CZ33324U1 - Hydrogel based on a cross-linked hydroxyphenyl derivative of hyaluronic acid - Google Patents

Description

The invention relates to a hydrogel based on a cross-linked hydroxyphenyl derivative of hyaluronic acid in admixture with chondroitin sulphate with an improved rate of degradation.

BACKGROUND OF THE INVENTION

Hyaluronic acid (also hyaluronan, HA) is a polysaccharide of the glycosaminoglycan family consisting of disaccharide units composed of D-glucuronic acid and Wacetyl-Dglucosamine. It is a polysaccharide that is readily soluble in an aqueous environment where, depending on molecular weight and concentration, it forms viscous solutions to viscoelastic hydrogels. HA is a natural component of the intercellular mass of tissues. By binding to specific cell surface receptors, the hyaluronan molecule is able to interact with cells in its environment and regulate their metabolic processes (Xu, Jha et al. 2012). For these reasons, materials containing hyaluronan or derivatives thereof are often used for the manufacture of formulations used in biomedical applications. Hydrogels based on hyaluronan undergo natural degradation by the action of specific enzymes (hyaluronidases), resp. by reactive oxygen species (ROS), resulting in their gradual absorption after their implantation into the body (Stem, Kogan et al. 2007).

Many types of hydrogels containing covalently cross-linked hyaluronan have been developed to achieve more mechanically resistant materials and to slow down their biodegradation. Such hydrogels are used as materials for the viscosupplementation of synovial fluid, soft tissue augmentation, serve as support structures for cell culture and implantation, and the like (Tognana, Borrione et al. 2007, Buck li, Alam et al. 2009, Li, Raitcheva et al. 2012, Salwowska, Bebenek et al. 2016).

Various types of hyaluronan derivatives have also been developed in the past that are capable of undergoing a sol-gel transition under physiological conditions in situ (Burdick and Prestwich 2011, Prestwich 2011). For example, phenolic derivatives of hyaluronan can be used for this purpose. Calabro et al. (Calabro, Akst et al. 2008, Lee, Chung et al. 2008, Kurisawa, Lee et al. 2009) describe in EP1587945B1 and EP1773943B1 a process for preparing phenolic hyaluronan derivatives by reacting carboxyls present in the D-glucuronic acid structure of hyaluronan with aminoalkyl- phenol derivatives such as tyramine. Hyaluronan amides are the product of this reaction (Darr and Calabro 2009).

Crosslinking of phenolic hyaluronan derivatives may be initiated by the addition of peroxidase (eg horseradish peroxidase) and a dilute hydrogen peroxide solution. Horseradish peroxidase (HRP, EC 1.11.1.7) is currently used as a catalyst for organic and biotransformation reactions (Akkara, Senecal et al. 1991, Higashimura and Kobayashi 2002, Ghan, Shutava et al. 2004, Shutava, Zheng et. al., 2004, Veitch 2004). Hydrogels based on hydroxyphenyl derivatives of hyaluronan can be used as injectable matrices for the controlled release of biologically active substances or as materials suitable for cell culture and implantation (Kurisawa, Lee et al. 2010). Wolfova et al. describe in CZ303879 a hyaluronan-tyramine conjugate comprising an aliphatic linker interposed between the polymer chain and tyramine. The presence of an aliphatic linker allows for greater cross-linking efficiency and imparts greater elasticity to the web.

Chondroitin sulfate (ChS) is another representative of glycosaminoglycans, which is often used to prepare materials for use in the treatment of degenerative diseases, such as osteoarthritis (OA). The ChS chain consists of disaccharide units composed of Wacetylgalktosamine (GalNAc)

And iduronic acid (IdoA). The disaccharide CHS units may be sulfated at the 4 and 6 positions of GalNAc and optionally at the 2-position of IdoA. Chondroitin sulphate is a linear, sulphated and negatively charged glycosaminoglycan composed of repeating monomer units of A-acetyl-Dgalactosamine and D-glucuronic acid interconnected by β (1 -> 3) and β (1 -> 4) Oglycosidic bonds (structural formula of chondroitin sulphate see below).

where

R ¹ is H or Na,

R ² is H, O-SO ₂ -OH or O-SO ₂ -ONa

The source of chondroitin sulfate is animal connective tissue, where it binds to proteins and thus forms part of proteoglycans. Sulfation of chondroitin is effected by sulfotransferases at different positions and different proportions. The unique pattern of sulfation of individual positions in the polymer chain encodes the specific biological activity of chondroitin sulfate. It is an important building block of cartilage in the joints, which provides compressive resistance and restores balance in the composition of the joint lubricant (Baeurle SA, Kiselev MG, Makarova ES, Nogovitsin EA 2009. Polymer 50: 1805). Chondroitin sulfate and glucosamine together used as a dietary supplement for the treatment or prevention of osteoarthritis, also in humans (e.g. Flextor® Advance Nutraceutics, Ltd.) or animals (e.g. ^dog Geloren ®, Contipro Pharma Ltd.). From a pharmaceutical point of view, chondroitin sulfate is considered to be a delayed onset of pain-relieving effect in degenerative joint disease (Aubry-Rozier B. 2012. Revue Médicale Suisse 14: 571).

In vitro and in vivo studies have shown that ChS inhibits the effect of hyaluronidases. The inhibitory effect of ChS on enzymes is due to the formation of electrostatic (ionic) interactions. It has also been shown that ChS is able to capture ROS and thereby protect against degradation of the extracellular matrix component (Bali, Cousse et al. 2001, Xiong and Jin 2007).

The use of a combination of hyaluronan and chondroitin sulfate for the preparation of a composition for protecting human or animal cells and tissues from traumatization is described in EP0136782 (1983). Similarly, US6051560 (1992) discloses the use of a mixture of hyaluronan and chondroitin sulfate as viscosupplementing materials during ophthalmic procedures. WO030417024 discloses a viscous composition comprising a therapeutically effective amount of a ChS and HA mixture for the manufacture of a medicament for treating a joint of a human with cartilage damage caused by chondromalacia or OA grade I and II, which uses intraarticular administration of the composition. We also find in the patent literature documents describing a composition for parenteral administration suitable for preventing and treating articular cartilage damage in humans or animals, comprising a therapeutically effective amount of chondroitin sulfate, hyaluronan and glucosamine (WO2004034980, 2002). EP2219595 describes a formulation based on polysaccharides, especially glycosaminoglycans, and mixtures thereof with flavonoids, which form hydrogels with extended biodegradation times. Said document also discloses hyaluronan-containing hydrogel, a hyaluronan derivative cross-linked with butanediol 1,4-diglycidyl ether and ChS, which exhibits increased resistance to degradation by the enzyme hyaluronidase.

