RU2145874C1 - Method of manufacturing necroectomic dressing - Google Patents

Abstract

FIELD: surgical facilities. SUBSTANCE: raw material from pancreas of slaughtered livestock completed with small intestine mucous tissue, sodium bicarbonate, and sodium chloride is utilized. Biomass is diluted with water, thermally equilibrated, placed into refrigerator for water extraction of enzymes, filtered off, and squeezed. Residue is mixed with more water and refiltered off. Aqueous extracts are combined and benzoic and boric acids are added. Biomass is used to impregnate hygroscopic material, which is then subjected to lyophilic drying. Product in the form of plates is tightly packed into polyethylene bags. EFFECT: improved therapeutic effect of dressings. 2 cl, 5 tbl, 2 ex

Description

 The invention relates to medicine and can be used in surgical practice for the treatment of deep wounds with the presence of necrotic areas of the skin and tissues adjacent to it, as well as sluggish purulent wounds and burns.

 In modern surgical practice, various necrolytic agents are used in the treatment of thermal burns and purulent wounds with the presence of extensive devitalized skin areas and tissues adjacent to it. They are resorted to in cases where total or tensile necrectomy is not indicated, and spontaneous and prolonged rejection of necrotic tissue is undesirable.

 Known methods allow to obtain drugs used for these purposes, with a narrow range of enzymatic action, with low proteolytic activity, often packaged in extremely small dosages and in dosage forms that are inconvenient for use. This group of drugs also includes chymopsin. Its production method is based on the chromatographic separation of proteins extracted and salted from crushed pancreatic tissue of slaughter cattle. It contains a mixture of chymotrypsin and trypsin. Shiny flakes or powder of white or white with a faint yellowish tint. Easily soluble in water and isotonic sodium chloride solution; pH of a 0.2% aqueous solution of 4.5 to 6.5. In the treatment of purulent wounds and bedsores, 0.025-0.05 g (25-50 mg) of chymopsin is dissolved in 10-50 ml of 0.25 N. novocaine solution; the solution is moistened with sterile wipes, which are applied to the wound surface for 8 hours or more. For burns of the III degree, a thin layer of chymopsin is applied (in the form of powder) and covered with a dressing soaked in an isotonic sodium chloride solution or in a 0.25% solution of novocaine in borate buffer (pH 8.6). M.D. Mashkovsky Medicines. M .: New wave, 1996, v. 2, p. 59-60.

 In the same way, collagenase, trypsin, chymotrypsin, asperase and pankipsin / MHP / are obtained from the pancreas of farm animals, and from bacterial cultures: profesim, terrilithin, lecozyme, caripazin, terridekase, lysoamidase, etc., which are widely used for enzymatic necrectomy ( I.Kuzin, V.K.Sologub, V.V. Yudenich // Burn disease. - M .: Medicine, 1982, 159 pp. /; M.D. Mashkovsky Medicines. M.: New wave, 1996 ., t.2, pp. 59-66. The disadvantages of all these drugs are: a narrow range of enzymatic action, small and poor good packaging, creating a number of inconveniences in their application and an extremely limited period of effective action.

 The known method adopted by us for the prototype / Instructions for use "Pankipsin" Approved in 1989 by the Ministry of Health of the MPR /, allows to obtain the drug - Pankipsin / MPR / from the pancreas of small cattle by mechanical grinding, fractional salting out, dialysis, protein separation on chromatographic columns, and then freeze-drying the eluate. Using a multi-stage process, a powdery preparation is obtained containing trypsin and chymotrypsin, which is packaged in 30 mg vials.

 However, the existing method for producing an enzyme-containing non -rectomotic agent, pankipsin, does not allow it to be widely used in the treatment of deep and extensive burn injuries, as well as sluggish purulent wounds due to the low proteolytic activity with respect to connective tissue structures and extremely limited purchases of it from MNR.

 The mild proteolytic effect of the drug on connective tissue is due to the fact that it lacks elastase / pancreatopeptidase E /. Its effect is due only to the presence of trypsin and chymotrypsin, which poorly hydrolyze peptide bonds in the connective tissue.

 The manufacturer’s instructions for the Pankipsin ointment provide an option for producing an ointment for the treatment of thermal burns and purulent wounds with the inclusion of 0.25 g of this drug per 100 g of ointment on a lanolin base / Instructions for use of Pankypsin approved by the Ministry of Health of the MPR /. The proposed recipe can hardly be considered appropriate, because per 1 g of ointment accounts for 2.5 mg of pankipsin. This amount of the drug is clearly insufficient to obtain a highly effective necrectomy drug. In addition, for the preparation of 100 g of ointment, it is necessary to extract the contents of 10 vials, which creates certain inconveniences. Pancypsin solutions in various solvents are even less promising since the contents of one bottle can handle an extremely limited area of the dressing. The disadvantage of pankipsin is its short-term effect due to the very fast inactivation of enzymes.

