RU2750000C1 - Method for synthesis of modified hyaluronan and application thereof in medicine, including in endoprosthetics - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to the field of biotechnology and medicine, including endoprosthetics, namely, to a method for producing a biopolymer based on sodium hyaluronate and to an application thereof in various fields of medicine. The proposed method for producing a biopolymer based on a modified copolymer of sodium hyaluronate includes creation of cross-linking covalent bonds between the residues of D-glucuronic acid and DN-acetylglucosamine comprised in sodium hyaluronate by introducing two or more cross-linking agents of different molecular weights in a sodium hydroxide solution, wherein each cross-linking agent is independently characterised by a molecular weight within the range from 500 to 8000 Da, wherein the molar ratio of sodium hyaluronate and crosslinking agents is 1:200 to 1:600, wherein each of the agents constitutes (polypropylene glycol)diglycidyl ether. The stage of creating cross-linking covalent bonds is then stopped and the free epoxy groups are bound by means of adding glycine. Also proposed is a biopolymer produced by the described method for use as an endoprosthesis of synovial fluid of a specific structural formula.

EFFECT: invention allows to produce a biopolymer based on a modified sodium hyaluronate copolymer, ensuring most complete replacement of endoprosthetic biological fluid while maintaining the basic natural properties, including structural and transport functions, additionally characterised by low toxicity.

12 cl, 13 dwg, 3 tbl, 10 ex

Description

Technology area

The present invention relates to the field of production of medical devices, biotechnology and medicine, including endoprosthetics, and in particular to a method for producing a biopolymer based on a modified copolymer of sodium hyaluronate and its use in various fields of medicine.

State of the art

Biopolymers based on polysaccharides and their modified derivatives, in particular, based on hyaluronic acid (hereinafter referred to as HA), are used in various fields of biotechnology, medicine, general and aesthetic, in particular surgical practice and endoprosthetics, they have such important properties as biodegradability in living tissues over time, and the rate of the biodegradation process depends on the HA derivative or a complex composition that includes it. These biopolymers must meet such important criteria as biocompatibility, non-immunogenicity, bioinertness and hypoallergenicity. It is known from the prior art that as such a biopolymer, a polysaccharide is widespread — sodium hyaluronate, an anionic glycosaminoglycan.

Glycosaminoglycans (GAGs) are linear polysaccharides formed from repeats of disaccharides consisting of amino sugars - hexosamines and uronic acid residues (D-glucuronic or L-iduronic). Hyaluronic acid (HA) ([D-glucuronic acid (1-β-3), N-acetyl-D-glucosamine (1-β-4)] n) (Fig. 1), which is characterized by the absence of sulfate groups, is a biopolymer a wide range and is widely used in various fields of medicine. This macromolecule is most often referred to as hyaluronan, because of the many different forms that the HA molecule can take under physiological conditions (a form of its own acid, and a salt such as sodium hyaluronate) [1].

This biopolymer is widespread in nature, being identified in the soft tissues of vertebrates (for example, joints, synovial fluid, skin, vitreous of the eye, umbilical cord) [2], in algae [3], in molluscs [4], and also in cultured eukaryotic cell lines and some prokaryotes, where it acts as a protective capsule surrounding the cell [5]. HA, present in all vertebrates, is the main component of the extracellular matrix: in the vitreous humor of the human eye (0.1 mg / ml) and in the synovial joint fluid (3-4 mg / ml).

In vertebrates, HA performs a wide range of functions: in the skin, it maintains tissue hydration [8]; in cartilage, it nourishes chondrocytes, regulates water exchange and ion content, maintains the physical properties of tissue and regulates the interaction of cartilage cells with the intercellular substrate. Biological effects associated with the interaction of the GK-receptor cause changes in the rate of cell proliferation, migration, and angiogenesis [9, 10]. In addition, an overproduction of HA is observed in diseases associated with inflammation, fibrosis, and cancer. Recently, direct evidence indicates the involvement of HA in cancer metastasis [11, 12]. The molar mass of natural hyaluronan ranges from a few hundred Daltons to 8 million Daltons, but the average value ranges from 1 million to 2 million Daltons.

Hyaluronan, having good solubility in water, is energetically stable [13] and can exist in various conformational states (elongated chains, relaxed helices, rod-like structures, etc.). The great ability of HA to retain water is due to its hydrophilic chemical nature. This structure (Fig. 2) is supported by intramolecular hydrogen bonds, which give the macromolecule a certain rigidity and promote the formation of hydrophobic regions on its surface, which makes it possible to cross-link adjacent HA molecules, as well as its interaction with cell membranes and lipid components. Long polymer molecules of hyaluronic acid form fibrils, networks, stacks [14]. The viscoelastic nature refers to the rheological behavior of aqueous HA solutions, which exhibit the elasticity of the gel in combination with the viscosity of the liquid.

Even at low concentrations of hyaluronan, intramolecular interaction occurs, an ordered and relatively stable three-dimensional structure in the form of plates and tubes appears, although the polysaccharide molecules in solution continue to be in motion [17].

Due to the aforementioned highlighted properties, the development and commercialization of HA-based products is in constant flux. HA is mainly used in the treatment of osteoarthritis, in cosmetics, in ophthalmology, in aesthetic medicine, in surgery and wound healing, as well as in local drug delivery and tissue engineering [16, 18].

In some of the aforementioned applications, HA is used in its natural form, namely in a linear form. However, for many purposes, modifications to the main body are required. Crosslinks initiated in a linear form are divided into physical (formed as a result of electrostatic interaction or hydrogen bonds) and chemical (covalent bonds). The number of covalent crosslinks affects the properties of the obtained hydrogels, such as the degree of swelling, mechanical strength and elasticity, permeability, characteristics associated with the migration of dosage forms and small molecules through them [19].

