RU2582702C1 - Method for preparing composition based on modified sodium hyaluronate, composition based on modified sodium hyaluronate and use thereof - Google Patents

Abstract

FIELD: chemistry; medicine.

SUBSTANCE: invention relates to a method for preparing a composition based on modified sodium hyaluronate and use thereof in various fields of medicine, veterinary medicine and cosmetology. Method of preparing a composition based on modified copolymer of sodium hyaluronate and heparin comprises forming transverse covalent bonds between hydroxyl groups residues of D-glucuronic acid and D-N-acetylglucosamine included into composition of sodium hyaluronate, and residues of α-D-glucosamine and uronic acid related to heparin, by introducing active cross-linking bifunctional agent - (polyethylene glycol) diglycidyl ether (PEGDE) in an alkaline medium. After chemical copolymerisation, reaction with cross-linking agent is stopped. Obtained reaction mixture is neutralised to pH 7.0, then subjected to tangential filtration using a membrane with cut-off threshold 0.22 mcm, concentrated and stirred until complete homogenisation. Starting material used is animal-derived sodium hyaluronate of pharmacopoeial purity, subjected to tangential ultrafiltration to remove low-molecular fragments capable of triggering inflammatory response.

EFFECT: biocompatibility, biodegradability and non-immunogenicity of modified compositions based on sodium hyaluronate allows use thereof in various fields of medicine and veterinary medicine, including surgery and arthroplasty, orthopedics, traumatology, ophthalmology, dermatology, aesthetic medicine and cosmetology.

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Description

The present invention relates to the field of the pharmaceutical industry, biotechnology and medicine, and in particular to a method for producing a composition based on modified sodium hyaluronate and its use in various fields of medicine, veterinary medicine and cosmetology.

The essence of the invention consists in a method for producing a composition based on a modified copolymer of sodium hyaluronate and heparin, which consists in creating transverse covalent bonds between the hydroxyl groups of the residues of D-glucuronic acid and DN-acetylglucosamine, which are part of sodium hyaluronate, and the residues of α-D-glucosamine and uronic acids related to heparin by introducing an active crosslinking bifunctional agent - (polyethylene glycol) diglycidyl ether (PEGDE) in an alkaline medium. After the chemical copolymerization process, the reaction with the crosslinking agent is stopped. The resulting reaction mixture was neutralized to pH 7.0, then subjected to tangential filtration using a membrane with a cutoff threshold of 0.22 μm, concentrated and stirred until complete homogenization.

Sodium hyaluronate of non-animal origin of pharmacopoeial purity, subjected to tangential filtration to get rid of low molecular weight fragments capable of initiating an inflammatory response, is used as a feedstock.

The biocompatibility, biodegradability and non-immunogenicity of the composition based on modified sodium hyaluronate allow its use in various fields of medicine and veterinary medicine, including surgery and endoprosthetics, orthopedics, traumatology, ophthalmology, dermatology, aesthetic medicine and cosmetology.

Description of the invention

FIELD OF THE INVENTION

The invention relates to the field of the pharmaceutical industry, biotechnology and medicine, namely, to a method for producing a composition based on modified sodium hyaluronate and its use in various fields of medicine, veterinary medicine and cosmetology.

State of the art

Currently, polysaccharides are widely used in various fields of medicine, veterinary medicine and cosmetology, because they possess such important characteristics as biocompatibility, non-immunogenicity, and biodegradability.

The main component of the proposed composition is the polysaccharide hyaluronan, which belongs to the group of non-sulfated glycosaminoglycans. It is one of the main components of the extracellular matrix, is part of the connective, epithelial and nerve tissues, is found in many biological fluids (synovial fluid, saliva, etc.), is produced by some bacteria as part of the external polysaccharide capsule (for example, Streptococcus, Pasteurella). From an evolutionary point of view, hyaluronan is a conservative molecule and is found in all vertebrates.

