WO2021043349A1

WO2021043349A1 - Hydrogel based on crosslinked hydroxyphenyl derivative of hyaluronic acid

Info

Publication number: WO2021043349A1
Application number: PCT/CZ2020/050065
Authority: WO
Inventors: Evgeniy Toropitsyn; Martin Pravda; Lenka KOVAROVA; Julie BYSTRONOVA; Vladimir Velebny
Original assignee: Contipro AS
Current assignee: Contipro AS
Priority date: 2019-09-06
Filing date: 2020-09-03
Publication date: 2021-03-11
Anticipated expiration: 2022-03-06
Also published as: SK9649Y1; SK500272022U1; FR3104945A3; AT18104U1; CZ33324U1; FR3104945B3; DE212020000715U1

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

Hydrogel based on crosslinked hydroxyphenyl derivative of hyaluronic acid Field of invention

Invention relates to hydrogel based on crosslinked hydroxyphenyl derivative of hyaluronic acid in the mixture with chondroitin sulfate with improved degree of degradation.

Hyaluronic acid (also hyaluronan, HA) is a polysaccharide from the group of glycosaminoglycan that is composed of disaccharide units composed of D-glucuronic acid and A-acctyl-D-glucosaminc. It relates to a polysaccharide that is well soluble in aqueous environment, where it forms viscous solutions up to viscoelastic hydrogels depending on molecular mass and concentration. HA is a natural component of intercellular tissue matrix. Molecule of hyaluronan is able to interact with surrounding cells and regulate their metabolic processes (Xu, Jha et al. 2012) through binding to specific cell surface receptors. The materials containing hyaluronan or optionally its derivatives are therefore often used for manufacturing of preparations used in biomedicinal applications. Hydrogels based on hyaluronan undergo in the organism natural degradation through the action of specific enzymes (hyaluronidases), eventually by acting of reactive oxygen species (ROS), thanks to which their gradual absorption in the organism occurs upon their implantation (Stern, Kogan et al. 2007).

For obtaining mechanically more resistant materials and to slow down their biodegradation, a number of hydrogel types containing covalently crosslinked hyaluronan was developed. Such hydrogels are used as materials for viscosupplementation of synovial fluid, augmentation of soft tissues, serve as scaffold structures for culture and implantation of cells, 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 developed, that are able to undergo sol-gel transition under physiological conditions in situ (Burdick and Prestwich 2011, Prestwich 2011). For example, phenolic derivatives of hyaluronan may 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 documents EP1587945B1 and EP1773943B1 protocol for preparation of phenolic derivatives of hyaluronan by reaction of carboxyls present in the structure of D- glucuronic acid of hyaluronan, with aminoalkyl-derivatives of phenol, e. g. tyramine. Hyaluronan amides (Darr and Calabro 2009) are product of this reaction.

Crosslinking of phenolic derivatives of hyaluronan may be initiated by addition of peroxidase (e.g. horseradish peroxidase) and diluted solution of hydrogen peroxide. Horseradish peroxidase (Horseradish peroxidase, HRP, E.C.1.11.1.7.) is presently used as catalyst of 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 may be used as injection applicable matrix for controlled release of biologically active compounds or as materials suitable for culture and implantation of cells (Kurisawa, Lee et al. 2010). Wolf et al. describe in document CZ303879 conjugate of hyaluronan and tyramine containing aliphatic linker inserted between chains of polymer and tyramine. The presence of aliphatic linker enables higher efficiency of crosslinking reaction and provides the net with higher elasticity.

Chondroitin sulfate (ChS) is another member of glycosaminoglycans, that is often used for preparation of materials for use in treatment of degenerative diseases, e.g. osteoarthrosis (OA). Chain ChS is made of disaccharide units composed of N- acct y 1 ga 1 ac to s a m i nc (GalNAc) and iduronic acid (IdoA). Disaccharide units ChS may be sulfated in position 4 and 6 of GalNAc and optionally in position 2IdoA. Chondroitin sulfate is a linear, sulfated and negatively-charged glycosaminoglycan composed of repeating monomer units of A-acctyl-D- galactosamine and D-glucuronic acid interconnected with b(1 3) a b(1 4) Oglycosidic bonds (for structure formula of chondroitin sulfate, see below).

