RU2137486C1 - Human placenta allogenic preparation showing anti-inflammatory, reparative, antibacterial, wound-healing effect and method of its preparing - Google Patents

Abstract

FIELD: medicine, pharmacy. SUBSTANCE: invention relates to technology of medicinal preparations preparing. Invention proposes the human placenta allogenic preparation showing an anti-inflammatory, reparative, antibacterial and wound-healing effect that contains human placenta high-molecular proteins and glucoseaminoglycans (molecular mass is above 10 000 Da) and also glycerol and purified water at the following ratio of components, wt. -%: placenta proteins (molecular mass is above 10 000 Da) 5.0-5.5; glucoseamino-glycans (molecular mass is above 10 000 Da) 1.0-1.5; glycerol 50.0-55.0, and purified water - the rest. Method of preparing the human placenta allogenic preparation involves extraction of biomaterial with 40% alcohol for 15 min followed by homogenization and extraction with purified water (as measured 1:1), centrifugation at 3 000 rev/min and separation of supernatant. The latter is purified and concentrated on ultrafiltering unit working as dynamic filter and glycerol and purified water are added. EFFECT: simplified method, improved quality of the preparation. 2 cl, 12 tbl, 4 ex

Description

 The present invention relates to medicine, namely to the technology for producing drugs.

 The methods used to obtain biogenic preparations, in particular human placenta, are expensive and difficult to implement.

 There are known placenta preparations that are currently used to stimulate the regeneration of wound surfaces of various etiologies, as well as those used to treat peptic ulcer of the stomach and duodenum of various etiologies, both domestic and foreign, have in most cases limitations or contraindications in their appointment and use . (M. D. Mashkovsky. Medicines. In two parts. Part I., Part II. - 12th ed., Revised., Rev. And add. - M.: New wave, 1996).

 The disadvantages of these drugs and methods and preparation can be attributed to their abiogenicity (for non-protein preparations), often, low wound surface regeneration effect, weak bactericidal or antimicrobial properties, insufficient anti-inflammatory effect, high cost and inaccessibility for the general population, as well as losses active substances in the process of obtaining. Currently, there is a need for new highly effective agents for treating wounds of various etiologies and pathogenesis, with minimal contraindications and side effects.

 There is a method of obtaining aqueous extraction of the placenta, which consists in the following: a placenta obtained after normal delivery and tested for hepatitis, HIV infection and syphilis are washed with purified water and filled with 40% ethyl alcohol until the material is completely covered and incubated for 15 minutes. After the specified time, the placenta is removed from the alcohol solution and the alcohol solution is removed. The placenta thus treated is ground and homogenized. The homogenate is poured with purified water at a rate of 1: 1. Biomass extraction is carried out with stirring. After extraction of the biomass, the aqueous layer (supernatant) is separated.

Fine crystalline ammonium sulfate was added to the obtained supernatant to obtain a concentration of 60% of a saturated solution. The mixture was kept at + 4 ^° C for 2 hours to form a precipitate. The coagulated protein is separated by centrifugation at 3000 rpm for 15 minutes. The supernatant is drained, the resulting protein precipitate is dissolved in a minimum amount of purified water and dialyzed in a flow dialyzer to completely remove traces of ammonium sulfate. The duration of dialysis is at least 24 hours. After dialysis, the protein concentration in the dialyzed solution is determined by the Lowry method. After this, a preparation is obtained with a protein content of 110-120 mg / ml, introducing 50.0 (wt.%) Glycerol and purified water to 100.0 (wt.%), D. Freifelder. Physical biochemistry. Moscow, 1986.

 The aim of the invention is to develop an optimal method for producing an allogeneic preparation of human placenta, improving its quality and accelerating the healing of wound surfaces of various etiologies and pathogenesis.