In terms of chemical structure, hyaluronan isolated from various sources is identical - it is a polyanionic, linear heteropolysaccharide built of regularly alternating residues of D-glucuronic acid and DN-acetylglucosamine connected by β-1,3- and β-1,4-glycosidic bonds (Fig. 1 ) The number of disaccharide repeats forming one hyaluronan molecule varies depending on the source, isolation procedure, and determination method. Natural hyaluronan has a molecular weight of from several hundred daltons to ten million daltons (an average of 1-2 million Da).

The hyaluronan molecule has a large number of hydrogen bonds that form both inside the macromolecule between adjacent carbohydrate residues and between neighboring molecules, and in water solution also with water molecules (Fig. 2). The resulting structure is energetically stable [1] and can exist in various conformational states (elongated chains, relaxed spirals, rod-like structures, etc.). Hyaluronan chains interacting with each other to form fibrils, networks, and stacks [2].

Under physiological conditions (pH ≈ 7), the carboxyl groups of hyaluronan are dissociated, and the polymer molecule has a negative high-density charge that attracts a mass of osmotically active cations of alkali metals, magnesium, calcium, etc., due to which one molecule of hyaluronan can bind a huge amount of water (up to 1000 times its own weight). Accepting extended conformations, which occupy a large volume relative to their mass, hyaluronan even at very low concentrations forms highly viscous aqueous solutions, and at relatively high concentrations (1-4 wt.%) - pseudogels, solutions of colossal viscosity. Even at low concentrations of hyaluronan, intermolecular interaction and structuring takes place — an intermolecular three-dimensional cellular mesh structure is formed, which determines the unique properties of hyaluronan [3].

The cross-links formed in biopolymers are divided into physical (formed as a result of electrostatic interaction or hydrogen bonds) and chemical (covalent bonds). The density of cross-linking directly affects the fundamental properties of hydrogels, such as the degree of swelling, mechanical strength and elasticity, permeability, diffusion characteristics [4].

The big disadvantage of using native hyaluronan is the instability of drugs in vivo due to the rapid degradation of hyaluronidases and free radicals, which reduces the molecular weight of hyaluronan and changes its visco-elastic properties and physical characteristics. Therefore, an extremely urgent task is the creation of more stable forms of hyaluronan. One approach to this problem is covalent crosslinking of hyaluronan using various reagents, for example, carbodiimides (US 7879818, US 20070224277), divinyl sulfone (US 4582865, US 4605601, US 4636524, EP 0939086), polyfunctional epoxides (US 471616) US 4772419, US 4716154), including 1,4-butanediol diglycidyl ether (WO 2013021249, US 4716224), polyalcohols (US 4957744), aldehydes (US 4713448, US 5128326, US 4582865), biscarbodiimides (US 5356883), polycarboxylic acids (EP 0718312-c), complex O′-alkyl carbonate-substituted pyridine (EP 2408823), oligo - or polypeptide (EP 2094736), polyfunctional aziridine (US 20050222081).

The advantages of cross-linked forms of sodium hyaluronate are greater stability compared to non-cross-linked hyaluronan due to the smaller surface area available for enzymatic degradation and, therefore, prolonged residence at the injection site in vivo (up to several months or even years), improved visco-elastic properties .

In addition, many cross-linking agents require the use of excessive amounts, which makes it difficult to predict the degree of crosslinking of sodium hyaluronate and its subsequent purification. Some crosslinking agents, for example carbodiimides and aldehydes, are toxic, and the presence of even trace amounts of crosslinking agents in compositions for medical use is unacceptable.

Based on the analysis of literature data and experiments to determine the toxic effects in animal models, (polyethylene glycol) diglycidyl ether (PEGDE) was chosen as a bifunctional cross-linking agent for the modification of sodium hyaluronate - it is widely used in pharmacology, an inexpensive, easy to use, highly effective and safe reagent, in small quantities, not toxic to humans and animals. No patented counterparts were found.