The use of linear forms of HA in vivo leads to a rapid loss of molecular weight, a change in viscoelastic properties and structure due to instability due to the action of hyaluronidase enzymes, free radicals and temperature. To solve the above problems, it is possible to implement an approach based on the creation of cross-links within and intermolecular bonds in the HA polymer molecule. This circumstance reduces the rate of biodegradation, implies an increase in the time of its location in tissues and its planning, depending on the type of modification. These derivatives can be obtained using various reagents, for example, carbodiimides [US 7879818, US 20070224277], divinyl sulfone derivatives [US 4582865, US 4605601, US 4636524, EP 0939086], substituted oxiranes [US 4716224, US 4772419, US 4716154], 1,4-butanediol diglycidyl ether (BDDE) [WO 2013021249, US 4716224], polyhydric alcohols [US 4957744], organic aldehydes [US 4713448, US 5128326, US 4582865], biscarbodiimides [US 5356883], carboxylic acid polymers [EP 0718 , carbonate-substituted pyridine [EP 2408823], oligo- or polypeptide [EP 2094736], derivatives of aziridine [US 20050222081].

Thus, if the linear form of HA is used for subcutaneous injection, then the beneficial effect quickly disappears in a certain period of time. The modified form of HA, on the contrary, is less susceptible to chemical and enzymatic hydrolysis, shows long-term stability in vivo, thus prolonging the beneficial effect of its use [16]. Modification processes, especially crosslinking, significantly improve the specific mechanical properties of the material [16].

However, it is worth mentioning that as a result of the use of cross-linking agents of various nature, there is a need for their timely deactivation, since as a result of polymer degradation they can be released while maintaining toxic properties.

Polypropylene glycol (PPG) is a non-toxic, clear and colorless odorless liquid that is widely used in the food, pharmaceutical and cosmetic industries as a direct and indirect additive. The liquid has low vapor pressure and low viscosity. Commercial polypropylene glycol is usually obtained from water and propylene oxide obtained from organic and inorganic sources.

Polypropylene glycol has been designated GRAS (Generally Recognized as Safe) by the US Food and Drug Administration because it is easily metabolized in the human body as carbohydrates. In the food and pharmaceutical industries, PPGs have been in use for over 50 years. The inert chemical serves as a solvent and stabilizer as it does not react or alter the ingredients in the final product.

Thus, the development of new approaches to the creation of biopolymers (gels) based on modified HA is an urgent and important task.

Disclosure of invention

The objective of the present invention is to develop a new effective method for producing a biopolymer based on a modified spatially cross-linked copolymer of sodium hyaluronate, which promotes a complete replacement of the endoprosthetic biological fluid while maintaining basic natural properties, including structural and transport functions.

The technical result of the present invention is the development of a new effective method for producing a biopolymer based on a modified copolymer of sodium hyaluronate, which promotes the most complete replacement of endoprosthetic biological fluid while maintaining basic natural properties, including structural and transport functions. The specified method of obtaining a biopolymer is highly scalable, allows you to obtain a hypoallergenic biopolymer with a high degree of purity and a low level of endotoxin content, that is, characterized by low toxicity. In addition, this method makes it possible to regulate the creation of cross-linking in order to obtain a biopolymer with a spatial structure, capable of retaining and / or releasing proteins, that is, to carry out a depositing transport function. The biopolymer obtained by the claimed method can be widely used in medicine, including surgery and endoprosthetics, orthopedics, traumatology, ophthalmology, and aesthetic medicine.

The specified technical result is achieved by implementing a method for producing a biopolymer based on a modified copolymer of sodium hyaluronate, including the following stages:

- the creation of cross-linking covalent bonds between the residues of D-glucuronic acid and DN-acetylglucosamine, which are part of sodium hyaluronate, by introducing two or more cross-linking agents of different molecular weights, in different ratios in the basic medium, each of which is (polypropylene glycol) diglycidyl ether ;

- stopping the stage of creating cross-linked covalent bonds and binding free epoxy groups by adding glycine.

In particular embodiments, each crosslinking agent has a molecular weight ranging from 500 to 8000 Da.

In particular embodiments of the invention, the molar ratio of sodium hyaluronate and crosslinking agents is 1: 200 to 1: 600. In more specific embodiments, the molar ratio of sodium hyaluronate to crosslinking agents is 1: 400.

In particular embodiments of the invention, after the step of adding glycine, the reaction mixture is stirred at a temperature of 4-25 ° C and for 10-12 hours.

In particular embodiments of the invention, the resulting reaction mixture, after the stage of stopping and binding of epoxy groups, is neutralized to a pH of 7.0 ± 0.5, dialyzed in a flow system with a physiological phosphate buffer, where the pH takes on a value of 7.3 ± 0.2.

In particular embodiments of the invention, the molecular weight of sodium hyaluronate is from 1 × 10 ⁶ Da to 2 × 10 ⁶ Da.

In particular embodiments of the invention, the covalent crosslinking step is carried out in a temperature range of 0 ° C to 10 ° C.

In particular embodiments, the covalent crosslinking step is carried out for 18-22 hours.

In particular embodiments of the invention, the crosslinking agents can be administered simultaneously, sequentially or over a period of time, in particular over a period of 3-5 hours.

In particular embodiments of the invention, the molar mass of the resulting biopolymer does not exceed 5000 kDa.

The present invention also includes a biopolymer based on a modified sodium hyaluronate copolymer intended for use as a synovial fluid endoprosthesis and obtained by the method of the invention. In particular embodiments of the invention, the molar mass of the biopolymer does not exceed 5000 kDa. In particular embodiments of the invention, the biopolymer has a degree of crosslinking of 10 ± 3%.

Detailed Disclosure of the Present Invention

Brief description of the drawings.

