Biotechnological method of obtaining a wound healing drug

 

(57) Abstract:

The invention relates to biotechnology and medicine. A method of obtaining a wound healing drug carry out the cultivation of strains - producers of the lower fungus can trispora (+) T (-) T followed by the separation of biomass. The biomass is treated with chemical reagents and dialysis. After dialysis to liofilizirovannom or reliabilitybased product add aminoglycoside antibiotic (gentamicin or tobramycin) and water. The method provides an increase in the yield of the final product and its antimicrobial activity. 1 C.p. f-crystals.

The invention relates to the field of biotechnology and medicine and can be used to produce the substance "Memoran" wound healing drugs with increased antibacterial activity.

In recent years the pharmaceutical industry has increased interest in the establishment of wound healing drugs. This interest is due to the following reasons:

a progressive increase in injuries among the population,

the ability to predict medical drugs with specific biological properties thanks to advances in the chemistry of natural polymers and postproduction chemical synthesis of biologically active compounds, with high regenerative capacity and the absence of toxic effects [2].

Among the natural compounds promising natural polysaccharides having the above properties and differentiated by the absence of an immune response against drug wound healing drugs, created on the basis of protein (collagen).

Of the known medicines most active proliferation of fibroblasts called polysaccharides, in particular polyaminoamide - chitin and especially chitosan [3]. The latter is used to create burns drugs based on chitin and chitosan crabs. This drug was called Beschitin-W[4]. Chitin-based basidiomycetes also created the drug "mycoton" [5] , which according to the authors can be widely used in medicine as an immunomodulatory agent. Chitin and chitosan are present in mycelium phycomycetes, such as ((can trispora). On the basis of this producer was developed biotechnology for the production of wound-drug - Micorn" [6].

On the basis of clinical trials and further testing of pilot batches of the drug has been approved for medical use on the basis of p the properties, high biocompatibility and has been recommended for the treatment of burns IIIA and IIIB degree, training RAS to autodermoplastike, accelerate epithelization nonhealing wounds and superficial burns. Active early this drug are chitin and chitosan phycomycetes, which stimulate the proliferation of fibroblasts in a greater degree than polyaminoamide obtained from crabs [7].

However, the disadvantage of these drugs with high healing ability is insufficient antibacterial activity against pathogenic microorganisms, RAS, especially Staphilococcus aureus and Pseudomonas aeruginosa.

The closest to the goal is a method for wound healing drug "Mihran, 4.0 g, namely substance "Memoran" [6, 7], according to which filamentous fungi belonging to the order Phycomycetes - can trispora And-732-3 (+) and a-732-3 (-) grown in submerged culture.

Boiling water helps to remove water-soluble unbound covalent or ionic bonds compounds: proteins, carbohydrates, organic acids and other Processing 1 N. lye with detergent gives you the opportunity to delete some of the strongly bound dilaceration proteins, lipids and polishe alkali with ethanol leads to further destruction of the cytoplasmic content of the mycelium of fungi, 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 microorganisms.

The purpose of this invention is to develop a new way of getting from chitin-containing fungal mycelium, wound healing drugs with enhanced antibacterial activity, expanding the number of producers and increase the yield of the final product.

The essence of the invention lies in the fact that filamentous fungi belonging to the order Phycomycetes, family process specifications have been issued, the strains can trispora T (+) and T (-) is cultivated in submerged culture. Grown biomass is separated from the environment of the cultivation and heated in a water bath at a temperature of 98oC for one hour. The filtered biomass homogenized and treated with 1 N. lye with detergent for 12 hours at a temperature of 28oC and vigorous stirring. The next cleaning stage includes re-homogenization of the target product and its treatment with 1% ethanol 0.3 N. alkali at a temperature of 98oC for 1 h Then the target product on the KTU add aminoglycoside antibiotic substances of nature: gentamicin or tobramycin. Prepare solutions of gentamicin or tobramycin and add them in such a concentration that the daily dose was 6 mg and 8 mg per 1 kg of body weight.