An additional aspect of the application of the invention is the possibility of using cross-linked sodium hyaluronate as a delivery vehicle and depot for additional drug components, for example, growth factors. Hyaluronan can retain protein factors solely due to weak non-specific electrostatic interactions. To achieve linear kinetics of the release of growth factor seems unlikely. Therefore, heparin is included in the composition of the proposed composition as an additional component, since it is able to retain protein factors due to strong affinity interactions, which increases their stability in vivo and allows linear kinetics of release of growth factors to be achieved.

Heparin is a sulfated intracellular glycosaminoglycan, the molecule of which is represented by several polysaccharide chains linked to a common protein core. Heparin polysaccharide chains consist of repeating disaccharides connected with 1,4-glycosidic bonds - α-D-glucosamine and uronic acid. The protein core consists mainly of serine and glycine residues; approximately two-thirds of serine residues are linked to polysaccharide chains. Due to the presence of a significant amount of negatively charged sulfate and carboxyl groups, the heparin molecule is a strong natural polyanion capable of forming complexes with many polycationic protein and synthetic compounds that carry a total positive charge.

Thus, the copolymerization of sodium hyaluronate with heparin makes it possible to use the claimed composition not only as a therapeutic agent, but also as a biopolymer carrier for targeted delivery (to a specific affected organ / tissue) and controlled prolonged release of additional biologically active and medicinal components, in t .h. protein nature.

Disclosure of invention

Describes a method for producing a modified copolymer of hyaluronate and heparin, which consists in creating transverse covalent bonds between the hydroxyl groups of the residues of D-glucuronic acid and DN-acetylglucosamine, which are part of sodium hyaluronate, and the residues of α-D-glucosamine and uronic acid related to heparin, by the introduction of an active crosslinking bifunctional agent - (polyethylene glycol) diglycidyl ether (PEGDE) in an alkaline medium (Fig. 3). After the chemical copolymerization process, the reaction with the crosslinking agent is stopped. The resulting reaction mixture was neutralized to pH 7, then subjected to tangential filtration using a membrane with a cutoff threshold of 0.22 μm.

(Polyethylene glycol) diglycidyl ether - an inexpensive, easy to use, safe crosslinking agent that is actively used in pharmaceuticals due to the absence of toxic effects on the body; the formula is C ₃ H ₅ O ₂ - (C ₂ H ₄ O) _n -C ₃ H ₅ O (Fig. 4), where the monomer is an ethylene glycol molecule, the average molar mass is 526 g / mol.

The starting material may be sodium hyaluronate of any molecular weight (preferably 10-200 kDa) subjected to a tangential filtration procedure for additional purification and disposal of low molecular weight fragments capable of initiating an inflammatory response. Then, sodium hyaluronate is dissolved in a sodium hydroxide solution — this is necessary for the reaction with PEGDE and a decrease in the viscosity of the solution, which facilitates the mechanical mixing and homogenization of the reaction solution.

The cross-linking bifunctional agent PEGDE is added to a solution of sodium hyaluronate and heparin, maintaining the temperature from 0 to 30 ° C. The reaction time is within 18-24 hours at each stage. PEGDE interacts with the hydroxyl groups of sodium hyaluronate and heparin mainly at the 6th position of the glycosidic backbone at alkaline pH. The resulting copolymer is characterized by the presence of sulfated groups and predicted characteristics.

An important advantage of the invention is the ability to control both the degree of copolymerization of sodium hyaluronate with heparin, and the degree of cross-linking of sodium hyaluronate by changing the amount of crosslinking agent added to the reaction mixture, which allows to obtain a wide range of hyaluronan derivatives that differ in their characteristics, such as viscoelastic properties, mechanical strength, etc., for specific medical, veterinary and cosmetic tasks.

The desired degree of transverse covalent crosslinking (percentage of hydroxyl groups involved in crosslinking) is fully reproducible, as evidenced by NMR data. Therefore, compositions based on modified sodium hyaluronate, obtained according to the same protocols, have constant physicochemical characteristics.

