RU2776015C1 - Local haemostatic antibacterial agent - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to the pharmaceutical industry, namely, to a local haemostatic antibacterial agent for suppressing massive bleeding. Local haemostatic antibacterial agent for suppressing massive bleeding, constituting a macromolecular complex of a chitosan salt and an antibacterial agent, wherein the chitosan salt is bound to the antibacterial agent by intermolecular hydrogen bonds, and 2,3-bis-(hydroxymethyl)-quinoxalin-1,4-di-N-oxide with a mass fraction from 0.5% to 20% acts as an antibacterial agent, the chitosan salt is selected from chitosan formate, acetate, citrate, glycolate, or lactate.

EFFECT: above agent is effective in suppressing massive bleeding, exhibits high antibacterial activity.

3 cl, 3 tbl, 6 ex

Description

The invention relates to medicine, specifically to agents used to stop external bleeding from large blood vessels, and can be used in emergency and extreme situations, as well as in hospital conditions where anaerobic or mixed infection is possible, as a local hemostatic antibacterial agent.

It is known [RU 2665950] a hemostatic antibacterial agent containing a polysaccharide associated with an antibiotic agent and a method for its preparation, which is a supramolecular complex in the form of a powder containing oxidized powdered cellulose and vancomycin, bound through non-covalent intermolecular hydrogen bonds, intended to stop bleeding in surgery, as well as to provide local antiseptic action and regeneration of damaged tissues. The disadvantage of the proposed tool is a limited range of applications only in surgery to stop diffuse and profuse bleeding and the inability to use it to stop massive bleeding.

It is known [RU 2694372] a hemostatic anti-inflammatory agent containing a polysaccharide - chitosan succinate and calendula extract, in the form of a water-based ointment, additionally containing lecithin, glycerol, lidocaine, dioxidine (2,3-bis-(hydroxymethyl)-quinoxaline-1,4 -di-N-oxide) and a preservative designed to stop local venous, parenchymal and capillary bleeding, as well as to provide a local antiseptic effect in first aid and emergency care. However, the drug is not able to stop intense arteriovenous bleeding.

It is known [RU 2675631] a local hemostatic agent for stopping massive bleeding, containing polysaccharides - chitosan salt, sodium carboxymethylcellulose and an antibiotic selected from the group of fluoroquinolones (ciprofloxacin or pefloxacin or lomefloxacin), in the form of a napkin made of non-woven material with a composition applied to its surface - powder from a mechanical mixture of the listed components, characterized by a high sorption capacity for blood, a short time for the onset of hemostasis and increased antimicrobial activity. The disadvantage of the proposed tool is its low antibacterial activity against resistant forms of staphylococci resistant to fluoroquinolones, and against gram-negative bacteria of the Enterobacteriaceae, Pseudomonas families, which are characterized by multiple resistance to antibacterial drugs. The disadvantage of the tool is also the complexity of its manufacturing technology, as in the preparation of compositionally homogeneous mixtures of powders with an antibiotic content of less than 1% wt. (it is possible to localize most of the antibiotic powder in a separate volume of the mixture), and when applying a mixture of powders to the surface of a non-woven material.

Patent [RU 2519220] discloses the use of a local hemostatic agent to stop massive bleeding, characterized by high sorption capacity for blood, short time of onset of hemostasis and high antimicrobial activity, containing a polysaccharide - chitosan salt, which is selected from a number of polydisperse powders of hydrochloride, hydrobromide, formate, acetate , succinate, citrate, glycolate, or chitosan lactate, and polyhexamethylene guanidine hydrochloride of the formula:

where R=H, C ₁₈ H ₃₇ or C ₁₇ H ₃₃ , n=10-20, while the chitosan salt and polyhexamethylene guanidine hydrochloride are covalently crosslinked with a polyfunctional compound from a number of glycidyl ethers in the following ratio of these components, wt. %: chitosan salt 75.0-95.0, polyhexamethylene guanidine hydrochloride 4.0-20.0, polyfunctional compound 1.0-5.0. This technical solution, the closest in terms of features to the claimed invention, is taken as a prototype.

