RU2698801C1 - Method of pharmacological correction of post-traumatic disorders of immunity and reparative processes in experiment - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to experimental pharmacology, dermatology, surgery, and can be used for treating long-term non-healed scalp infected wounds requiring pharmacological correction (stimulation) of immunity and reparative processes. Method is achieved by using pharmacological agents, characterized in that a synthetic low-molecular immunomodulator of peptide nature is prescribed – preparation Moliksan^® subcutaneously in dose of 30 mg/kg to experimental animals daily one time throughout the period of antibacterial therapy with levoflocacine 21 mg/kg. Disclosed is a method for pharmacological correction of post-traumatic disorders of immunity and reparative processes in mammals.

EFFECT: invention extends the range of methods for pharmacological correction of immunity and reparative processes.

1 cl, 6 tbl

Description

The invention relates to medicine, in particular to experimental pharmacology, dermatology, surgery, and can be used to treat long-term non-healing scalped infected wounds that require pharmacological correction (stimulation) of the immune system and reparative processes.

Currently, in the treatment of diseases requiring stimulation of the immune system and reparative processes, in particular, infected scalp wounds, an important role is given to the correct selection of antibacterial and immunocorrective therapy.

Existing means and regimens of therapy are not always effective and are characterized by a large number of contraindications and side effects.

There is a method of treating pyoderma with autohemotherapy, which consists in the fact that the blood of the patient himself, taken from a vein, is injected intramuscularly to him after two days on the third one sequentially in the amount of 3, 4, 5 and 6 ml [Skin diseases / Garibas // Medical Bulletin. - Linmed.ru, 06/09/2007. - 8 p.].

The disadvantages of this method are the need for repeated puncture of the vein and low efficiency in the chronic course of the pathological process.

The known Method of external use of a leukocyte serum preparation that has an immunomodulating effect on the body and stimulates tissue regeneration [I.G. Jitava, E.I. Bueverova, E.V. Brazil and others The use of leukocyte serum in the treatment of long-term non-healing trophic ulcers of the lower leg // Surgery. Magazine them. N.I. Pirogov. - 1998. - No. 1. - S. 37-39].

The disadvantage of this method is its relative complexity with low efficiency in the treatment of purulent wounds.

A known method of treating nonhealing wounds in an experiment, comprising a combination of ozone and EHF-therapy, in which daily until complete healing, gaseous ozone is applied to the wound surface with a concentration of 5 mg / l at the outlet of the apparatus and a gas flow rate of 1 l / min for 30 min and locally irradiated with millimeter waves of the EHF range with a white noise spectrum in the frequency range 53.57-78.33 GHz for 20 min (RF patent No. 2349326, IPC A61K 33/00, A61N 5/02. Publ. March 20, 2009).

The disadvantages of this method include a narrow spectrum of effects of ozone and EHF-therapy, mainly on gram-positive microorganisms (staphylococcus, streptococcus); instability of ozone molecules, especially in the presence of a large number of organic components of necrotic wound detritus, toxicity of ozone when it enters the respiratory tract; the possibility of treating only superficial wounds, because deep wounds and fistulous passages cannot be treated in this way.

A known method of treating infected wounds in animals, including cleansing the skin from contamination and applying to the skin an alcoholic solution of glue BF-8, the film is removed after wound healing (patent No. 2383333, publ. 03/10/2010)

The disadvantage of this method is its applicability for the treatment of only small linear paresis, uncomplicated purulent-inflammatory processes

To study the rate of wound healing by secondary intention, the planimetric method of L.N. Popova, based on recording the rate of reduction of the wound surface in time. The percentage reduction in wound area per day was determined by the formula, where: S is the wound area in the previous measurement; Sn is the area of the wound during subsequent measurement; t is the number of days between the previous and subsequent measurements [RF patent No. 2460553, 2012].