Another advantage provided by the present invention is the ability to include additional components in the composition based on sodium hyaluronate, including of protein nature, for targeted delivery to the damaged organ / tissue and controlled sustained release. For example, such additional components can be bone morphogenetic proteins (bone morphogenetic proteins, BMP), used for targeted regeneration of bone and cartilage tissue in orthopedics and traumatology [5]. In the composition of the copolymer of sodium hyaluronate and heparin, BMP can be delivered directly to the joint affected by osteoarthritis and, released for a long time, contribute to the directed regeneration of hyaline cartilage and bone tissue, subject to destructive changes. The possibility of using a hydrogel of hyaluronic acid with heparin for the controlled delivery of bone morphogenetic protein BMP-2 was shown in the works of Chi et al. [6] and Bhakta et al. [7].

An additional advantage provided by the present invention is the ability to sterilize compositions based on derivatives of cross-linked sodium hyaluronate (for example, thermal sterilization up to 120 ° C or gas sterilization with ethylene oxide) without changing the technological properties.

The composition based on the modified sodium hyaluronate according to the invention is biocompatible, non-immunogenic, resistant to degradation by enzymes, which allows its use in various fields of medicine, veterinary medicine and cosmetology:

1) in rheumatology, orthopedics and traumatology as a means for intraarticular administration of prolonged action in the treatment of osteoarthritis of degenerative-dystrophic and traumatic genesis of various localization (mainly large synovial joints - knee and hip), as well as in military and orthopedic surgery and sports medicine as a means to restore the health of joints, in particular after injuries of various origins and at increased loads;

2) in ophthalmology as part of eye drops for the treatment of dry eye syndrome, in the surgical treatment of ophthalmic diseases, eye injuries, corneal injuries as a viscoprotector, endothelial cell protector during operations;

3) in veterinary medicine as a means for the treatment of non-infectious arthritis of the synovial joints, as well as for restoring the joints after injuries and at increased loads, for example, in racehorses;

4) as a biopolymer carrier for targeted delivery (to a specific affected organ / tissue) and controlled prolonged release of additional biologically active and medicinal components, including protein nature;

5) in aesthetic medicine as intradermal microimplants, injection preparations, filling material in plastic surgery and instrumental cosmetology for aesthetic correction and endoprosthetics of problem areas;

6) in cosmetology as moisturizing agents, the basis for various cosmetic compositions for skin care: creams, gels, lipsticks, lotions and other products.

Brief description of graphic images:

FIG. 1. The structural formula of the disaccharide unit of the hyaluronan molecule.

FIG. 2. The structural formula of the tetrasaccharide portion of the hyaluronic acid molecule, consisting of two repeating disaccharide units: A - in the absence of an aqueous environment, B - in the most energetically favorable conformation (in an aqueous environment) (according to Scott J.E. [2] with changes).

FIG. 3. The chemical scheme for the modification of sodium hyaluronate using PEGDE.

FIG. 4. The structural formula of the crosslinking agent PEGDE.

The implementation of the invention

An object of the present invention is to provide a composition based on a modified copolymer of sodium hyaluronate and heparin by introducing an active crosslinking bifunctional agent - (polyethylene glycol) diglycidyl ether (PEGDE) in an alkaline medium.

The implementation of the invention will consider typical examples.

Example 1

In a flask equipped with a 250 ml overhead stirrer, 25 ml of a 1% sodium hydroxide solution and 30 mg of sodium hyaluronate were placed, then 200 μl of 10% heparin was added. The mixture was allowed to stir for 3 hours at + 15 ° C (200 rpm). Then, 750 μl of PEGDE (ρ = 1.14 g / cm ³ ) was added to the reaction mixture. The reaction was carried out for another 24 hours with stirring (160 rpm, + 5 ° C).

The reaction was stopped by adding 3 ml of methanol and allowed to stir for 10 hours at + 5 ° C (30 rpm). The resulting gel was transferred to a 500 ml flask, 500 ml of ddH ₂ O was added and neutralized with a solution of 1 M hydrochloric acid to pH 7.0. Then, 50 ml of physiological phosphate buffer (2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.0) was added to the gel. The resulting product was subjected to tangential filtration using a membrane with a cutoff threshold of 0.22 μm. Upon reaching a refractive index of n ₂₀ = 1.3535, gel concentration was continued to a concentration of 20 mg / ml.

