SK9649Y1 - Hydrogel based on crosslinked hydroxyphenyl derivative of hyaluronic acid - Google Patents

Abstract

Hydrogel based on a crosslinked hydroxyphenyl derivative of hyaluronic acid containing molecules of a hydroxyphenyl derivative of hyaluronic acid, HA-TA, or its pharmaceutically acceptable salt of a general formula (I) where n is in the range 2-7500 and where R1 is H+ or ion of alkali salt or salt of alkali earth metal and R2 is OH or tyramine substituent of general formula (II): whereas within one molecule of the hydroxyphenyl derivative of hyaluronic acid or its pharmaceutically acceptable salt of the general formula (I) is at least one R2 tyramine substituent of the general formula (II) and whereas at least two tyramine substituents of the general formula (II) are connected through covalent bond in any ortho position of phenyl groups, and it further contains chondroitin sulfate or its pharmaceutically acceptable salt selected from the group comprising alkali salt or salt or alkali earth metal.

Description

The field of technology

The technical solution concerns a hydrogel based on a cross-linked hydroxyphenyl derivative of hyaluronic acid mixed with chondroitin sulfate with an improved degradation rate.

Current state of the art

Hyaluronic acid (also hyaluronan, HA) is a polysaccharide from the group of glycosaminoglycans, which consists of disaccharide units composed of D-glucuronic acid and N-acetyl-D-glucosamine. It is a polysaccharide that is easily soluble in water, where, depending on the 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 surroundings and regulate their metabolic processes (Xu, Jha et al. 2012). For these reasons, materials containing hyaluronan or its derivatives are often used for the production of preparations used in biomedical applications. Hyaluronan-based hydrogels undergo natural degradation in the body by the action of specific enzymes (hyaluronidase), or by the action of reactive oxygen species (ROS), which results in their gradual absorption after implantation in the body (Stern, Kogan et al. 2007).

A range of types of hydrogels containing covalently cross-linked hyaluronan was developed to achieve mechanically more resistant materials and to slow down their biodegradation. Such hydrogels are used as materials for viscosupplementation of synovial fluid, augmentation of soft tissues, serve as supporting structures for cell cultivation and implantation, etc. (Tognana, Borrione et al. 2007, Buck Ii, Alam et al. 2009, Li, Raitcheva et al. 2012, Salwowska, Bebenek et al. 2016).

In the past, various types of hyaluronan derivatives were also developed, which are able to undergo the sol-gel transition under physiological conditions in situ (Burdick and Prestwich 2011, Prestwich 2011). For these purposes it is possible to use e.g. phenolic derivatives of hyaluronan. Calabro et al. (Calabro, Akst et al. 2008, Lee, Chung et al. 2008, Kurisawa, Lee et al. 2009) describe in EP1587945B1 and EP1773943B1 a procedure for the preparation of phenolic derivatives of hyaluronan by the reaction of carboxyls present in the D-glucuronic acid structure of hyaluronan with aminoalkyl derivatives phenol, e.g. tyramine. The product of this reaction is hyaluronan amides (Darr and Calabro 2009).

Cross-linking of phenolic derivatives of hyaluronan can be initiated by the addition of a peroxidase (e.g. horseradish peroxidase) and a dilute solution of hydrogen peroxide. Horseradish peroxidase (HRP, E.C.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. coll. 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 cultivation and implantation (Kurisawa, Lee et al. 2010). Wolfová et al. describe in CZ303879 a conjugate of hyaluronan and tyramine containing an aliphatic linker inserted between the polymer chain and tyramine. The presence of an aliphatic linker enables a higher efficiency of the cross-linking reaction and gives the network higher elasticity.

Chondroitin sulfate (ChS) is another representative of glycosaminoglycans, which is often used for the preparation of materials intended for use in the treatment of degenerative diseases, e.g. osteoarthritis (OA). The ChS chain is formed by disaccharide units composed of N-acetylgalactosamine (GalNAc) and iduronic acid (IdoA). ChS disaccharide units can be sulfated in positions 4 and 6 of GalNAc and possibly also in position 2 of IdoA. Chondroitin sulfate is a linear, sulfated and negatively charged glycosaminoglycan composed of repeating monomeric units of N-acetyl-D-galactosamine and D-glucuronic acid interconnected by β(1^3) and β(1^4) O-glycosidic bonds (see structural chondroitin sulfate formula).

where

R ¹ is H or Na,

R ² is H, O-SO 2 -OH or O-SO 2 -Ona.

