucoid and 10.0 g/l of sodium chloride; 25,0 g/l of ovomucoid and 9.0 g/l of sodium chloride; 50.0 g/l of ovomucoid and 9.0 g/l of sodium chloride; 0.9 g/l of ovomucoid and 9.0 g/l of sodium chloride, respectively.

P R I m e R s 13-14. (S 15-24. The experiment is carried out according to example 7. Used enzymes, protein inhibitors of proteases, substrates for determination of activity and activity measurement results are shown in table.2.

ANTIPROTEINASES DRUG-based proteinase inhibitor protein nature, sodium chloride and water for injection, characterized in that as a proteinase inhibitor it contains ovomucoid of protein duck eggs in the following ratio of components, g/l:

Ovomucoid of protein duck eggs from 0.5 to 50.0

Sodium chloride - 8,5 - 10,0

Water for injection Up to 1 l

 

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