 The method is carried out abroad, but is not provided for in domestic conditions.

 The aim of the invention is: to obtain a highly effective drug for enzymatic necrectomy of devitalized connective tissue structures and underlying tissues in patients with burn damage and purulent wounds.

Tasks:
- increase the duration of the drug;
- expansion of indications for the use of dressings;
- reduction of production costs;
- creating a simple and easy-to-use tool;
- reduction of treatment time.

The essence of the invention is to obtain a multienzyme preparation with increased specific proteolytic activity, due to the preparation of an enzyme-containing composition from the pancreas with the addition of a homogenate of the mucous membrane of the small intestine, sodium chloride and sodium bicarbonate, which contribute to the activation of pancreatic and intestinal proteinases at 1.5 - 2, 0 hour incubation, followed by 16 to 20 hours extraction at + 4 - 6 ^o C, separation of the liquid fraction of the composition, impregnation with a hygroscopic about the material which, after freeze drying, is sealed.

 A nonrectomy effect is achieved due to the presence in the preparation of a complex of activated proteinases / trypsin elastase, chymotrypsin, carboxypeptidase, aminopiptidase, etc. /, extracted from the pancreas and small intestine mucosa of slaughtered animals and the addition of substances providing the necessary reaction of the solution / bicarbonate of soda /, isotonicity / table salt /, preservation and antibacterial protection / benzoic and boric acid /.

 The method is as follows: they take fresh or defrosted pancreas of slaughtered animals (preferably beef, it contains less fatty inclusions than pork), they are mechanically freed from visible fat accumulations and crushed to a homogeneous state. After that, they take sections of the small intestine (preferably the duodenum, with the highest content of proteinases) of 30-40 cm, washed with running water and scraped off the mucous membrane. Then a mixture is prepared from homogenates of the pancreas and the mucosa of the small intestine, sodium bicarbonate, sodium chloride and water.

The mixture is thoroughly mixed and placed in a thermostat at 36-37 ^o C for 1.5-2.0 hours to activate the enzymes, then the mixture is again mixed and placed in a refrigerator with +4 - 5 ^o C for 16-20 hours for extraction of enzymes from tissues.

 Bicarbonate soda is introduced into the mixture to obtain the required pH value (7.3-8.1), which promotes the conversion of proenzymes into enzymes / trypsinogen into trypsin, chymotrypsinogen into chymotrypsin, pro-elastase into elastase, etc. /, then bicarbonate soda provides the necessary reaction of the medium and in the final product.

 For a more complete extraction of enzymes from homogenized raw materials, the mixture is shaken for 20-30 minutes / for example, in the apparatus "Labor" 2124 /, and then it is filtered and squeezed through 2 - 3-layer cheesecloth or rubbed through a No. 2 sieve. In this case, excess connective tissue structures / vessels, ducts, and other ballast components / are separated. Distilled water (2: 1 ratio) was added to the solid precipitate, stirred and the filtering procedure was repeated. Both filtrates are mixed, a homogeneous liquid mass is obtained, to which benzoic and boric acids are added and mixed thoroughly. They are reliable preservatives, and boric acid is also a means of chemical necrectomy / M.I. Kuzin et al. / Burn disease M .: Medicine. - 1982. - 159 p. /.

 This homogeneous mass is evenly applied with a thickness of 3-4 mm on absorbent tissue, for example pieces of gauze previously cut / 5 x 5 cm, 10 x 10 cm / and laid out in 2-3 layers on the bottom of the baking sheets from a sublimation unit. Then the baking sheets are placed in a freeze drying chamber. Preservatives and freeze drying ensure reliable sterilization. Therefore, after drying, flexible dense plates of light yellow or pale pink color are obtained. They are put in plastic bags, and sealed by sealing.