Figure 1. Structural formula of the monomeric unit of the hyaluronan molecule.

Figure 2. Structural formula of a tetramer of a hyaluronic acid molecule in an aqueous environment in solution.

Figure 3. Chemical scheme of the modification of hyaluronan by means of PPHDE for two molecular weights.

Figure 4. General structural formula of the crosslinking agent PPHDE.

Figure 5. Interaction of the free epoxy group with glycine.

Figure 6. MTT analysis of cytotoxicity of the sample "Giruanana plus" (HA) and a sample of biopolymer (X1) obtained by the method according to the invention. The Y-axis indicates a dimensionless value: the cytotoxicity index (CI), expressed in quotient from the division of the number of cells in the experiment by the number of cells in the control.

Figure 7. Morphology of cells of peritoneal exudate (M1 and M2 correspond to the population of macrophages, Ly - to lymphocytes):

(A) in control;

(B) after i.p. administration of the Giruanana plus (HA) sample;

(C) after i.p. administration of a biopolymer sample (X1) obtained by the method according to the invention.

Figure 8. Deposition of HA (A) and X1 (B) in adipose cells 7 days after subcutaneous administration. HA and X1 are labeled with rhodamine B (red), the nuclei are colored blue. Scale 37 microns.

Figure 9. Capture by macrophages of the peritoneal cavity of the sample HA (A) or X1 (B) 7 days after the intravenous injection of fillers. Macrophages are stained with antibodies with CD11b (green), fillers are stained with rhodamine B (red), nuclei are colored blue. Scale 5-6 microns.

Figure 10. Biodistribution of sample HA (A-B) and X1 (E-C) 10 days after i.p. injection of 200 μl of sample with rhodamine B (red). A, D adipose tissue, scale 17 microns; B, E - kidneys, scale 17 μm (inset, scale 120 μm); B, F - liver, scale 30 microns, H - liver, scale 111 microns; D - liver, intact mouse. Cell nuclei are colored blue.

Figure 11. Biodegradation of a sample of HA (A) and X1 (B) by human skin cells (HaCaT line) on day 9 of spheroid cultivation. Scale 22 microns. Spheroids were obtained on an anti-adhesive substrate.

Figure 12. Biodegradation of HA (A, C) and X1 (B, D) in A / Sn mice 20 days after intraperitoneal injection (A, B) or in the area of fat pad No. 3 of the mouse. Scale 45 microns. Nuclei are stained with Hoechst (blue), cell membranes - with phalloidin (green).

Figure 13. The presence of a fluorescent signal in the region of rhodamine (FL2) in splenocytes of mice line A / Sn 20 days after intraperitoneal injection (A, B) or in the area of fat pad # 3 (C, D).

Definitions and terms

For a better understanding of the present invention, below are some of the terms used in the present description of the invention.

In the description of this invention, the terms "includes" and "including" are interpreted as meaning "includes, among other things". These terms are not intended to be construed as “consists only of”.

In the description of this invention, the term "cleavage or depolymerization" refers to structural changes in a biopolymer (hyaluronate) due to the influence of environmental, chemical or thermal factors that entail a decrease in the average molecular weight of the biopolymer, including during autoclaving, leading to a change in rheological properties ...

In the description of this invention, the term "degradation" refers to a change in physicochemical properties resulting from the action of temperatures, acids, or alkalis, as well as the action of enzymes in vitro , in particular, huluronidase. Degradation of biopolysaccharides occurs mainly through the cleavage of glycosidic bonds by acid catalytic hydrolysis.

In the description of this invention, the term "biodegradation" refers to a change in physicochemical properties that occurs over a certain time, as a result of exposure to factors of the immune system and the enzymatic activity of the interstitial and intercellular environment, where the implant was placed in vivo .

The term "molecular weight (average molecular weight)" is the statistical average of the relative molecular weights of the macromolecules that make up the biopolymer.

The term "oligomer" in this document refers to a molecule in the form of a chain of a small number of identical constituent units.

The term "monomer" in this document refers to a molecule (disaccharide), an elementary structural unit of which a biopolymer is composed as a result of its lining up in a chain with multiple repetitions.

In the description of this invention, the terms "hyaluronan" refers to a non-sulfated glycosaminoglycan, which is a linear, unbranched polymer built from a repeating disaccharide moiety of hyaluronic acid, consisting of D-glucuronide-β- (1,3) - (N-acetyl) -glucosamine and linked by a β (1-4) glycosidic bond.

(Polypropylene glycol) diglycidyl ether is an affordable and easy-to-use, safe cross-linking agent, the general formula is C ₃ H ₅ O ₂ -OCH (CH ₃ ) CH ₂ ) _n -C ₃ H ₅ O (Fig. 4), where the monomers for The spacing of HA chains are polypropylene glycol molecules, with molar masses from 500 to 8000000 Da.

Implementation of the invention

The method of obtaining a biopolymer based on a modified copolymer of sodium hyaluronate according to the invention consists in creating transverse covalent ether bonds between the residues of D-glucuronic acid and DN-acetylglucosamine, which are part of sodium hyaluronate, as a result of the introduction of two or more cross-linking bifunctional agents - (polypropylene glycol) diglycidyl ethers (PPGDE) with different molar masses and in different proportions in the basic environment (Fig. 3). The main raw material for the process is hyaluronic acid, its sodium salt can be obtained, in particular, by the method of biotechnological synthesis based on bacterial strains ( Streptococcus, Bacillus subtilis ). After the chemical copolymerization process, the resulting reaction mixture is neutralized to a pH of about 7, the remaining free epoxy groups are deactivated with glycine (Fig. 5), then the resulting biopolymer is subjected to flow dialysis, homogenized and, after filling in glass vials or pre-filled syringes, sterilized with steam. The use of the stage of deactivation of epoxy groups with glycine allows obtaining a safe product characterized by low toxicity. This removes glycine residues during the dialysis step.