A method of obtaining an allogeneic preparation of a human placenta with anti-inflammatory, reparative, antimicrobial and wound healing effects is as follows: a placenta obtained after normal birth and tested for hepatitis, HIV infection and syphilis is placed in sterile glass wide-necked containers with a volume of 1000 - 3000 cm ³ with a tight-fitting lid. Store the material in a refrigerator at a temperature of 5 - 7 ^o C for 24 hours until the desired amount is accumulated. After the accumulation of the required amount of placenta, it is thoroughly washed with purified water and transferred to an enamel tray on a baking sheet with holes of 2-3 mm to remove water. The biomaterial is transferred to a glass desiccator with a capacity of 2000 cm ³ , poured with 40% ethyl alcohol until the material is completely coated and incubated for 15 minutes. After the specified time, the placenta is removed from the alcohol solution and placed in an enamel tray on a baking sheet with a hole diameter of 2-3 mm to remove the alcohol solution.

The placenta is transferred to an enamel tray and crushed with a scalpel to a particle size of 30x50 mm. Coarsely ground material is passed through an electric meat grinder and, then, the resulting biomass is placed in a homogenizer with a capacity of 5000 cm ³ homogenized at 14000 rpm for 30 minutes. The homogenate is transferred to a wide-necked glass container with a volume of 10,000 cm ³ and poured with purified water at a rate of 1: 1. The biomass extraction is carried out with stirring for 15 minutes with an MR-25 paddle mixer at 50 rpm. After extraction, the biomass is transferred to a K-70 centrifuge glass with a volume of 1000 cm ³ and centrifuged at 3000 rpm for 30 minutes. The aqueous layer (supernatant) is separated from the precipitate by decantation in a sterile glass container.

 The resulting supernatant is fed to an ultrafiltration unit AMP-4u-20. The ultrafiltration unit AMP-4u-20 works on the principle of a dynamic filter with a fixed filtering partition. The supernatant to be separated is fed into the pressure channels of the apparatus, where it moves tangentially to the surface of the MIFIL-20 and UPM-20 membrane. Part of the supernatant is filtered off and into the collection vessel through the drainage channels. The unfiltered portion of the supernatant is returned to the product container, from where the supernatant is again pumped into the apparatus. Thus, the product circulates in a closed loop until the desired concentration of high molecular weight components (molecular weight above 10,000 D) and the desired degree of purification of the drug are achieved. The tangential flow above the membrane prevents the formation of sediment on its surface, causing clogging of the pores and a decrease in the filtration rate. The minimum productivity of the AMP-4u-20 apparatus at a pressure of 0.1 MPa is 20-30 liters per hour.

The concentration of high molecular weight protein is determined by the Lowry method. Its content should be 110-120 mg / ml. The supernatant concentrate is placed in plastic bags (GOST 42-21-2-85) pre-sterilized by gamma radiation from isotopes ⁶⁰ Co and ¹³⁷ Co 2.5 Mrad, for 15 minutes or UV radiation for 30-45 minutes at a wavelength 254-257 nm followed by their sealing.

Packets of supernatant concentrate are placed in a refrigerator and frozen at -24 ^° C for the duration of the HIV and Australian antigen tests. After receiving positive tests, the concentrate is thawed at room temperature and transferred to a sterile glass container. A calculated amount of glycerol is added to the supernatant concentrate. The resulting mass is thoroughly mixed.

The finished product is packaged with A-2 liquid dispenser (TU 64-1-2828-81) in sterile NS-2 brand bottles with a capacity of 20 cm ^{3 and} sealed with IR-119 rubber stoppers for running in aluminum caps. Vials with the drug are pasteurized by the tindalization method in accordance with the Global Fund X edition, p. 991.

 The resulting compositions were tested for the quantitative content of protein (according to the Lowry method) and glucosaminoglycans (according to the Dische method) (Table N1), antimicrobial (Table N 2), reparative (Table NN 3, 4, 5) activity and the treatment of ulcers and erosion of the gastrointestinal tract of the disease (table. N 6).