In order to determine the necrolytic ability of the proposed drug, the main biochemical parameters were established in a multienzyme non-fracturing dressing / PNP /. To do this, 2-3 samples are taken from each batch of PNP received. 1 cm of PNP is cut off and each piece is brought into a separate tube, 5 ml of distilled water are added and placed in a refrigerator with + 4 ^o C. The contents of the tubes are periodically shaken. After 2 hours, the extracts were checked:
1) The total proteolytic activity / according to the Anson method, in the modification of P.G. Storozhuk and V.L. Krishtopa // Sat.mater. to scientific confer. Young scientists of the Kuban. - Krasnodar. - 1969. - S. 115-116 /;
2) Trypsin activity / according to the Erlanger method, modified by V.A. Sheternikova // Biochemical research methods in the clinic. / Ed. A.A. Pokrovsky. - M .: Medicine. - 1969. - S. 210-211 /.

3) Elastase activity / according to the method of P.G. Storozhuk and I.I. Pavlyuchenko. - A rationalization proposal. N 96 1989, Kuban Medical Institute /;
4) The amount of mg of dry matter:
5) The amount of mg protein / according to Kjeldahl, in the modification of Conway // Biochemical methods in the clinic. / Under the editorship of A.A. Pokrovsky. - M .: Medicine. - 1969. - S. 77-78 /.

6) the pH of the solution;
As an example, the results of a study of three samples of one of the batches of PNP / table. 1/.

In order to prove a higher necrolytic effectiveness of PNP than other non-fracturing drugs, its main biochemical parameters are compared with those of terrilithin and pankypsin. To do this, the contents of the vial of the drug pankipsin / 25 mg /, as well as terrilitin / 10 mg / were dissolved in 10 ml of physiological saline and treated with this solution of 100 cm ^{2 of a} three-layer gauze. The comparison results are presented in table. 2.

 From this table it can be seen that the PNP has an advantage over the dressing treated with pankypsin. So, in it, tryptic activity is 13% more, and elastase activity is 15 times more. As for the dressing treated with terrilithin, compared with it in the PNP, the tryptic activity is more than 45, and the elastase is 100 times.

In order to determine the proteolytic effect of enzymes in PNP on devitalized tissues, the decreasing activity of proteinases at various time intervals in a simulated system close to wound conditions was studied. To do this, pieces of PNP in 3 cm ² each were weighed into three test tubes / which weighed 117, 121, 125 mg /. To each of them was added 15 ml of distilled water. After 2 hours of extracting the enzymes from the gauze base (at + 4 ^° C), the initial levels of trypsin and elastase were determined in the extracts. Then the tubes were transferred to a water thermostat / + 37 ^o C /, from which samples were periodically taken for analysis. The results of these studies are presented in table. 3.

 It can be seen from this table that in PNP, tryptic activity gradually decreases and by the end of 7 hours it drops by 90%. Elastolytic activity in the middle of the experiment even increases, and at the end of the experiment it is approximately the same as the original one.

To compare the non-fracturing effect of PNP / according to the application / with pankipsin and terralitin, similar experiments were performed, which are presented in table. 4 and 5
From the table. Figures 4 and 5 show that after 3 hours the elastolytic activity in PNP slightly increases, while in terrilithin it increases more than 2 times, then it gradually decreases. In pancypsin, a decrease begins from the first hour and goes to the end of the experiment. Pancypsin has the highest trypsin activity, but it is detected only during the first hour. In terrilithin, it is 15 times smaller, but it has already been detected within 3 hours, while in PNP it is the lowest, but remains during the entire 7-hour experiment. These data indicate that enzymes associated with concomitant proteins, for example in PNP, are more stable during thermostating than in purified enzyme preparations, for example, in pancypsin.

 The method of producing PNP was tested in laboratory conditions at the Department of Biological Chemistry of the Kuban State Medical Academy.

Example 1. For multienzyme non-fracturing dressings, biomass was first prepared. To do this, they took: 1) fresh beef pancreas, removed the accumulation of fat, passed through a meat grinder, and then subjected to homogenization; 2) segments of the small intestine / 30-40 cm / cattle, freed from accumulations of fat and turned out by the mucous membrane, were thoroughly washed in running water and scraped off the mucous membrane with a blunt knife, which was further homogenized. After that weighed:
Pancreatic homogenate - 750 g
Homogenate of the intestinal mucosa - 70 g
Sodium bicarbonate (bicarbonate of soda) - 35 g
Salt - 35 g
Distilled water - 600 ml
The resulting biomass / ratio, respectively: 9.0: 09: 0.4: 0.4: 7.0 / was thoroughly mixed and placed in a thermostat for 1.5 hours at 37 ^° C. Stirring was repeated every 20-30 minutes. Then the tank with biomass was transferred to the refrigerator at + 4 ^o C. After 16 hours, the biomass was filtered through 2 layers of gauze and squeezed slightly. 1/3 by weight of distilled water was added to a solid precipitate (750 g). Mix thoroughly and repeat the filtration procedure. As a result of these two operations, 1050 ml of a pale pink extract was obtained. An additional 1.0 g of benzoic acid and 1.0 g of boric acid were added to it. Thoroughly mixed and spread on cheesecloth, previously cut into pieces of 10x10 cm and laid in 3 layers on the bottom of the baking sheets from the sublimation unit. Biomass was applied in such a way as to completely soak gauze. Using 1050 ml of biomass, 960 cm ² gauze was treated. After that, the trays were placed in a freeze-drying chamber, and their contents were subjected to freeze drying.