The starting material for the implementation of the method according to the invention is pharmacopoeially pure sodium hyaluronate with a molecular weight in the range from 1 × 10 ⁶ Da to 2 × 10 ⁶ Da. Then hyaluronan is dissolved in a sodium hydroxide solution, which is a prerequisite for the reaction with PPHDE and a decrease in the final viscosity of the solution, which in turn improves the rheological properties of the solution and makes it possible for its uniform mixing and homogenization.

Cross-linking agents PPGDE separately, or a mixture in a certain proportion is added to the sodium hyaluronate solution, the reaction is carried out in the temperature range from 0 ° C to 10 ° C. The reaction at each stage (including the stage of HA dissolution, the stage of cross-linking (creation of cross-links), the stage of stopping the reaction, neutralization and dialysis) lasts no more than 24 hours.

The main feature of the invention is the ability to accurately control (in particular, through the method of one-dimensional ( ¹ H, ¹³ C) and two-dimensional ( ¹ H- ¹ H COZY, ¹ H- ¹³ C HSQC) NMR spectroscopy, as well as through Raman spectra) the reaction conditions polymerization, which will make it possible to obtain hyaluronan derivatives with unique properties, the spatial structure of the molecular network that affects the protein interaction, namely, with the ability to control the exchange and release of proteins. The method according to the invention allows you to accurately change the rheological properties of the obtained biopolymer to ensure the performance of natural functions of the prosthetic biological fluid, in particular, as an endoprosthesis of synovial fluid, while maintaining the properties of unhindered penetration into the spongy intercellular structure of the cartilage to deliver nutrient components to chondrocytes. In other words, the method according to the present invention produces a biopolymer with a defined pore size that allows transport of proteins. Thus, the method according to the invention makes it possible to obtain a biopolymer, which is characterized not only by the necessary rheological properties, but also by transport functions similar to those for biological fluids, in particular synovial fluid.

The rate of interaction of hyaluronic acid and epoxy groups of cross-linking agents is not a linear characteristic. The required percentage of crosslinking, in particular 10 ± 3%, according to the method according to the invention was monitored by means of NMR methods such as: ¹ H-NMR, ¹³ C-NMR, ¹ H- ¹ H COZY, ¹ H- ¹³ CHSQC, ¹ H- ¹³ CHMBC. Thus, the method according to the invention makes it possible to industrially obtain a biopolymer with reproducible physicochemical characteristics, with the possibility of controlling the structure and amount of free epoxy groups.

The main advantage of this method (double crosslinking) is the ability to obtain biopolymers similar in rheological properties to human biological fluid, while controlling the average molecular weight of the obtained polymer crosslinked chains. For example, when replacing synovial fluid with a biopolymer obtained by the method according to the invention, there is no change in the locomotion of the joint where the endoprosthesis of the synovial fluid was placed, and there are no obstacles to penetration into the intercellular space, namely into the pores of the cartilage tissue. In this connection, there is no interruption in the supply of cartilage tissue with this type of therapy.

The biopolymer based on modified sodium hyaluronate according to the invention is biocompatible, non-immunogenic, more resistant to enzymatic degradation than unmodified hyaluronan, which allows it to be used in various fields of general medicine, in particular:

1) in rheumatology, orthopedics and traumatology as a means for intra-articular administration of prolonged action in the treatment of osteoarthritis of degenerative-dystrophic and post-traumatic genesis of various localization (not only large joints, namely the knee and hip, but smaller ones, such as the shoulder and elbow joints) ... With intra-articular injections of cross-linked and linear forms of HA, hyaluronan was found to have a therapeutic effect in osteoarthritis. Several studies have been conducted to investigate the effects showing that HA is able to suppress cartilage degeneration, protect the soft tissue of the joint surfaces, normalize the rheological properties of synovial fluid, and reduce pain perception [20, 21, 18].

2) in ophthalmology as an active ingredient for moisturizing the eyes, the biopolymer according to the invention can also be used in the surgical treatment of ophthalmic diseases, eye injuries, corneal injuries as a viscoprotector, protector of endothelial cells during operations;

3) as a depositing agent for injection implantation into the affected tissue or organ with the subsequent release of biologically active substances and drugs: both small molecules and therapeutic biopolymers, in particular, of a peptide nature;

4) in aesthetic medicine as dermal fillers for non-surgical augmentation of soft tissues, including for smoothing wrinkles and increasing the volume of soft tissues, such as lips and breasts.

5) in cosmetics as a moisturizing component. The use of a biopolymer obtained by the method according to the invention can help to moisturize the skin and thus restore elasticity, thereby reducing the depth of wrinkles. In addition, this biopolymer can stimulate epidermal cells to proliferate and divide, as well as help protect human skin from ultraviolet radiation.

6) application for local drug delivery.

The implementation of the method for producing a copolymer with an intermolecular and intramolecular crosslinked structure can be demonstrated in the following examples.

Example 1.

In a flask equipped with an overhead stirrer with a volume of 50 ml was placed 25 ml of 1% sodium hydroxide solution and 1.3 g of sodium hyaluronate. The mixture was left stirring for 3 h at + 20 ° C (160 rpm). Then, 1.5 ml of PPHDE (M = 1500 Da, ρ = 1.10 g / cm ³ ) and 4.0 ml of PPHDE (M = 640 Da, ρ = 1.06 g / cm ³ ) were added to the reaction mixture. The reaction was carried out for another 20 h with stirring (160 rpm, + 4 ° C).