SK 9649 Υ1

The source of chondroitin sulfate is animal connective tissues, where it binds to proteins and thus forms part of proteoglycans. Sulfation of chondroitin is carried out by means of sulfotransferases in different positions and in different representation. The unique pattern of sulfation of individual positions in the polymer chain encodes the specific biological activity of chondroitin sulfate. The latter is an important building block of the cartilage in the joints, which provides pressure resistance and restores the balance in the composition of the joint lubricant (Baeurle SA, Kiselev MG, Makarova ES, Nogovitsin EA 2009. Polymer 50: 1805). Chondroitin sulfate is used together with glucosamine as a nutritional supplement for the treatment or prevention of osteoarthritis in humans (e.g. Flextor®, Advance Nutraceutics, Ltd.) or animals (e.g. Geloren ^dog ®, Contipro Pharma, Ltd.). From a pharmaceutical point of view, chondroitin sulfate is considered a drug with a delayed onset of pain relief 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 action of hyaluronidases. The inhibitory effect of ChS on enzymes is caused by the formation of electrostatic (ionic) interactions. It has also been demonstrated that ChS is able to trap ROS and thereby protect against degradation of extracellular matrix components (Balí, Cousse et al. 2001, Xiong and Jin 2007).

EP0136782 (1983) describes the use of a combination of hyaluronan and chondroitin sulfate for the preparation of an agent for the protection of human or animal cells and tissues against trauma. Similarly, document US6051560 (1992) describes the use of a mixture of hyaluronan and chondroitin sulfate as viscosupplementation materials during ophthalmological procedures. Patent WO030417024 describes a viscous composition containing a therapeutically effective amount of a mixture of ChS and HA for the manufacture of medicaments for the treatment of joints in people with cartilage damage caused by chondromalacia or grade I and II OA, which uses intra-articular administration of the mixture. In the patent literature, there are also documents that describe a preparation for parenteral administration suitable for the prevention and treatment of articular cartilage damage in humans or animals, which consists of a therapeutically effective amount of chondroitin sulfate, hyaluronan and glucosamine (WO2004034980, 2002). Document EP2219595 describes a formulation based on polysaccharides, especially glycosaminoglycans, and their mixture with flavonoids, which forms hydrogels with extended biodegradation time. The mentioned document also describes a hydrogel containing hyaluronan, a derivative of hyaluronan cross-linked with butanediol 1,4-diglycidyl ether and ChS, which shows increased resistance to degradation by the action of the hyaluronidase enzyme.

The essence of the technical solution

The technical solution relates to a hydrogel based on a cross-linked hydroxyphenyl derivative of hyaluronic acid, the essence of which is that it contains molecules of a hydroxyphenyl derivative of hyaluronic acid (HA-TA) or its pharmaceutically acceptable salt according to the general formula (I)

____________________________________________0), where n is in the range of 2 to 7,500 and where R ¹ is H ⁺ or an ion of an alkaline salt or an alkaline earth salt and R ² is OH or a tyramin substituent according to the general formula (II):

whereas within one molecule of the hydroxyphenyl derivative of hyaluronic acid or its pharmaceutically acceptable salt according to the general formula (I) at least one R ² is a tyramine substituent according to the general formula (II) and while at least two tyramine substituents according to the general formula (II) are connected via a covalent bond in any orthoposition of the phenyl groups, and further contains

SK 9649 Υ1 chondroitin sulfate or a pharmaceutically acceptable salt thereof selected from the group consisting of alkaline salts or alkaline earth salts.

Alkaline salts or alkaline earth salts of the hydroxyphenyl derivative of hyaluronic acid according to general formula (I) or chondroitin sulfate are preferably selected from the group containing Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ .

The concentration of chondroitin sulfate or its pharmaceutically acceptable salt is in the range of 0.5 to 50 mg/ml hydrogel according to the technical solution, preferably in a concentration of 1 to 20 mg/ml, 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 technical solution.

According to another advantageous embodiment of the technical solution, the hydrogel further contains hyaluronic acid or its pharmaceutically acceptable salt 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 technical solution.

The covalent bond can be within one molecule of the hyaluronic acid derivative according to the general formula (I) in any of the orthopolophenyl groups of at least two tyramine substituents of the general formula (II) that are found in this molecule. This is the so-called intramolecular cross-linking. Also, the covalent bond can be in any orthoposition of the phenyl groups of at least two tyramine substituents of the general formula (II), which are found in different molecules of the hyaluronic acid derivative according to the general formula (I). This represents an interconnected network between molecules of the HA derivative.

