RU2756316C1 - Local hemostatic antibacterial agent, method for production thereof, and medical product based thereon - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to a hemostatic antibacterial agent containing modified natural polysaccharides bound with polyguanidine hydrochloride exhibiting antibacterial properties, to methods for producing the agent and medical products based thereon, to methods for application thereof.

EFFECT: technical result of the proposed invention is production of a stable supramolecular complex by means of immobilising polyguanidine hydrochloride on oxidised cellulose or oxidised starch, exhibiting an effective hemostatic and bactericidal activity.

4 cl, 6 ex, 3 tbl

Description

The invention relates to medicine, namely to a hemostatic antibacterial agent containing modified natural polysaccharides associated with a biocidal substance, methods for its production, as well as medical products using polysaccharides associated with antibacterial compounds. Designed to stop bleeding arising from traumatic organ damage and during surgical operations.

The proposed tool can be used to stop local bleeding resulting from gunshot, shrapnel, knife wounds during military conflicts, car accidents, natural and man-made disasters. Timely, effective stopping of bleeding at the prehospital stage is one of the main issues of military field and emergency medicine.

Bleeding during surgery and nosocomial infection in the postoperative period are among the most pressing problems in surgery. According to the world literature, bleeding increases the duration of hospitalization by an average of 4 days, and the cost of treatment by 20%.

The methods of combating bleeding that exist today have a number of significant disadvantages: diathermoelectrocoagulation leads to tissue burns, systemic transfusion of blood components can be accompanied by infectious and immune complications, and local hemostatic agents do not fully meet the requirements of surgeons.

Known hemostatic materials based on fibrin, which, due to their animal origin, can lead to an anaphylactic reaction to their use (Beierlein W., Scheule AH, Dietrich W., Ziemer G. “Forty years of clinical aprotirin use: a review of 124 hypersensitivity reaction "// Ann. Thorac. Surg. 2005, 19, P. 741-748).

Side effects from known collagen-based hemostatic materials are rare and most commonly include allergic reactions, adhesion formation and inhibition of postoperative wound healing. Bone wax is a material that is difficult for the body to absorb. In experiments by various authors, it was found that bone wax is one of the risk factors for sternal infection, and its use inhibits tissue regeneration (Wellsz T., Armstrong JK, Cambidge J., et. Al. “The effect of a soluble polymer and bone wax on sternal healing in an animal model "// Ann. Thorac. Surg, 2005, 85, P. 1776-80).

The problem of prevention of nosocomial infections is no less acute. Thus, in the United States, according to the estimates of the Center for Disease Control and Prevention, about 1.7 million cases of nosocomial infections caused by all types of microorganisms, leading or accompanying 9,900 deaths, have been recorded annually. In Europe, according to the results of hospital studies, mortality from nosocomial infections is about 25 000 cases per year. No less urgent is the problem of reinfection of wound surfaces with hospital strains from the hands of medical personnel, bed linen, sutures and dressings in groups of patients in intensive care units, etc. %.

An effective solution to the problem of bleeding arising from traumatic organ injuries and during surgical operations, and the addition of a nosocomial infection in the postoperative period, is to simultaneously use a combination of hemostatic and antiseptic agents to stop bleeding.

The known invention proposes a hemostatic sponge based on collagen and hydroxyapatite as a drug, which may additionally contain gentamicin or lincomycin (see patent RU 2034572, A61L 15/32, A61L 15/44, 03.11.1992).

The disadvantage of this composition is the use of antibiotics gentamicin or lincomycin, which are not effective against frequent causative agents of nosocomial infections, such as MRSA (Staphylococcusaureus), MRSE (Staphylococcusepidermidis), ampicillin and aminoglycoside resistant, S.

It is known to use hemostatic gauze based on oxidized cellulose and the antibiotic vancomycin in the form of a "sandwich": vancomycin powder is placed between two layers of oxidized cellulose in the form of gauze (Kauko M., Liman T., Topsu S. "Alow-Costand Simple Method to Stop Intraoperative Leakage -Type Bleeding: Use of the Vancomycin-Oxidized Regenerated Cellulose (ORC) Sandwich "// Journal of Investigative Surgery, 2006, 19, N5, P. 323-327).

The disadvantages of this method are the duration of stopping bleeding and the uneven distribution of the antibiotic vancomycin over the surface of the hemostatic gauze, which increases the risk of infection and inflammation.