The reaction was stopped by adding 0.2 g of glycine, previously dissolved in 1 ml of ddH ₂ O (sterile ultrapure water) and left stirring for 10 h at + 4 ° C (30 rpm). The resulting biopolymer was neutralized with a 1 M hydrochloric acid solution to pH 7.0, then transferred to a dialysis bag (Viking, 12 kDa). The biopolymer was dialyzed by changing the physiological phosphate buffer 4 times (PBS, 1 L, 2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.2 ± 0.2). The resulting product was brought with the required amount of buffer to a concentration of 20 mg / ml. The resulting biopolymer was stirred with an overhead stirrer for another 2 h at 30 rpm for its complete homogenization at a temperature of + 4 ° C.

Example 2.

In a flask equipped with an overhead stirrer with a volume of 50 ml was placed 25 ml of 1% sodium hydroxide solution and 2 g of sodium hyaluronate. The mixture was left stirring for 3 h at + 10 ° C (160 rpm). Then, 1.0 ml of PPHDE (M = 1500 Da, ρ = 1.10 g / cm ³ ) and 4.0 ml of PPHDE (M = 640 Da, ρ = 1.06 g / cm ³ ) were added to the reaction mixture. The reaction was carried out for another 24 h with stirring (160 rpm, + 10 ° С).

The reaction was stopped by the addition of 0.4 g of glycine, previously dissolved in 1 ml of ddH ₂ O, and the mixture was left stirring for 12 h at + 4 ° C (30 rpm). The resulting biopolymer was neutralized with a 1 M hydrochloric acid solution to pH 7.0, then transferred to a dialysis bag (Viking, 12 kDa). The biopolymer was dialyzed by changing the physiological phosphate buffer 4 times (PBS, 1 L, 2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.2 ± 0.2). The resulting product was brought with the required amount of buffer to a concentration of 30 mg / ml. The resulting biopolymer was stirred with an overhead stirrer for another 2 h at 30 rpm for its complete homogenization at a temperature of + 4 ° C.

Example 3.

In a flask equipped with an overhead stirrer with a volume of 50 ml was placed 25 ml of 1% sodium hydroxide solution and 0.9 g of sodium hyaluronate. The mixture was left stirring for 3 h at + 20 ° C (160 rpm). Then, 2.0 ml of PPHDE (M = 1500 Da, ρ = 1.10 g / cm ³ ) and 4.0 ml of PPHDE (M = 640 Da, ρ = 1.06 g / cm ³ ) were added to the reaction mixture. The reaction was carried out for another 18 h with stirring (160 rpm, + 10 ° C).

The reaction was stopped by the addition of 0.2 g of glycine, previously dissolved in 1 ml of ddH ₂ O, and the mixture was left stirring for 10 h at + 4 ° C (30 rpm). The resulting biopolymer was neutralized with a 1 M hydrochloric acid solution to pH 7.0, then transferred to a dialysis bag (Viking, 12 kDa). The biopolymer was dialyzed by changing the physiological phosphate buffer 4 times (PBS, 1 L, 2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.2 ± 0.2). The resulting product was brought with the required amount of buffer to a concentration of 15 mg / ml. The obtained biopolymer was stirred by means of an overhead stirrer for another 2 h at 30 rpm for its complete homogenization at a temperature of + 4 ° C.

Example 4.

In a flask equipped with an overhead stirrer with a volume of 50 ml was placed 25 ml of 1% sodium hydroxide solution and 1.4 g of sodium hyaluronate. The mixture was left stirring for 3 h at + 20 ° C (160 rpm). Then 3.0 ml of PPHDE (M = 1500 Da, ρ = 1.10 g / cm ³ ) and 4.0 ml of PPHDE (M = 640 Da, ρ = 1.06 g / cm ³ ) were added to the reaction mixture. The reaction was carried out for another 18 h with stirring (160 rpm, + 4 ° C).

The reaction was stopped by the addition of 0.2 g of glycine, previously dissolved in 1 ml of ddH ₂ O, and the mixture was left stirring for 10 h at + 4 ° C (30 rpm). The resulting biopolymer was neutralized with a 1 M hydrochloric acid solution to pH 7.0, then transferred to a dialysis bag (Viking, 12 kDa). The biopolymer was dialyzed by changing the physiological phosphate buffer 4 times (PBS, 1 L, 2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.2 ± 0.2). The resulting product was brought with the required amount of buffer to a concentration of 20 mg / ml. The resulting biopolymer was stirred with an overhead stirrer for another 2 h at 30 rpm for its complete homogenization at a temperature of + 4 ° C.

Example 5.

In a flask equipped with an overhead stirrer with a volume of 50 ml was placed 25 ml of 1% sodium hydroxide solution and 1.4 g of sodium hyaluronate. The mixture was left stirring for 3 h at + 20 ° C (160 rpm). Then 0.2 ml of PPHDE (M = 1500 Da, ρ = 1.10 g / cm ³ ) and 4.0 ml of PPHDE (M = 640 Da, ρ = 1.06 g / cm ³ ) were added to the reaction mixture. The reaction was carried out for another 18 h with stirring (160 rpm, + 4 ° C).

The reaction was stopped by the addition of 0.2 g of glycine, previously dissolved in 1 ml of ddH ₂ O, and the mixture was left stirring for 10 h at + 4 ° C (30 rpm). The resulting biopolymer was neutralized with a 1 M hydrochloric acid solution to pH 7.0, then transferred to a dialysis bag (Viking, 12 kDa). The biopolymer was dialyzed by changing the physiological phosphate buffer 4 times (PBS, 1 L, 2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.2 ± 0.2). The resulting product was brought with the required amount of buffer to a concentration of 20 mg / ml. The resulting biopolymer was stirred with an overhead stirrer for another 2 h at 30 rpm for its complete homogenization at a temperature of + 4 ° C.

Example 6.

