Wound healing composition

 

(57) Abstract:

The invention relates to medicine, namely to drugs and compositions for the treatment of surgical wounds, burns, lesions, erosions, trophic ulcers, bedsores, etc., especially complicated by purulent-inflammatory processes. The invention consists in that the composition consists of base and active agent-containing interleukin-1, and further comprises a superoxide dismutase. As the basis of the composition comprises water, saline, aqueous solutions of polyethylene oxide. The invention provides high efficiency and can be used to treat a wide range of diseases. 5 C.p. f-crystals, 6 PL.

The invention relates to medicine, namely to drugs and compositions for the treatment of surgical wounds, burns, lesions, erosions, trophic ulcers, bedsores, etc., especially complicated by purulent-inflammatory processes.

There are a large number of drugs in various forms, capsules for oral administration (tribenoside vinsol aescusan), solutions for impregnation napkins and tampons (alisol, esterized, carolien, sea buckthorn oil), ointments (Mashkovsky M. D. Medicines, M, Medicine,tx2">

The lack of preparations for internal use is the presence of side effects, lack of efficacy, thus forcing me to use them, usually in complex therapy.

Also known drug solcoseryl (Medicines. Directory, the Firm Alkaloid, Skopje, Yugoslavia), which is a suspension of the extract blood of cattle, which is not disclosed in oil-based. Solcoseryl ointment in the form recommended for the fresh appearance of granulations on the surface of the ulcer obliterative arterial diseases, venous ulcers, burns, etc.

The disadvantage of the drug is low efficiency, severe disease drug substitute for antibiotics, since the addition of antibiotics in solcoseryl ointment and jelly is not recommended.

Known topical wound healing compositions on various body basics - fats, lanolin, waxes, vaseline, grease and other (Kogan, J. Technology of medicinal forms, Medgiz, 1992, S. 209-244). As a rule, ointment contain the active principle and fat basis. So, bromatology ointment includes 0.3% menthol, 5% boric acid and 54.5% of vaseline ointment Postavy - as an active start - up 17.3% streptocide.

The disadvantage of these ointments is the low efficiency for the treatment of purulent wounds.

The closest analogues to the claimed composition and the achieved effects are drugs used in the form of a gel, for example aqueous solutions of interleukin-1 with carboxymethyl cellulose (CMC), are applied by impregnation their napkins (CBL. a PCT application 89/05653, 1989, class A 61 K 37/02).

The disadvantage of these drugs is the high consumption of expensive drugs, the limited application for pathologies that require long-term treatment of difficult and extensive purulent secretions.

The problem solved by the present invention was to create a wound healing drug, with increased efficiency. This problem is solved by using as the active agent a mixture of interleukin-1 and superoxide dismutase (SOD) in the following ratio of ingredients:

interleukin-1 - 0.01-10 ng/ml

superoxide dismutase - 0.03-0.3 wt.%

aqueous base - rest

As the basis of the composition comprises water or aqueous solutions, such as saline or a solution of polyethylene oxide (PEO) with a molecular mass of 400-1500 D, etc..

The use of lower doses of IL-1 h is the possibility of full compensation damaging effect of atomic oxygen. The introduction of higher concentrations of IL-1 and SOD is possible, but not advisable for economic reasons.

The method of obtaining the composition consists of two stages. At the first stage receives a composition based on water or saline. To do this, prepare a solution of SOD in physiological solution with a given estimated concentration of the active start, for example, and then adds in the necessary quantities of a concentrated solution of the substance of recombinant IL-1 person to achieve the required concentrations. Due to the relatively low stability of aqueous solutions of SOD and IL-1 composition do not more than a day before use or further processing, or give it the stabilizing additives, such as human serum albumin (CSA) to 1%. If necessary, the next step to that obtained with the aforementioned method to the solution is added a water-soluble polymer or an aqueous solution.

Features of the new composition is a creation of conditions for a combination of possible regulation of inflammatory responses by interleukin-1 (IL-1) and provide the protective functions of tissue using SOD. IL-1 also stimulates the function of leukocytes, endothelial function, and C is the local anti-infective functions in full, induces their activation, enhancing chemotaxis, degranulation and production of superoxide anion, stimulates the functional activity of T - and b-lymphocytes, proliferation of fibroblasts and connective tissue metabolism.

The production of superoxide anion is crucial in the fight against penetrated by microorganisms, however, results in the case of hyperactively leukocytes to undesirable consequences associated with damage to its own tissues free forms of oxygen.

The use of SOD helps to neutralize the generated superoxide radicals. However, for effective operation of the system as a whole was required to create a composition which would provide a stable coexistence of both components of the active principle while their penetration into the wound through the surface layers of secretions.