The disadvantage of the prototype is that the proposed hemostatic agent has insufficient antibacterial activity against anaerobic or mixed infections, since the polyhexamethyleneguanidine hydrochloride included in its composition as an additive that increases the antimicrobial activity of the composition loses its bactericidal properties as a result of the covalent cross-linking reaction with a polyfunctional compound from a number of glycidyl ethers. In addition, polyhexamethyleneguanidine hydrochloride can be a toxic hazard, as it has a general toxic effect that can cause functional disorders in the nervous system, blood system, lungs, liver, kidneys [Muratova N.M. Tosicological and hygienic aspects of the action of polyhexamethyleneguanidine hydrochloride - a new drug of the polyguanidine series. Abstract of the dissertation for the degree of candidate of medical sciences, Irkutsk, 1994].

The technical objective of the present invention is the development of a local hemostatic agent with increased antibacterial activity against gram-positive, non-fermentative gram-negative and enterobacteria, which is important in providing first and primary health care to protect the life and health of people exposed to damaging factors that cause massive bleeding combined with the possibility of purulent-inflammatory diseases of the skin and soft tissues.

The invention is directed to the creation of a local hemostatic agent based on polysaccharides - chitosan salts in combination with the active pharmaceutical substance of the antimicrobial agent - 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide, which enhances the bactericidal properties of chitosan.

The task of the claimed invention is solved by immobilization of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide on macromolecules of chitosan salt.

The technical result is achieved by the proposed local hemostatic antibacterial agent for stopping massive bleeding, which is a macromolecular complex of a chitosan salt and an antibacterial agent, characterized in that the chitosan salt is associated with an antibacterial agent through intermolecular hydrogen bonds, and 2.3 acts as an antibacterial agent. -bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide with a mass fraction of 0.5% to 20%, chitosan salt is selected from formate, acetate, citrate, glycolate, or chitosan lactate.

It is advisable that the local hemostatic antibacterial agent for stopping massive bleeding is presented in the form of granules, sponges or films.

Preferably, a topical hemostatic antibacterial agent for stopping massive bleeding has antibacterial activity against gram-positive, non-fermentative gram-negative, and enterobacteria.

The declared content of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide in the composition of the macromolecular complex is selected in order to achieve maximum antibacterial activity of the local hemostatic agent. When the mass fraction of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide is less than 0.5%, the effect of increasing the bactericidal action is not manifested. An increase in the mass fraction of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide by more than 20% does not give a synergistic effect of the bactericidal effect of the hemostatic composition and is not economically feasible.

The effectiveness of the hemostatic and bactericidal action of the agent does not depend on the type of anion selected for salt formation of acids.

The essence of the invention lies in the fact that the macromolecular complex of chitosan salt and 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide in the selected range of component ratios causes a synergistic effect of bactericidal action in combination with the effect of local hemostatic action .

The 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide introduced into the composition of the claimed local hemostatic agent, which is the substance of the dioxidine drug, has a high antibacterial activity against a wide range of microorganisms, including anaerobic ones. The mechanism of the antibacterial action of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide is associated with damage to bacterial cells due to the activation of free radical mechanisms that induce the formation of reactive oxygen species, leading, in particular, to irreversible changes in the structure nucleotide, and already under the influence of subinhibitory concentrations. Under anaerobic conditions, an increase in the antibacterial activity of the drug by 8-128 times was noted, depending on the type of pathogen [Padeyskaya E.N. Antibacterial drug dioxidine: features of biological action and significance in the treatment of various forms of purulent infection. Infections and antimicrobial therapy. Complex issues of antimicrobial chemotherapy. 2001; 3:5]. When applied topically for the treatment of wounds, 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide does not have irritating properties, and, along with an antibacterial effect, it contributes to a faster course of reparative processes and wound epithelialization.

At the same time, chitosan salts, being the basis of a number of medical products designed to stop massive bleeding, are produced by foreign manufacturers [Celox (Medtrade Products Ltd. (UK), NetSop Bandage PRO (Tricol Biomedical INC (USA), Revul) ( Yuria-Pharm, (UA)] and domestic [Gemoflex-Combat (Inmed LLC), Gepoglos (Lumi LLC), Ellarga (Nobr LLC)] companies, has insufficient “soft” [US20170258959 ] antibacterial activity.

The authors of the invention demonstrated for the first time that the macromolecular complex of chitosan salt and 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide, along with the properties of an effective hemostatic agent, has an increased antibacterial activity against anaerobic or mixed infection. The mechanism of this action is targeted delivery and prolonged release of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide on the surface of bacterial cells during electrostatic interaction of cationic chitosan with a negatively charged surface of bacterial cells.

