RU2155812C1 - Biotechnological method of wound-healing preparation preparing - Google Patents

Abstract

FIELD: biotechnology, medicine. SUBSTANCE: method of preparing the wound-healing preparation is carried out by culturing strain-producers of lower fungus Blakeclea trispora (+)T and (-)T followed by biomass separation. Biomass is treated with chemical reagents followed by dialysis. After dialysis aminoglycoside antibiotic (gentamycin or tobramycin) and water are added to lyophilized or nonlyophilized product. EFFECT: increased yield of end product, enhanced antibacterial activity. 2 cl, 4 ex

Description

 The invention relates to the field of biotechnology and medicine and can be used to obtain wound healing preparations with increased antibacterial activity from the substance "Mikoran".

In recent years, the pharmaceutical industry has seen an increase in interest in the creation of wound healing drugs. This interest is due to the following reasons:
progressive increase in injuries among the population,
the ability to predict medicines with certain biomedical properties due to advances in the chemistry of natural polymers and advances in biotechnology and mycology [1],
the ability to use natural biologically active compounds with high regenerative ability and the absence of toxic effects when creating drugs instead of chemical synthesis products [2].

 Among natural compounds, natural polysaccharides with the above properties and favorably characterized by the absence of an immune response from medicinal wound healing preparations based on protein (collagen) are promising.

 Of the known drugs, the most active proliferation of fibroblasts is caused by polysaccharides, in particular polyaminosaccharides - chitin and especially chitosan [3]. The latter are used to create anti-burn drugs based on chitin and chitosan of crabs. This drug is called Beschitin-W [4]. On the basis of chitin of basidiomycetes, the drug Mikoton was also created [5], which, according to the authors, can be widely used in medicine as an immunomodulating agent. Chitin and chitosan are also found in the mycelium of lower fungi, for example, mucous (Blakeslea trispora). Based on the use of this producer, a biotechnology for producing a wound healing preparation, Mikoran, was developed [6].

 Based on clinical trials and further testing of pilot batches, this drug was approved for medical use on the basis of order of the Ministry of Health of the Russian Federation No. 368 of 10/28/96. The drug showed high wound healing activity, lack of toxic properties, high biocompatibility and was recommended for the treatment of IIIa and IIIb degree burns, preparation of wounds for autodermaplasty, acceleration of epithelization of long-healing wounds and superficial burns. The active principle of this drug is chitin and chitosan of lower fungi, which stimulate the proliferation of fibroblasts to a greater extent than polyaminosaccharides obtained from crabs [7].

 However, the disadvantage of these drugs with high wound healing ability, is the lack of antibacterial activity against pathogenic microflora of wounds, especially Staphilococcus aureus and Pseudomonas aeruginosa.

 Closest to the goal is a method for producing wound healing preparation "Mikoran, 4.0 g", namely the substance "Mikoran" [6, 7], according to which the mycelial fungi belonging to the order Phycomycetes - Blakeslea trispora A-732-3 (+ ) and A-732-3 (-) are grown in submerged culture.

 Boiling with water helps to remove water-soluble unbound covalent or ionic compounds: proteins, carbohydrates, organic acids, etc. Processing 1 N. alkali with a detergent makes it possible to remove part of the strongly bound alkali-soluble proteins, lipids and polysaccharides. The breakdown of intermolecular bonds in biopolymers is significantly facilitated by the introduction of a detergent. Treatment with alkali with ethanol leads to further removal of the cytoplasmic contents of fungal mycelium, including proteins, carbohydrates, and especially fat-soluble compounds.

 The disadvantage of this method is the low yield of the final product and the lack of antibacterial activity against pathogenic microflora.

 The aim of the present invention is to develop a new method for producing chitin-containing, wound healing preparations with increased antibacterial activity from mycelium fungi, expanding the number of producers used and increasing the yield of the final product.

The essence of the invention lies in the fact that the mycelial fungi belonging to the order Phycomycetes, the family Choanephoraceae, strains Blakeslea trispora T (+) and T (-) are cultivated in an immersed culture. The grown biomass is separated from the cultivation medium and heated in a water bath at a temperature of 98 ^o C for one hour. The filtered biomass is homogenized and treated with 1 N. alkali with the addition of detergent for 12 hours at a temperature of 28 ^o C with vigorous stirring. The next stage of purification involves re-homogenization of the target product and its processing with 1% ethanol in 0.3 N. alkali at a temperature of 98 ^o C for 1 h. Next, the target product is dialyzed for 12 hours against distilled water and antibiotic substances of aminoglycoside nature: gentamicin or tobramycin are added to the lyophilized or non-lyophilized product. Solutions of gentamicin or tobramycin are prepared and added in such a concentration that the daily dose is 6 mg and 8 mg per 1 kg of body weight, respectively.