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

According to the technical solution, such hydrogels show increased resistance against biodegradation processes arising from the action of hydrolytic enzymes and reactive oxygen species.

According to a preferred embodiment, the weight average molar mass (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 , preferably 8 x 10 ⁵ g.mol ¹ . PI is in the range of 1 to 3.

According to another embodiment of the technical solution, the degree of substitution (DS) of the hydroxyphenyl hyaluronan derivative of general 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 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, the hydrogel contains hyaluronan (HA) or its pharmaceutically acceptable salt with a Mw in the range of 5 x 10 ⁴ to 2.5 x 10 ⁶ g.mol ¹ , preferably 1.5 x 10 ⁶ to 2.5 x 10 ⁶ g. mol ¹ , preferably 2.0 x 10 ⁶ g.mol ¹ .

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

Overview of images on drawings

fig. 1: Comparison of the rate of degradation of ChS-supplemented HA solutions by ROS

fig. 2: Comparison of the rate of degradation of materials by ROS

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

Implementation examples

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

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

The infrared spectra of the prepared derivatives were obtained by the FT-IR method.

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

Example 1

Synthesis of the tyramine derivative of HA (HA-TA)

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

6-[(tert-Butoxycarbonyl)amino]hexanoic acid (1.00 g, 4.3 mmol) was dissolved in 50 mL of tetrahydrofuran (THF). 1,1'-carbodiimidazole (0.70 g, 4.3 mmol) was added to the acid solution. The mixture was heated to 50°C for sixty minutes. Then the reaction vessel was flushed with an inert gas. Tyramine (0.59 g, 4.3 mmol) was added to the reaction mixture. The mixture was further heated for another 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 to the solution. The solution was heated under 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)

1H NMR (D2O, ppm) δ: 1.17 (m, 2H, Y-CH2-hexanoic acid); 1.48(m, 2 H, β-CH2-hexanoic acid); 1.58 (m, 2 H, δ-CH2-hexanoic acid); 2.17 (t, 2 H, - CH 2 -CO-); 2.73 (m, 2H, -CH 2 -Ph); 2.91 (m, 2H, -CH 2 -NH 2 ); 3.42 (m, 2H, -CH2-NH-CO-); 6.83 (d, 2H, arom); 7.13 (d, 2H, arom).

¹³ C NMR(D2O, ppm) δ: 24 (γ-C-hexanoic acid); 26 (δ-C-hexanoic acid); 33 (β-C-hexanoic acid); 35 (-C-CO-); 39 (-C-NH 2 ); 40 (C-Ph); 63 (-C-NH-CO-); 115 (C3 arom); 126 (C1 arom); 130 (C2 arom.); 153 (C4 arom.); 176 (-CO-).

Preparation of aldehyde derivative (HA-CHO)

Hylauronan (10.00 g, M _w . = 2 x 10 ⁶ g.mol ^-1 ) was dissolved in 750 ml of a 2.5% (w/w) Na2HPO4 solution. 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 thorough homogenization of the solution, 3 ml of NaClO solution (10-15% available O2) was added to the reaction mixture. The reaction continued with constant stirring for 15 min. The reaction was terminated by the addition of 100 ml of a 40% propan-2-ol solution. The product was purified by ultrafiltration and isolated by precipitation with propan-2-ol.

ID (KBr): 3417, 2886, 2152, 1659, 1620, 1550, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm' ¹ . 1H NMR (D2O) δ: 2.01 (s, 3H, CH3-), 3.37-3.93 (m, hyaluronan backbone), 4.46 (s, 1H, anomer), 4.54 (s , 1H anomer, -O-CH(OH)-), 5.27 (geminal glycol -CH- (OH)2).

a) Preparation of tyraminated HA derivative with C ₆ spacer (Mw = 3 x 10 ⁵ g.mol ^-1 , DS = 2%)

The aldehyde derivative HA (= 3 x 10 ⁵ g.mol ^-1 , DS = 9%) (5.00 g) was dissolved in 500 ml of demineralized water. Using acetic acid, the pH of the solution was adjusted to 3. 6-amino-N-[2-(4-hydroxyphenyl)ethyl]hexanamide (intermediate (I)) (1.25 g, 5 mmol) was added to the HA-CHO solution. The mixture was stirred for 2 hours at room temperature. Picoline-borane complex (0.270 g, 2.5 mmol) was then added to the reaction mixture. The mixture was stirred for another 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).