where

R¹ is H or Na, R² is H, O-SOi-OH or O-SOi-ONa

Animal connective tissues are source of chondroitin sulfate, where it binds proteins and forms thus part of proteoglycans. Sulfatation of chondroitin occurs with sufotransferases in various positions and in various representation. Unique formula of sulfatation of individual position in polymer chain codes specific biologic activity of chondroitin sulfate. It is an important building block of cartilage in joints, that provides resistance to pressure and restores balance in composition of synovial fluid (Baeurle, S. A., Kiselev M. G., Makarova E. S., Nogovitsin E. A. 2009. Polymer 50: 1805). Chondroitin sulfate is together with glucosamine used as dietary supplement for treating or prevention of development of osteoarthritis in humans (e.g. Flextor®, Advance Nutraceutics, Ltd.) or in animals (e.g. Geloren^dog®, Contipro Pharma, Ltd.). From pharmaceutical point of view, chondroitin sulfate is considered as drug with delayed onset of action of pain relief in degenerative joint disorder (Aubry-Rozier B. 2012. Revue Medicale Suisse 14:571).

In vitro and in vivo study showed that ChS inhibits the effect of hyaluronidases. Inhibition effect of ChS on enzymes is caused by formation of electrostatic (ionic) interactions. It was also proved that ChS is able to capture ROS and thus protect from degradation the components of extracellular matrix (Bali, Cousse et al. 2001, Xiong and Jin 2007).

The use of combination of hyaluronan and chondroitin sulfate for preparation of means for protection of human or animal cells and tissues from trauma is described in document EP0136782 (1983). Similarly, document US6051560 (1992) describes the use of mixture of hyaluronan and chondroitin sulfate as viscosupplementation materials during opthtalmological surgery. Patent W0030417024 describes viscous composition containing therapeutically efficient amount of mixture of ChS and HA for production of drugs for treating joints in humans with damaged cartilage caused by chondromalacia or OA of degree I and II, that uses intra articular administration of the mixture. Documents may be also found in patent literature, that describe means for parenteral administration suitable for prevention and treatment of damage of joint cartilage in humans or animals, that is composed of therapeutically efficient amount of chondroitin sulfate, hyaluronan and glucosamine (W02004034980, 2002). Document EP2219595 describes formulation based on polysaccharides, especially glycosaminoglycans and their mixtures with flavonoids that forms hydrogels with prolonged time of biodegradation. The mentioned document describes also hydrogel containing hyaluronan, hyaluronan derivative crosslinked with butanediol 1,4- diglycidyl ether and ChS that shows increased resistance to degradation caused by enzyme hyaluronidase.

Summary of the inventionn Invention relates to hydrogel based on crosslinked hydroxyphenyl derivative of hyaluronic acid, whose subject-matter is that it contains molecules of hydroxyphenyl derivative of hyaluronic acid (HA-TA) or its pharmaceutically acceptable salt of a general formula I

where n is in the range 2 to 7500 and where R¹ is H⁺ or an ion of alkali salt or salt of alkaline earth metal and R² is OH or tyramine substituent of a general formula II:

whereas in one molecule of hydroxyphenyl derivative of hyaluronic acid or its pharmaceutically acceptable salt according to the general formula I, is at least one R² tyramine substituent of the general formula II and whereas at least two tyramine substituents of general formula II are connected with covalent bond in any ortho position of phenyl groups, and it further contains chondroitin sulfate or its pharmaceutically acceptable salt selected from the group containing alkali salts or salts of alkali earth metal. Alkali salts or salts of alkali metal of hydroxyphenyl derivative of hyaluronic acid of the general formula I or chondroitin sulfate are preferably selected from the group comprising Na⁺, K⁺, Ca²⁺, Mg²⁺.

Concentration of chondroitin sulfate or its pharmaceutically acceptable salt is in the range 0.5 to 50 mg/mL hydrogel according to the invention, preferably in concentration 1 to 20 mg/mL, more preferably 5 mg/mL.

The content of the crosslinked hydroxyphenyl derivative of hyaluronan is in the range 5 to 30 mg/mL, preferably 10 mg/mL hydrogel according to the invention. According to another preferred embodiment of the invention, the hydrogel further contains hyaluronic acid or its pharmaceutically acceptable salt in concentration 1 to 20 mg/mL, preferably 5 to 10 mg/mL, more preferably 5 mg/mL hydrogel according to the invention.