The concentration of sodium hyaluronate was determined by the following method. Five polypropylene tubes of 1.5 ml volume were sequentially weighed on an analytical balance, and their weight was recorded. 50 μl of gel was placed in each tube; the tubes were centrifuged on a centrifuge evortex for 20 s at 2800 rpm. The tubes were placed in a thermostat and kept at 90 ° C for 20 min with the lids open. Each tube with dried samples was weighed on an analytical balance, the weight of the corresponding empty tube was subtracted from the obtained value, the arithmetic average value of the mass of dry residue from five samples was determined. The mass of dry residue in the sample should be (1.4 ± 0.2) mg, which corresponds to a concentration of sodium hyaluronate of 20 mg / ml.

The resulting concentrated product was stirred by overhead stirrer for another 2 hours at 30 rpm for complete homogenization.

Example 2

In a flask equipped with a 250 ml overhead stirrer, 25 ml of a 1% sodium hydroxide solution and 25 mg of sodium hyaluronate were placed, then 250 μl of 10% heparin was added. The mixture was allowed to stir for 3 hours at + 20 ° C (200 rpm). Then, 400 μl of PEGDE (ρ = 1.14 g / cm ³ ) was added to the reaction mixture. The reaction was carried out for another 18 hours with stirring (160 rpm). Then, 50 ml of a solution containing 0.5% sodium hyaluronate solution, 0.005% heparin and 0.05% sodium hydroxide was added to the mixture, after which another 400 μl of PEGDE was added. The reaction mixture was allowed to stir for 24 hours at + 20 ° C (160 rpm).

The reaction was stopped by adding 4 ml of ethanol (96%) and allowed to stir for another 10 hours at + 10 ° C (30 rpm). The resulting gel was transferred to a 500 ml flask, 425 ml of ddH ₂ O was added and neutralized with a solution of 1 M hydrochloric acid to pH 7.0. Then, 50 ml of physiological phosphate buffer (2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.0) was added to the gel. The resulting product was subjected to tangential filtration using a membrane with a cutoff threshold of 0.22 μm. Upon reaching a refractive index of n ₂₀ = 1.3535, the gel was continued to concentrate to a concentration of 20 mg / ml (for the procedure for determining the concentration of sodium hyaluronate, see Example 1). The resulting concentrated product was stirred by overhead stirrer for another 2 hours at 30 rpm for complete homogenization.

Example 3

In a flask equipped with a 250 ml overhead stirrer, 25 ml of a 1% sodium hydroxide solution and 25 mg of sodium hyaluronate were placed, then 250 μl of 10% heparin was added. The mixture was allowed to stir for 3 hours at + 15 ° C (200 rpm). Then, 756 μl of PEGDE (ρ = 1.14 g / cm ³ ) was added to the reaction mixture. The reaction was carried out for another 24 hours with stirring (160 rpm).

The reaction was stopped by adding 3 ml of methanol and allowed to stir for another 10 hours at + 10 ° C (30 rpm). The resulting gel was transferred to a 500 ml flask, 500 ml of ddH ₂ O was added and neutralized with a solution of 1 M hydrochloric acid to pH 7.0. Then, 50 ml of physiological phosphate buffer (2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.0) was added to the gel. The resulting product was subjected to tangential filtration using a membrane with a cutoff threshold of 0.22 μm. Upon reaching a refractive index of n ₂₀ = 1.3535, gel concentration was continued to a concentration of 20 mg / ml (for a method for determining the concentration of sodium hyaluronate, see Example 1). The resulting concentrated product was stirred by overhead stirrer for another 2 hours at 30 rpm for complete homogenization.

