RU2369398C2 - Human fibroblast-based supernatant medicine with wound healing activity - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: medicine with wound healing activity is produced on the base of supernantant of human fibroblasts and it additionally contains human plasma O (I) of Rh-positive, group, regular insulin, natrium nuclinate, amino acid complex Inphezol 100, fat concentrate Lipofundin MST/LST 20%, salts, glucose and vitamins.

EFFECT: high-efficiency complex medicine with wound healing activity without any agents prohibited for clinical use.

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Description

The invention relates to medicine, namely to agents with reparative and regenerative effects on wounds of various origins and used in surgery, obstetrics and gynecology, dermatology.

Attempts are known to use, on the one hand, donor plasma and its components ("Patent No. 2222317;" Drug and method for the seamless treatment of wounds ", No. 2002134900), on the other hand, cell culture supernatant or its fractions ("Means for changing the rate of growth or reproduction of cells, a method for its preparation, a method for stimulating wound healing or treatment of burns, a method for correcting a cosmetic defect, a method for inhibiting skin aging and a method for stimulating hair growth", Patent No. 228) 0459; "Therapeutic agent for dermatosis and covering material for treating dermatosis using concentrated solution of extracellular matrix", patent No. JP2004339107). However, the use of cell-conditioned culture medium in clinical medicine is currently not possible, because some components of the media are not registered in the State Pharmacopoeia and, accordingly, do not have permission for clinical use. The use of whole plasma promotes the formation of dense fibrin plaque on the wound, which can not only stimulate repair, but, along with other protein, lipid and carbohydrate components of the plasma, contribute to the development of microbial inflammation and worsen the condition of the wound. At the same time, the release of only certain components from plasma, for example, fibronectin or thrombin, deprives the drug of other biologically active plasma components that are stimulators of the growth and reproduction of the epithelium and extracellular matrix production (growth factors, cytokines, hormones).

The closest is: "A substance with anti-inflammatory, immunomodulating and wound healing activity" (Patent for invention No. 2300384 from 06/10/2007). The specified tool has a major drawback - it contains a culture medium having the above limitation for medical use.

The objective of the invention is the creation of a highly effective complex drug with wound healing activity and not containing substances not approved for clinical use. The task is achieved by the most accurate reproduction of the composition of the culture medium through the use of registered official preparations. In addition, in order to enhance the reparative activity of the fibroblast supernatant, a number of drugs with reparative activity (insulin, sodium nucleinate), as well as diluted donor plasma, were added to the final product, which ensured the preservation of sufficient concentrations of biologically active substances, but avoided the previously mentioned negative aspects application of whole plasma. The composition of the wound healing agent is as follows:

- air-conditioned confluent culture normal human fibroblasts daily supernatant in 0.9% isotonic solution sodium chloride 300 ml - complex of amino acids "Infezol 100" 5 ml - fat concentrate "Lipofundin MCT / LST 20%" 5 ml - glucose 20 ml of 5% solution - calcium gluconate 5 ml of 10% solution - potassium chloride 10 ml 4% - magnesium sulfate 0.4 ml of a 25% solution - sodium bicarbonate 55 ml of 4% solution - vitamin C 1 ml of 5% solution nicotinamide 1 ml of 2.5% solution - pyridoxine chloride 1 ml of 5% solution - thiamine chloride 1 ml of 2.5% solution - leucovorin 1 ml of 1% solution - riboflavin 1 ml of 1% solution - human blood plasma 0 (I) of the rhesus group - positive 75 ml - simple insulin 200 units - sodium nucleinate 50 ml of 0.25% solution - isotonic sodium chloride 0.9% up to 1 l

A cooking technology has been developed for the proposed composition. The transplanted lines of normal human fibroblasts (NPFs) were obtained by the non-enzymatic method. Pieces of tissue (pieces of dermis, lungs, spongy bone containing red bone marrow) were mechanically disaggregated to fragments of 0.1-0.2 mm ³ , MEM culture medium supplemented with 10% serum of cow embryos was added. Tissue fragments were resuspended in the indicated medium, introduced into plastic culture bottles and cultured in a humid atmosphere with 5% CO ₂ . Within 1-3 days, fibroblasts migrated from tissue fragments and culture bottles were attached to the bottom. After that, tissue fragments with medium from the vials were removed, and fresh culture medium was added to the attached cells. After the NPF monolayer was formed at the bottom of the culture flasks, the spent nutrient medium was removed, the cells were washed three times from the residual medium with a sterile isotonic 0.9% sodium chloride solution heated to 37 ° C, and then the composition of amino acids, fats, glucose, salts was sterilely added to the cells and vitamins, diluted in 0.9% isotonic sodium chloride solution in the above ratios, and re-cultured for 24 hours under standard conditions. After a day, the thus obtained conditioned NPF supernatant was discarded and used to prepare a wound healing agent. Before the clinical use of the biomaterial, the fibroblast donor was examined for the absence of syphilis, HIV, hepatitis B and C pathogens, and the culture itself was examined for the presence of intracellular pathogens (Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma genitalium, Mycoplasma hominis, Herpesx virus simplex and II, Cytomegalovirus, Human papillomavirus type 16 and 18), during the detection of which all biomaterial was rejected.