Covalent bond may be in one molecule of derivative of hyaluronic acid of the general formula I in any ortho position of phenyl groups of at least two tyramine substituents of the general formula II that are in this molecule. It is referred to as intramolecular crosslinking. Covalent bond may be also in any ortho position of phenyl groups of at least two tyramine substituents of the general formula II, that are in different molecules of the derivative of hyaluronic acid of the general formula I. It represents interconnected crossling among molecules of the derivative HA.

An example of crosslinked hydroxyphenyl derivative of hyaluronan. (crossHA-TA) is schematically shown below, see a formula III:

Such hydrogels according to the invention show increased resistance to biodegradable processes of hydrolytic enzymes and reactive oxygen species.

According to a preferred embodiment is the weight average molar mass (Mw) of hydroxyphenyl derivative of hyaluronan of the general formula I in the range 5 x 10⁴ to 1.5 x 10⁶ g.mol ¹, preferably 2.5 x 10⁵ to 1 x 10⁶ g.mol ¹, more preferably 8 x 10⁵ g.mol ¹. PI is in the range 1 to 3. According to another embodiment of the present invention is the degree of substitution (DS) of hydroxyphenyl derivative of hyaluronan of general formula I in the range 0.5 to 10%, preferably 1 to 4%, more preferably 1%. According to another preferred embodiment is Mw of chondroitin sulfate in the range 5 x 10³ to 95 x 10³ g.mol ¹, further preferably 10 x 10³ to 40 x 10³ g.mol ¹.

According to a preferred embodiment, the hydrogel contains hyaluronan (HA) or its pharmaceutically acceptable salt of Mw in the range 5 x 10⁴ to 2.5 x 10⁶ g.mol ¹, preferably 1.5 x 10⁶ to 2.5 x 10⁶ g.mol ¹, more preferably 2.0 x 10⁶ g.mol ¹.

Such hydrogels according to the invention may be used in cosmetics, medicine and regenerative medicine, especially for preparation of materials for tissue regeneration, tissue augmentation, scaffold for tissue engineering preparation, as matrix for controlled release of biologically active agents and drugs and viscosupplementation of synovial fluid.

Brief description of the drawings.

Fig. 1: Comparison of degradation rate of solutions HA with addition of ChS with ROS Fig. 2: Comparison of degradation rate of materials with ROS Fig: 3: Cumulative degradation of hydrogel [%] BTH 30 U/mg

Examples of embodiment of the invention

DS = degree of substitution = 100 % * molar amount of modified disaccharide units of hyaluronan / molar amount of all disaccharide units of hyaluronan derivative. Degree of substitution was determined with ^XH HMR spectroscopy. Weight average molar mass (Mw) and polydispersity index (PI) were determined with SEC- MALLS.

Infrared spectra of prepared derivatives were obtained with FT-IR.

Chondroitin sulfate in pharmaceutical quality from Bioiberica, ES was used for injection administration. Example 1

Synthesis of tyramine derivative of HA (HA-TA)

Synthesis of 6-amino-A-l 2(4hvdiOxyphcnyl )cthyl Ihcxanamidc 6 -[(terc. butoxycarbonyl)amino]hexanoic acid (1.00 g, 4.3 mmol) was dissolved in 50 mL tetrahydrofuran (THF). 1 .1 'carbodiimidazolc (0.70 g, 4.3 mmol) was added to the solution of the acid. The mixture was heated to 50 °C for sixty minutes. The reaction container was then washed with 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 ethylacetate. The solution was washed with 150 mL purified water (divided into three parts). Organic layer was dried over molecular sieve. Ethylacetate was removed by distillation under reduced pressure. The residue was dissolved in 50 mL MeOH and 2 mL of trifluoracetic acid (TFA) were added to the solution. The solution was heated for 6 hours under reflux. The solvent was removed by distillation under reduced pressure. The residue was dissolved in 50 mL of ethylacetate. The solution was washed with 150 mL of purified water (divided into three parts). Organic layer was dried over molecular sieve. Ethylacetate was removed by distillation under reduced pressure. m = 0.75 g (70% theory)

¹ H NMR (D2O, ppm) d: 1,17 (m, 2 H, y-CH2-hexanoic acid); l,48(m, 2 H, P-CH2-hexanoic acid); 1,58 (m, 2 H, 5-CH₂-hexanoic acid); 2,17 (t, 2 H, -CH2-CO-); 2,73 (m, 2 H, -CH₂-Ph); 2,91 (m, 2 H, -CH2-NH2); 3,42 (m, 2 H, -CH2-NH-CO-); 6,83 (d, 2 H, arom); 7,13 (d, 2 H, arom).