 The effectiveness of the treatment of skin wounds of various etiologies and pathogenesis was carried out on outbred white rats weighing 180-220 g of both sexes, in the autumn-spring period, obtained from the laboratory animal farm RAMS "Rappalovo". Three groups of animals were identified, 50 each. in each group. The wounds of animals of the first group were treated daily with an allogeneic drug, the wounds of animals of the second group were treated with the drug obtained by the method of obtaining the prototype, the third group was the control and did not receive treatment.

Cut skin wounds were applied to the lateral surface of the rat body with an area of 20-25 mm ² , which were copied and during the experiment were compared with the results obtained (table N3).

A thermal burn was applied to the depilated surface of rat skin. The water temperature was 98-100 ^o C. The resulting burn wounds were copied and during the experiment were compared with the results obtained (table N4).

 Cut skin wounds were infected with a culture fluid containing strains of Staphylococcus aureus, Pseudomonas aeruginosa, Proteus and Escherichia coli (0.5 billion pathogens per wound) (table No. 5).

 The antiulcer activity of an allogeneic drug was studied using a model of chronic "vinegar ulcers". Two groups of animals were identified, 100 each. in each group. The first group received treatment with an allogeneic drug, the second group did not receive treatment and was a control. The dose of drug administration is 200 mg / kg 2 times a day. On 10, 15, 20, and 25 days of the study, 25 animals were selected from each group, which were killed with chloroform. The condition of the mucosa was evaluated on a 5-point scale, according to the guidelines for evaluating antiulcer properties - 0 points - 100% recovery, 5 points - 0% recovery (table No. 6) (Anichkov S.V., Zavodskaya I.S.- M .: 1987).

 An analysis of the data obtained allowed us to determine the optimal technology for obtaining an allogeneic preparation of human placenta, which provides the most pronounced therapeutic effect due to the preservation of high molecular weight components of the placenta in an active form.

 The effectiveness of the use of an allogeneic preparation of human placenta in the treatment of wound surfaces, duodenal ulcers and stomach, various etiology and pathogenesis was studied using in vivo methods in comparison with domestic and foreign drugs. Studies were conducted on various models of wounds: models of cut skin wounds; burn wound models; models of infected skin wounds, in the autumn-spring period on outbred white rats weighing 180-220 g of both sexes, obtained from the laboratory animal laboratory RAMS "Rappalovo".

 Example No. 1.

Seven groups of animals were identified, 100 each. in each group. Large wounds with an area of 20-25 mm ² were applied to the lateral surface of the rat's body, which were copied and compared during the experiment with the results obtained.

 The wounds of animals of the first group were treated daily with an allogeneic preparation, the second group of animals with 10% methyluracil ointment (Unguentum Methyluracili 10%). Manufacturer - Russia. The wounds of the third group were treated with Solcoseryl ointment (Unguentum "Solcoseryl") - manufacturer - Switzerland, the fourth group - with the enzymatic preparation of the protein nature "Lysocim" ("Lysocim"), in the form of a 0.05% solution in 0.25% novocaine solution ( manufacturer - Russia), wounds of the fifth group - ointment "Dermazin" (Unguentum "Dermasin") - manufacturer - Yugoslavia, wounds of the sixth group were treated with ointment "Sulfargin" (Unguentum "Sulfarginum") - manufacturer - Russia.

 Wounds without treatment served as control.

 The treatment results are shown in table N7.

 According to the results of the studies presented in the table, we can conclude that when treating cutaneous skin wounds with an allogeneic preparation of human placenta, the regeneration process ends on the 15th day of treatment. Similar results were obtained in the treatment with the drug "Lysozyme" ("Lysocim"). In other cases, recovery occurred on the 19-20th day of treatment.

 In the control group of animals on the 20th day, the percentage of skin surface regeneration was 82.8% ± 1.1%.

 Example No. 2.

Seven groups of animals were identified, 100 each. in each group. A thermal burn was applied to the depilated surface of rat skin. The water temperature was 98-100 ^o C. The resulting burn wounds were copied and during the experiment were compared with the results.