The main objective of the invention is to simplify and reduce the cost of treating infected lacerated and scalped wounds using the Molixan ^® drug for pharmacological correction of post-traumatic immunosuppression and activation of reparative processes in the body

The problem is achieved by the method of treatment and / or / weakening of the immunosuppressive state, which consists in the fact that every day, once for 2 weeks, against the background of antibiotic therapy with levofloxacin, a pharmacological agent is administered. Moreover, the drug Molixan ^® from the group of chemically pure low molecular weight immunomodulators of peptide nature is used as a pharmacological agent. Moliksan ^{® is} approved for clinical use as an immunomodulating agent with hepatoprotective activity. Registration certificate No. 001355/02 - dated 14.07.2011 Moliksan ^®

The closest in technical essence to the proposed invention is the method described in the invention ["A method for the treatment of diseases requiring stimulation of immunity and reparative processes (patent No. 2517060, published 05.27.2014), and" in a method for treating infected wounds in an experiment "(patent No. 2460553, published September 10, 2012) including a preliminary wound toilet, dressing containing iron and copper nanoparticles, and daily dressing changes; a dressing containing 0.9% sodium chloride (NaCl) sous in isotonic solution is applied to the wound enziyu iron nanoparticles at a concentration of 0.1 mg / ml and copper nanoparticles at a concentration of 0.001 mg / ml, obtained when exposed to a plasma flow having a temperature 5000-6000 K. In this case, the dispersibility of copper nanoparticles is 30 nm, and iron nanoparticles - 70 nm.

The disadvantage of this invention is the complexity of the proposed method and the high toxicity of iron and copper nanoparticles when using the proposed dressings.

The technical result of our proposed method is to solve the problem of creating a method for the pharmacological correction of post-traumatic immunity disorders using daily subcutaneous administration of a pharmacopeia immunomodulator - Molixan ^® preparation to experimental animals against a 2-week antibiotic therapy with levomoxacin.

The solution of the technical problem is achieved by the fact that in the method of pharmacological correction of post-traumatic immunity disorders and reparative processes, the experimental animals are prescribed a synthetic low molecular weight immunomodulator - the drug Molixan® subcutaneously at a dose of 30 mg / kg daily for the entire period of antibiotic therapy with levofloxacin at a dose of 21 mg / kg.

The claimed method has novelty in comparison with the prototype, differing from it by such an essential feature as the effectiveness of the method of pharmacological correction is evaluated on a more adequate model of post-traumatic immunosuppression with inhibition of reparative processes. As a means of pharmacological correction, a synthetic low molecular weight peptide pharmacopeia drug Molixan ^{® was used} . In the invention, the prototype examined the immunostimulating effectiveness of subcutaneous 3-fold injection of leukocyte blood serum of the patient.

The advantages of our proposed method are that it is economically more profitable due to the lower cost of the domestic preparation Molixan ^® . In addition, the claimed method is characterized by ease of execution and high efficiency.

The applicant is not aware of technical solutions that have the specified distinguishing feature that provides the desired result, so he believes that the claimed method meets the criterion of "inventive step"

The claimed method can find wide application in medicine, namely, in experimental pharmacology, dermatology, surgery in the development of new immunostimulating drugs, and therefore meets the criterion of "industrial applicability".

The inventive method is implemented as follows: The problem is solved using the proposed method for the treatment of scalped infected wounds that require stimulation of the immune system and reparative processes, which includes the use of the drug Molixan ^® . The essence of the proposed method lies in the fact that, as an independent method of treatment, or as a supplement to any other traditional method, in case of a protracted or complicated course of the disease, subcutaneous injections of the drug Molixan ^® are carried out daily 1 time per day at a dose of 30 mg / kg throughout the entire period basic (base), in particular, antibiotic therapy with levofloxacin at a dose of 21 mg / kg.

The proposed method for the treatment of post-traumatic scalped infected wounds that require stimulation of the immune system and reparative processes, including the use of the drug Molixan ^® is as follows.

An example implementation of the proposed method in the treatment of scalped infected wounds in experimental animals.

An experimental study was performed on white outbred male rats weighing 180-220 g from the Rappolovo cattery of the Russian Academy of Sciences. Animals were kept under standard vivarium conditions on a normal diet, with free access to water.

Experimental animals were divided into 4 groups of 10 animals each.

Group 1 - control of the wound process of a scalped infected wound;

Group 2 - scalped infected wound + treatment with levofloxacin intragastrically 1 time per day for 14 days at a dose of 21 mg / kg body weight;

Group 3 - animals with a scalped infected wound treated with Molixan ^® at a dose of 30 mg / kg subcutaneously daily for 14 days

Group 4 - animals with a scalped infected wound treated with subcutaneous molixan and levofloxacin intragastrically at doses of 30 mg / kg and 21 mg / kg, respectively, poorly effective when used separately.