¹³C NMR(D20, ppm) d: 24 (g-C- hexanoic acid); 26 (d-C- hexanoic acid); 33 (b-C- hexanoic acid); 35 (-C-CO-); 39 (-C-NH2); 40 (C-Ph); 63 (-C-NH-CO-); 115 (C3 arom); 126 (Cl arom); 130 (C2 arom.); 153 (C4 arom); 176 (-CO-).

Preparation of aldehydic derivative (HA-CHO)

Hyaluronan (10.00 g, Mw = 2 x 10⁶ g.mol ¹) was dissolved in 750 mL 2.5% (w/w) solution Na₂HP0₄.12H₂0. The solution was cooled down to 5 °C. 2.60 g NaBr and 0.05 g 4- acetamido-2,2,6,6-tetramethylpiperidine-l-oxyl was added to the solution. Following a thorough homogenization of the solution were 3 mL of solution NaCIO (10-15% available Ch) added to reaction mixture. Reaction proceeded under stirring for 15 min. The reaction was quenched by addition of 100 mL 40% solution of propan-2-ol. Product was purified by ultrafiltration and isolated by precipitation with propan-2-ol. IR (KBr): 3417, 2886, 2152, 1659, 1620, 1550, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm ¹.

NMR (D _Q) d: 2,01 (s, 3 H, CH -), 3,37 - 3,93 (m, skelet of hyaluronan), 4,46 (s, 1H, anomer), 4,54 (s, 1H anomer., -O-CH(OH)-), 5,27 (geminal glycol -CH-(OH)2). a) Preparation of tyramine derivative of HA with Ce spacer (Mw ~ 3 x 10⁵ g.mol ¹, DS ~ 2 %)

Aldehyde derivative HA (~ 3 x 10⁵ g.mol ¹, DS = 9 %) (5.00 g) was dissolved in 500 mL demineralized water. pH of the solution adjusted to 3 using acetic acid. 6-amino-/V-[2-(4- hydroxyphenyl)ethyl]hexanamide (intermediate (I)) (1.25 g, 5 mmol) was added to the solution of HA-CHO. The mixture was stirred for 2 hours at room temperature. Complex picoline-borate (0.270 g, 2.5 mmol) was then added to reaction mixture. The mixture was stirred for another 12 hours at room temperature. The product was purified by ultrafiltration and isolated from retentate by precipitation with propan-2-ol. Precipitate was dehumidified and the residual propan-2-ol was removed by drying in hot-air drier (40 °C, 3 days).

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

NMR (D₂0) d: 1,25 (t, 2 H, g -CH₂- aminohexanoic acid), 1,48 (m, 2 H, d -CH₂- aminohexanoic acid)l,51 (m, 2 H, b -CH₂- aminohexanoic acid), 2,01 (s, 3 H, CH3-), 2,65 (m, 2H, Ph-CH₂-), 2,73 (m, 2H, e-CH₂- aminohexanoic acid), 3,37 - 3,93 (m, skelet 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 10⁵ g.mol ¹ DS (^lH NMR): 2,1 % b) Preparation of tyramine derivative of HA with Ce spacer (Mw ~ 8 x 10⁵ g.mol ¹, DS ~ 1 %)

Aldehyde derivative of HA (Mw ~ 8 x 10⁵ g.mol ¹, DS ~ 5 %) (5.00 g) was dissolved in 500 mL demineralized water. pH was adjusted to 3 using acetic acid. 6-amino- V-[2-(4- hydroxyphenyl)ethyl]hexanamide (intermediate (I)) (0.625 g, 2.5 mmol) was added to the solution of HA-CHO. The mixture was stirred for 2 hours at room temperature. Picoline- borate complex (0.270 g, 2.5 mmol) was then added to the mixture. The mixture was stirred for another 12 hours at room temperature. The product was purified by ultrafiltration and isolated from retentate by precipitation with propan-2-ol. Precipitate was dehumidified and the residual propan-2-ol was removed by drying in hot-air drier (40 °C, 3 days).