The essence of the technical solution

The present invention relates to a hydrogel based on a cross-linked hydroxyphenyl derivative of hyaluronic acid, the nature of which is that it contains molecules of a hydroxyphenyl derivative of hyaluronic acid

The acid (HA-TA) or a pharmaceutically acceptable salt thereof of formula (I)

wherein n is in the range of 2 to 7500 and wherein R ¹ is H ⁺ or an alkali or alkaline-earth salt ion and R ² is OH or a tyramine substituent of formula II:

(Π), wherein within one molecule of the hydroxyphenyl derivative of hyaluronic acid or a pharmaceutically acceptable salt thereof of formula I, at least one R ² is a tyramine substituent of formula II, and wherein at least two tyramine substituents of formula II are linked via a covalent bond in any ortho position of phenyl groups, and further comprising chondroitin sulfate or a pharmaceutically acceptable salt thereof selected from the group consisting of alkali or alkaline earth salts.

The alkali or alkaline earth salts of the hydroxyphenyl derivative of hyaluronic acid of formula I or chondroitin sulfate are preferably selected from the group consisting of Na ⁺ , IC ⁺ , Ca ²⁺ , Mg ²⁺ .

The concentration of chondroitin sulfate or a pharmaceutically acceptable salt thereof is in the range of 0.5 to 50 mg / ml of the hydrogel according to the invention, preferably at a concentration of 1 to 20 mg / l, more preferably 5 mg / ml.

The content of the cross-linked hydroxyphenyl derivative of hyaluronan is in the range of 5 to 30 mg / ml, preferably 10 mg / ml of the hydrogel according to the invention.

According to another preferred embodiment of the invention the hydrogel further comprises hyaluronic acid or a pharmaceutically acceptable salt thereof in a concentration of 1 to 20 mg / ml, preferably 5 to 10 mg / ml, more preferably 5 mg / ml of the hydrogel according to the invention.

The covalent bond may be within one molecule of the hyaluronic acid derivative of formula I at any ortho position of the phenyl groups of at least two tyramine substituents of formula II present in that molecule. This is called intramolecular crosslinking. Also, the covalent bond may be in any ortho position of the phenyl groups of at least two tyramine substituents of formula II, which are found in different molecules of the hyaluronic acid derivative of formula I. This represents an interconnected network between the HA derivative molecules.

An example of a covalently cross-linked hydroxyphenyl derivative of hyaluronan. (crossHA-TA) is shown schematically below, see Formula III:

(ΙΠ)

Such hydrogels according to the invention exhibit increased resistance to biodegradation processes resulting from the action of hydrolytic enzymes and reactive oxygen species.

According to a preferred embodiment, the mean molecular (Mw) of the hydroxyphenyl derivative of hyaluronan according to the general formula I is in the range of 5 x 10 ⁴ to 1.5 x 10 ⁶ g.mol · ¹ , preferably 2.5 x 10 ⁵ to 1 x 10 ⁶ g. mol ¹ , more preferably 8 x ^{10 5} g.mol · ¹ . It drinks in the range of 1 to 3.

According to another embodiment of the invention, the degree of substitution (DS) of the hydroxyphenyl derivative of the hyaluronan of formula I is in the range of 0.5 to 10%, preferably 1 to 4%, more preferably 1%.

According to another preferred embodiment, the Mw of the chondroitin sulfate is in the range of 5 x 10 ³ to 95 x 10 ³ g.mol · ¹ , further preferably 10 x 10 ³ to 40 x 10 ³ g.mol · ¹ .

According to a preferred embodiment of the hydro gel comprising hyaluronan (HA) or a pharmaceutically acceptable salt of Mw in the range of 5 x 10 ⁴ to 2.5 x 10 ⁶ g.mol · ^1, preferably 1.5 XLO ⁶ to 2.5 x 10 ⁶ g.mol · ¹ , more preferably 2.0 x 10 ⁶ g.mol · ¹ .

Such hydrogels according to the invention can be used in cosmetics, medicine and regenerative medicine, in particular for preparing tissue regeneration materials, tissue augmentation, tissue engineering scaffolds, as a matrix for the controlled release of biologically active substances and drugs and viscosupplementation of synovial fluid.

Brief description of the pictures

Giant. 1: Comparison of degradation rate of HA solutions with addition of ChS by ROS

Giant. 2: ROS comparison of material degradation rate

Giant. 3: Cumulative degradation of hydrogel [%] BTH 30 U / mg

Examples of technical solution

DS = degree of substitution = 100% * molar amount of modified disaccharide units of hyaluronan / molar amount of all disaccharide units of hyaluronan derivative. The degree of substitution was determined by 1 H NMR spectroscopy.

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The weight-average molar mass (Mw) and the polydispersity index (PI) were determined by the SEC-MALLS method.

Infrared spectra of prepared derivatives were obtained by FT-IR method.

Pharmaceutical grade chondroitin sulfate was used for injection from Bioiberica, ES.

Example 1

Synthesis of tyramine derivative HA (HA-TA)

Synthesis of 6-amino-N- [2- (4-hydroxyphenyl) ethyl] hexanamide

6 - [(tert-butoxycarbonyl) amino] hexanoic acid (1.00 g, 4.3 mmol) was dissolved in 50 mL tetrahydrofuran (THF). To the acid solution was added 1,1'-carbodiimidazole (0.70 g, 4.3 mmol). The mixture was heated to 50 ° C for sixty minutes. The reaction vessel was then purged with an inert gas. To the reaction mixture was added tyramine (0.59 g, 4.3 mmol). The mixture was further heated for an additional 2 hours. THF was then removed by distillation under reduced pressure. The residue was dissolved in 50 mL of ethyl acetate. The solution was washed with 150 ml of purified water (divided into three parts). The organic layer was dried over a molecular sieve. Ethyl acetate was removed by distillation under reduced pressure. The residue was dissolved in 50 mL of MeOH and 2 mL of trifluoroacetic acid (TFA) was added. The solution was heated at reflux for 6 hours. The solvent was removed by distillation under reduced pressure. The residue was dissolved in 50 mL of ethyl acetate. The solution was washed with 150 ml of purified water (divided into three parts). The organic layer was dried over a molecular sieve. Ethyl acetate was removed by distillation under reduced pressure, m = 0.75 g (70% of theory)

1 H NMR (D 2 O, ppm) δ: 1.17 (m, 2H, γ-C 1 H-hexanoic acid); 1.48 (m, 2H, 3-CH 2 -hexanoic acid); 1.58 (m, 2H, [beta] -CIF-hexanoic acid); 2.17 (t, 2H, -CH 2 -CO-); 2.73 (m, 2H, -CH 2 Ph); 2.91 (m, 2H, -CH 2 -NH 2); 3.42 (m, 2H, -CH 2 -NH-CO-); 6.83 (d, 2H, arom); 7.13 (d, 2H, arom).