Example 4

In a flask equipped with a 250 ml overhead stirrer, 25 ml of a 1% sodium hydroxide solution and 25 mg of sodium hyaluronate were placed, then 250 μl of 10% heparin was added. The mixture was allowed to stir for 3 hours at + 15 ° C (200 rpm). Then, 577 μl of PEGDE (ρ = 1.14 g / cm ³ ) was added to the reaction mixture. The reaction was carried out for another 18 hours with stirring (160 rpm). Then, 50 ml of a solution containing 0.5% sodium hyaluronate solution, 0.005% heparin and 0.05% sodium hydroxide was added to the mixture, after which another 577 μl of PEGDE was added. The reaction mixture was allowed to stir for 24 hours at + 15 ° C (160 rpm).

The reaction was stopped by adding 4 ml of ethanol (96%) and allowed to stir for another 10 hours at + 5 ° C (30 rpm). The resulting gel was transferred to a 500 ml flask, 425 ml of ddH ₂ O was added and neutralized with a solution of 1 M hydrochloric acid to pH 7.0. Then, 50 ml of physiological phosphate buffer (2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.0) was added to the gel. The resulting product was subjected to tangential filtration using a membrane with a cutoff threshold of 0.22 μm. Upon reaching a refractive index of n ₂₀ = 1.3535, gel concentration was continued to a concentration of 20 mg / ml (for a method for determining the concentration of sodium hyaluronate, see Example 1). The resulting concentrated product was stirred by overhead stirrer for another 2 hours at 30 rpm for complete homogenization.

Example 5

In a flask equipped with a 250 ml overhead stirrer, 25 ml of a 1% sodium hydroxide solution and 50 mg of sodium hyaluronate were placed, then 100 μl of 10% heparin was added. The mixture was allowed to stir for 3 hours at + 15 ° C (200 rpm). Then, 855 μl of PEGDE (ρ = 1.14 g / cm ³ ) was added to the reaction mixture. The reaction was carried out for another 18 hours with stirring (160 rpm). Then, 50 ml of a solution containing 0.5% sodium hyaluronate solution, 0.005% heparin and 0.05% sodium hydroxide was added to the mixture, after which another 425 μl of PEGDE (ρ = 1.14 g / cm ³ ) was added. The reaction mixture was allowed to stir for 36 hours at + 10 ° C (160 rpm).

The reaction was stopped by adding 4 ml of ethanol (96%) and allowed to stir for another 10 hours at + 5 ° (30 rpm). The resulting gel was transferred to a 500 ml flask, 425 ml of ddH ₂ O was added and neutralized with a solution of 1 M hydrochloric acid to pH 7.0. Then, 50 ml of physiological phosphate buffer (2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.0) was added to the gel. The resulting product was subjected to tangential filtration using a membrane with a cutoff threshold of 0.22 μm. Upon reaching a refractive index of n ₂₀ = 1.3535, gel concentration was continued to a concentration of 20 mg / ml (for a method for determining the concentration of sodium hyaluronate, see Example 1). The resulting concentrated product was stirred by overhead stirrer for another 2 hours at 30 rpm for complete homogenization.

Example 6

In a flask equipped with a 250 ml overhead stirrer, 25 ml of a 1% sodium hydroxide solution and 10 mg of sodium hyaluronate were placed, then 100 μl of 10% heparin was added. The mixture was allowed to stir for 3 hours at + 15 ° C (200 rpm). Then, 395 μl of PEGDE (ρ = 1.14 g / cm ³ ) was added to the reaction mixture. The reaction was carried out for another 10 hours with stirring (160 rpm). Then, 50 ml of a solution containing 0.5% sodium hyaluronate solution, 0.005% heparin and 0.05% sodium hydroxide was added to the mixture, after which another 282 μl of PEGDE (ρ = 1.14 g / cm) was added. The reaction mixture was allowed to stir for 18 hours at + 15 ° C (160 rpm).