To prepare a wound healing agent for the subsequent clinical use, in a sterile 1000 ml glass bottle, to 300 ml of a confluent culture-conditioned NPF supernatant was added 75 ml of Rh-positive donor plasma, which was obtained at Kursk OSPK. Then 200 IU of simple insulin, 50 ml of 0.25% sodium nucleinate solution were sterilely added and the total volume was adjusted to 1000 ml with 0.9% sodium chloride solution.

The effect of the drug on the timing of epithelization of a burn wound.

Example 1

A study of the effectiveness and safety of the proposed drug in patients with thermal burn IIIa degree. 12 patients with a burn of the indicated depth located on the front and side surfaces of the abdominal wall were selected for the study. A sterile napkin impregnated with a solution of 0.05% furatsilin was placed on the left side surface (control 1), Dermazin ointment was applied to the anterior abdominal wall, which is silver sulfadiazine on a hydrophilic basis and recommended for the treatment of superficial and borderline burns (control 2), on the right side surface - napkins impregnated with the proposed composition. During the day, wipes with solutions were irrigated with appropriate preparations (furatsilin and the test composition) to prevent their complete drying out. Under intravenous anesthesia, every 48 hours, dressings with a wound toilet and a change of napkins were performed. When a scab was formed on a wound, it was tangentially excised in the operating room. The average period of self-epithelization of a burn wound at the site of use of the drug we offer was (M ± tm) 10.83 ± 1.23 days, in the control 1-17.83 ± 1.27 days, and in the control 2-14.42 ± 1 , 04 days.

Thus, the composition proposed by us statistically significantly (p <0.05) reduced the time of spontaneous epithelization of a border burn both in comparison with a standard antiseptic solution and in comparison with modern antimicrobial ointment, which is part of the standard therapy for this injury.

Example 2

A study of the effectiveness and safety of the proposed drug in patients with trophic ulcers of the lower leg against varicose veins and post-thrombophlebitis syndrome was conducted. For the study, 15 patients with a single leg ulcer were selected. The selection condition was the absence of purulent inflammation, allergic reactions to components of the wound healing agent and protein preparations in the anamnesis. The average life of the ulcer was (M ± tm) 2.80 ± 1.17 months. The average ulcer area (M ± tm) is 18.73 ± 3.69 cm ² . All patients previously underwent standard treatment with antimicrobial ointments (Levosin or Levomekol) and one of the reparative preparations: Actovegin gel or Curiosin without any visible effect.

After the toilet of the ulcer with a 0.1% sodium hypochlorite solution and drying with a sterile gauze swab, a sterile napkin impregnated with the proposed composition was applied to the ulcer. During the day, the napkin was irrigated with the test composition to prevent it from completely drying out. Dressings were changed daily with a dressing toilet. The final performance evaluation was performed after 28 days. For statistical processing of the results, the paired student criterion was used. As a result of treatment, the average ulcer area decreased by (M ± tm) 13.27 ± 3.04 cm and amounted to (M ± tm) 5.47 ± 2.46 cm ² (p <0.001). In 4 patients, complete epithelization of trophic ulcers was noted.

Thus, the composition we offer contributes to a statistically significant reduction in the area of trophic ulcers caused by venous insufficiency, previously refractory to standard treatment.

Claims (1)

A tool with wound healing activity based on the supernatant of normal human fibroblasts, characterized in that it additionally contains human blood plasma of the O (I) group, Rh positive, insulin simple, sodium nucleate, amino acid complex “Infesol 100”, lipofundin MCT / LST fat concentrate 20% ”, salts, glucose and vitamins in the following ratio of components:
conditioned daily confluent culture of normal human fibroblasts daily supernatant on 0.9% isotonic sodium chloride solution 300 ml amino acid complex "Infezol 100" 5 ml fat concentrate "Lipofundin MCT / LST 20%" 5 ml glucose 20 ml of 5% solution calcium gluconate 5 ml of 10% solution potassium chloride 10 ml of 4% solution magnesium sulfate 0.4 ml of a 25% solution sodium bicarbonate 55 ml of 4% solution vitamin C 1 ml of 5% solution nicotinamide 1 ml of 2.5% solution pyridoxine chloride 1 ml of 5% solution thiamine chloride 1 ml of 2.5% solution leucovorin 1 ml of 1% solution riboflavin 1 ml of 1% solution human blood plasma of the O (I) group Rh-positive 75 ml insulin simple 200 units sodium nucleinate 50 ml of 0.25% solution 0.9% isotonic sodium chloride solution up to 1 l