 The first part of the claimed method is similar to the biotechnological method of obtaining the substance "Mikoran" and its dosage form "Mikoran 4.0 g", on which VFS was obtained (7). However, for this purpose, the (+) and (-) T strains of B.trispora (8) were first used as producer. Strains (+) and (-) T of B. trispora differed from B. trispora A-732-3 (+) and A-732-3 (-) strains (prototype) in the following ways: (1) high growth rate, allowing reduce the process of growing mycelium from 96 to 86 hours; (2) an increase in biomass yield, respectively, from 15 g / l to 20 g / l; (3) (+) and (-) T strains had thickened cell walls, more resistant to the action of lytic enzymes, and contained 1.5 times more polyaminosaccharides (chitin and chitosan). The indicated properties of (+) and (-) Т strains of B.trispora provided during the biotechnological process large and stable yields of the final product by an average of 20-25%.

The drug with the addition of gentamicin is given the name "Mikogent" (MG), with the addition of tobramycin - "Mikotobr" (MT). The resulting preparations are applied in an even thin layer at the rate of 8-10 mg per 1 cm ^{2 of the} burned surface, preferably immediately after a burn injury and then after excision of the scab and then every day for two days. According to biomedical tests, already on the 7th day, the general condition of animals with the introduction of these drugs improves markedly, and the study of the dynamics of insemination of wounds showed a decrease in the content of microbes by 30-40% compared with the control.

Example 1
Strains of the mycelial fungus Blakeslea trispora T (+) and T (-) are grown on a medium of the following composition: soy flour - 4%, corn flour -1.73%, KH ₂ PO ₄ - 0.05%; pH of the medium is 5.8; sterilization is carried out at 1 atmosphere for 30 minutes The resulting biomass is separated from the growing medium and in an amount of 20 g (biomass humidity 70-80%) is subjected to homogenization 3 times for 3 minutes to remove cytoplasmic contents. Next, a homogenized biomass containing fragments of mycelium is placed in a flask, a four-fold volume of water is added and heated for 1 h at 98 ^° C, without boiling. The biomass is filtered off, divided into 4 equal parts and placed in 2.5 L flasks, to which 0.5 L of a 0.2% solution of detergent in 1 N NaOH is added. As a detergent, lotus laundry detergent is used. Flasks with biomass are kept under vigorous stirring for 12 h at a temperature of 28 ^o C. Next, to neutralize add 0.5 l of 1 N. HCl. The precipitate is washed with hot water (98 ^o C), then cold water to pH. Microscopic control of the removal of the cytoplasmic contents of the mycelium is carried out. The next stage of processing includes re-homogenization of the target product, its processing with 1% ethanol in 0.3 N. alkali at a temperature of 98 ^o C for 1 h with vigorous stirring. Then neutralize 0.3 N. hydrochloric acid and washed with hot and cold water to pH water. At this stage of processing, the microscopic control should show a significant loss of the cytoplasmic content of the mycelium and the predominance of the fraction of cell walls, otherwise the alkaline treatment must be repeated. The next stage of treatment is dialysis against distilled water (the target product is placed in a plastic bag) for 12 hours to remove traces of alkali and other low molecular weight compounds.

The resulting preparation is not lyophilized and antibiotics are added to it at the rate of: 12 mg of gentamicin in 10 ml of water is added to 26 g of crude substance (2.5 g by dry weight). The resulting mixture was thoroughly triturated in a mortar and then lyophilized. The resulting preparation is subjected to grinding and is used to treat burn wounds. About 125 mg of a preparation containing a daily dose of gentamicin (0.6 mg for a rat weighing 200 g) is applied to an experimental burn with an area of 25 cm ² .

Biomedical tests [9] on male rats weighing 120-150 g showed that wounds seeded after thermal damage of S.aureus and P.aeruginosa (2 • 10 ⁶ bacteria in 1 ml) in the treatment of MG show a pronounced wound healing and antimicrobial effect of the drug.

 Burns in animals of the experimental and control groups were carried out in an open way. MG was used immediately after injury, then after excision of the scab and then for the next two days every day. On day 7, the reduction in the area of wounds was 8% of the initial value, and in the control, the area of wounds not only did not change, but even increased by 2-5%, by 15 days the difference in reduction in the area of wounds compared to the control group of animals was 15-20 %, on day 21 the difference in wound area was 30-35%.

 On the 7th day after applying the burn in the control group, secondary necrosis is most pronounced, which is accompanied by perifocal inflammation of the soft tissues and the appearance of the first signs of suppuration of wounds. In the treatment of MG, the clinical picture is diametrically opposite - the wound compartment is poor and serous, areas of secondary necrosis are barely distinguishable, a thin layer of granulation tissue appears on a large area of wounds, and in some cases a thin border of red epithelium is noted. These data indicate that the wound healing effect of MG manifests itself at the very beginning of the wound healing process. Histological examination revealed significant differences in wound morphology already on the 7th day. Under the action of MG, a clearly more favorable course of the wound process is noted. Secondary necrosis is very mild and is superficial. A viable layer is represented by granulation tissue, which was characterized by a small number of capillaries, the presence of moderately swollen fibroblasts, and a significant number of fibrous structures. Unlike the control, the border between the affected and healthy tissues is clear, with no inflammatory infiltrates in the tissues. The presence of granulation tissue is already noted for 3-4 days.