Ή NMR (D₂0) d: 1,25 (t, 2 H, g -CH₂- aminohexanoic acid), 1,48 (m, 2 H, d -CH₂- aminohexanoic acid) 1,51 (m, 2 H, b -CH₂- aminohexanoic acid), 2,01 (s, 3 H, CH3-), 2,65 (m, 2H, Ph-CH₂-), 2,73 (m, 2H, e-CH₂- aminohexanoic acid), 3,37 - 3,93 (m, skelet 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 10⁵ g.moT¹

DS NMR) 1,1 % c) Preparation of tyramine derivative of HA with Ce spacer (Mw ~ 1.5 x 10⁶ g.mol ¹, DS ~ 0.5 %)

Aldehyde derivative of HA (Mw ~ 1.5 x 10⁶ g.mol ¹, DS ~ 0.5 %) (5.00 g) was dissolved in 500 mL demineralized water. pH was adjusted to 3 using acetic acid. 6-amino- V-[2-(4- hydroxyphenyl)ethyl]hexanamide (intermediate I)) (0.625 g, 2.5 mmol) was added to solution HA-CHO. The mixture was stirred for 2 hours at room temperature. Picoline-borate complex (0.270 g, 2.5 mmol) was then added to reaction mixture. The mixture was stirred for another 12 hours at room temperature. The product was purified by ultrafiltration and isolated from retentate by precipitation with propan-2-ol. Precipitate was dehumidified and the residual propan-2-ol was removed by drying in hot-air drier (40 °C, 3 days).

NMR (D₂0) d: 1,25 (t, 2 H, g -CH₂- aminohexanoic acid), 1,48 (m, 2 H, d -CH₂- aminohexanoic acid) 1,51 (m, 2 H, b -CH₂- aminohexanoic acid), 2,01 (s, 3 H, CH3-), 2,65 (m, 2H, Ph-CH₂-), 2,73 (m, 2H, e-CH₂- aminohexanoic acid), 3,37 - 3,93 (m, skelet 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 10⁶ g.mol ¹ DS (^ln NMR): 0.5 % d) Preparation of tyramine derivative of HA with Ce spacer (Mw ~ 5 x 10⁴ g.moT¹, DS ~

10 %)

Aldehyde derivative of HA Mw ~ 5 x 10⁴ g.mol ¹, DS ~ 10 %) (5,00 g) was dissolved in 500 mL demineralized water. pH was adjusted to 3 using acetic acid. 6-amino- V-[2-(4- hydroxyfenyl)ethyl]hexanamide (intermediate I)) (0.625 g, 2.5 mmol) was added to solution HA-CHO. The mixture was stirred for 2 hours at room temperature. Picoline-borate complex (0.270 g, 2.5 mmol) was then added to reaction mixture. The mixture was stirred for another 12 hours at room temperature. The product was purified by ultrafiltration and isolated from retentate by precipitation with propan-2-ol. Precipitate was dehumidified and the residual propan-2-ol was removed by drying in hot-air drier (40 °C, 3 days).

IR (KBr): 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm⁴.

SEC MALLS: Mw = 5 x 10⁴ g.mol ¹ DS (^ln NMR): 10 %

Example 2

Preparation of hydrogels based on hydroxyphenyl derivative of HA-TA at concentration 20 mg/mL Hydrogels were prepared by mixing two precursor solutions A and B, for whose preparation had been used aqueous solution of NaCl (9 g/L). Lor preparation of precursor solutions was used hydroxyphenyl derivative of HA-TA of Mw = 2.78 x 10⁵ g.mol ¹ and DS 2.1 %. The composition of the solutions is shown in the following table (Table 1).

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

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

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

Example 3

Preparation of hydrogels containing ChS in concentration 0.5 mg/mL Hydrogels were prepared by mixing two precursor solutions A and B, for whose preparation had been used aqueous solution of NaCl (9 g/L). Hydroxyphenyl derivative of HA-TA of Mw = 2.78 x 10⁵ g.moT¹ and DS 2.1% and ChS of Mw = 10 x 10³ - 40 x 10³ g.mol ¹ was used for preparation of precursor solutions. Composition of the solutions is shown in the following table (Table 3).