 The wounds of animals of the first group were treated daily with an allogeneic preparation. As comparison preparations were used: for animals of the second group - methyluracil ointment 10% (Unguentum Methyluracili 10%). Manufacturer - Russia; for animals of the third group - ointment "Solcoseryl" (Unguentum "Solcoseryl"). Manufacturer - Switzerland; for animals of the fourth group - an enzymatic preparation of protein nature "Lysozyme" ("Lysocim"), in the form of a 0.05% solution in a 0.25% solution of novocaine. Manufacturer - Russia; for animals of the fifth group - ointment "Dermazin" (Unguentum "Dermasin"). Manufacturer - Yugoslavia; for animals of the sixth group - ointment "Sulfargin" (Unguentum "Sulfarginum"). Manufacturer - Russia.

 Wounds without treatment served as control.

 The treatment results are shown in table N8.

 As a result of the experiments, it was found that the treatment of burn wounds with an allogeneic preparation, 10% methyluracil ointment, and the enzymatic preparation of the protein nature “Lysozyme” allows to obtain 100% regeneration of the skin surface on the 20th day of treatment. In the control group of animals, this indicator was 73.1% ± 0.8% on the 20th day of treatment.

 In addition, visual observation of the regeneration of the wound surface showed that with the use of the drug, the healing process went without the formation of scar tissue (killoid formations), and in the wounds there was no suppuration and edema characteristic of burn surfaces.

 Example No. 3.

Seven groups of animals were identified, 100 each. in each group. Large wounds with an area of 20–25 mm ² were applied to the lateral surface of the rat’s body, which were copied and compared during the experiment with the results obtained.

 Wounds were infected with Staphylococcus aureus culture fluid (0.5 billion pathogens per wound). The wounds of animals of the first group were treated daily with an allogeneic preparation. The wounds of animals of the remaining groups were treated: animals of the second group - 10% methyluracil ointment (Unguentum Methyluracili 10%); animals of the third group - ointment "Solcoseryl" (Unguentum "Solcoseryl"); animals of the fourth group - an enzymatic preparation of protein nature "Lysozyme" ("Lysocim"), in the form of a 0.05% solution in a 0.25% solution of novocaine; animals of the fifth group - ointment "Dermazin" (Unguentum "Dermasin"); animals of the sixth group - ointment "Sulfargin" (Unguentum "Sulfarginum").

 The control was infected wounds without treatment.

 The treatment results are shown in table N 9.

 From the results presented in table No. 9, we can conclude about the effectiveness of the use of allogeneic preparation of human placenta in the treatment of wounds, seeded Staphylococcus aureus. The effectiveness of treatment in this case is 100.0% on the 14th day of treatment. Similar results were obtained with the use of ointments "Dermazin", "Sulfargin" and the drug "Lysozyme".

 Example No. 4.

 Determination of the effectiveness of the use of an allogeneic preparation on a model of infected skin wounds with cultures of Staphylococcus aureus (Staphylococcus aureus), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Proteus (Proteus vulgaris), E. coli (Escherichia coli).

Seven groups of animals were identified, 100 each. in each group. Large wounds with an area of 20-25 mm ² were applied to the lateral surface of the rat's body, which were copied and compared during the experiment with the results obtained. Wounds were infected with a culture fluid containing strains of Staphylococcus aureus, Pseudomonas aeruginosa, Proteus and Escherichia coli (0.5 billion pathogens per wound). The wounds of animals of the first group were treated daily with an allogeneic preparation. As comparison preparations were used: for animals of the second group - methyluracil ointment 10% (Unguentum Methyluracili 10%); For animals of the third group - ointment "Solcoseryl" (Unguentum "Solcoseryl"); for animals of the fourth group - an enzyme preparation of protein nature "Lysozyme"("Lysocim"), in the form of a 0.05% solution in 0.25% solution of novocaine; for animals of the fifth group - ointment "Dermazin" (Unguentum "Dermasin"): for animals of the sixth group - ointment "Sulfargin" (Unguentum "Sulfarginum").