The reaction was stopped by adding 4 ml of ethanol (96%) and allowed to stir for another 10 hours at + 5 ° C (30 rpm). The resulting gel was transferred to a 500 ml flask, 425 ml of ddH ₂ O was added and neutralized with a solution of 1 M hydrochloric acid to pH 7.0. Then, 50 ml of physiological phosphate buffer (2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.0) was added to the gel. The resulting product was subjected to tangential filtration using a membrane with a cutoff threshold of 0.22 μm. Upon reaching a refractive index of n ₂₀ = 1.3535, gel concentration was continued to a concentration of 20 mg / ml (for a method for determining the concentration of sodium hyaluronate, see Example 1). The resulting concentrated product was stirred by overhead stirrer for another 2 hours at 30 rpm for complete homogenization.

Example 7

In a flask equipped with a 250 ml overhead stirrer, 25 ml of a 1% sodium hydroxide solution and 10 mg of sodium hyaluronate were placed, then 100 μl of 10% heparin was added. The mixture was allowed to stir for 3 hours at + 15 ° C (200 rpm). Then, 635 μl of PEGDE (ρ = 1.14 g / cm ³ ) was added to the reaction mixture. The reaction was carried out for another 24 hours with stirring (160 rpm). The reaction was stopped by adding 4 ml of ethanol (96%) and allowed to stir for another 12 hours at + 5 ° C (30 rpm). The resulting gel was transferred to a 500 ml flask, 300 ml of ddH ₂ O was added and neutralized with a solution of 1 M hydrochloric acid to pH 7.0. Then, 50 ml of physiological phosphate buffer (2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.0) was added to the gel. The resulting product was subjected to tangential filtration using a membrane with a cutoff threshold of 0.22 μm. Upon reaching a refractive index of n ₂₀ = 1.3535, gel concentration was continued to a concentration of 20 mg / ml (for a method for determining the concentration of sodium hyaluronate, see Example 1). The resulting concentrated product was stirred by overhead stirrer for another 2 hours at 30 rpm for complete homogenization.

Example 8

In a flask equipped with a 250 ml overhead stirrer, 25 ml of a 1% sodium hydroxide solution and 40 mg of sodium hyaluronate were placed, then 50 μl of 10% heparin was added. The mixture was allowed to stir for 3 hours at + 15 ° C (200 rpm). Then, 587 μl of PEGDE (ρ = 1.14 g / cm ³ ) was added to the reaction mixture. The reaction was carried out for another 24 hours with stirring (160 rpm).

The reaction was stopped by adding 4 ml of ethanol (96%) and allowed to stir for another 12 hours at + 5 ° C (30 rpm). The resulting gel was transferred to a 500 ml flask, 300 ml of ddH ₂ O was added and neutralized with a solution of 1 M hydrochloric acid to pH 7.0. Then, 50 ml of physiological phosphate buffer (2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.0) was added to the gel. The resulting product was subjected to tangential filtration using a membrane with a cutoff threshold of 0.22 μm. Upon reaching a refractive index of n ₂₀ = 1.3535, gel concentration was continued to a concentration of 20 mg / ml (for a method for determining the concentration of sodium hyaluronate, see Example 1). The resulting concentrated product was stirred by overhead stirrer for another 2 hours at 30 rpm for complete homogenization.

Example 9

In a flask equipped with a 250 ml overhead stirrer, 25 ml of a 2% sodium hydroxide solution and 30 mg of sodium hyaluronate were placed, then 300 μl of 10% heparin was added. The mixture was allowed to stir for 3 hours at + 15 ° C (200 rpm). Then, 400 μl of PEGDE (ρ = 1.14 g / cm ³ ) was added to the reaction mixture. The reaction was carried out for another 18 hours with stirring (160 rpm). Then, 50 ml of a solution containing 0.5% sodium hyaluronate solution, 0.005% heparin and 0.05% sodium hydroxide was added to the mixture, after which another 400 μl of PEGDE was added. The reaction mixture was allowed to stir for 24 hours at + 15 ° C (160 rpm).