During the research it was found that in the patented range of concentrations of IL-1 and SOD in a day form in aqueous solutions in appreciable quantities relatively stable complex that reduces the effectiveness of SOD and IL-1 compared with an individual introduction of ingredients. However, the decrease in activity of kanemasa complex or falls into components when introduced into the body, or allows member components sufficiently to make the necessary regulatory functions. Thus, the reaction medium affects the absolute SOD activity, but not on the General course of events. To improve the activity and stability of the complex, it is advisable to have in an aqueous solution additives water-soluble polymers of the type PEO.

Example 1. Cooking preparations of IL-1 and SOD

For preparation to 100 ml of SOD in physiological solution with the concentration of the active start 100 u/ml was added in the desired amounts of a concentrated solution of the substance of recombinant IL-1 person to achieve the required concentrations. The resultant preparations were subjected to sterilizing filtration through filters with a pore diameter of 0.22 μm and used for testing for the presence of the enzymatic activity of SOD and the biological activity of IL-1 in cell cultures. The solutions were prepared not later than 1 day before the production of biological experiments. Characteristics of the fluids used are shown in table 1 (preparations 5 and 9 contain additional 0.3 and 1.0% CSA).

Effect of IL-1 and SOD lymphocyte proliferation in the thymus of mice was performed to test indukti is POZITIV thymus of mice CBA was scored by the method of cervical dislocation. Under aseptic conditions took their timony, which are then homogenized in a medium Needle using needles of different diameters. Received a suspension of thymocytes resuspendable in 1 ml of eagle medium and counted in the camera Goryaeva. Medium Needle containing 4% fetal serum, and drove cell concentration up to 10 million/ml In wells of 96-well plate to cultivation contributed studied cultivation of drugs IL-1 and SOD and 100 μl of the previously prepared suspension of thymocytes. In culture also contributed to the preparation of concanavalin A dose of 0.1 μg/ml as an inducer of cell proliferation. Cultivation continue for 72 hours in a CO2 incubator. 18 hours before the end of cultivation in all wells were made on 40 Bq3H-thymidine in the volume of 10 µl. At the end of the cultivation timesfannie culture from all wells was transferred using a harvester on filters, which are then dried. The filters were placed in vials with scintillation fluid and determined the inclusion of3H-thymidine by thymocytes each culture, using a scintillation counter (RACKBETA 1217). Data for each of the three parallel cultures were averaged and received data according to the level of inclusion of3H-thymidine from R and, as the stabilizer of blood using heparin at a concentration of 20 u/ml of the Chemiluminescence enhanced by lyuminola, was assessed in whole blood on the luminometer company LKB model 1251. Determination was made as follows: into a cuvette for chemiluminescence poured into 0.1 ml of whole blood was added 0.6 ml of buffered saline solution (pH 7.2), 0.1 ml of the study drug, 0.2 ml of a solution lyuminola at a concentration of 10-5M if measured induced chemiluminescence in the cuvette were added to the inductor in a volume of 0.1 ml as an inductor using formalistic acetate (PMA), which is the most powerful activator of redox processes in faguoqitirute cells. The chemiluminescence was measured in the "Lumina" within 30 minutes by two parameters: sitesome in mV/min and the maximum chemiluminescence in mV.

Generation of superoxidation was assessed by the activity of the recovery process, salts of tetrazole. The test was performed with neutrophil fraction of leukocytes. The selection of neutrophils was carried out according to standard methods described by Boyum, using gradient ficoll-urografin (beats. density 1078), followed by hypotonic shock. The reaction was carried out in 96-LUN is the target of tetrazole nitro-blue in buffered physiological solution (pH of 7.2) was layered in card then added 0.1 ml of study drug in appropriate concentrations, were placed in the incubator for 1 hour, removed the supernatant, the monolayer cells were dried, fixed with methanol. The elution of granules formazan formed in the reaction, carried out with a mixture of alkali and dimethyl sulfoxide. The reaction was taken into account on the device MultiScan (firm BioRad) at a wavelength of 595 nm. The reaction was evaluated in terms of extinction. If the effect of the drug was studied on the background of the inductor, the inductor used opsonizing zymosan.

The results of studies on the combined influence of the drug on the proliferation of thymocytes are shown in table 2.

According to the information provided in part of the complex products there is a change in the biological activity of IL-1, namely the reduction of its ability to activate cells involved in the inflammatory response, indicating that the interaction of the ingredients together.

The redox potential of cells was assessed in parallel in the two tests: chemiluminescence of whole blood with lyuminola, allowing to determine the oxidative burst in total, ve given the Oia microcinema, giving the possibility to measure the level of production of superoxide anion.

Study of the combined influence of the drug on the generation of superoxidation was conducted in 3 experiments. It was evaluated the influence of the drug as a spontaneous response rate, and cells, activated opsonization simhasanam. The results of the reaction are presented in table 3.