Thus, the proposed local hemostatic antibacterial agent for stopping massive bleeding is a macromolecular complex of chitosan salt and 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide of the proposed formula

where: n is the number of chitosan salt units;

m is the number of units of the macromolecular complex (MCC) in the polymer chain of the chitosan salt,

R is the alkyl residue of the acid.

The invention is implemented as follows. These examples illustrate, but do not limit the proposed technical solution.

Example 1

951.6 ml of purified water according to FS 42-2619-97 is poured into a 2-liter round-bottom flask. The rotor of the ULTRA-TURREX T-25 digital disperser is lowered into the water and mixing is turned on, smoothly increasing the rotor speed to 1000 rpm. With stirring, 22.0 g of chitosan according to TU 9289-067-00472121 with a degree of deacetylation of 90%, a molecular weight of 150-200 kDa, a particle size of 0.18-1.25 mm, a moisture content of 9.5 wt. %, 25 g of glacial acetic acid grade Ch according to GOST 61-75 and 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide according to LSR-002744/09 in an amount of 0.12 g. the mixture is heated to a temperature of 45-50°C and stirring is continued for 0.5-1 hour until the ingredients are completely dissolved. The resulting homogeneous transparent hydrogel is cooled to room temperature, poured into stainless steel trays siliconized on the inner surface with a layer of 10 mm thick and placed on the shelves of freeze dryers TG-50, in which freeze-drying is carried out. Upon completion of the process in the trays, sponges of chitosan acetate with 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide are obtained with a quantitative yield, having a surface density of 27 mg/cm ² , water absorption capacity of 40- 50 g/g and a mass fraction of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide 0.5%.

Example 2

According to example 1, characterized in that the sample of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide is 1.3 g, and its mass fraction in the final product is 5.2%.

Example 3

According to example 1, characterized in that the weight of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide is 4.0 g, and its mass fraction in the final product is 16.0%.

Example 4

According to example 1, characterized in that the weight of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide is 5.0 g, and its mass fraction in the final product is 20.0%.

Example 5

100 g of chitosan according to TU 9289-067-00472121 with a degree of deacetylation of 90%, a molecular weight of 150-200 kDa, a particle size of 0.25-0.42 mm, a content of moisture 9.5 wt. % and pour 475 g of ethyl alcohol according to GOST R 51652-2000 rectified from food raw materials "Basic". Turn on the stirrer and bring the temperature in the water bath to 40-43°C. With stirring, 43.5 g of 80% glycolic acid grade Ch according to TU 6-09-4679-78 is added to the flask, containing 2,3-bis-(hydroxymethyl)-quinoxalin-1,4-di- N-oxide according to LSR-002744/09 in the amount of 6 g. The resulting suspension is kept at a temperature in a water bath of 40-43°C and stirring for 1.5 hours. At the end of mixing, pour the contents of the flask onto a filter funnel (Schott filter, class 3) mounted on a Bunsen flask and filter under vacuum with a water jet pump. At the end of the filtration, the precipitate is completely reloaded back into the flask, and 475 g of ethanol are poured into the same place. The suspension is heated and maintained at a temperature in a water bath of 40-43°C and stirring for 1 hour. The contents of the flask are again filtered. The resulting wet sediment is transferred to a sheet of filter paper 0.5 × 0.5 m in a fume hood, leveling with a layer of 5-10 mm thick, and left overnight for drying. The dried granules are placed in a 1-liter round-bottom flask and dried at a temperature in a water bath of 48-50°C in a water-jet pump vacuum at a residual pressure of not more than 15 mm Hg. within 3 hours. Granules of chitosan glycolate with 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide are obtained with a yield of 130-135 g, bulk density of 0.28-0.32 g/cm ³ , absorption capacity of water 30-40 g/g and a mass fraction of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide 5.2%.

Example 6

Homogeneous transparent hydrogel, obtained according to example 4, is poured into stainless steel trays siliconized on the inner surface with a layer of 5 mm thick and placed on the shelves of an oven, in which the hydrogel is dried at a temperature of 45°C and atmospheric pressure to constant weight. Upon completion of the process in the trays, transparent, colorless or with a yellowish tinge, swelling films of chitosan acetate with 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide, with a thickness of 110 microns with a surface density of 14 mg/cm ² and a mass fraction of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide 20%.