ID (KBr): 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm' ¹ . 1H NMR (D2O) δ: 1.25 (t, 2H, γ-CH2- aminohexanoic acid), 1.48 (m, 2H, δ- CH2- aminohexanoic acid) 1.51 (m, 2H, P -CH2-aminohexanoic acid), 2.01 (s, 3 H, CH3-), 2.65 (m, 2H, Ph-CH2-), 2.73 (m, 2H, ε-CH2 aminohexanoic acid), 3, 37 - 3.93 (m, hyaluronan skeleton), 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 (1H NMR): 2.1%

b) Preparation of tyraminated HA derivative with C6 spacer (Mw = 8 x 10 ⁵ g.mol ¹ , DS = 1%)

The aldehyde derivative HA (Mw = 8 x 10 ⁵ g.mol ¹ , DS = 5%) (5.00 g) was dissolved in 500 ml of demineralized water. Using acetic acid, the pH of the solution was adjusted to 3. 6-amino-N-[2-(4-hydroxyphenyl)ethyl]hexanamide (intermediate (I)) (0.625 g, 2.5 mmol) was added to the HA-CHO solution. The mixture was stirred for 2 hours at room temperature. Picoline-borane complex (0.270 g, 2.5 mmol) was then added to the reaction mixture. The mixture was stirred for another 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 from moisture and residual propan-2-ol by drying in a hot air oven (40 °C, 3 days).

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

1H NMR (D2O) δ: 1.25 (t, 2H, γ-CH2- aminohexanoic acid), 1.48 (m, 2H, δ- CH2- aminohexanoic acid), 1.51 (m, 2H, e-CH2- aminohexanoic acid), 2.01 (s, 3 H, CH3-), 2.65 (m, 2H, Ph-CH2-), 2.73 (m, 2H, ε-CH2 aminohexanoic acid), 3 .37 - 3.93 (m, hyaluronan skeleton), 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

DS (1H NMR): 1.1%

c) Preparation of tyraminated HA derivative with C6 spacer (Mw = 1.5 x 10 ⁶ g.mol ^-1 , DS = 0.5%)

The aldehyde derivative of HA (Mw = 1.5 x 10 ⁶ g.mol ¹ , DS = 0.5%) (5.00 g) was dissolved in 500 ml of demineralized water. Using acetic acid, the pH of the solution was adjusted to 3. 6-amino-N-[2-(4-hydroxyphenyl)ethyl]hexane-amide (intermediate (I)) (0.625 g, 2.5 mmol) was added to the HA-CHO solution ). The mixture was stirred for 2 hours at room temperature. Picoline-borane complex (0.270 g, 2.5 mmol) was then added to the reaction mixture. The mixture was stirred for another 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 ^o C, 3 days).

ID (KBr): 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm' ¹ . 1H NMR (D2O) δ: 1.25 (t, 2 H, γ-CH2- aminohexanoic acid), 1.48 (m, 2 H, δ- CH2- aminohexanoic acid) 1.51 (m, 2 H, β -CH2- aminohexanoic acid), 2.01 (s, 3 H, CH3-), 2.65 (m, 2H, Ph-CH2-), 2.73 (m, 2H, ε-CH2- aminohexanoic acid), 3.37 - 3.93 (m, hyaluronan skeleton), 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 (1H NMR): 0.5%

d) Preparation of tyraminated HA derivative with C6 spacer (Mw = 5 x 10 ⁴ g.mol ¹ , DS = 10%)

The aldehyde derivative of HA (Mw = 5 x 10 ⁴ g.mol ^-1 , DS = 10%) (5.00 g) was dissolved in 500 ml of demineralized water. Using acetic acid, the pH of the solution was adjusted to 3. 6-amino-N-[2-(4-hydroxy-phenyl)ethyl]hexanamide (intermediate (I)) (0.625 g, 2.5 mmol) was added to the HA-CHO solution ). The mixture was stirred for 2 hours at room temperature. Picoline-borane complex (0.270 g, 2.5 mmol) was then added to the reaction mixture. The mixture was stirred for another 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).

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

1H NMR (D ₂ O) δ: 1.25 (t, 2 H, y-CH ₂ - aminohexanoic acid), 1.48 (m, 2 H, δ- CH ₂ - aminohexanoic acid), 1.51 (m , 2 H, e-CH2- of aminohexanoic acid), 2.01 (s, 3 H, CH3-), 2.65 (m, 2H, Ph-CH2-), 2.73 (m, 2H, ε-CH2 -aminohexanoic acid), 3.37 - 3.93 (m, hyaluronan skeleton), 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 (1H NMR): 10%

Example 2

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

Hydrogels were prepared by mixing two precursor solutions A and B, for their preparation an aqueous solution of NaCl (9 g/l) was used. The hydroxyphenyl derivative HA-TA with Mw = 2.78 x 10 ⁵ g.mol ^-1 and DS 2.1% was used for the preparation of precursor solutions. The composition of the solutions is shown in the following table (Table 1).