 The control was infected wounds without treatment.

 The treatment results are shown in table N10.

 According to the results of these experiments, we can conclude that the allogeneic preparation of human placenta can be attributed to highly effective drugs recommended in the treatment of wounds and wound surfaces of various etiologies that have undergone mixed microbial contamination. The regeneration process was completed on the 14th day of treatment. Similar results were obtained using ointment "Dermazin" (Unguentum "Dermasin"), ointment "Sulfargin" (Unguentum "Sulfarginum") and the drug "Lysozyme".

 The sensitivity of microorganisms to an allogeneic preparation of human placenta was studied on strains that are most often found in purulent infections using a two-layer agar method in Petri dishes.

The control was Petri dishes with nutrient media containing, instead of the drug, a similar amount of a 50% glycerol solution. Colony counting was carried out after 48 hours and finally 5 days after incubation at a temperature of 30-35 ^o C.

 The growth suppression efficiency was: Staphylococcus aureus 52.1 ± 1.3%; Streptococcus agalactiae - 67.6 ± 1.7%; Klebsinella azaenae - 44.2 ± 1.3%; Pseudomonas aeruginosa - 43.7 ± 1.1%: Escherichia coli - 53.5 ± 1.4%; Bacteria of the Enterobacteriaceae family - 39.7 ± 1.6%; Bacillus subtilis - 80.3 ± 1.2%; Proteus vulgaris - 73.1 ± 1.5%.

 The microbiological activity of the drug in relation to these microorganisms was determined by the method of paper disks. The research results are presented in table No. 11.

 The antiulcer activity of the drug was studied on the model of chronic "vinegar ulcers". The experiment was carried out in spring on white outbred rats weighing 180-220 g. Of both sexes, obtained from the laboratory animal nursery RAMS "Rappalovo". Six animal groups were identified, 100 each. in each group. The dose of the drug was 200 mg / kg 2 times a day. On 10, 15, 20, and 25 days of the study, 25 animals were selected from each group, which were killed with chloroform. The condition of the mucosa was evaluated on a 5-point scale - 0 points - 100% recovery, 5 points - 0% recovery, according to the guidelines for evaluating antiulcer properties (S.V. Anichkov, I.S. Zavodskaya. - M .: 1987) .

 The first group of animals was treated with an allogeneic drug, the second group with Methyluracil, the third group with Solcoseryl, the fourth group with Cimetidine, the fifth group with Carbenoxolone, the sixth group was not treated and was a control. The research results are presented in table No. 12.

 As a result of studies, it was found that the allogeneic preparation of human placenta has a pronounced antiulcer effect. The effectiveness of the treatment of "vinegar ulcers" of the gastric mucosa with the drug (table N12, 1st group of animals) coincides with the same indicators of the preparations "Cimetidine" and "Carbenoxolone" (table N12, 4th and 5th groups of animals, respectively).

 During the experiment, visual observation of the wound surface showed that when using the drug to treat stomach ulcers, the process of regeneration and restoration of the surface occurs without the formation of coarse scar tissue.

 Thus, as a result of the studies, it was found that in the treatment of cutaneous skin wounds with an allogeneic preparation, the regeneration process ends on the 15th day of treatment. In the control group of animals on the 20th day, the percentage of skin surface regeneration was 82.8% ± 1.1%.

 Treatment of burn wounds with the drug made it possible to obtain 100% regeneration of the skin surface on the 20th day of treatment without the formation of killoid tissue. Similar results were obtained using 10% methyluracil ointment, an enzyme preparation of the protein nature Lysozyme, but in these cases, the formation of scar tissue was observed. In the control group of animals, this indicator was 73.1% ± 0.8% on the 20th day of treatment.