At the first stage of the experiment, a model of a purulent wound was first obtained as follows according to the method described in the invention (patent for invention No. 2510083, publication date: March 20, 2014). After pretreatment of the skin under aseptic conditions, under anesthesia, on the shaved area of the skin in the interscapular region of the rats, the skin with subcutaneous tissue was excised in the form of a square 2 × 2 cm (400 mm) along the contour. The edges and bottom of the wound were crushed by Kocher clamp. A gauze swab was added to the wound with a suspension of the daily culture of a polyantibiotic-resistant clinical strain of Staphylococcus aureus at a dose of 2 billion microbial bodies in 1 ml of physiological saline. The wound in animals of the control and experimental groups was covered with a gauze dressing moistened with an isotonic solution, glued with a plaster to prevent drying of the wound. In animals in the interscapular region, an abscess formed with all the characteristic signs of purulent inflammation: there was swelling and flushing of the skin in the area of the wound, swelling, pus was released in some animals. The following parameters of the course of the wound process were taken into account: the presence and nature of the inflammatory reaction, the condition of the edges and bottom of the wound, the timing of cleansing the wound from necrotic tissues and the appearance of granulations, the nature of granulation tissue, the timing of the onset of wound epithelization. To study the rate of wound healing by secondary intention, the planimetric method of L.N. Popova, based on recording the rate of reduction of the wound surface in time. The percentage reduction in wound area per day was determined by the formula, where: S is the wound area in the previous measurement; Sn is the area of the wound during subsequent measurement; t is the number of days between the previous and subsequent measurements [RF patent No. 2460553, publication 2012].

Studies have shown that the use of molixan together with the antibiotic levofloxacin ensured almost complete healing of infected scalped wounds by 10-12 days of observation. It should be noted that in groups of animals treated with the antibiotic or immunocorrector molixan, the processes of cleansing wounds from necrotic tissues and the appearance of granulations separately proceeded significantly (1.5 times slower) than under the conditions of their joint use.

Note: * - p <0.05; ** - p <0.01; *** - p <0.001 - significant in relation to the control group.

The protective efficacy of the studied immunomodulating (molixan) and antibacterial (levofloxacin) preparations in the context of their isolated and complex use was evaluated in experiments on outbred male mice infected with Staphylococcus aureus at doses of 10 or 100 LD ₅₀ . The number of each experimental and control groups is 10 individuals. Evaluated preparations or mixtures thereof were administered to animals per os, in a volume of 0.5 ml. The antibacterial drug levofloxacin, as well as Molixan, were used in doses equivalent to single human doses. The results of the studies are shown in table 3.

Based on the data presented in table 3, the maximum 100% level of protection was observed in the treatment of levofloxacin in animals infected with 10 LD _{50 of the} pathogen. With an increase in the infecting dose to 100 LD _50, this indicator decreased to 50%. Molixan, in isolation, was expected to be ineffective against Staphylococcus aureus.

The physical mixture of levofloxacin with molixan provided a protection level of up to 100% (10 LD ₅₀ ) and 80% (100 LD ₅₀ ), respectively. It should be noted that at a higher infectious dose, the protective effect was 30% higher than from the use of levofloxacin alone.

It is known that the integrity of the skin and epithelium of the oral mucosa when exposed to mechanical, chemical and physical (radiation) stimuli is largely determined by the state of the systems of proliferation and differentiation of epithelial cells. The determinants, in the first place, include growth factors, such as epidermal growth factor (EGF) and interleukins. Considering the fact that there is practically no data in the literature on the dynamics of the level of epidermal growth factor when exposed to damaging factors of a scalped infected wound, one of the objectives of this study was to study the effect of post-traumatic infected wounds on the content of epidermal growth factor (EGF) in the blood of animals, and also pro- and anti-inflammatory cytokines IL-10 and IL-4.