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

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

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 2), where crossHA-TA is a covalently cross-linked hydroxyphenyl derivative of hyaluronan.

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

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

Example 3

Preparation of hydrogels containing ChS with a concentration of 0.5 mg/ml

Hydrogels were prepared by mixing two precursor solutions A and B, for the preparation of which an aqueous solution of NaCl (9 g/l) was used. The hydroxyphenyl derivative HA-TA with Mw = 2.78 x 10 ⁵ g.mol ^-1 and DS 2.1% and ChS with Mw = 10 x 10 ³ - 40 x 10 ³ g.mol ^-1 were used for the preparation of precursor solutions. . The composition of the solutions is shown in the following table (Table 3).

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

Solution A SolutionB 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

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 4), where crossHA-TA is a covalently cross-linked hydroxyphenyl derivative of hyaluronan.

Table 4 Final composition of hydrogels containing ChS with a concentration of 0.5 mg/ml

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

Example 4

Preparation of hydrogels containing ChS with a concentration of 3.3 mg/ml

Hydrogels were prepared by mixing two precursor solutions A and B, for the preparation of which an aqueous solution of NaCl (9 g/l) was used. The hydroxyphenyl derivative HA-TA with Mw = 2.78 x 10 ⁵ g.mol ^-1 and DS 2.1% and ChS with Mw = 10 x 10 ³ - 40 x 10 ³ g.mol ^-1 were used for the preparation of precursor solutions. . The composition of the solutions is shown in the following table (Table 5).

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

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

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), where crossHA-TA is a covalently cross-linked hydroxyphenyl derivative of hyaluronan.

Table 6 Final composition of hydrogels containing ChS with a concentration of 3.3 mg/ml

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

Example 5

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

Hydrogels were prepared by mixing two precursor solutions A and B, for the preparation of which an aqueous solution of NaCl (9 g/l) was used. The hydroxyphenyl derivative HA-TA with Mw = 2.78 x 10 ⁵ g.mol ^-1 and DS 2.1% and ChS with Mw = 10 x 10 ³ - 40 x 10 ³ g.mol ^-1 were used for the preparation of precursor solutions. . The composition of the solutions is shown in the following table (table 7).

Table 7 Composition of precursor solutions for the preparation of hydrogels containing ChS with a concentration of 10 mg/ml

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

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

Table 8 Final composition of hydrogels containing ChS with a concentration of 10 mg/ml

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

Example 6

Preparation of hydrogels containing ChS with a concentration of 50 mg/ml

Hydrogels were prepared by mixing two precursor solutions A and B, for the preparation of which an aqueous solution of NaCl (9 g/l) was used. Hydroxyphenyl derivative HA-TA with Mw = 2.78 x 10 ⁵ g.mol ^-1 and DS 2.1% and ChS with Mw = 10 x 10 ³ - 40 x 10 ³ g.mol' ¹ were used for the preparation of precursor solutions. . The composition of the solutions is shown in the following table (table 9).

Table 9 Composition of precursor solutions for the preparation of hydrogels containing ChS with a concentration of 50 mg/ml

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

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

Table 10 Final composition of hydrogels containing ChS with a concentration of 50 mg/ml

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

Example 7

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

The preparation of hydrogels containing non-cross-linked hyaluronic acid included 3 basic steps:

1. Preparation of a hydrogel 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 derivative HA-TA with Mw = 8.09 x 105 g.mol-1 and DS 1.1% was used for the preparation of precursor solutions. The composition of the solutions is shown in the following table (table 11).

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

Solution A Solution B HRP................................... ........12.8 mU/ml H2O2........................ .......0.6 mmol/l HA-TA........................ ........20 mg/ml HA-TA........................ ........20 mg/ml NaCl................................ ........8 g/l NaCl........................ ........8 g/l KCl............................ ........0.2 g/l KCl............................ ........0.2 g/l Na2HPO4.12H2O........ ........2.85 g/l Na2HPO4.12H2O....... ........2.85 g/l KH2PO4................... ........0.2 g/l KH2PO4................... ........0.2 g/l

By mixing solution A and solution B in a 1:1 ratio, a hydrogel was prepared with the composition shown in the following table (table 12).