The reaction was stopped by adding 5 ml of methanol and allowed to stir for another 10 hours at + 5 ° C (30 rpm). The resulting gel was transferred to a 1000 ml flask, 600 ml of ddH ₂ O was added and neutralized with a solution of 1 M hydrochloric acid to pH 7.0. Then, 50 ml of physiological phosphate buffer (2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.0) was added to the gel. The resulting product was subjected to tangential filtration using a membrane with a cutoff threshold of 0.22 μm. Upon reaching a refractive index of n ₂₀ = 1.3535, gel concentration was continued to a concentration of 20 mg / ml (for a method for determining the concentration of sodium hyaluronate, see Example 1). The resulting concentrated product was stirred by overhead stirrer for another 2 hours at 30 rpm for complete homogenization.

Example 10

In a flask equipped with a 250 ml overhead stirrer, 25 ml of a 0.5% sodium hydroxide solution and 25 mg of sodium hyaluronate were placed, then 250 μl of 10% heparin was added. The mixture was allowed to stir for 12 hours at + 8 ° C (200 rpm). Then, 625 μl of PEGDE (ρ = 1.14 g / cm ³ ) was added to the reaction mixture. The reaction was carried out for another 24 hours with stirring (160 rpm). To the mixture was added 50 ml of a solution containing 0.5% sodium hyaluronate solution, 0.005% heparin and 0.05% sodium hydroxide, after which another 625 μl of PEGDE was added. The reaction mixture was allowed to stir for 24 hours at + 15 ° C (160 rpm).

The reaction was stopped by adding 4 ml of methanol and allowed to stir for another 12 hours at + 5 ° C (30 rpm). The resulting gel was transferred to a 500 ml flask, 400 ml of ddH ₂ O was added and neutralized with a solution of 1 M hydrochloric acid to pH 7.0. Then, 50 ml of physiological phosphate buffer (2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.0) was added to the gel. The resulting product was subjected to tangential filtration using a membrane with a cutoff threshold of 0.22 μm. Upon reaching a refractive index of n ₂₀ = 1.3535, gel concentration was continued to a concentration of 20 mg / ml (for a method for determining the concentration of sodium hyaluronate, see Example 1). The resulting concentrated product was stirred by overhead stirrer for another 2 hours at 30 rpm for complete homogenization.

List of sources used

1. Scott J.E. Secondary structures in hyaluronan solutions: chemical and biological implications. The biology of hyaluronan // Ciba Found. Symp - 1989 - Vol. 143 - P. 6-15.

2. Khabarov V.N., Boykov P.Ya., Selyanin M.A. Hyaluronic acid: preparation, properties, application in biology and medicine - M .: Practical medicine, 2012 - 224 p.

3. Beloded A.V. Microbiological synthesis and degradation of hyaluronic acid by bacteria p. Streptococcus: dis. Cand. biol. Sciences - M., 2008 .-- S. 174.

4. Lolvmcin A.M., Peppcis N.A. Molecular analysis of interpolymer complexation in graft copolymer networks // Polymer. - 2000 - Vol. 41. - No. 1. - P. 73-80.

5. Sykaras N., Lynne A. Opperman bone morphogenetic proteins (BMPs): how do they function and what can they offer the clinician // J. Oral Sci. - 2003. - Vol. 45. - P. 57-73.

6. Xu X., Jha A.K, Duncan R.L., Jia X.Q. Heparin-decorated, hyaluronic acid-based hydrogel particles for the controlled release of bone morphogenetic protein 2 // Acta Biomater - 2011 - Vol. 7. - P. 3050-3059.

7. Bhakta G., Rai B., Lim Z.X. et al. Hyaluronic acid-based hydrogels functionalized with heparin that support controlled release of bioactive BMP-2 // Biomaterials - 2012. - Vol. 33 - P. 6113-6122.

Claims (1)

A method of obtaining a composition based on modified sodium hyaluronate and heparin, which consists in creating transverse covalent bonds between the hydroxyl groups of the residues of D-glucuronic acid and DN-acetylglucosamine, which are part of sodium hyaluronate, and the residues of α-D-glucosamine and uronic acid related to heparin , by introducing an active crosslinking bifunctional agent - (polyethylene glycol) diglycidyl ether (PEGDE) in an alkaline medium.

Images