¹³ C NMR (D 2 O, ppm) δ: 24 (γ-C-hexanoic acid); 26 (δ-C-hexanoic acid); 33 (β-Chexanoic acid); 35 (-C-CO-); 39 (-C-NH ₂ ); 40 (C-Ph); 63 (--C - NH - CO--); 115 (C3 arom); 126 (Cl arom); 130 (C2 arom.); 153 (C4 arom); 176 (-CO--).

Preparation of aldehyde derivative (HA-CHO)

Hyaluronan (10.00 g, M _w. = 2 × 10 ^{6 1} gmol) was dissolved in 750 ml of 2.5% (w / w) solution of Na2HPO4. 12 H2O. The solution was cooled to 5 ° C. 2.60 g of NaBr and 0.05 g of 4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxyl were added to the resulting solution. After thoroughly homogenizing the solution, 3 mL of NaClO solution (10-15% available Cl 2) was added to the reaction mixture. The reaction was continued with stirring for 15 min. The reaction was quenched by the addition of 100 mL of a 40% propan-2-ol solution. The product was purified by ultrafiltration and isolated with propan-2-ol precipitate.

IR (KBr): 3417, 2886, 2152, 1659, 1620, 1550, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm ^-1 .

1 H NMR (D 2 O) δ: 2.01 (s, 3 H, CH 3 -), 3.37-3.93 (m, skeleton of hyaluronan), 4.46 (s, 1H, anomer), 4.54 (s 1 H anomer, -O-CH (OH) -, 5.27 (geminal glycol -CH- (OH) 2).

a) Preparation of the tyramine derivative HA with Ce spacer (Mw 3 x 10 ⁵ g.mol ¹ , DS ~ 2%)

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Aldehyde derivative HA 3 x 10 ⁵ g.mol ¹ , DS = 9%) (5.00 g) was dissolved in 500 ml demineralized water. The pH of the solution was adjusted to 3 with acetic acid. To the HACHO solution was added 6-amino-N- [2- (4-hydroxyphenyl) ethyl] hexanamide (intermediate (I)) (1.25 g, 5 mmol). The mixture was stirred at room temperature for 2 hours. Picolin-borane complex (0.270 g, 2.5 mmol) was then added to the reaction mixture. The mixture was stirred for an additional 12 hours at room temperature. The product was purified by ultrafiltration and isolated from the retentate by precipitation with propan-2-ol. The precipitate was freed of moisture and residual propan-2-ol by drying in a hot air oven (40 ° C, 3 days).

IR (KBr): 3425, 2983, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945.893 cm ^-1 .

II NMR (D 2 O) δ: 1.25 (t, 2,, γ -CH 2 -aminohexanoic acid), 1.48 (m, 2 Η, δ -CH 2 aminohexanoic acid) 1.51 (m, 2 Η, β -CH 2) aminohexanoic acid), 2.01 (s, 3H, CH 3 -),

2.65 (m, 2H, PI1-CH2-), 2.73 (m, 2H, g-CIL-aminohexanoic acid), 3.37-3.93 (m, skeleton of hyaluronan), 4.46 (s, 1H, anomer), 4.54 (s, 1H anomer, -O-CH (OH) -), 6.59 (d, 2H, arom.), 7.01 (d, 2H, arom.).

SEC MALLS: Mw - 2.78 x 10 ⁵ g.mol ¹

DS CHNMR): 2.1%

b) Preparation of tyraminated HA derivative with Ce spacer (Mw & lt ^; 8x10 ⁵ g.mol ¹ , DS ~ 1%) Aldehyde HA derivative (Mw < 8 x 10 ⁵ g.mol ¹ , DS ~ 5%) (5.00 g was dissolved in 500 ml of demineralized water. The pH of the solution was adjusted to 3 with acetic acid. To the HACHO solution was added 6-amino-N- [2- (4-hydroxyphenyl) ethyl] hexanamide (intermediate (I)) (0.625 g, 2.5 mmol). The mixture was stirred at room temperature for 2 hours. Picolin-borane complex (0.270 g, 2.5 mmol) was then added to the reaction mixture. The mixture was stirred for an additional 12 hours at room temperature. The product was purified by ultrafiltration and isolated from the retentate by precipitation with propan-2-ol. The precipitate was freed of moisture and residual propan-2-ol by drying in a hot air oven (40 ° C, 3 days).

IR (KBr): 3425. 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm ^-1 .

II NMR (D 2 O) δ: 1.25 (t, 2 Η, γ -CH 2 -aminohexanoic acid), 1.48 (m, 2 Η, δ -CH 2 aminohexanoic acid) 1.51 (m, 2 Η, β -CH 2 aminohexanoic acid), 2.01 (s, 3H, CH 3 -),

2.65 (m, 2H, PI1-CH2-), 2.73 (m, 2H, g-CIL-aminohexanoic acid), 3.37-3.93 (m, skeleton of hyaluronan), 4.46 (s, 1H, anomer), 4.54 (s, 1H anomer, -O-CH (OH) -), 6.59 (d, 2H, arom.), 7.01 (d, 2H, arom.).

SEC MALLS: Mw = 8.09 x 10 ⁵ g.mol ¹

PS (ΊΙ NMR): 1.1%

c) Preparation of a tyraminated HA derivative with a Ce spacer (Mw 1,5 1.5 x 10 ⁶ g.mol ¹ , DS «0.5%) Aldehyde HA derivative (Mw 1,5 1.5 x 10 ⁶ g.mol ¹ , DS 0 0) (5%) was dissolved in 500 ml of demineralized water. The pH of the solution was adjusted to 3 with acetic acid. To the HACHO solution was added 6-amino-N- [2- (4-hydroxyphenyl) ethyl] hexanamide (intermediate (I)) (0.625 g, 2.5 mmol). The mixture was stirred at room temperature for 2 hours. Picolin-borane complex (0.270 g, 2.5 mmol) was then added to the reaction mixture. The mixture was stirred for an additional 12 hours at room temperature. The product was purified by ultrafiltration and isolated from the retentate by precipitation with propan-2-ol. The precipitate was freed of moisture and residual propan-2-ol by drying in a hot air oven (40 ° C, 3 days).