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

Composition of the cross-linked crossHA-TA derivative HRP................................... ........0.24 U/ml crossHA-TA............... ..........20 mg/ml NaCl................................ ..........8 g/l KCl............................ .........0.2 g/l Na2HPO4.12H2O........ .............2.85 g/l KH2PO4................... .........0.2 g/l

2. Preparation of hyaluronan solution

A HA solution with a concentration of 5 mg/ml was prepared by dissolving native hyaluronan with a Mw of 1.91 x 10 ⁶ g.mol ^-1 in PBS.

3. Homogenization of hydrogel and hyaluronan solution

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

Table 13 Final composition of the hydrogel, containing HA with a concentration of 5 mg/ml

Final composition HRP................... .0.12 U/ml crossHA-TA........ .....10 mg/ml HA................... ..5 mg/ml NaCl................... ..8 g/l KCl................... ..0.2 g/l Na2HPO4.12H2O. .....2.85 g/l KH2PO4............... .0.2 g/l

Example 8

Preparation of hydrogels containing HA with a concentration of 20 mg/ml

The preparation of hydrogels containing non-cross-linked hyaluronic acid included 3 basic steps:

1. Preparation of a hydrogel 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 derivative HA-TA with Mw = 8.09 x 10 ⁵ g.mol ^-1 and DS 1.1% was used for the preparation of precursor solutions. The composition of the solutions is shown in the following table (table 14).

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

Solution A Solution B HRP................................... .........12.8 mU/ml H2O2........................ ........0.6 mmol/l HA-TA........................ ...........20 mg/ml HA-TA........................ .........20 mg/ml NaCl................................ .........8 g/l NaCl................................ .........8 g/l KCl............................ ........0.2 g/l KCl............................ .......0.2 g/l Na2HPO4.12H2O........ ...........2.85 g/l Na2HPO4.12H2O....... ...........2.85 g/l KH2PO4................... ........0.2 g/l KH2PO4................... ........ 0.2 g/l

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

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

Composition of the cross-linked crossHA-TA derivative HRP................... .....0.24 U/ml crossHA-TA.......... .......20 mg/ml NaCl................... .....8 g/l KCl........................ ...0.2 g/l Na2HPO4.12H2O... ........2.85 g/l KH2PO4................... .....0.2 g/l

2. Preparation of hyaluronan solution

A HA solution with a concentration of 40 mg/ml was prepared by dissolving native hyaluronan with 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 cross-linked crossHA-TA derivative and HA solution in a ratio of 1:1 with subsequent homogenization of the mixture. The final composition of the material is shown in the following table (table 16).

Table 16 Final composition of the hydrogel, containing HA with a concentration of 20 mg/ml

Final composition HRP................... .....0.12 U/ml crossHA-TA............ ........10 mg/ml HA................... .....20 mg/ml NaCl...................... .....8 g/l KCl.......................... ....0.2 g/l Na2HPO4.12H2O.... .......2.85 g/l KH2PO4................... .....0.2 g/l

Example 9

Preparation of hydrogels containing HA with a concentration of 5 mg/ml and ChS with a concentration of 5 mg/ml

The preparation of hydrogels containing non-cross-linked hyaluronic acid and chondroitin sulfate included 3 basic steps:

1. Preparation of a hydrogel 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 derivative HA-TA with Mw = 8.09 x 10 ⁵ g.mol ^-1 and DS 1.1% was used for the preparation of precursor solutions. The composition of the solutions is shown in the following table (table 17).

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

Solution A Solution B HRP................................... ...........12.8 mU/ml H2O2........................ .........0.6 mmol/l HA-TA........................ .............20 mg/ml HA-TA..................... .........20 mg/ml NaCl................................ ...........8 g/l NaCl........................ .........8 g/l KCl............................ ........0.2 g/l KCl.......................... ......0.2 g/l Na2HPO4.12H2O........ .............2.85 g/l Na2HPO4.12H2O...... ............2.85 g/l KH2PO4................... ...........0.2 g/l KH2PO4................... .......0.2 g/l

By mixing solution A and solution B in a ratio of 1:1, a hydrogel was prepared with the composition shown in the following table (table 18).