Known antimicrobial composition containing a complex of oxidized regenerated cellulose, covalently bound to gentamicin, which has potential nephrotoxicity, ototoxicity and can cause neuromuscular blockade (see patent RU 2465917, A61K 41/48, A61P 31/00, publ. 10.11.2012 , bulletin No. 31).

The disadvantage of the composition is its low efficiency against frequent causative agents of nosocomial infections, such as MRSA, MRSE, enterococci, ampicillin and aminoglycoside resistant enterococci.

The closest in technical essence to the proposed invention is a hemostatic antibacterial agent, which is a supramolecular complex in the form of a powder containing oxidized powdered cellulose and vancomycin, linked through non-covalent intermolecular hydrogen bonds. The tool is intended for stopping bleeding in surgery, as well as for providing local antiseptic action and regeneration of damaged tissues (see patent RU 2665950, A61P 7/04, A61P 31/04, A61K 31/7036, A61K 38/04, A61K 47/38 , A61K 9/14, publ. 09/05/2018, bul. No. 25).

The disadvantage of this composition is the use of antibiotics, since in connection with the growth of resistant strains of microorganisms that cause nosocomial infections, a limited number of antibiotics remain active against them. In addition, the use of certain antibiotics for etiotropic therapy requires a long preliminary isolation of the infectious agent to determine its sensitivity to antibiotics.

Also the closest in technical essence to the present invention is a local hemostatic antibacterial agent for stopping massive bleeding, containing chitosan salt and polyhexamethylene guanidine hydrochloride, covalently cross-linked with a polyfunctional compound from a number of glycidyl ethers. The agent has a high sorption capacity for blood, a short time of onset of hemostasis and high antimicrobial activity (see patent RU 2519220, A61K 31/722, A61K 31/155, A61L 15/22, A61L 26/00, A61P 7/04, publ. 10.06.2014, bul. No. 16).

The disadvantage of this tool is the use of chitosan, the production of which from raw materials is long and expensive. In addition, a sufficiently long time is required for the complete resorption of chitosan.

The objective of the invention is to obtain an effective hemostatic agent with bactericidal properties, including polyguanidine hydrochloride and a natural modified polysaccharide based on cellulose or starch, the creation of a hemostatic antibacterial agent for providing first and emergency medical care for open wounds and surgical interventions on parenchymal vessels and blood vessels. apply in the form of powder, solution, granules or tablets.

The technical result of the present invention is to obtain a stable supramolecular complex by immobilizing polyguanidine hydrochloride on natural modified polysaccharides. The advantages of using modified polysaccharides to create a hemostatic agent are their high sorption capacity for blood, a short time of onset of hemostasis, rapid resorption, antimicrobial effect against a wide range of pathogenic organisms due to the low pH created by carboxyl groups, as well as the availability and cheapness of the raw material base.

The polyguanidine hydrochloride introduced into the composition of the hemostatic agent exhibits an antibacterial effect and has such properties as non-toxicity, biocompatibility and biodegradability.

Thus, a hemostatic antibacterial agent has an effective hemostatic and bactericidal effect, excludes an infectious complication in the postoperative period caused by gram-positive flora.

The invention is aimed at creating a hemostatic antibacterial agent of local action to reduce the time to stop bleeding and reduce the intensity of blood loss.

Water-soluble polyguanidine hydrochloride is obtained by polycondensation of diamines and guanidine hydrochloride in accordance with the scheme (Stelmakh S.A., Bazaron L.U., Mognonov D.M.On the mechanism of polycondensation of hexamethylenediamine and guanidine hydrochloride. - Journal of Applied Chemistry, Issue - 2010. Vol. 83 2. - S. 344-345.):

The structure of the obtained polyguanidine hydrochloride is confirmed by the data of IR spectroscopy. Stretching vibrations of free amino groups are observed in the region of 3270 and 3160 cm ^-1. Two bands in the region of 2930 and 2855 cm ^-1 correspond to stretching asymmetric and symmetric vibrations of methylene groups. The band at 1630 cm ^-1 is a characteristic band of guanidine salts.

Oxidized cellulose is obtained by oxidizing cellulose powder with a solution of nitrogen dioxide in an inert solvent (hexane) (Grigoriev I.A., Morozov S.V., Tkacheva N.I., Pankrushina N.A., Chernyavsky AM, Tarkova A.R. Patent of the Russian Federation No. 2563279, 2014. Bull. Fig. No. 26, 2015):

The content of carboxyl groups is 20-21%, the content of bound nitrogen does not exceed 0.5%. The molecular weight of oxidized cellulose is 31.8 ± 6.1 kDa. In the IR spectrum, a signal of the carbonyl carbon is observed at 1735 cm ^-1 , in the NMR spectrum, the signal of the carbon of the carboxyl group in the region of a weak field with a chemical shift of 133 ppm.