IR (KBr): 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm ^-1 .

-6GB 33324 U1

1 H NMR (D 2 O) δ: 1.25 (t, 2 Η, γ -CH 2 -aminohexanoic acid), 1.48 (m, 2 Η, δ -CH 2 -aminohexanoic acid) 1.51 (m, 2 Η, β -CH 2) aminohexanoic acid), 2.01 (s, 3H, CH 3 -),

2.65 (m, 2H, PI1-CH2-), 2.73 (m, 2H, s-C1-aminohexanoic acid), 3.37-3.93 (m, skeleton of hyaluronan), 4.46 (s, 1H, anomer), 4.54 (s, 1H anomer, -O-CH (OH) -), 6.59 (d, 2H, arom.), 7.01 (d, 2H, arom.).

SEC MALLS: Mw = 1.5 x 10 ⁶ g.mol ¹

DS CHNMR1: 0.5%

d) Preparation of a tyraminated HA derivative with a Ce spacer (Mw ~ 5 x 10 ⁴ g.mol ¹ , DS «10%) Aldehyde HA derivative (Mw ~ 5 xl () ⁴ g.mol ¹ , DS 10 10%) (5, 00 g) was dissolved in 500 ml of demineralized water. The pH of the solution was adjusted to 3 with acetic acid. To the HACHO solution was added 6-amino- N - [2- (4-hydroxyphenyl) ethyl] hexanamide (intermediate (I)) (0.625 g, 2.5 mmol). The mixture was stirred at room temperature for 2 hours. Picolin-borane complex (0.270 g, 2.5 mmol) was then added to the reaction mixture. The mixture was stirred for an additional 12 hours at room temperature. The product was purified by ultrafiltration and isolated from the retentate by precipitation with propan-2-ol. The precipitate was freed of moisture and residual propan-2-ol by drying in a hot air oven (40 ° C, 3 days).

IR (KBr): 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm ^-1 .

II NMR (D 2 O) δ: 1.25 (t, 2 Η, γ -CH 2 -aminohexanoic acid), 1.48 (m, 2 Η, δ -CH 2 aminohexanoic acid) 1.51 (m, 2 Η, β -CH 2 aminohexanoic acid), 2.01 (s, 3H, CH 3 -),

2.65 (m, 2H, Ph-CH 2 -), 2.73 (m, 2H, s-CH 2 -aminohexanoic acid), 3.37-3.93 (m, skeleton of hyaluronan), 4.46 (s, 1H, anomer), 4.54 (s, 1H anomer, -O-CH (OH) -), 6.59 (d, 2H, arom.), 7.01 (d, 2H, arom.).

SEC MALLS: Mw = 5 x 10 ⁴ g.mol ¹

DS (11 ¹ H NMR): 10%

Example 2

Preparation of hydrogels based on hydroxyphenyl derivative HA-TA at a concentration of 20 mg / ml

The hydrogels were prepared by mixing two precursor solutions A and B using an aqueous solution of NaCl (9 g / L). The hydroxyphenyl HA-TA derivative of Mw = 2.78 x 10 ⁵ g.mol ¹ and DS 2.1% was used to prepare the precursor solutions. The composition of the solutions is shown in the following table (Table 1).

Solution A Solution B HRP ......................... ............... 0.48 U / ml H2O2 ...................... .............. 2.0 mmol / l HA-TA .............. 20 mg / ml HA-TA .................. .............. 20 mg / ml NaCl ........................ ........... 9 g / l NaCl ...................... ........... 9 g / l

Table 1: Composition of precursor solutions for the preparation of hydrogels based on the hydroxyphenyl derivative HA-TA at a concentration of 20 mg / ml

By mixing solution A and solution B in a 1: 1 ratio, the hydrogels were prepared with the final composition shown in the following table (Table 2), wherein the crossHA-TA is a covalently crosslinked hydroxyphenyl derivative of hyaluronan.

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Composition of hydrogels HRP ................................. ......... 0.24 U / ml crossHA-TA .................... 20 mg / ml NaCl ................................ ........... 9 g / l

Table 2: Final composition of hydrogels based on the hydroxyphenyl derivative HA-TA at a concentration of 20 mg / ml

Example 3

Preparation of hydrogels containing 0.5 mg / ml ChS

The hydrogels were prepared by mixing two precursor solutions A and B using an aqueous solution of NaCl (9 g / L). The hydroxyphenyl derivative HATA of Mw = 2.78 x 10 ⁵ g.mol · ¹ and DS 2.1% and CHS of Mw = 10 x 10 ³ - 40 x 10 ³ g.mol · ¹ were used to prepare the precursor solutions. The composition of the solutions is shown in the following table (Table 3).

Solution A Solution B HRP ........................... ................ 0.48 U / ml H2O2 .......................... ................ 2,0 mmol / l HA-TA ....................... 20 mg / ml HA-TA ..................... 20 mg / ml ChS ........................... 0.5 mg / ml ChS .......................... ............... 0,5 mg / ml NaCl ........................... ................ 9 g / l NaCl .......................... ................. 9 g / l

Table 3: Composition of the precursor solutions for the preparation of hydrogels containing 0.5 mg / ml ChS

By mixing solution A and solution B in a 1: 1 ratio, the hydrogels were prepared with the final composition shown in the following table (Table 4), wherein the crossHA-TA is a covalently cross-linked hydroxyphenyl derivative of hyaluronan.

Composition of hydrogels HRP ................................. 0.24 U / ml crossHA-TA .................... 20 mg / ml ChS ................................. 0.5 mg / ml NaCl ................................ ............. 9 g / l

Table 4: Final hydrogel composition containing 0.5 mg / ml CHS

Example 4

Preparation of hydrogels containing 3.3 mg / ml CHS

The hydrogels were prepared by mixing two precursor solutions A and B using an aqueous solution of NaCl (9 g / L). The hydroxyphenyl derivative HATA of Mw = 2.78 x 10 ⁵ g.mol ¹ and DS 2.1% and CHS of Mw = 10 x 10 ³ - 40 x 10 ³ g.mol · ¹ were used to prepare the precursor solutions. The composition of the solutions is shown in the following table (Table 5).