In a flask equipped with an overhead stirrer with a volume of 50 ml was placed 12.5 ml of 1% sodium hydroxide solution and 0.65 g of sodium hyaluronate. The mixture was left stirring for 3 h at + 20 ° C (160 rpm). Then, 1.5 ml of PPHDE (M = 1500 Da, ρ = 1.10 g / cm ³ ) was added to the reaction mixture, the reaction was carried out for 6 h with stirring. Then, additionally, a mixture of 12.5 ml of 1% sodium hydroxide solution and 0.65 g of sodium hyaluronate was placed in the reaction volume, left stirring for an hour (160 rpm, + 4 ° C). Then 4.0 ml of PPHDE was added (M = 640 Da, ρ = 1.06 g / cm ³ ). The reaction was carried out for another 20 h with stirring (160 rpm, + 4 ° C).

The reaction was stopped by the addition of 0.2 g of glycine, previously dissolved in 1 ml of ddH ₂ O, and the mixture was left stirring for 10 h at + 4 ° C (30 rpm). The resulting biopolymer was neutralized with a 1 M hydrochloric acid solution to pH 7.0, then transferred to a dialysis bag (Viking, 12 kDa). The biopolymer was dialyzed by changing the physiological phosphate buffer 4 times (PBS, 1 L, 2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.2 ± 0.2). The resulting product was brought with the required amount of buffer to a concentration of 20 mg / ml. The resulting biopolymer was stirred with an overhead stirrer for another 2 h at 30 rpm for its complete homogenization at a temperature of + 4 ° C.

Example 7.

In a flask equipped with an overhead stirrer with a volume of 50 ml was placed 12.5 ml of 1% sodium hydroxide solution and 1.00 g of sodium hyaluronate. The mixture was left stirring for 3 h at + 20 ° C (160 rpm). Then, 1.5 ml of PPHDE (M = 1500 Da, ρ = 1.10 g / cm ³ ) was added to the reaction mixture, the reaction was carried out for 6 h with stirring. Then, additionally, a mixture of 12.5 ml of 1% sodium hydroxide solution and 0.40 g of sodium hyaluronate was placed in the reaction volume, left stirring for an hour (160 rpm, + 4 ° C). Then 4.0 ml of PPHDE was added (M = 640 Da, ρ = 1.06 g / cm ³ ). The reaction was carried out for another 20 h with stirring (160 rpm, + 4 ° C).

The reaction was stopped by the addition of 0.2 g of glycine, previously dissolved in 1 ml of ddH ₂ O, and the mixture was left stirring for 10 h at + 4 ° C (30 rpm). The resulting biopolymer was neutralized with a 1 M hydrochloric acid solution to pH 7.0, then transferred to a dialysis bag (Viking, 12 kDa). The biopolymer was dialyzed by changing the physiological phosphate buffer 4 times (PBS, 1 L, 2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.2 ± 0.2). The resulting product was brought with the required amount of buffer to a concentration of 20 mg / ml. The resulting biopolymer was stirred with an overhead stirrer for another 2 h at 30 rpm for its complete homogenization at a temperature of + 4 ° C.

Example 8.

In a flask equipped with an overhead stirrer with a volume of 50 ml was placed 12.5 ml of 1% sodium hydroxide solution and 0.65 g of sodium hyaluronate. The mixture was left stirring for 3 h at + 20 ° C (160 rpm). Then 3.0 ml of PPHDE (M = 1500 Da, ρ = 1.10 g / cm ³ ) was added to the reaction mixture, the reaction was carried out for 6 h with stirring. Then, additionally, a mixture of 12.5 ml of 1% sodium hydroxide solution and 0.65 g of sodium hyaluronate was placed in the reaction volume, left stirring for an hour (160 rpm, + 4 ° C). Then 4.0 ml of PPHDE was added (M = 640 Da, ρ = 1.06 g / cm ³ ). The reaction was carried out for another 20 h with stirring (160 rpm, + 4 ° C).

The reaction was stopped by the addition of 0.2 g of glycine, previously dissolved in 1 ml of ddH ₂ O, and the mixture was left stirring for 10 h at + 10 ° C (30 rpm). The resulting biopolymer was neutralized with a 1 M hydrochloric acid solution to pH 7.0, then transferred to a dialysis bag (Viking, 12 kDa). The biopolymer was dialyzed by changing the physiological phosphate buffer 4 times (PBS, 1 L, 2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.2 ± 0.2). The resulting product was brought with the required amount of buffer to a concentration of 20 mg / ml. The resulting biopolymer was stirred with an overhead stirrer for another 2 h at 30 rpm for its complete homogenization at a temperature of + 5 ° C.

Example 9.

In a flask equipped with an overhead stirrer with a volume of 50 ml was placed 25 ml of 1% sodium hydroxide solution and 0.9 g of sodium hyaluronate. The mixture was left stirring for 4 h at + 10 ° C (160 rpm). Then, 4.0 ml of PPHDE (M = 1500 Da, ρ = 1.10 g / cm ³ ) and 2.5 ml of PPHDE (M = 640 Da, ρ = 1.06 g / cm ³ ) were added to the reaction mixture. The reaction was carried out for another 24 h with stirring (160 rpm, + 10 ° С).

The reaction was stopped by the addition of 0.2 g of glycine, previously dissolved in 1 ml of ddH ₂ O, and the mixture was left stirring for 10 h at + 4 ° C (30 rpm). The resulting biopolymer was neutralized with a 1 M hydrochloric acid solution to pH 7.0, then transferred to a dialysis bag (Viking, 12 kDa). The biopolymer was dialyzed by changing the physiological phosphate buffer 4 times (PBS, 1 L, 2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.2 ± 0.2). The resulting product was brought with the required amount of buffer to a concentration of 15 mg / ml. The resulting biopolymer was stirred with an overhead stirrer for another 2 h at 30 rpm for its complete homogenization at a temperature of + 4 ° C.