Oxidized starch is obtained by oxidation of water-soluble powdered starch with nitrogen dioxide in an inert solvent hexane at a temperature of 18-20 ° C (Butrim S.M., Bildyukevich TD, Yurkshtovich T.P. Studying the kinetics of starch oxidation in the system nitric oxide (IV) - carbon tetrachloride. - Journal of Applied Chemistry. - 2001. - T. 74, Issue 12. - From 2046-2050):

The content of carbonyl groups in the obtained samples of oxidized starch is 22.7-25.8%, the nitrogen content is 0.3-0.4%. In the IR spectrum of oxidized starch, there is an absorption band at 1747 cm ^-1 , characteristic of carboxyl groups. The molecular weight of oxidized starch is determined by gel permeation chromatography, which is Mw 5.4 ± 1.1 kDa.

The problem of obtaining an effective hemostatic agent with bactericidal properties is solved by immobilizing polyguanidine hydrochloride on oxidized powdered cellulose or oxidized powdered starch under mild conditions, excluding retrogradation of polysaccharides.

To improve immobilization and the formation of more stable and stable supramolecular complexes, the stage of swelling of modified polysaccharides in water is added, which ensures the availability of their functional groups.

The content of polyguanidine hydrochloride in supramolecular complexes containing oxidized starch or oxidized cellulose associated with polyguanidine hydrochloride due to non-covalent dispersion (donor-acceptor) forces and intermolecular hydrogen bonds is from 15 to 20 wt. %. The resulting agent has a hemostatic and antibacterial effect and can be used to stop diffuse bleeding from parenchymal organs and vascular anastomoses.

A medical device based on a hemostatic antibacterial agent containing oxidized starch or oxidized cellulose associated with polyguanidine hydrochloride can be a fine powder, a solution / suspension of the powder in a sterile solvent (distilled water, saline), as well as granules or tablets. Such granules or tablets can be used to stop local bleeding in case of gunshot, shrapnel, knife wounds during military conflicts, car accidents, natural and man-made disasters.

The essence of the invention is illustrated by the following examples.

Example 1. 0.75 g of oxidized cellulose is added to a glass flask to 4.0 ml of water, the mixture is stirred for 1 hour, then a solution of 0.25 g of polyguanidine hydrochloride in 1.0 ml of water is added. The mixture is stirred for 20-26 hours at a temperature of 25-35 ° C. During the mixing time, the oxidized cellulose swells and the availability of its functional groups increases, which contributes to the immobilization of polyguanidine hydrochloride on the oxidized cellulose. The resulting suspension is separated in a centrifuge into solid and liquid parts. The solid part is dried at a temperature of 25-30 ° C to constant weight for 48-54 hours and ground in a mortar. A beige powder is obtained, which is a supramolecular complex of oxidized cellulose with immobilized polyguanidine hydrochloride. The yield is 70%.

The molecular weight of the obtained complex was determined by gel permeation chromatography, which is Mw 13.6 ÷ 2.7 kDa. Elemental analysis data of the complex,%: found C, 38.4; H, 6.0; N, 3.7; Cl 1.3, the ratio of oxidized cellulose and polyguanidine hydrochloride in the complex is from 1: 5 to 1: 6. In the IR spectrum of the complex, bands of stretching and bending vibrations characteristic of oxidized cellulose and polyguanidine hydrochloride molecules 1736 and 1638 cm ^-1, respectively, are identified.

Example 2. 1.5 g of oxidized starch and 7 ml of distilled water are placed in a glass flask and incubated with vigorous stirring for 10 hours. During mixing, the oxidized starch swells and the availability of its functional groups increases. 0.3 g of polyguanidine hydrochloride is added to the viscous solution and the mixture is stirred vigorously at a temperature of 25-30 ° C until the polyguanidine hydrochloride is completely dissolved within 3-5 hours. The resulting viscous solution is dried in a porcelain cup at a temperature of 30-40 ° C for 24-28 hours. The yield is 83%.

The resulting pale beige powder is a supramolecular complex of oxidized starch with immobilized polyguanidine hydrochloride.