The effect of molixan on the content of cytokines, the state of epidermal growth factor receptors and the activity of the mitogen-activated protein kinase system in the peripheral blood of white outbred rats with a scalped infected wound

1. As you know, a special place among the products of activated phagocytes is occupied by antimicrobial peptides (AMPs), which are, on the one hand, natural endogenous antibiotics and, on the other hand, signal molecules involved in the activation of immune cells and tissue repair [Trotti, A. Mucositis incidence, seventy and associated outcomes in patients with head and neck cancer receiving radiotherapy with or without chemotherapy: a systemic literature review / A. Trotti, LA Bellm, J.B. Epstein [et al.] // Radiother. Oncol. - 2003. - Vol. 66, No. 3. - P. 253-263; Yang, D. Mammalian defensins in immunity: more than just microbicidal / D. Yang, A. Biragyn, L.W. Kwak, J.J. Oppenheim // Trends Immunol. - 2002. - Vol. 23. - P. 291-296.]. Assuming that impaired AMP synthesis may be one of the causes of immune dysfunction in the presence of scalped infected wounds. To date, the peptides LL37 (hCAP18), β-defensin (hBD-3) and α-defensin (HNP 1-3) are the only identified human AMPs exhibiting immunoregulatory and antimicrobial activity against gram-negative and gram-positive bacteria, and also candida and viruses. Based on the foregoing, the main objective of this section was to study the serum of animal cationic antimicrobial peptides α-defensin (HNP 1-3) in case of skin lesions with the development of infected wounds, as well as to assess the possibility of regulating AMP expression using the organic salt of glutathione disulfide - the drug molixan .

The results of this research phase are shown in table 4.

Note: * p <0.05 - compared with the initial level (by student criterion);

# p <0.05 - compared with the group of infits. wound (by student criterion);

the number of animals in each group n = 10.

As can be seen from the data presented in table 4, the formation of an infected wound in experimental animals reduces the amount of α-defensin HNP 1-3 (on average by 30%) in rat blood compared to the group of "intact animals". The use of the organic salt of glutathione disulphide and inosine of the drug molixan helped to restore almost to the initial level of concentration of the studied antimicrobial peptide. Apparently, such shifts in the content of α-defensin HNP 1-3 contributed to an increase in the antibacterial activity of levofloxacin.

The results of a study of regulatory cytokines in the blood of animals with an infected scalp wound are presented in table 5.

Table 5 - Effect of molixan on the content of EGF, IL-lβ and IL-4 in the blood serum of white outbred rats with an infected wound, pg / ml

Note: Note: * p <0.05 - compared with the initial level (by student criterion); # p <0.05 - compared with the group of infits. wound (by student criterion); the number of animals in each group n = 10.

As can be seen from table 5, infection of the scalped wound in animals of the control group was accompanied by an increase in blood levels of EGF and IL-lβ.

It would seem that an increase in the level of regulatory peptides (EGF and IL-lβ) in the blood of animals exposed to traumatic effects is good for the body and may indicate the activation of reparative processes. However, clinical observations indicate the opposite: with an increase in the blood concentration of animals, the concentration of the studied cytokines did not increase the severity of manifestations of activation of reparative processes. The use of the antibiotic levofloxacin and the drug molixan as a treatment for wound infections contributed to the activation of reparative processes and normalization of the concentration of the studied peptides.

Note: * p <0.05 - compared with the background (by student criterion)

In vitro studies on A431 cell culture, it was found that the drug molixan, introduced into the incubation medium, stimulates the EGF receptor within 8 hours of observation (Table 6). The nature of the activation of the receptor was expressed in a small (on average 3 times) increase in the level of phosphorylation of receptor proteins during the first 5 minutes, followed by a decrease to the control value by 30 minutes (first wave). This was followed by an increase in activation (11 times on average) after 1 h, followed by a decrease after 4 h and reaching a maximum after 8 h (second wave).

The obtained experimental data (table 6) indicate that the use of molixan contributed to the restoration of the sensitivity of the receptor to the epidermal growth factor with the subsequent activation of reparative processes in a scalped wound. In addition, it should be noted that according to the current opinion, an increase in the functional activity of the above-mentioned growth factor is crucial in overcoming antibiotic resistance.

According to two parameters: the rate of decrease in the area of the wound and the rate of elimination of the contaminating pathogen from the wound, it can be concluded that the use of a mixture of molixan and levofloxacin with the indicated concentrations is effective for treating purulent wounds caused by polyantibiotic-resistant strains of Staphylococcus aureus.

Thus, the claimed method significantly reduces the elimination time of the contaminating pathogen, accelerates the regeneration and complete healing of wounds.

Claims (1)

A method of pharmacological correction of post-traumatic immunity disorders and reparative processes in the experiment by using pharmacological agents, characterized in that the experimental animals are prescribed a synthetic low molecular weight immunomodulator - the drug Moliksan ^® subcutaneously at a dose of 30 mg / kg daily for the entire period of antibacterial therapy with levofloxacin at a dose of 21 mg / kg