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Solution A Solution B HRP ........................... ................. 0.48 U / ml H2O2 ............................ ............. 2,0 mmol / l HA-TA ....................... 20 mg / ml HA-TA ........................ ........... 20 mg / ml ChS ........................... ............... 3.3 mg / ml ChS ............................. ........... 3.3 mg / ml NaCl ........................... ................ 9 g / l NaCl ............................ .............. 9 g / l

Table 5: Composition of precursor solutions for the preparation of hydrogels containing 3.3 mg / ml ChS

By mixing solution A and solution B in a 1: 1 ratio, hydrogels were prepared with the final composition shown in the following table (Table 6), wherein the crossHA-TA is a covalently crosslinked hydroxyphenyl derivative of hyaluronan.

Composition of hydrogels HRP ................................ 0.24 U / ml crossHA-TA .................... 20 mg / ml ChS ................................. ............ 3.3 mg / ml NaCl ................................ ............. 9 g / l

Table 6: Final hydrogel composition containing 3.3 mg / ml CHS

Example 5

Preparation of hydrogels containing ChS at a concentration of 10 mg / ml

The hydrogels were prepared by mixing two precursor solutions A and B using an aqueous solution of NaCl (9 g / L). Hydroxyphenyl derivative HATA of Mw = 2.78 x 10 ⁵ g.mol ¹ and DS 2.1% and CHS of Mw = 10 x 10 ³ - 40 x 10 ³ g.mol ¹ were used to prepare the precursor solutions. The composition of the solutions is shown in the following table (Table 7).

Solution A Solution B HRP ........................... ................. 0.48 U / ml H2O2 .......................... ............... 2,0 mmol / l HA-TA ....................... 20 mg / ml HA-TA ...................... 20 mg / ml ChS ........................... ................. 10 mg / ml ChS .......................... 10 mg / ml NaCl ........................... ................. 9 g / l NaCl .......................... ................. 9 g / l

Table 7: Composition of precursor solutions for the preparation of hydrogels containing CHS at a concentration of 10 mg / ml

By mixing solution A and solution B in a 1: 1 ratio, the hydrogels were prepared with the final composition shown in the following table (Table 8), wherein the crossHA-TA is a covalently cross-linked hydroxyphenyl derivative of hyaluronan.

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Composition of hydrogels HRP ................................ ......... 0.24 U / ml crossHA-TA .................... 20 mg / ml ChS ................................. ........... 10 mg / ml NaCl ................................ ........... 9 g / l

Table 8: Final hydrogel composition containing CHS at 10 mg / ml

Example 6

Preparation of hydrogels containing 50 mg / ml ChS

The hydrogels were prepared by mixing two precursor solutions A and B using an aqueous solution of NaCl (9 g / L). Hydroxyphenyl derivative HATA of Mw = 2.78 x 10 ⁵ g.mol ¹ and DS 2.1% and CHS of Mw = 10 x 10 ³ - 40 x 10 ³ g.mol ¹ were used to prepare the precursor solutions. The composition of the solutions is shown in the following table (Table 9).

Solution A Solution B HRP ....................... .................... 0.48 U / ml H2O2 ......................... ................ 2,0 mmol / l HA-TA 20 mg / ml HA-TA ..................... 20 mg / ml ChS ........................ 50 mg / ml ChS .......................... 50 mg / ml NaCl ....................... ..................... 9 g / l NaCl ......................... ................. 9 g / l

Table 9: Composition of precursor solutions for the preparation of hydrogels containing 50 mg / ml of CH

Mixing solution A and solution B in a 1: 1 ratio prepared the hydrogels with the final composition shown in the following table (Table 10), where the crossHA-TA is a covalently cross-linked hydroxyphenyl derivative of hyaluronan.

Composition of hydrogels HRP ................................ 0.24 U / ml crossHA-TA .................... 20 mg / ml ChS ................................. 50 mg / ml NaCl ................................ ............. 9 g / l

Table 10: Final hydrogel composition containing 50 mg / ml CHS

Example 7

Preparation of hydrogels containing HA at a concentration of 5 mg / ml

The preparation of hydrogels containing uncrosslinked hyaluronic acid included 3 bases

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1) Preparation of hydrogels containing cross-linked crossHA-TA derivative

The hydrogel containing the cross-linked crossHA-TA derivative was prepared by mixing two precursor solutions A and B, which were prepared by dissolving the individual components in phosphate buffered saline (PBS). The hydroxyphenyl HA-TA derivative of Mw = 8.09 x 10 ⁵ g.mol ¹ and DS 1.1% was used to prepare the precursor solutions. The composition of the solutions is shown in the following table (Table 11).

Solution A Solution B HRP ............................... 12.8 mU / ml H ₂ O ₂ ........................... HA-TA .......................... 20 mg / ml HA-TA ....................... 20 mg / ml NaCl .............................. .......... 8 g / l NaCl ............................ ....... 8 g / l KC1 ............................... ......... 0.2 g / l KC1 ............................. ........ 0.2 g / l For ₂ HPO ₄ .12H ₂ O ........... .......... 2.85 g / l For ₂ HPO ₄ .12H ₂ O ......... ....... 2.85 g / l KH ₂ PO ₄ ......................... .......... 0.2 g / l KH ₂ PO ₄ ........................ ....... 0.2 g / l

Table 11: Composition of precursor solutions for the preparation of a cross-linked derivative of crossHA-TA

By mixing 1: 1 of solution A and solution B, the hydrogel of the following table was prepared (Table 12).

Composition of cross-linked derivative crossHA-TA HRP ............................. ........... 0.24 U / ml CrossHA-TA ............... 20 mg / ml NaCl ............................. ........... 8 g / l KC1 .............................. ............ 0.2 g / l For ₂ HPO ₄ .12H ₂ O .......... ........... 2.85 g / l KH ₂ PO ₄ ........................ ........... 0.2 g / l

Table 12: Composition of cross-linked crossHA-TA derivative

2) Preparation of the hyaluronan solution

A 5 mg / ml HA solution was prepared by dissolving native hyaluronan of Mw 1.91x10 ⁶ g.mol ¹ in PBS.

3) Homogenization of hydrogels and hyaluronan solution

The final hydrogel was prepared by mixing the cross-linked crossHA-TA derivative and the HA solution in a ratio of 1: 1 followed by homogenization of the mixture. The final material composition is shown in the following table (Table 13).