The ratio of oxidized starch and polyguanidine hydrochloride in the complex is from 1: 0.15 to 1: 0.20. In the IR spectrum there is an absorption band of the carboxyl group at 1735 cm ^-1 . The value of the hypsochromic shift is small, because the energy of intermolecular interaction is comparable to the energy of dispersion interaction and donor-acceptor bond caused by the overlapping of molecular orbitals due to the lone electron pair of the polyguanidine hydrochloride heteroatom and carbonyl groups that accept an electron in the p-orbital.

For biological tests, the resulting product is sterilized by radiation.

Example 3. A medical device is a fine powder obtained as described in examples 1 and 2, as well as granules or tablets obtained by direct cold pressing of this powder in defatted sterile molds at 25-30 ° C and a specific pressure of 40-50 MPa ... After pressing, the resulting product is packed in a sealed plastic bag.

For the manufacture of medical devices in the form of a 4-6% solution (for a composition of oxidized starch with polyguanidine hydrochloride) or a suspension (for a composition of oxidized cellulose with polyguanidine hydrochloride), 0.5 g of a hemostatic antibacterial agent in the form of a powder is placed in a sterile mixing container.

Then add 8-12 ml of sterile solvent (distilled water or saline) and mix with a sterile instrument. The ratio of dry hemostatic antibacterial agent to solvent is 1:16 to 1:24 (gram: ml).

Example 4. The study of the hemostatic properties of medical devices in the form of a solution or suspension in in vitro experiments is carried out using the method for determining the hemostatic activity of liquid hemostatic agents developed at the Scientific Center for Cardiovascular Surgery. A.N. Bakuleva (Mezhneva V.V., Samsonova N.I., Pliushch M.G., Kostava V.T. "Experimental studies of the hemostatic activity of liquid hemostatic agents" // Bulletin of the scientific center of cardiovascular surgery named after A. N. Bakuleva RAMS. Cardiac and vascular diseases, 2007, 8 (2), 71-75).

For research, rat blood is used, taken from the femoral artery of animals and stabilized with a 3.8% sodium citrate solution in a ratio of 9: 1. The degree of hemostatic activity is determined in a Petri dish by adding dropwise 0.1 ml of the test agent in the form of a solution or suspension to 1 ml of stabilized blood with stirring with a glass rod. Time of blood clot formation is determined by a stopwatch.

The results of the study of the hemostatic properties of oxidized cellulose, oxidized starch and their compositions with polyguanidine hydrochloride in in vitro experiments are presented in table. 1.

As follows from the data in Table 1, a medical device based on oxidized starch and polyguanidine hydrochloride has the maximum hemostatic effect in an in vitro system.

Example 5.

To assess the hemostatic properties of the proposed agent in vivo, experiments are carried out on 39 white WISTAR female rats weighing 300-350 g. Animals are divided into 5 groups:

Group 1 (control, n = 11);

Group 2 (oxidized cellulose, n = 7);

Group 3 (oxidized cellulose + polyguanidine hydrochloride, n = 7);

Group 4 (oxidized starch, n = 7);

Group 5 (oxidized starch + polyguanidine hydrochloride, n = 7).

Experiments are performed according to the technique described in the patent.

"A method for identifying and comparatively assessing the hemostatic activity of hemostatic matrix preparations of local action" (see patent RU 2127428, G01N 33/48, G01N 33/49, publ. 03/10/1999).

The operations are performed under intramuscular anesthesia (xylanit, 0.6 mg / kg). The rats undergo an upper midline laparotomy, the left lobe of the liver is removed into the wound, a cut wound (length 10 mm, depth 3 mm) is performed with a sharp blade and a hemostatic material in the form of a powder weighing 7 mg is applied to it, depending on the animal belonging to the corresponding group.

The criteria for the manifestation of hemostatic properties in the studied samples are considered to be a decrease in the time to stop bleeding (sec.) And a decrease in the intensity of blood loss by the mass of outflowing blood (mg) in comparison with the indicators of spontaneously stopping bleeding (control). The time to stop bleeding is determined by the cessation of blood flow from the wound, the amount of blood loss is determined by weighing a gauze swab before and after soaking it with blood.

The results of the study of the hemostatic properties of the proposed agents, the results of the control test (without the use of hemostatic agents), as well as comparative data of the hemostatic properties of oxidized cellulose and oxidized starch are shown in Table 2.