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

Cross-linked composition crossHA-TA derivative HRP................................... ...........0.24 U/ml crossHA-TA................ ..............20 mg/ml NaCl................................ ............8 g/l KCl............................ ..........0.2 g/l Na2HPO4.12H2O........ ...............2.85 g/l KH2PO4................... ..........0.2 g/l

2. Preparation of a solution containing hyaluronan and chondroitin sulfate

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

3. Homogenization of hydrogel and solution containing hyaluronan and chondroitin sulfate

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

Table 19 Final composition of the hydrogel, HA with a concentration of 5 mg/ml and ChS with a concentration of 5 mg/ml

Final composition HRP................... .....0.12 U/ml crossHA-TA.......... .........10 mg/ml HA........................ .....5 mg/ml ChS................... .......5 mg/ml NaCl................... ......8 g/l KCl........................ ...0.2 g/l Na2HPO4.12H2O.... .........2.85 g/l KH2PO4................... ......0.2 g/l

Example 10

Preparation of hydrogels containing HA with a concentration of 5 mg/ml and ChS with a concentration of 10 mg/ml

The preparation of hydrogels containing non-cross-linked hyaluronic acid and chondroitin sulfate included 3 basic steps:

1. Preparation of a hydrogel 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 derivative HA-TA with Mw = 8.09 x 10 ⁵ g.mol ^-1 and DS 1.1% was used for the preparation of precursor solutions. The composition of the solutions is shown in the following table (table 20).

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

Solution A HRP...................................12.8 mU/ml HA-TA...............................20 mg/ml NaCl...................................8 g/l KCl...................................0.2 g/l Na2HPO4.12H20...................2.85 g/l KH2PO4............................0.2 g/l Solution B H2O2...................................0.6 mmol/l HA-TA...............................20 mg/ml NaCl...................................8 g/l KCl...................................0.2 g/l Na2HPO4.12H20...................2.85 g/l KH2PO4............................0.2 g/l

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

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

Composition of the cross-linked crossHA-TA derivative HRP................................... ...........0.24 U/ml crossHA-TA................. ..............20 mg/ml NaCl................................ ............8 g/l KCl................................... ..........0.2 g/l Na2HPO4.12H2O.......... ..............2.85 g/l KH2PO4........................ ..........0.2 g/l

2. Preparation of a solution containing hyaluronan and chondroitin sulfate

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

3. Homogenization of hydrogel and solution containing hyaluronan and chondroitin sulfate

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

Table 22 Final composition of the hydrogel, HA with a concentration of 5 mg/ml and ChS with a concentration of 10 mg/ml

Final composition HRP................................... ...........0.12 U/ml crossHA-TA................ ..............10 mg/ml HA................................... ............5 mg/ml ChS............................... ..............10 mg/ml NaCl................................ ...........8 g/l KCl................................... .........0.2 g/l Na2HPO4.12H2O......... ............2.85 g/l KH2PO4................... ..........0.2 g/l

Example 11

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

Hydrogels were prepared by mixing two precursor solutions A and B, for the preparation of which an aqueous solution of NaCl (9 g/l) was used. The hydroxyphenyl derivative HA-TA with Mw = 1.5 x 10 ⁶ g.mol ^-1 and DS 0.5% was used for the preparation of precursor solutions. The composition of the solutions is shown in the following table (table 23).

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

Solution A Solution B HRP............................0.12 U/ml HA-TA.........................5 mg/ml NaCl...................9 g/l H2O2...................................0.5 mmol/l HA-TA...............................5 mg/ml NaCl...................................9 g/l

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

Table 24 Final composition of hydrogels based on the hydroxyphenyl derivative HA-TA with a concentration of 5 mg/ml

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

Example 12

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

Hydrogels were prepared by mixing two precursor solutions A and B, for the preparation of which an aqueous solution of NaCl (9 g/l) was used. The hydroxyphenyl derivative HA-TA with 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).

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

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

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

Table 26 Final composition of hydrogels based on the hydroxyphenyl derivative HA-TA with a concentration of 30 mg/ml

Composition of hydrogels HRP................................... ....0.6 U/ml crossHA-TA................... ....30 mg/ml NaCl................................... .....9 g/l

Example 13

Degradation of ChS-supplemented HA solutions by ROS

To compare the rate of degradation of HA solutions with ChS by ROS, solutions of hyaluronan in phosphate buffer (PBS) with different concentrations of ChS were prepared. Hyaluronic acid with Mw 1.91 x 106 g.mol ^-1 and ChS with 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 ChS, solution E 20 mg/ml HA and 5 mg/ml ChS, solution F 20 mg/ml HA and 20 mg/ml ChS.

Conducting a degradation experiment

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

fig. 1 shows the degradation of hyaluronan chains, which is expressed by the percentage decrease in the viscosity of the solutions over time. From fig. 1 shows the effect of ChS concentration on the rate of hyaluronan degradation. 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, decreased by 97% after 3 hours compared to the initial value before degradation, of solution B (with the addition of 0.5 mg/ml ChS) by 89%, of 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) by only 8%.