The chemical structure of the resulting macromolecular complex is confirmed by high-resolution IR Fourier spectroscopy data from a comparative analysis of the spectra of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide powder, films of chitosan acetate and chitosan acetate with the inclusion of 20 % 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide (example 6). The spectrum of a transparent chitosan acetate film containing no crystalline particles containing 20% 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide contains an absorption band at 1322 cm ^-1 , indicating the presence of unsubstituted structural elements N→O of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide, 774 cm ^-1 band of out-of-plane CH vibrations for aromatic structure, 1024 cm ^-1 band for 1,2-substituted aromatic cycles. Absorption band at 1405 cm ^-1 indicating the presence of carboxylate anions in the spectrum [N.V. Afanaseva et al. Molecular mobility of chitosan and features of its interaction with montmorillmonite in composite films according to dielectric and IR spectroscopy data, Macromolecular Compounds, Series A, 2013, volume 55, no. 12, p. 1437-1447], in a doublet with a band of 1378 cm ^-1 indicates a hydrogen bond between the carboxylate anion of chitosan acetate and the proton of the HOCH ₂ group of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide .

For microbiological and hemostatic tests, samples of granules, sponges and films are placed in sealed packaging bags made of packaging material according to TU 2242-002-81659324-2010 and sterilized by the radiation method with a sterilizing radiation dose of 10-15 kGy.

The following examples illustrate but do not limit the proposed technical solution.

Determination of the antibacterial activity of the declared local hemostatic antibacterial agent was carried out according to GOST R ISO 20743-2012 “Textile materials. Determination of antibacterial activity "by the method of disks for samples according to example 1 and the method of" wells "for samples according to example 5 using a collection of clinical strains of microorganisms of the Institute of Surgery. A.V. Vishnevsky - the main causative agents of surgical infection.

The collection includes both Gram-positive and Gram-negative microorganisms.

Gram-positive microorganisms were represented by staphylococci: S. aureus, S. epidermidis, both methicillin-sensitive (MSSA, MSSE) and methicillin-resistant (MRSA, MRSE), as well as the Enterococcus spp. group, which included E. faecalis and E. faecium.

Gram-negative microorganisms of anaerobic or mixed infection were represented by 2 groups: non-fermenting gram-negative bacteria P.aeruginosa and A. baumanii and enterobacteria from the Enterobacteriaceae family - E. Coli, Klebsiella spp. and P. mirabilis.

The following samples were used as objects for determining antimicrobial activity:

- chitosan acetate sponges with a mass fraction of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide (dioxidine) 0.5%, 5.2%, 16.0% and 20.0% ( code name "Hitokol-D"),

- chitosangicolate granules with a mass fraction of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide (dioxidine) 5.2% (code name "Biohem").

Samples were used as objects of comparison:

- chitosan acetate sponges that do not contain 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide (code name "Chitokol"),

- chitosan lactate granules according to RU 2519220, not containing 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide (code name "Hemochit"),

- Airlaid non-woven material made of cellulose and synthetic fibers according to TU 8390-001-1342972701-2011, impregnated with 1% solution of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide for external use ("Dioxidin -solution") with a mass fraction of 4.8%,

- the drug "Dioxidin-ointment" for external use per. number: P N002534/01 with a mass fraction of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide 5.0%.

The results of the assessment of the antibacterial activity of samples according to the invention (code name "Chitokol-D" and "Biohem") and reference samples "Chitokol", "Gemohit", "Dioxidin-solution" and "Dioxidin-ointment" are presented in tables 1, 2, 3 .

The data in Table 1 show that the samples according to the invention "Khitokol-D" and the comparison samples have antibacterial activity against staphylococci and enterococci, although half of the strains of both Staphylococcus aureus and epidermal Staphylococcus aureus were methicillin-resistant, i.e. multiresistant, and E. faecim and E. faecalis were sensitive only to the antibiotic vancomycin. At the same time, "Khitokol-D" in the range from 0.5 wt. % up to 20.0 wt. % exhibits higher activity than the Chitokol sample and the dioxidine solution sample separately.