Table 3: Composition of precursor solutions for preparation of hydrogels containing ChS at concentration 0.5 mg/mL.

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

Table 4: Final composition of hydrogels containing ChS at concentration 0.5 mg/mL Example 4

Preparation of hydrogels containing ChS in concentration 3.3 mg/mL

Hydrogels were prepared by mixing two precursor solutions A and B, for whose preparation had been used aqueous solution of NaCl (9 g/L). For preparation of precursor solutions was used hydroxyphenyl derivative HA-TA of Mw = 2.78 x 10⁵ g.moT¹ and DS 2.1% and ChS of Mw = 10 x 10³ - 40 x 10³ g.moT¹. Composition of the solutions is shown in the following table (Table 5).

Table 5: Composition of precursor solutions for preparation of hydrogels containing ChS at concentration 3.3 mg/mL By mixing solution A and solution B in ratio 1:1 were prepared hydrogels with final composition shown in the following table (Table 6), where crossHA-TA is covalently crosslinked hydroxyphenyl derivative of hyaluronan.

Table 6: Final composition of hydrogels containing ChS at concentration 3.3 mg/mL. Example 5

Preparation of hydrogels containing ChS in concentration 10 mg/mL

Hydrogels were prepared by mixing two precursor solutions A and B, for whose preparation had been used aqueous solution of NaCl (9 g/L). For preparation of precursor solutions was used hydroxyphenyl derivative HA-TA of Mw = 2.78 x 10⁵ g.moT¹ and DS 2.1% and ChS of Mw = 10 x 10³ - 40 x 10³ g.moT¹. Composition of the solutions is shown in the following table (Table 7).

Table 7: Composition of precursor solutions for preparation of hydrogels containing ChS at concentration 10 mg/mL

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

Table 8: Final composition of hydrogels containing ChS in concentration 10 mg/mL. Example 6

Preparation of hydrogels containing ChS in concentration 50 mg/mL

Hydrogels were prepared by mixing two precursor solutions A and B, for whose preparation had been used aqueous solution of NaCl (9 g/L). For preparation of precursor solutions was used hydroxyphenyl derivative HA-TA of Mw = 2.78 x 10⁵ g.mol ¹ and DS 2.1% and ChS of Mw = 10 x 10³ - 40 x 10³ g.mol ¹. Composition of the solutions is shown in the following table (Table 9).

Table 9: Composition of precursor solutions for preparation of hydrogels containing ChS at concentration 50 mg/mL

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

Table 10: Final composition of hydrogels containing ChS in concentration 50 mg/mL.

Example 7

Preparation of hydrogels containing HA in concentration 5 mg/mL

Preparation of hydrogels containing non- crosslinked hyaluronic acid comprised 3 basic steps: 1) Preparation of hydrogel containing crosslinked derivative crossHA-TA

Hydrogel containing crosslinked derivative crossHA-TA was prepared by mixing two precursor solutions A and B, that had been prepared by dissolving of individual components in phosphate buffered saline (PBS). For preparation of precursor solutions was used hydroxyphenyl derivative of HA-TA of Mw = 8.09 x 10⁵ g.mol ¹ and DS 1.1 %. Composition of solutions is shown in the following table (Table 11).

able 11: Composition of precursor solutions for preparation of crosslinked derivative crossHA-TA

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

Table 12: composition of crosslinked derivative of HA-TA 2) Preparation of hyaluronan solution

Solution of HA in concentration 5 mg/mL was prepared by dissolving native hyaluronan of Mw 1.91 x 10⁶ g.mol ¹ in PBS. 3) Homogenization of hydrogel and solution of hyaluronan

Final hydrogel was prepared by mixing crosslinked derivative crossHA-TA and solution HA in ratio 1:1 with the following homogenization of the mixture. Final composition of the material is shown in the following table (Table 13).