- 11 GB 33324 U1

Final composition HRP .............................. 0.12 U / ml crossHA-TA .................. 10 mg / ml HA ................................ 5 mg / ml NaCl ............................... .......... 8 g / l KC1 ............................... ........... 0.2 g / l For ₂ HPO ₄ .12H ₂ O ........... .......... 2.85 g / l KH ₂ PO ₄ .......................... .......... 0.2 g / l

Table 13: Final hydrogel composition containing 5 mg / ml HA

Example 8

Preparation of hydrogels containing HA at 20 mg / ml

The preparation of hydrogels containing uncrosslinked hyaluronic acid involved 3 basic steps:

1) Preparation of hydrogels containing cross-linked derivative of crossHA-TA

The hydrogel containing the cross-linked crossHA-TA derivative was prepared by mixing two precursor solutions A and B, which were prepared by dissolving the individual components in phosphate buffered saline (PBS). The hydroxyphenyl HA-TA derivative of Mw = 8.09 x 10 ⁵ g.mol ¹ and DS 1.1% was used to prepare the precursor solutions. The composition of the solutions is shown in the following table (Table 14).

Solution A Solution B HRP ............................... 12.8 mU / ml H ₂ O ₂ ............................ ....... 0.6 mmol / l HA-TA .......................... 20 mg / ml HA-TA ........................ 20 mg / ml NaCl .............................. .......... 8 g / l NaCl ............................ ....... 8 g / l KC1 ............................... ........... 0.2 g / l KC1 ............................. ........ 0.2 g / l For ₂ HPO ₄ .12H ₂ O ........... .......... 2.85 g / l For ₂ HPO ₄ .12H ₂ O ......... ....... 2.85 g / l KH ₂ PO ₄ ......................... .......... 0.2 g / l KH ₂ PO ₄ ....... 0.2 g / l

Table 14: Composition of precursor solutions for the preparation of cross-linked derivative of crossHA-TA

By mixing solution A and solution B in a 1: 1 ratio, a hydrogel of the composition shown in the following table was prepared (Table 15).

- 12GB 33324 U1

Composition of cross-linked derivative crossHA-TA HRP ............................. ........... 0.24 U / ml crossHA-TA ................ 20 mg / ml NaCl ............................ ........... 8 g / l KC1 .............................. ............ 0.2 g / l For ₂ HPO ₄ .12H ₂ O ......... .......... 2.85 g / l KH ₂ PO ₄ ........................ ........... 0.2 g / l

Table 15: Composition of cross-linked crossHA-TA derivative

2) Preparation of the hyaluronan solution

A 40 mg / ml HA solution was prepared by dissolving native hyaluronan of Mw 1.91 x 10 ⁶ g.mol ¹ in phosphate buffer (PBS).

3) Homogenization of hydrogel and hyaluronan solution

The final hydrogel was prepared by mixing the cross-linked crossHA-TA derivative and the HA solution in a ratio of 1: 1 followed by homogenization of the mixture. The final material composition is shown in the following table (Table 16).

Final composition

HRP ........................................ 0.12U / ml crossHA-TA. ......................... 10 mg / ml

HA .......................................... 20mg / ml

NaCl8 g / l

KC1 ......................................... 0,2 g / l

For ₂ HPO ₄ .12H ₂ O ..................... 2.85 g / l

KH ₂ PO ₄ ................................... 0.2 g / l

Table 16: Final hydrogel composition containing HA at 20 mg / ml

Example 9

Preparation of hydrogels containing 5 mg / ml HA and 5 mg / ml ChS

The preparation of hydrogels containing uncrosslinked hyaluronic acid and chondroitin sulfate involved 3 basic steps:

1) Preparation of hydrogels containing cross-linked derivative of crossHA-TA

- 13 GB 33324 U1

The hydrogel containing the cross-linked crossHA-TA derivative was prepared by mixing two precursor solutions A and B, which were prepared by dissolving the individual components in phosphate buffered saline (PBS). The hydroxyphenyl HA-TA derivative of Mw = 8.09 x 10 ⁵ g.mol ¹ and DS 1.1% was used to prepare the precursor solutions. The composition of the solutions is shown in the following table (Table 17).

Solution A Solution B HRP ............................... H ₂ 0 ₂ ............................. ....... 0.6 mmol / l HA-TA ........................... 20 mg / ml HA-TA ....................... 20 mg / ml NaCl ............................... .......... Bg / 1 NaCl ............................ ....... Bg / 1 KC1 ............................... ........... 0.2 g / l KC1 ............................. ........ 0.2 g / l For ₂ HPO ₄ .12H ₂ O ............ .......... 2.85 g / l For ₂ HPO ₄ .12H ₂ O ......... ....... 2.85 g / l KH ₂ PO ₄ ......................... .......... 0.2 g / l KH ₂ PO ₄ ........................ ....... 0.2 g / l

Table 17: Composition of precursor solutions for the preparation of cross-linked derivative of crossHA-TA

By mixing 1: 1 of solution A and solution B, the hydrogel of the following table was prepared (Table 18).

Composition of cross-linked derivative crossHA-TA HRP ............................. ........... 0.24 U / ml crossHA-TA ................ 20 mg / ml NaCl ............................. ........... Bg / 1 KC1 .............................. ............ 0.2 g / l For ₂ HPO ₄ .12H ₂ O ......... ........... 2.85 g / l KH ₂ PO ₄ ........................ ........... 0.2 g / l

Table 18: Composition of cross-linked crossHA-TA derivative

2) Preparation of a solution containing hyaluronan and chondroitin sulfate

A 10 mg / ml HA solution and a 10 mg / ml ChS solution was prepared by dissolving native hyaluronan Mw 1.91 x 10 ⁶ g.mol ¹ and chondroitin sulfate ChS Mw = 10x10 ³ - 40 x 10 ³ g.mol ¹ in phosphate buffer (PBS).

3) Homogenization of hydrogels and solution containing hyaluronan and chondroitin sulfate

The final hydrogel was prepared by mixing the cross-linked crossHA-TA derivative and a 1: 1 solution of HA and CHS followed by homogenization of the mixture. The final composition of the material is shown in the following table (Table 19).

Final composition

HRP ........................................ 0.12 U / ml

- 14GB 33324 U1 crossHA-TA .......................... 10 mg / ml

HA .......................................... 5 mg / ml

ChS ....................................... 5 mg / ml

NaCl ......................................... Bg / 1

KC1 ......................................... 0,2 g / l

For ₂ HPO ₄ .12H ₂ O ..................... 2.85 g / l

KH ₂ PO ₄ ................................... 0.2 g / l

Table 19: Final composition of hydrogel, HA at 5 mg / ml and ChS at 5 mg / ml

Example 10

Preparation of hydrogels containing HA at 5 mg / ml and ChS at 10 mg / ml

The preparation of hydrogels containing uncrosslinked hyaluronic acid and chondroitin sulfate involved 3 basic steps:

1) Preparation of a hydrogel containing a cross-linked crossHA-TA derivative

The hydrogel containing the cross-linked crossHA-TA derivative was prepared by mixing two precursor solutions A and B, which were prepared by dissolving the individual components in phosphate buffered saline (PBS). The hydroxyphenyl HA-TA derivative of Mw = 8.09 x 10 ⁵ g.mol ¹ and DS 1.1% was used to prepare the precursor solutions. The composition of the solutions is shown in the following table (Table 20).