As follows from the experimental results, presented in table 3, the combined form of the drug, including interleukin-1 and SOD at a concentration of 0.3%, exert inhibitory effects on spontaneous and stimulated simhasanam products of superoxidation. The preparation containing a physiological concentration of SOD - 0,03%, had no effect on the generation of oxygen radicals.

It was also investigated the effect of combined drugs on Lomonosovsky the chemiluminescence of whole blood. Preparations were examined in the same concentrations as in the previous experiments.

As follows from table 4, the combined product also had an inhibitory effect on the chemiluminescence of whole blood. Moreover, as in the previous series of experiments, the most pronounced inhibitory of apuleia interleukin-1 in the same concentrations did not cause reduction products of oxygen radicals.

As a result of experiments it was shown that claimed the drug caused the suppression of the production of oxygen radicals detected by the two tests: a chemiluminescence and recovery of salts of tetrazole, making it a potentially promising for the treatment of acute inflammatory processes.

Example 2 preparation and properties of a product based on PEO.

50 g of PEO with an average molecular weight of 400 L and 50 g of PEO with an average molecular weight of 1500 D thoroughly mixed, and to the parts of the resulting mass was added to 0.5 ml product N-6, manufactured by the method of example 1 to the content of SOD - 3%, IL-1 - 1 ng/ml, and for control instead of the drug N 6 solutions separately containing an equivalent amount of IL-1 or SOD.

The preparation of the study on their impact on the course of experimental burns in rats. While conducting experiments used outbred rats (males) weighing 200-220 g anesthetized with a mixture of calypsol and droperidola (70 and 1 mg/kg of body weight, respectively) modeling of skin burns by hot water. For this purpose, the animals on the back cut hair with scissors and shaved remnants of hair using a razor. Burns caused by the attachment of the tube with hot water (to

Burn wounds were covered with gauze napkins, which were attached to the skin with the silk sutures. Shift ointment preparations was carried out on a daily basis. Skin impedance was determined on the test and control areas of the skin at frequencies of 50, 100 or 1000 Hz to burn, immediately after the burn, after 1,3, 7 and 10 days after burn. To calculate the polarization ratio as the ratio of the impedance at low frequency (50 Hz) to its value at high (1000 Hz).

Obtained during the measurement values of the coefficient of polarization (CP) are given in table 5.

As the table shows, the impact of hot water, led to the destruction of the surface layers of the skin, which in turn has led to a sharp decrease in the ratio of polarization to 1,784.

In the control group this indicator in future continue to decline (to 1,330,17 on the 7th day) and remained the lowest in comparison with other points to the end of the experiment (10 days was 1.590.36). Apparently, this was due to the fact that polystylene-1500 has pronounced osmoticheskoe and only 10 days rose to the level 1,960,11.

In the case of the use of superoxide dismutase (as well as a mixture of interleukin-1 + SOD) showed the highest values of CP, especially in the later stages.

Thus, on the basis of the obtained results we can conclude that the pronounced therapeutic effect have two drugs: SOD and combined drug SOD+IL-1, which is confirmed by the values of the polarization coefficient of the skin of rats. However, when using a combined preparation containing SOD+IL-1, wound on the surface much faster was formed multilayered epithelium, than at use of preparations containing only SOD or only IL-1. This allows us to conclude that it is a combination drug containing SOD and IL-1 in the proposed concentrations, is the most promising dosage form.

The use of integrated product in the form of daily applications in the area of burn significantly improved the dynamics of the healing of the lesion compared with the control group animals. Moreover, the combined drug was significantly more effective than the preparations of IL-1 or SOD used for the treatment of this form of burn separately.

According to the obtained result of the Oia and contributes less destruction of tissue and the predominance of reparative processes over necrotic compared to control and drugs containing only IL-1 or SOD.

Example H. the Influence of nature and concentration of the individual components on the effectiveness of the composition.

According to the method of example 2 were obtained drugs with different concentrations of components.

Effectiveness was determined by the method described in example 2, according to the changes of the values of the polarization of the skin of rats on day 10 after application of the burn and the use of drugs.

The results are shown in table 6.

1. Wound healing composition consisting of base and active agent-containing interleukin-1, characterized in that it further comprises a superoxide dismutase in the following ratio of ingredients:

Interleukin-1 is 0.01-10 ng/ml

Superoxide dismutase - 0.03 to 0.3 wt.%

Basis - Rest

2. Wound healing composition under item 1, characterized in that as the Foundation it contains aqueous solutions.

3. Wound healing composition under item 1, characterized in that aqueous solutions it contains water.

4. Wound healing composition under item 1, characterized in that aqueous solutions it contains saline.

5. RA is ing solutions of polyethylene oxide with a molecular mass of 400-1500 D.

6. Wound healing composition in PP.1-4, characterized in that it further comprises albumin to 1 wt.%.

 

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