The first part of the claimed method similar biotechnological method for producing a substance "Mihran and its dosage forms "Mihran 4.0 g", which received VFS (7). However, for this purpose, first used as a producer (+) and (-) T strains of B. trispora (8). Strains (+) and (-) T B. trispora differed from strains of B. trispora And-732-3 (+) and a-732-3 (-) (prototype) according to the following criteria: (1) high growth rate to reduce the process of growing mycelium from 96 to 86 hours; (2) increased biomass yield, respectively, with 15 g/l to 20 g/l; (3) (+) and (-) T strains had thickened cell wall, more resistant to the action of lytic enzymes, and contained 1.5 times more polyaminoacids (chitin and chitosan). The specified properties of the (+) and (-) T strains of B. trispora was provided during the biotechnological process is large and stable yields of final product, on average, 20 to 25%.

The drug with the addition of gentamicin given the name "Ecogent" (MG), with the addition of tobramycin - "Ecotop" (MT). The obtained preparations put a thin layer of smooth at the rate of 8-10 mg the Oia scab and then daily for two days. According to medical-biological tests on day 7, the General condition of the animals with the introduction of these drugs is significantly improved, and the study of the dynamics of contamination of the wounds showed a decrease in the content of microbes by 30-40% compared to control.

Example 1

Strains of the filamentous fungus can trispora T (+) and T (-) is grown on a medium of the following composition: soy flour - 4%, corn flour -1,73%, KH2PO4- a 0.05%; pH of the medium - 5,8; sterilization is carried out at 1 atmosphere for 30 minutes the resulting biomass is separated from the environment of cultivation and in the amount of 20 g (biomass moisture 70-80%) is subjected to homogenization 3 times for 3 minutes to remove the cytoplasmic content. Next gomogenizirovannogo biomass containing fragments of mycelium was placed in the flask, add four times the volume of water and heated 1 h at 98oC without boiling. Biomass is filtered, divided into 4 equal parts and placed in a flask at 2.5 l where add 0.5 l of 0.2% solution of detergent in 1 n NaOH. As a detergent use detergent "Lotus". Flasks with biomass incubated with vigorous stirring for 12 h at a temperature of 28oC. Next to neutralize add 0.5 liters of 1 N. HCl. The precipitate is washed GM cytoplasmic content of the mycelium. The next stage of treatment involves re-homogenization of the target product, its processing 1% ethanol 0.3 N. alkali at a temperature of 98oC for 1 h under vigorous stirring. Then neutralize 0,3 N. hydrochloric acid and washed with cold and hot water until the pH of the water. At this stage of processing microscopic control should show a substantial loss of cytoplasmic content of the mycelium and the dominated fraction of the cell wall, otherwise the alkaline treatment must be repeated. The next stage of processing - dialysis against distilled water (target product is placed in a plastic bag) for 12 h to remove traces of alkali and other low molecular weight compounds.

The resulting product is not subjected to lyophilization and add to it the antibiotics from the calculation: to 26 g of crude substance (2.5 g dry weight) contributed 12 mg gentamicin in 10 ml of water. The resulting mixture was triturated in a mortar and then lyophilizer. The resulting preparation is subjected to grinding and is used to treat burn wounds. On the experimental burn area of 25 cm2put about 125 mg of the drug containing the daily dose of gentamicin (0.6 mg for rats weighing 200 g).

Burns in animals of the experimental and control groups was conducted in an open way. MG was applied immediately after the injury, then after excision of a scab and then two days later every day. On day 7, the reduction in the area of RAS was 8% of the original value, while in the control area of RAS has not only not changed, but even increased by 2-5%, to 15 days the difference in the reduction in the area of RAS compared with the control group of animals was 15-20% at 21 days the difference in the area of RAS was 30-35%.