The drying mode was reduced to the fact that at first in the chamber the temperature gradually decreased over a period of 10 hours to -40 ^o C with intensive ventilation. In the next 12 hours, it was gradually raised to +15 ^o C, and then for another 12 hours to +25 ^o C. At this temperature, the process continued until complete drying for another 12 hours. Dried napkins in sterile conditions, 1 piece each was transferred to plastic bags, which were immediately sealed.

 Dressings were tested in the Burn Center of the Regional Clinical Hospital in Krasnodar for 63 patients. Moreover, the results showed that the treatment time was reduced by 30-40% compared with the use of the prototype. Due to the greater activity of the enzymes trypsin, chymotrypsin, elastase, dressings have been more effective in the treatment of intractable, sluggish purulent-septic processes. It is noted that the use of the proposed dressings does not cause side effects in the form of allergic reactions. Particularly relevant is the solution to the problem of increasing the period of use. Tests have shown that the activity of enzymes practically does not decrease for 5 years.

Example 2. To obtain the PNP, first biomass was prepared, for this weighed:
Pancreatic homogenate - 800 g
Homogenate of the small intestine mucosa - 80 g
Sodium Bicarbonate - 40 g
Salt - 40 g
Distilled water - 640 ml
The resulting biomass / ratio: 10.0: 1.0: 0.5: 0.5: 8.0 / was thoroughly mixed and transferred to a thermostat for 2 hours at 37 ^° C. Stirring was repeated every 30-40 minutes. Then the tank with biomass was placed in a refrigerator at +4 ^o C for 20 hours. After that, the biomass was filtered through cheesecloth into 2 layers and squeezed slightly. To a solid precipitate (800 g), 300 ml of distilled water was added, mixed and filtered again. As a result of these two operations, 1,100 ml of a light pink extract was obtained. An additional 1 g of benzoic and boric acid was added to it. The resulting extract was applied to pieces of three-layer gauze, previously cut into the shape of the trays of the sublimation unit. This biomass was able to soak about 1 m ² gauze base. After that, the trays were placed in a sublimation chamber and their contents were subjected to freeze drying. The drying mode is exactly the same as in example 1. The dried napkins under sterile conditions were cut into pieces of approximately 5.0 and 7.5 cm ² , put into plastic bags and sealed.

 The main advantage is: the possibility of domestic production, and not the purchase of foreign currency abroad / in the Mongolian People's Republic /; a wider spectrum of action on necrotic tissue than that of the prototype "pankypsin": lengthening the duration of the prolitic effect on devitalized tissues due to incomplete purification from ballast proteins and immobilization on absorbent tissue; / simplicity and ease of use / dressing is removed from a plastic bag, wetted with distilled water and applied to the wound. The proposed method has signs of significant novelty in the manufacture of multienzyme non-fracturing dressings / PNP / for the treatment of deep burn wounds. PNP has a good necrolyzing property, significantly reduces pus excretion, enhances granulation and epithelization, which accelerates the transition to the subsequent stages of treatment and shortens the length of hospital stay.

Claims (2)

1. A method of obtaining a non-fracturing dressing for the treatment of burn and purulent wounds, comprising extracting enzyme-containing fractions from the pancreas and freeze drying, characterized in that to activate the enzymes in the isolated pancreatic fraction add homogenous mucosa of the small intestine, sodium chloride, bicarbonate soda and water, incubated for 1.5 - 2 hours, from tissue enzymes after incubation extracted with brine once within 16 - 20 h at 4 - 6 ^o C, a liquid fraction is separated from the precipitate with water REMOVE MISFED cabins enzymes filtrates were combined, impregnated with a mixture of water-absorbent fabric, for example gauze, and after freeze drying are hermetically packaged.  2. The method according to claim 1, characterized in that before impregnating the absorbent tissue, benzoic and boric acids are introduced into the mixture of filtrates as antibacterial agents.

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