 The results of the study of the antibacterial properties of MG showed that this drug has significantly greater antibacterial activity in relation to the control (by 7.2% by 35.2%; by 21 days - 30.4%).

 Example 2. The same as in example 1, but gentamicin is added to the lyophilized preparation. After that, the substance with the antibiotic is ground in a mortar or homogenized in a homogenizer, and then subjected to freeze drying. Tests on nutrient agar using disks impregnated with gentamicin showed that the antibacterial activity of MG against S. aureus and P. aeruginosa is lower by 30-40% than if the drug was prepared according to example 1.

 Example 3. The same as in example 1, but tobramycin at a concentration of 8 mg / kg of body weight of the animal is used as an antibiotic based on: 16 mg of tobramycin in 10 ml of water is added to 26 g of crude substance with a moisture content of 90%.

 Example 4

 The same as in example 3, but the solution of tobramycin is added to the lyophilized preparation. After that, the mixture is ground in a mortar or homogenized in a homogenizer, and then subjected to freeze drying.

 Unlike MG, the wound healing effect of MT appears only on the 7th day and amounts to 7-8% in relation to the control, where the area of wounds increased by 2-3%. On the next 15 and 21 days, a reduction in the area of wounds under the influence of MT compared with the control was not observed. However, the contamination of wounds in the presence of MT is also less than in the control lot, and the increase in antibacterial activity is 7-18%, on day 15 - 20%. Histology showed that the scale of secondary necrosis is greater than with MG, but the latter was not, as in control, susceptible to purulent dissection. Revealed purulent inflammation, not marked by MT, in contrast to control is very superficial. The presence of new granulations and in terms of maturity is less than under the action of MG, but significantly exceeds the control.

Thus, the result of a comprehensive examination (clinical evaluation of the wound healing process, study of antibacterial properties, histological picture of experimental burn wounds) of the action of antibiotic-containing compositions based on the substance "Mikoran" indicate a positive effect on wound healing. The degree and nature of the effect is that the biostimulating effect of Mikoran is enhanced by the antibacterial effect of the aminoglycoside antibiotics included in the preparation. The most effective combination of Mikoran with gentamicin:
epithelization of superficial burns by 15 days, which is 7 days earlier than in the control,
the appearance of a distinct border of the marginal epithelium and the first granulations with deep burns already on the 7th day,
superficial nature or absence of phenomena of secondary necrosis,
more rapid maturation of granulations compared to control,
consistently high and greater than in the control and under the action of MT, the rate of reduction of the size of deep burn wounds.

 By reducing the microbial contamination of wounds below a critical level, the MG preparation excludes the possibility of the development of microbial invasion and progressive necrosis, which in turn creates favorable conditions for maximizing the biostimulating effect of the Mikoran 4.0 g preparation.

Literature
1. Modern approaches to the development of effective dressings and polymer implants. Abstracts of the 1st International Conference. M., 1992.

 2. Modern approaches to the development of effective dressings, suture materials and polymer implants. Materials of the 11th International Conference, M., 1995.

 3. Dixon B. Using fungal dressing to heal wounds. Biotechnology, 1995, v. 13, p. 120-121.

 4. Japans Unitika commercializes new chitin product. Bioprocess.Technol. 1988, v. 10, N 8, p. 4.

 5. Gorovoj L., Burdukova L. Chitin-containing material produced from fungi: medical application perspectives, I Inter. conferense of the european chitin Soc., Brest, 1995, p. 22.

 6. Feofilova E. P., Tereshina V. M., Alekseev A. A., Grishina I. A. A method of obtaining a wound healing drug. RF patent, N 2086247, 1994.

 7. VFS 42-2737, VFS 42-2738.

 8. Feofilova EP. Progress in the field of experimental mycology as the basis for the creation of modern biotechnology. Microbiology, 1997, v. 66, p. 302-309.

 9. Report on the results of a biomedical study of the effect of antibiotic-containing compositions of the substance "Mikoran" on the healing process of burn wounds. Institute of Surgery A.V. Vishnevsky RAMS, Center for Thermal Lesions. M., 1997.

Claims (2)

 1. A method of obtaining a wound healing preparation, comprising cultivating strains of producers of the lower fungus Blakeslea trispora, separating biomass, its subsequent treatment with chemical reagents, dialysis and freeze drying, characterized in that the producers use (+) T and (-) T strains of Blakeslea trispora, after dialysis, an aminoglycoside antibiotic and water are added to the lyophilized or non-lyophilized product.  2. The method according to p. 1, characterized in that as an aminoglycoside antibiotic use gentamicin or tobramycin.