Solution A Solution B HRP ............................... 12.8 mU / ml H ₂ O ₂ ............................ ....... 0.6 mmol / l HA-TA .......................... 20 mg / ml HA-TA ........................ 20 mg / ml NaCl .............................. .......... 8 g / l NaCl ............................ ....... 8 g / l KC1 ............................... ......... 0.2 g / l KC1 ............................. ........ 0.2 g / l For ₂ HPO ₄ .12H ₂ O ........... .......... 2.85 g / l For ₂ HPO ₄ .12H ₂ O ......... ....... 2.85 g / l KH ₂ PO ₄ ......................... .......... 0.2 g / l KH ₂ PO ₄ ........................ ....... 0.2 g / l

Table 20: Composition of precursor solutions for the preparation of a cross-linked derivative of crossHA-TA

By mixing solution A and solution B in a 1: 1 ratio, a hydrogel having the composition shown in the following table (Table 21) was prepared.

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Composition of cross-linked derivative crossHA-TA HRP ............................. ........... 0.24 U / ml crossHA-TA ................ 20 mg / ml NaCl ............................. ........... 8 g / l KC1 .............................. ............ 0.2 g / l For ₂ HPO ₄ .12H ₂ O .......... ........... 2.85 g / l KH ₂ PO ₄ ........................ ........... 0.2 g / l

Table 21: Composition of the cross-linked crossHA-TA derivative

2) Preparation of a solution containing hyaluronan and chondroitin sulfate

A 10 mg / ml HA solution and a 20 mg / ml ChS solution were prepared by dissolving native hyaluronan of Mw 1.91 x 10 ⁶ g.mol ¹ and chondroitin sulfate of Mw = 10 x 10 ³ - 40 x 10 ³ g. mor ¹ in phosphate buffer (PBS).

3) Homogenization of hydrogels and solution containing hyaluronan and chondroitin sulfate

The final hydrogel was prepared by mixing the cross-linked crossHA-TA derivative and a 1: 1 solution of HA and CHS followed by homogenization of the mixture. The final material composition is shown in the following table (Table 22).

Final composition HRP .............................. 0.12 U / ml crossHA-TA .................. 10 mg / ml HA ................................ 5 mg / ml ChS ............................... 10 mg / ml NaCl .............................. .......... 8g / l KC1 ............................... ........... 0.2 g / l For ₂ HPO ₄ .12H ₂ O ........... .......... 2.85 g / l KH ₂ PO ₄ ......................... .......... 0.2 g / l

Table 22: Final composition of hydrogels, HA at 5 mg / ml and ChS at 10 mg / ml

Example 11

Preparation of hydrogels based on hydroxyphenyl derivative HA-TA at a concentration of 5 mg / ml

The hydrogels were prepared by mixing two precursor solutions A and B using an aqueous solution of NaCl (9 g / L). A hydroxyphenyl derivative of HATA of Mw = 1.5 x 10 ⁶ g.mof ¹ and DS 0.5% was used to prepare the precursor solutions. The composition of the solutions is shown in the following table (Table 23).

- 16GB 33324 U1

Solution A Solution B HRP ........................... ................. 0.12 U / ml H2O2 ......................... ................ 0,5 mmol / l HA-TA ....................... 5 mg / ml HA-TA ..................... 5 mg / ml NaCl ........................... ................. 9 g / l NaCl .......................... ................. 9 g / l

Table 23: Composition of precursor solutions for the preparation of hydrogels based on the hydroxyphenyl derivative HA-TA at a concentration of 5 mg / ml

Mixing solution A and solution B in a 1: 1 ratio prepared the hydrogels of the final composition shown in the following table (Table 24), wherein the crossHA-TA is a covalently cross-linked hydroxyphenyl derivative of hyaluronan.

Composition of hydrogels HRP .................................. ........ 0.06 U / ml crossHA-TA ...................... 5 mg / ml NaCl .................................. .......... 9 g / l

Table 24: Final composition of 5 mg / ml hydroxyphenyl derivative of HA-TA based hydrogels

Example 12

Preparation of hydrogels based on hydroxyphenyl derivative HA-TA at a concentration of 30 mg / ml

The hydrogels were prepared by mixing two precursor solutions A and B using an aqueous solution of NaCl (9 g / L). The hydroxyphenyl derivative HATA of Mw = 5 x 10 ⁴ g.mol · ¹ and DS 10% was used for the preparation of precursor solutions. The composition of the solutions is shown in the following table (Table 25).

Solution A Solution B HRP ........................... ............ 1.2 U / ml H2O2 ........................ ............... 5 mmol / l HA-TA ....................... ............... 30 mg / ml HA-TA ..................... ................ 30 mg / ml NaCl ........................... ................ 9 g / l NaCl .......................... ................. 9 g / l

Table 25: Composition of precursor solutions for the preparation of hydrogels based on the hydroxyphenyl derivative HA-TA at a concentration of 30 mg / ml

By mixing solution A and solution B in a 1: 1 ratio, the hydrogels were prepared with the final composition shown in the following table (Table 26), wherein the crossHA-TA is a covalently cross-linked hydroxyphenyl derivative of hyaluronan.

Composition of hydrogels

HRP ........................................ 0.6 U / ml crossHA-TA. .......................... 30 mg / ml

- 17 GB 33324 U1

NaCl ........................................ 9 g / l

Table 26: Final composition of HA-TA hydroxyphenyl derivative hydrogels at a concentration of 30 mg / ml

Example 13

Degradation of HA solutions with addition of ChS by ROS

To compare the rate of degradation of HA solutions with ChS by ROS, solutions of hyaluronan in phosphate buffered saline (PBS) with different ChS concentrations were prepared. Hyaluronic acid of Mw 1.91 x 10 ⁶ g.mol ¹ and CHS of Mw = 10 x 10 ³ - 40 x 10 ³ g.mol ¹ were used to prepare the solutions. Solution A contained 20 mg / ml HA, solution B 20 mg / ml HA and 0.5 mg / ml CHS, solution C 20 mg / ml HA and 1 mg / ml ChS, solution D 20 mg / ml HA and 3 mg / ml ml CHS, solution E 20 mg / ml HA and 5 mg / ml ChS, solution E 20 mg / ml HA and 20 mg / ml ChS.