Table 13: Final composition of hydrogel containing HA in concentration 5 mg/mL Example 8

Preparation of hydrogels containing HA in concentration 20 mg/mL

Preparation of hydrogels containing non-crosslinked hyaluronic acid comprised 3 basic steps:

1) Preparation of hydrogel containing crosslinked derivative crossHA-TA Hydrogel containing crosslinked derivative crossHA-TA was prepared by mixing two precursor solutions A and B, that had been prepared by dissolving of individual components in phosphate buffered saline (PBS). For preparation of precursor solutions was used hydroxyphenyl derivative of HA-TA of Mw = 8.09 x 10⁵ g.mol ¹ and DS 1.1 %. Composition of the solutions is shown in the following table (Table 14).

Table 14: Composition of precursor solutions for preparation of crosslinked derivative crossHA-TA

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

Table 15: Composition of crosslinked derivative crossHA-TA

2) Preparation of hyaluronan solution

Solution of HA in concentration 40 mg/mL was prepared by dissolving native hyaluronan of Mw 1.91 x 10⁶ g.mol ¹ in phosphate buffer (PBS). 3) Homogenization of hydrogel and solution of hyaluronan

Final hydrogel was prepared by mixing crosslinked derivative crossHA-TA and solution HA in ratio 1:1 with following homogenization of the mixture. Final composition of the material is shown in the following table (Table 16).

Table 16: Final composition of hydrogel containing HA in concentration 20 mg/mL Example 9

Preparation of hydrogels containing HA in concentration 5 mg/mL and ChS in concentration 5 mg/mL

Preparation of hydrogels containing non- crosslinked hyaluronic acid and chondroitin sulfate comprised 3 basic steps:

1) Preparation of hydrogel containing crosslinked derivative crossHA-TA Hydrogel containing crosslinked derivative crossHA-TA was prepared by mixing two precursor solutions A and B, that had been prepared by dissolving of individual components in phosphate buffered saline (PBS). For preparation of precursor solutions was used hydroxyphenyl derivative of HA-TA of Mw = 8.09 x 10⁵ g.moT¹ and DS 1.1 %. Composition of solutions is shown in the following table (Table 17).

Table 17: Composition of precursor solutions for preparation of crosslinked derivative crossHA-TA

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

Table 18: Composition of crosslinked derivative crossHA-TA

2) Preparation of solution with content of hyaluronan and chondroitin sulfate Solution of HA of concentration 10 mg/mL and ChS in concentration 10 mg/mL was prepared by dissolving native hyaluronan of Mw 1.91 x 10⁶ g.mol ¹ and chondroitin sulfate of MwlO x 10³ - 40 x 10³ g.mol ¹ in phosphate buffer (PBS).

3) Homogenization of hydrogel and solution with content of hyaluronan and chondroitin sulfate

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

Table 19: Final composition of hydrogel containing HA in concentration 5 mg/mL and ChS in concentration 5 mg/mL

Example 10

Preparation of hydrogels containing HA in concentration 5 mg/mL and ChS in concentration 10 mg/mL

Preparation of hydrogels containing non-crosslinked hyaluronic acid and chondroitin sulfate comprised 3 basic steps:

1) Preparation of hydrogel containing crosslinked derivative crossHA-TA

Hydrogel containing crosslinked derivative crossHA-TA was prepared by mixing two precursor solutions A and B, that had been prepared by dissolving of individual components in phosphate buffered saline (PBS). For preparation of precursor solutions was used hydroxyphenyl derivative of HA-TA of Mw = 8.09 x 10⁵ g.mol ¹ and DS 1.1 %. Composition of the solutions is shown in the following table (Table 20).

Table 20: Composition of precursor solutions for preparation of crosslinked derivative crossHA-TA

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

Table 21: Composition of crosslinked derivative crossHA-TA

2) Preparation of solution with content of hyaluronan and chondroitin sulfate Solution of HA in concentration 10 mg/mL and ChS in concentration 20 mg/mL was prepared by dissolving native hyaluronan of Mw 1.91 x 10⁶ g.mol ¹ and chondroitin sulfate of MwlO x 10³ - 40 x 10³ g.mol ¹ in phosphate buffer (PBS).

Final hydrogel was prepared by mixing crosslinked derivative crossHA-TA and solution HA and ChS in ratio 1:1 with following homogenization of the mixture. Final composition of the material is shown in the following table (Table 22).