Example 10.

In a flask equipped with an overhead stirrer with a volume of 50 ml was placed 25 ml of 1% sodium hydroxide solution and 1.95 g of sodium hyaluronate. The mixture was left stirring for 3 h at + 20 ° C (160 rpm). Then, 1.0 ml of PPHDE (M = 1500 Da, ρ = 1.10 g / cm ³ ) and 4.5 ml of PPHDE (M = 640 Da, ρ = 1.06 g / cm ³ ) were added to the reaction mixture. The reaction was carried out for another 24 h with stirring (160 rpm, + 4 ° C).

The reaction was stopped by the addition of 0.2 g of glycine, previously dissolved in 1 ml of ddH ₂ O, and the mixture was left stirring for 12 h at + 4 ° C (30 rpm). The resulting biopolymer was neutralized with a 1 M hydrochloric acid solution to pH 7.0, then transferred to a dialysis bag (Viking, 12 kDa). The biopolymer was dialyzed by changing the physiological phosphate buffer 4 times (PBS, 1 L, 2 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ , 145.5 mM NaCl, pH 7.2 ± 0.2). The resulting product was brought with the required amount of buffer to a concentration of 30 mg / ml. The resulting biopolymer was mixed by means of an overhead stirrer for another 2 h at 30 rpm for its complete homogenization at a temperature of + 4 ° C.

Toxicity studies of the biopolymer of the invention

One of the main problems with the use of diglycyl ethers in the creation of cross-linking in HAs is the presence of oxirane groups remaining after the technological cycle of biopolymer preparation. The present invention proposes the introduction of an additional stage, including the treatment of the reaction mass of the crosslinked biopolymer with a glycine solution, this stage allows you to stop the polymerization reaction in a short time and additionally solve the problem of free epoxy groups that cause the toxicity of the resulting biopolymer, since as a result of isomerization, oxirane groups are isomerized to form aldehyde, which determines the toxicity of the biopolymer. As a result, glycine, as a nucleophilic agent, attaches and blocks the epoxy group. Using this technology, the resulting crosslinked biopolymer does not have a toxic effect.

Toxicity tests were carried out in comparison with the analogue of the synovial fluid endoprosthesis containing only HA solution without cross-linking, "Giruan plus". Also, the test result was a description of the effect of the drug on the mouse immune system and the provision of data with indicators of changes in marker cells, inflammatory reactions, the deposition effect of the drug and the assumption of ways of excretion.

In this experiment, the toxicity and biodistribution of the "Giruan plus" (HA) implant, a preparation containing HA without chemical modification at a concentration of 10 mg / ml, a phosphate-physiological buffer, and a synovial fluid endoprosthesis preparation ESL-HA-PPEGDE-1 (X1 ) - a preparation prepared according to the method according to the invention, which is a solution of 10 mg / ml of a cross-linked biopolymer (obtained according to example 2 of this document), phosphate-physiological buffer.

Viscous properties.

Both preparations are viscous, transparent, completely soluble in water, in the form of uniform drops pass through a 30Gx1 / 2 '' needle.

Toxicity in vitro ...

To assess toxicity, the preparations were diluted 8-fold and evaluated by the MTT method on the pancreatic carcinoma cell line T3M4. The experimental results showed that both drugs are low toxic (Fig. 6).

Toxicity in vivo ...

In vivo toxicity was assessed in A / Sn mice. Fillers were injected into mice intraperitoneally with a 30G needle in volumes of 50, 100, 150, and 200 μl per mouse. Mice were removed from the experiment after 7, 8 and 10 days. Peritoneal exudate and spleen were collected. The number and subpopulation composition of cells in the exudate and spleen were assessed. An intact mouse was used for comparison. The research results showed that the number of cells and subpopulation composition in the abdominal cavity is within normal limits (Fig. 7, Table 1).

Table 1. Subpopulation composition of peritoneal exudate on the 7th day after intravenous administration of the drug "Giruan plus" (HA) or the drug according to the invention (X1).

Control HA HA X1 X1 Norm The volume of the gel, including biopolymer, μl 0 200 fifty 200 fifty 0 Quantity, million 2 1.5 1.5 one 1.5 1-3 Macrophages M1,% nineteen 31 29 47 eighteen 15-50 Macrophages M2,% eight eight 10 10 eleven 5-15 Lymphocytes,% 61 47 47 44 53 50-75

Analysis of the number and subpopulation composition of the spleen showed insignificant changes, also within the normal range (Table 2). There is a decrease in the CD4 / CD8 ratio, but this is also within the normal range. No differences were found between samples HA and X1.

Table 2. Subpopulation composition of the spleen on day 7 after intraperitoneal administration of the drug "Giruan plus" (HA) or the drug according to the invention (X1).

Control HA HA X1 X1 R The volume of the gel, including biopolymer, μl 200 fifty 200 fifty Quantity, million 72 68 62 58 80 > 0.05 CD4 fourteen 12 13 12 13 > 0.05 CD8 nine 10 12 10 sixteen > 0.05 CD4 / CD8 1.6 1.2 1.1 1.2 0.8 > 0.05 CD19 55 56 56 fifty 47 > 0.05 CD62L ^high / CD44 ^low 71 72 72 63 68 > 0.05 CD62L ^low / CD44 ^high 12 12 eleven fourteen 13 > 0.05

Biodistribution of synovial fluid endoprosthesis preparations (ESF).