 Thus, as shown by studies of the regeneration of the wound surface, with the use of an allogeneic preparation, the healing process proceeds without the formation of scar tissue (killoid formations), and in the wounds, suppuration and edema characteristic of burn surfaces are not observed.

 Comparison of the results of treatment of infected skin wounds both in the case of monoculture and when infected with several pathogenic microorganisms, allows us to attribute the allogeneic drug to highly effective drugs recommended for the treatment of wounds and wound surfaces of various etiologies that have undergone microbial contamination. The regeneration process was completed on the 14th day of treatment. Similar results were obtained using ointment "Dermazin" (Unguentum "Dermasin"), ointment "Sulfargin" (Unguentum "Sulfarginum") and the drug "Lysozyme". In the control groups of animals, the percentage of regeneration of skin surfaces was 82.5% ± 1.7% and 84.8% ± 2.1% on the 20th day of treatment, respectively.

 It was found that the allogeneic drug has antimicrobial activity against the main pathogens of purulent infection.

 A distinctive advantage of the developed drug in the treatment of wound surfaces of various etiologies from known drugs is the combination of high bioavailability and biogenicity with a simple composition and relatively simple and cheap production technology.

 The content of high molecular weight proteins and glucosaminoglycans of human placenta as the main active components of the preparation provides a reduction in the terms of regeneration of the wound surface of various etiologies and pathogenesis without the formation of killoid tissue, while preventing the development of purulent infection, in contrast to the well-known drugs of domestic and foreign production.

 The proteo- and nucleotic activity of the protein components allows for the efficient maceration of damaged tissues of any etiology of damage, which contributes to the rapid cleaning of the wound surface. Lithium enzymes directly inhibit the development of wound microflora due to the proteolysis of the shells of microbes and the destruction of their nucleic acids.

 The presence of high molecular weight glucosaminoglycans inhibits hyaluronidase in wounds by switching their activity to an exogenous substrate, which slows down the process of glucosaminoglycan cleavage while restoring the integrity of damaged tissues. At the same time, glucosaminoglycans and their lysate serve as building components for tissues in the process of their regeneration.

 The presence of albumin in the drug promotes wound detoxification by binding toxins to albumin receptors. The high content of allogeneic protein in the preparation provides energetically favorable nutritional conditions for rapidly dividing fibroblasts, macrophages, perivascular cells, lymphocytes, which contributes to their functional activation and, as a result, stimulation of reparative processes in wound tissues of any etiology.

 Due to the activation of almost all cellular structures, starting from the first stages of regeneration (especially the cells of the basal layer - epidermal cells). The process of restoring the integrity of the tissues goes without the formation of killoid formations, with the achievement of a high cosmetic effect.

 The proposed drug does not have a sensitizing effect on the body, has no restrictions on the use of age. It can be used during pregnancy, for the treatment of deep wounds and burns.

Claims (1)

1. An allogeneic preparation of human placenta with wound healing, reparative, antiulcer, antimicrobial activity, including extractive substances of human placenta, characterized in that it contains high molecular weight proteins of the placenta and glucosaminoglycans with mol.m. over 10000 D, as well as glycerin and purified water in the following ratio of components, wt.%:
Proteins of the placenta with mol.m. over 10000 D - 5.0-5.5
Glucosaminoglycans of the placenta with a mol.m. over 10000 D - 1.0-1.5
Glycerin - 50.0-55.0
Purified Water - Else
2. A method of obtaining an allogeneic preparation of a human placenta with wound healing, reparative, antiulcer, antimicrobial activity, including keeping the biomaterial in 40% alcohol for 15 minutes, followed by homogenization and extraction with purified water at a rate of 1: 1, centrifugation at 3000 rpm / min and separation of the supernatant, characterized in that the obtained supernatant is purified, concentrated and filtered, using filters that allow you to allocate the components of the placenta from mol.m. over 10000 D, with the subsequent addition of the estimated amount of glycerin and purified water.

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