On day 7 after application of a burn in the control group, the most pronounced secondary necrosis, which is accompanied perifocal inflammation of the soft tissues and the appearance of the first signs of festering wounds. In the treatment of MG the clinical picture is of a diametrically opposite character - wound separation scarce and serous, areas of secondary necrosis subtle, over a large area of RAS appears thin layer of granulation tissue, and in some cases there is a thin red border of the epithelium. These data suggest that wound healing effect of MG is shown in the haunted differences in the morphology of RAS. Under the action of MG is observed clearly more favorable course of the wound process. Secondary necrosis is very weakly expressed and is superficial. Viable layer is represented by granulation tissue, which was characterized by a small number of capillaries, the presence of moderately swollen fibroblasts, a significant amount of fibrous structures. In contrast to the control, the boundary between diseased and healthy tissues clear, with inflammatory infiltrates in tissues was not observed. Indicates the presence of granulation tissue already for 3-4 days.

The results of the study of antibacterial properties of MG showed that this drug has a much higher antibacterial activity compared to control (on day 7 of 35.2%; for 21 days - 30,4%).

Example 2. Same as in example 1, but gentamicin is added to liofilizirovannom the drug. After this substance with antibiotic pounded in a mortar or homogenized in the homogenizer, and then subjected to freeze drying. Tested on nutrient agar disks impregnated with gentamicin showed that antibacterial activity of MG against S. aureus and P. aeruginosa, are 30-40% lower than if the drug prigot is ramitin at a concentration of 8 mg/kg of body weight of the animal based: to 26 g of crude substance with a humidity of 90% add 16 mg of tobramycin in 10 ml of water.

Example 4.

Same as in example 3, but the solution of tobramycin add to liofilizirovannom the drug. After that, the mixture is pounded in a mortar or homogenized in the homogenizer, and then subjected to freeze drying.

Unlike MG wound healing effect of MT appears only on day 7 and is 7-8% compared to the control, where the area of RAS increased by 2-3%. For the next 15 and 21 days, the reduction in the area of RAS under the action of MT compared to control was observed However, infection of wounds in the presence of MT less than in the control party, and the increase in antibacterial activity is on day 7 -18% 15 days - 20%. Histology showed that the incidence of secondary necrosis more than under the action of MG, however, the latter was not as in control, subject purulent stratification. Revealed purulent inflammation, it is not marked under the action of MT, in contrast to the control is very superficial. New granulation and maturity less than under the action of MG, but significantly higher than the control.

Thus, the result of a comprehensive examination (clinical assessment of wound healing, antibacterial study , on the basis of substance "Mihran indicate a positive effect on wound healing. The degree and nature of the influence is that biostimulating action of Midarana enhanced antibacterial effect is included in the preparation of aminoglycoside antibiotics. The most effective combination of Midarana with gentamicin:

epithelization of superficial burns to 15 days, which is 7 days earlier than in control

the appearance of a clear border edge of the epithelium and the first granulation with deep burns already on day 7,

superficial or absence of phenomena of secondary necrosis,

faster compared to control maturation of granulation,

consistently high and larger than in the control and under the action of MT the rate of reduction of the magnitude of deep burn wounds.

Reducing microbial colonization of wounds below the critical level of the drug is MG excludes the possibility of the development of microbial invasion and progressive necrosis, which in turn creates favorable conditions for the maximum realization of the biostimulating effect of the drug, "Mihran 4.0 g".

Literature

1. Modern approaches to the development of effective dressing devices and polymer implants. Abstracts of the 1st International conference. M., 1992.

2. Modern approaches to the development of the conference, M., 1995.

3. Dixon Century 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 C. 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 E. P. Progress in the field of experimental Mycology as the basis for modern biotechnology. Microbiology, 1997, T. 66, S. 302-309.

9. Report on the results of biomedical research impact antibioticotherapy compositions substance "Memoran" on the healing process of burn wounds. Institute of surgery. A. C. Wisniewski Russian Academy of medical Sciences, Center for thermal lesions. M., 1997.

1. A method of obtaining a wound healing drug, involving the cultivation of strains-producers of the lower fungus can trispora, branch biomass, its subsequent processing chemicals, dialysis and freeze drying, characterized in that as producers use (+) T (-) T trispora strains can, after dialysis to liofilizirovannom or reliabilitybased is the number of aminoglycoside antibiotic use gentamicin or tobramycin.

 

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