Performing a degradation experiment

The degradation rate was expressed as a percentage decrease in the viscosity of the solutions at a shear rate of 0.1 s ¹ from the initial value. Viscosity drop measurements were performed on a Kinexus Malvern rheometer in a cone-plate configuration. A 40 mm diameter cone with a 1 ° apex angle was used. Degradation of the material was carried out in 10 ml syringes, where 0.5 ml of 0.25 mmol / l CuSO ₄ solution was added to 9 ml of material followed by 0.5 ml of 2.5 mmol / l H2O2 solution. During the ongoing degradation of hydrogels, samples of material were taken at predetermined time intervals for which a viscosity was measured at 25 ° C and a shear rate of 0.1 s ¹ . The total degradation time was 3 h.

Giant. 1 shows the degradation of the hyaluronan chains, which is expressed as a percentage decrease in solution viscosity over time. FIG. 1 shows the effect of ChS concentration on hyaluronan degradation rate. As the concentration of ChS increases, the rate of degradation of HA by ROS decreases. The viscosity of solution A, which did not contain ChS, dropped by 97% after 3 hours from the initial value before degradation, solution B (with the addition of 0.5 mg / ml ChS) already by 89%, solution C to which 1 mg / ml was added ChS by 84%, solution D (20 mg / ml HA + 3 mg / ml ChS) by 46%, solution E (20 mg / ml HA + 5 mg / ml ChS) by 25% and solution F (20 mg / ml HA + 20 mg / ml ChS) only 08%.

Example 14

Degradation of hydrogels based on cross-linked crossHA-TA by ROS

Three types of materials were prepared to compare the rate of degradation of hydrogels by ROS.

Material A is a 20 mg / ml HA solution prepared by dissolving HA of Mw 1.91 x 10 ⁶ g.mol ¹ in PBS.

Materials B is a mixture of cross-linked derivative of crossHA-TA and non-cross-linked HA prepared as described in Example 7.

Materials C and D consisted of uncrosslinked HA, CHS and crossHA-TA and were prepared according to Examples 9 and 10.

The degradation rate was expressed as a percentage decrease in the viscosity of the materials at a shear rate of 0.1 s ¹ from the initial value. Viscosity drop measurements were taken on a rheometer

- 18 GB 33324 U1

Kinexus Malvern in cone-plate configuration. A 40 mm diameter cone with a 1 ° apex angle was used. Degradation of the material was carried out in 10 ml syringes, where 0.5 ml of 0.25 mmol / l CuSO ₄ solution was added to 9 ml of material followed by 0.5 ml of 2.5 mmol / l H2O2 solution. After certain degradation times, samples were taken for a viscosity at 25 ° C and a shear rate of 0.1 s ¹ . The total degradation time was 3 hours. 2, the presence of CHS in the prepared hydrogels (C - 5 mg / ml ChS; D - 10 mg / ml ChS) appears to increase their resistance to ROS.

Example 15

Enzymatic degradation of hydrogels based on cross-linked derivative of crossHA-TA

To determine the effect of the presence of ChS on the degradation rate of hydrogels based on crossHA-TA by bovine testicular hyaluronidase, three types of hydrogels were prepared:

A - hydrogels without addition of chondroitin sulfate, which were prepared according to the procedure in Example 2 B - hydrogels with addition of 3.3 mg / ml CHS, which were prepared according to the procedure in Example 4

C-hydrogels with addition of CHS at a concentration of 10 mg / ml, which were prepared according to the procedure of Example 5.

The rate of degradation was expressed as the percentage increase in the concentration of degradation products due to BTH.

Hydrogels were immersed in degradation medium (30 U / mg BTH hyaluronidase solution in 0.1 mg / ml bovine serum albumin (BSA) solution in 0.01 mol / l acetate buffer (OP) pH 5.3). The hydrogel degradation was carried out in an incubator at 37 ° C with stirring. Degradation medium samples with hydrogel degradation products were taken after certain time intervals. The concentration of the disaccharide HA units in the degradation medium was determined spectrophotometrically as the concentration of the Iglu Cosamine. Giant. 3 shows the increase in the concentration of hydrogel degradation products in the medium over time. FIG. it is clear that the presence of ChS in crossHA-TA-based hydrogels leads to a reduction in the rate of degradation of materials by hyaluronidase.

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-20GB 33324 U1

3280-3294.

PROTECTION REQUIREMENTS

Claims (5)

Hydrogel based on a cross-linked hydroxyphenyl derivative of hyaluronic acid, characterized in that it contains the hydroxyphenyl derivative of hyaluronic acid (HA-TA) or a pharmaceutically acceptable salt thereof according to the general formula I wherein n is in the range of 2 to 7500 and wherein R ¹ is H ⁺ or an alkali or alkaline-earth salt ion and R ² is OH or a tyramine substituent of formula II: (Π), wherein within one molecule of the hydroxyphenyl derivative of hyaluronic acid or a pharmaceutically acceptable salt thereof of formula I, there is at least one R ² tyramine substituent of formula II, and wherein at least two tyramine substituents of formula II are linked via a covalent bond in any ortho position of phenyl groups, and further comprising chondroitin sulfate or a pharmaceutically acceptable salt thereof selected from the group consisting of alkali or alkaline earth salts. Hydrogel according to claim 1, characterized in that the alkali or alkaline earth salts are selected from the group consisting of Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ . Hydrogel according to claim 1 or claim 2, characterized in that the concentration of chondroitin sulfate or a pharmaceutically acceptable salt thereof is in the range of 0.5 to 50 mg / ml of hydrogel, preferably in a concentration of 1 to 20 mg / l, more preferably 5 mg / ml. Hydrogel according to any one of claims 1 to 3, characterized in that the content of the cross-linked hydroxyphenyl derivative of hyaluronan is in the range of 5 to 30 mg / ml, preferably 10 mg / ml of hydrogel. Hydrogel according to any one of claims 1 to 4, characterized in that it further comprises hyaluronic acid or a pharmaceutically acceptable salt thereof in a concentration of 1 to 20 mg / ml hydrogel, preferably 5 to 10 mg / ml, more preferably 5 mg / ml hydrogel . 3 drawings