Example 14

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

To compare the rate of degradation of hydrogels by ROS, 3 types of materials were prepared. Material A is a HA solution with a concentration of 20 mg/ml, which was prepared by dissolving HA with a Mw of 1.91 x 10 ⁶ g.mol ^-1 in PBS.

Material B is a mixture of a cross-linked derivative of crossHA-TA and non-cross-linked HA, which was prepared according to Example 7.

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

The rate of degradation was expressed as a percentage decrease in the viscosity of the materials at a shear rate of 0.1 s ^-1 compared to the initial value. Viscosity drop measurements were performed on a Kinexus Malvern rheometer in a cone-plate configuration. A cone with a diameter of 40 mm with an apex angle of 1° was used. Degradation of the material took place in 10 ml syringes, where 0.5 ml of CuSO4 solution with a concentration of 0.25 mmol/l was added to 9 ml of the material, followed by 0.5 ml of H ₂ O ₂ solution with a concentration of 2.5 mmol/l. After certain degradation times, samples were taken, in which the viscosity was measured at a temperature of 25 °C and a shear rate of 0.1 s ^-1 . The total degradation time was 3 h. From fig. 2, it is clear that the presence of ChS in the prepared hydrogels (C - 5 mg/ml ChS; D - 10 mg/ml ChS) increases their resistance against the action of ROS.

Example 15

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

To determine the effect of the presence of ChS on the degradation rate of hydrogels based on crossHA-TA by the action of bovine testicular hyaluronidase, 3 types of hydrogels were prepared:

A - hydrogels without the addition of chondroitin sulfate, which were prepared according to the procedure in example 2,

B - hydrogels with the addition of ChS with a concentration of 3.3 mg/ml, which were prepared according to the procedure in example 4,

C - hydrogels with the addition of ChS with a concentration of 10 mg/ml, which were prepared according to the procedure in example 5.

The degradation rate was expressed as the increase in the concentration of degradation products caused by BTH, expressed as a percentage.

The hydrogels were immersed in the degradation medium (a solution of hyaluronidase BTH with an activity of 30 U/mg in a solution of bovine serum albumin (BSA) at a concentration of 0.1 mg/ml in 0.01 mol/l acetate buffer (OP) pH 5.3). Degradation of hydrogels took place in an incubator at 37 °C with simultaneous mixing. After certain time intervals, samples of the degradation medium with hydrogel degradation products were taken. The concentration of HA disaccharide units in the degradation medium was determined spectrophotometrically as the concentration of N -acetylglucosamine. fig. 3 shows the increase in the concentration of hydrogel degradation products in the medium over time. From fig. it is clear that the presence of ChS in hydrogels based on crossHA-TA leads to a decrease in the rate of degradation of the materials by the action of hyaluronidase.

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Claims (5)

CLAIMS FOR PROTECTION 1. A hydrogel based on a cross-linked hydroxyphenyl derivative of hyaluronic acid, characterized by the fact that it contains molecules of a hydroxyphenyl derivative of hyaluronic acid (HA-TA) or its pharmaceutically acceptable salt according to the general formula (I) (I), where n is in the range of 2 to 7,500 and where R ¹ is H ⁺ or an ion of an alkaline salt or an alkaline earth salt and R ² is OH or a tyramine substituent according to the general formula (II): wherein within one molecule of the hydroxyphenyl derivative of hyaluronic acid or its pharmaceutically acceptable salt according to the general formula (I) there is at least one R ² tyramine substituent according to the general formula (II) and while at least two tyramine substituents according to the general formula (II) are connected via a covalent bond in any ortho position of the phenyl groups, and further comprises chondroitin sulfate or a pharmaceutically acceptable salt thereof selected from the group consisting of alkali salts or alkaline earth salts. 2. Hydrogel according to claim 1, characterized in that the alkaline salts or alkaline earth salts are selected from the group containing Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ . 3. Hydrogel according to claim 1 or claim 2, characterized in that the concentration of chondroitin sulfate or its pharmaceutically acceptable salt is in the range of 0.5 to 50 mg/ml hydrogel, preferably in a concentration of 1 to 20 mg/ml, more preferably 5 mg/ Jr. 4. 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 the hydrogel. 5. Hydrogel according to any one of claims 1 to 4, characterized in that it further contains hyaluronic acid or its pharmaceutically acceptable salt in a concentration of 1 to 20 mg/ml hydrogel, preferably 5 to 10 mg/ml, more preferably 5 mg/ml hydrogel.