From the data in Table 2 it follows that the samples according to the invention "Khitokol-D" also exhibit antibacterial activity against non-fermenting gram-negative and enterobacteria, while the sample "Khitokol" is not active. At the same time, the sample "Khitokol-D" in the range of dioxidine concentrations from 0.5 wt. % up to 20.0 wt. % exhibits a higher activity in comparison with the sample solution of dioxidine.

According to Table 3, the sample according to the invention "Biohem" exhibits antibacterial activity against both gram-positive and non-fermenting gram-negative and enterobacteria, in contrast to the reference sample "Gemohit", which is inactive against non-fermenting gram-negative and enterobacteria. At the same time, "Biogem" exhibits higher activity against S. aureus than the samples "Hemohit" and "Dioxidin-ointment" separately and higher activity against S. aureus, E. coli and P. aeruginosa in comparison with the sample "Dioxidin-ointment". ointment".

Evaluation of the hemostatic effectiveness of the claimed agent was carried out by experimental study of stopping massive bleeding on 9 Large White pigs weighing 38.5-44.5 kg.

Anatomical and physiological parameters of the cardiovascular system (caliber of blood vessels, blood pressure, response to stress factors) ensured the validity of the experiment, which involved 9 animals.

The animals were divided into 2 groups, one experimental - 6 individuals and one control - 3 individuals. In 3 animals of the 1st group (n=3), samples of Biohem granules weighing 20 g were used to stop bleeding, and in 3 animals (n=3) samples of sponges Chitokol-D 90 × 45 × 8 mm in size were used to stop bleeding. in the amount of 6 pieces with a total weight of 9 g for each animal with a mass fraction of 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide in samples of granules and sponges of 5.2%.

The objective of the experiment was to study the effectiveness of "Biogem" and "Hitokol-D" on a standard model of external bleeding for a severe groin injury with damage to the vascular femoral bundle [Chepur S.V. On the choice of an external bleeding model for preclinical evaluation of the effectiveness of local hemostatic agents. Military Medical Journal, No. 7, 2016, p. 25-33]. In the control group, a pressure bandage was applied to the wound using a standard PPI-AV3 dressing.

After the wound was inflicted and the period of uncontrolled bleeding for 45-60 s, the Biohem granules were poured into the wound, and the Chitokol-D sponges were tightly placed one after the other in the wound, then manual compression was performed for 7 minutes, after which the adequacy of hemostasis was assessed by the number of recurrences of bleeding and the volume of blood loss. The duration of the acute experiment was 180 min.

The use of "Biohem" granules and "Khitokol-D" sponges made it possible to achieve stable primary hemostasis in 6 cases (100%). In 1 animal, after the resumption of bleeding, "Biohem" granules were reapplied with a positive effect. As a result, all animals in the experimental group survived until the end of the experiment. In the control group, all animals died prematurely, the recurrence rate of bleeding was 100%. The volume of blood loss in the experimental group was 400-550 ml, while in the control animals lost an average of 1570 ml.

Thus, the invention opens up the possibility of stopping massive bleeding with an effective local hemostatic agent containing a macromolecular complex of chitosan salt and 2,3-bis-(hydroxymethyl)-quinoxaline-1,4-di-N-oxide, which has increased antibacterial activity against gram-positive , non-fermentative gram-negative and enterobacteria that cause purulent-inflammatory diseases of the skin and soft tissues and can be used in emergency and extreme situations, as well as in hospital conditions where anaerobic or mixed infection is possible.

Claims (3)

1. A local hemostatic antibacterial agent for stopping massive bleeding, which is a macromolecular complex of a chitosan salt and an antibacterial agent, characterized in that the chitosan salt is associated with an antibacterial agent through intermolecular hydrogen bonds, and 2,3-bis-(hydroxymethyl) acts as an antibacterial agent )-quinoxaline-1,4-di-N-oxide with a mass fraction of 0.5% to 20%, the chitosan salt is selected from formate, acetate, citrate, glycolate, or chitosan lactate. 2. A local hemostatic antibacterial agent for stopping massive bleeding according to claim 1, characterized in that it is presented in the form of granules, sponges or films. 3. A local hemostatic antibacterial agent for stopping massive bleeding according to claim 1, characterized in that it has antibacterial activity against gram-positive, non-fermenting gram-negative and enterobacteria.