From the data obtained in the in vivo study, it follows that all samples have a pronounced hemostatic effect. The use of oxidized starch (group 4) reduces the time to stop bleeding relative to groups 1 and 2 by 48.7% and 8.3%, but inferior to the composition "oxidized cellulose + polyguanidine hydrochloride" (group 3). The use of oxidized starch reduces the mass of outflowing blood relative to groups 1, 2 and 3, respectively, by 61.9%, 44.8%, 23.4%. The highest hemostatic effect is possessed by the composition "oxidized starch + polyguanidine hydrochloride", which effectively reduces the time to stop bleeding by 63.2%, 34.3%, 17.4%, 28.3%, and the mass of the outflowing blood by 67.0% , 52.2%, 33.8%, 13.5% relative to the corresponding indicators in groups 1-4 of experimental animals.

The data obtained indicate a pronounced hemostatic activity of the claimed compositions in relation to stopping diffuse bleeding, a significant decrease in bleeding time and the volume of blood loss when simulating a wound of a parenchymal organ.

The use of the proposed tool does not require special preparation, it is enough to treat the wound with a powder or solution / suspension, or place granules or tablets in the wound, press down, organizing a slight surface pressure.

Example 6. The antibacterial properties of a medical device based on the proposed hemostatic antibacterial agent are confirmed by the conducted studies.

Opportunistic pathogenic microorganisms Escherichiacoli (E.coli), Bacilluscereus (B.cereus) are used as pathogenic test strains, which are model when conducting research in the field of microbiology and biotechnology.

Evaluation of antibacterial properties is carried out on the basis of the method of serial dilutions in agar. To obtain vegetative cells, cultures of E. coli and B. cereus are incubated in a liquid nutrient medium under aerobic conditions in a shaker-incubator BIOSANES-20 (180 min ^-1 , 37 ° C, for 24 hours).

Vegetative cells of each test object are prepared in sterile buffer solution from the corresponding one-day cultures by the limiting dilution method. The resulting bacterial suspensions contain from 10 ² to 10 ³ CFU / ml.

Under aseptic conditions, mix 20 ml of agar mesopatamia broth and 0.05 and 0.025 ml of a 1% solution of the test agent directly in a Petri dish. In parallel, prepare Petri dishes without hemostatic antibacterial agent (control).

After solidification of the agar broth, incoagulation is carried out with 0.1 ml of E. coli and B. cereus suspension on prepared solid culture media in five replicates and incubated in a thermostat at 35 ° C for 24 hours to count the surviving CFU. The data obtained are summarized in Table 3.

As can be seen from the data in Table 3, the cultivation of strains of multidrug-resistant microorganisms E. coli and B. cereus in a liquid nutrient medium with the addition of a hemostatic antibacterial agent with a concentration of 0.025 to 0.05 ml of a 1% solution showed a significant decrease in the number of viable cells in the experimental variants compared to with control.

The tool proposed in the present invention in comparison with the prototypes allows you to obtain the following advantages:

1. Opens up the opportunity to provide first and primary health care in stopping parenchymal, venous and arterial bleeding.

2. Is an effective hemostatic agent of local action based on a biocompatible, biodegradable composition of oxidized starch or oxidized cellulose with polyguanidine hydrochloride.

3. Has a high sorption capacity for blood, a short time of hemostasis onset and increased antimicrobial activity.

Claims (4)

1. Local hemostatic antibacterial agent, characterized in that the agent is a supramolecular complex in the form of a powder, containing a natural modified polysaccharide based on cellulose or starch and polyguanidine hydrochloride, bound by non-covalent intermolecular bonds, in which the content of polyguanidine hydrochloride is from 15 to 20. %. 2. A method of obtaining a local hemostatic antibacterial agent according to claim 1, characterized in that polyguanidine hydrochloride is added to a suspension of oxidized cellulose in distilled water, stirred for 20-26 hours at a temperature of 25-30 ° C for 48-54 hours. 3. A method of obtaining a local hemostatic antibacterial agent according to claim 1, characterized in that the oxidized starch is added to distilled water to form a viscous solution to which polyguanidine hydrochloride is added, stirred at a temperature of 25-30 ° C for 3-5 hours and dried at temperature 30-40 ° C. 4. A medical device that is a hemostatic antibacterial agent according to claim 1 in the form of a powder, 4-6% solution / suspension in distilled water or saline, granules or tablets obtained by direct cold pressing at 25-30 ° C and specific pressure 40-50 MPa.