In the course of this experiment, the biodistribution of preparations of the endoprosthesis of synovial fluid was studied with subcutaneous and intraperitoneal administration of a gel with the inclusion of a fluorescent label of rhodamine B. 200 μl. The mice were removed from the experiment after 8 and 10 days. No free gel was found at the injection site (sc or ip). The liver, spleen, kidneys, muscles, and adipose tissue were collected. The results of the experiments showed that the main depot are fat cells (Fig. 8). When administered intravenously, part of the gel is carried away by macrophages to the regional lymph nodes (Fig. 9) and the spleen, from where the gel enters the liver and kidneys (Fig. 10). Residues are excreted through urine (kidney) and feces (liver) (Table 3). No differences in biodistribution were found between HA and X1 samples.

Biodegradation of the synovial fluid endoprosthesis (ESF).

Biodegradation of the synovial fluid endoprosthesis was studied in 3D in vitro culture on cells of the HaCaT human keratinocyte line. The result of these studies showed that the samples are captured by cells and remain in them for at least 10 days without significant hydrolysis (Fig. 11).

Biodegradation of the synovial fluid endoprosthesis (ESF) in vivo :

Some of the mice were removed from the experiment after 20 days, and adipose tissue was taken from the injection site (abdominal cavity and in the area of subcutaneous injection). Analysis of adipose tissue was performed using histology. The results of the studies showed that after 20 days in the abdominal cavity and in the area of subcutaneous injection, the samples are partially hydrolyzed, but reliably detected (Fig. 12).

Table 3. Analysis of the biodistribution of the preparation "Giruan plus" (HA) and the preparation according to the invention (X1) on the 7-10th day after subcutaneous injection under paw No. 3 (sc) or intraperitoneally (ip).

Pick-up time 7 days 8 days 10 days Introduction sc sc sc sc ip ip ip ip Filler HA HA X1 X1 HA X1 HA X1 Volume of gel, including biopolymer, ml fifty* 200 fifty 200 fifty 200 fifty 200 Liver fifty 155 58 60 70 67 70 24 Kidney 23 116 13 28 five 51 82 29 Urine nd 504 nd 392 59 80 57 fifty Gastrointestinal tract 0 0 0 0 0 0 0 0 Lungs 0 0 0 0 0 0 0 0 Bile 0 0 0 0 0 0 0 0 Spleen 0 0 0 0 0 0 0 0 Blood 0 0 0 0 0 0 0 0

* Organs were homogenized in saline, an equal volume of 70% ethanol was added, rhodamine B was extracted for 12 hours, centrifuged at 15 thousand rpm for 20 min, the clarified supernatants were transferred to plates and the fluorescence was measured on a reader at a wavelength of 525 nm. Data on mean fluorescence intensity (MFI) minus control for intact mice are shown. Data are not normalized to organ weights.

Hydrolysis in tissues is associated with the capture of debris by macrophages, its hydrolysis and its partial transport to the spleen (Fig. 13) and to the liver. There were no differences in the hydrolysis and transport of the samples.

Thus, the studies carried out have shown that the preparations "Giruan plus" (HA) and the preparation according to the invention ESL-HA-PPEGDE-1 (X1) do not differ in toxicity, biodistribution and biodegradation, ways of excretion of intermediates and biodegradation products. As a consequence, it can be concluded that the method according to the present invention makes it possible to obtain the same low-toxicity biopolymer based on the modified copolymer of sodium hyaluronate as the hyaluronate itself.

Although the invention has been described with reference to the disclosed embodiments, it should be apparent to those skilled in the art that the specific experiments described in detail are provided for the purpose of illustrating the present invention only and should not be construed as in any way limiting the scope of the invention. It should be understood that various modifications are possible without departing from the spirit of the present invention.

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Claims (15)

1. A method for producing a biopolymer based on a modified sodium hyaluronate copolymer, comprising the following stages: - the creation of cross-linking covalent bonds between the residues of D-glucuronic acid and DN-acetylglucosamine, which are part of sodium hyaluronate, by introducing two or more crosslinking agents of different molecular weights in a sodium hydroxide solution, where each crosslinking agent is independently characterized by a molecular weight in the range from 500 to 8000 Da, and the molar ratio of sodium hyaluronate and crosslinking agents is 1: 200-1: 600, each of which is (polypropylene glycol) diglycidyl ether; - stopping the stage of creating cross-linked covalent bonds and binding free epoxy groups by adding glycine. 2. The method of claim 1, wherein the molar ratio of sodium hyaluronate to crosslinking agents is 1: 400. 3. The method according to claim 1, wherein after adding glycine, the reaction mixture is stirred at a temperature of 4-250 ° C and for 10-12 hours. 4. The method according to claim 1, in which the resulting reaction mixture after the stage of stopping and binding of epoxy groups is neutralized to a pH value of 7.0 ± 0.5, dialyzed in a flow system with a physiological phosphate buffer, where the pH takes on a value of 7.3 ± 0.2. 5. The method of claim 1, wherein the molecular weight of the sodium hyaluronate is 1 x 10 ⁶ to 2 x 10 ⁶ Da. 6. The method according to claim 1, wherein the step of creating cross-linking covalent bonds is carried out in a temperature range from 0 ° C to 10 ° C. 7. The method of claim 1, wherein the covalent crosslinking step is carried out for 18-22 hours. 8. The method of claim 1, wherein the crosslinking agents can be administered simultaneously, sequentially, or over a 3-5 hour interval. 9. The method of claim 1, wherein the molar mass of the biopolymer does not exceed 5000 kDa. 10. Biopolymer based on modified copolymer of sodium hyaluronate, intended for use as an endoprosthesis of synovial fluid and obtained by the method according to claim 1, characterized by the following structural formula:

11. The biopolymer according to claim 10, the molar mass of the biopolymer in which does not exceed 5000 kDa. 12. The biopolymer of claim 10, wherein the degree of crosslinking is 10 ± 3%.

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