Table 22: Final composition of hydrogel containing HA in concentration 5 mg/mL and ChS in concentration 10 mg/mL

Example 11

Preparation of hydrogels based on hydroxyphenyl derivative of HA-TA in concentration 5 mg/mL

Hydrogels were prepared by mixing two precursor solutions A and B, for whose preparation had been used aqueous solution of NaCl (9 g/L). For preparation of precursor solutions was used hydroxyphenyl derivative HA-TA of Mw = 1.5 x 10⁶ g.mol ¹ and DS 0.5%. Composition of the solutions is shown in the following table (Table 23).

Solution A Solution B

Table 23: Composition of precursor solutions for preparation of hydrogels based on hydroxyphenyl derivative HA-TA in concentration 5 mg/mL

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

Table 24: Final composition of hydrogels based on hydroxypheny derivative HA-TA in concentration 5 mg/mL

Example 12

Preparation of hydrogels based on hydroxyphenyl derivative HA-TA in concentration 30 mg/mL

Hydrogels were prepared by mixing two precursor solutions A and B, for whose preparation had been used aqueous solution NaCl (9 g/L). For preparation of precursor solutions was used hydroxyphenyl derivative HA-TA of Mw = 5 x 10⁴ g.moT¹ and DS 10%. Composition of solutions is shown in the following table (Table 25).

Table 25: Composition of precursor solutions for preparation of hydrogels based on hydroxyphenyl derivative HA-TA in concentration 30 mg/mL By mixing of solution A and solution B in ratio 1:1 were prepared hydrogels with final composition shown in the following table (Table 26) where crossHA-TA is covalently crosslinked hydroxyphenyl derivative of hyaluronan.

Table 26: Final composition of hydrogels based on hydroxypheny derivative HA-TA in concentration 30 mg/mL.

Example 13

Degradation of solutions HA with addition of ChS using ROS

For comparison of the rate of degradation of the solutions HA with ChS by the action of ROS were prepared solutions of hyaluronan in phosphate buffer (PBS) with various ChS concentration. Hyaluronic acid of Mw 1.91 x 10⁶ g.mol ¹ and ChS of Mw = 10 x 10³ - 40 x

10³ g.mol ¹ were used for preparation of 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. Accomplishment of the degradation experiment

Degradation rate was expressed as percentage decrease in viscosity of the solutions at shear rate 0.1 s ¹ versus initial value. Measurement of the viscosity decrease was carried out on reometer Kinexus Malvern in configuration cone -plate. Cone of diameter 40 mm with apex angle 1° was used. Material degradation proceeded in 10 mL syringes, where into 9 mL material was added 0.5 ml solution CuSC at concentration 0.25 mmol/L followed with 0.5 ml solution H2O2 at concentration 2.5 mmol/L. During ongoing hydrogel degradation were in predetermined time intervals withdrawn samples of the material, in which the viscosity at 25 °C and shear rate 0.1 s ¹ was measured. Total period of degradation was 3 h.

Fig. 1 shows degradation of chains of hyaluronan that is expressed in percentage decrease of viscosity of the solution in time. From Fig. 1, the influence of ChS concentration on the hyaluronan degradation rate can be seen. The HA degradation rate caused by ROS decreases with growing ChS concentration. Viscosity of solution A that contained no ChS decreased in 3 hours by 97% versus initial value before degradation, solution B (with the addition of 0.5 mg/mL ChS) by 89%, solution C, in which 1 mg/mL ChS was added 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 L (20 mg/mL + 20 mg/mL ChS) only by 8 %.

Example 14

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

3 types of materials were prepared for comparison of the rate of degradation of hydrogels by the action of ROS.

Material A is solution of HA at concentration 20 mg/mL that was prepared by dissolving HA of Mw 1.91 x 10⁶ g.mol ¹ in PBS.

Material B is a mixture of crosslinked derivative crossHA-TA and non-crosslinked HA that was prepared according to example 7.

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

Degradation rate was expressed as percentage decrease in viscosity of the materials at shear rate 0.1 s ¹ versus initial value. Measurement of the decrease of the viscosity was carried out on reometer Kinexus Malvern in configuration cone-plate. Cone of diameter 40 mm with apex angle 1° was used. Material degradation proceeded in 10 mL syringes, where into 9 mL material was added 0.5 ml solution CuSCL at concentration 0.25 mmol/L followed with 0.5 ml solution H2O2 at concentration 2.5 mmol/L. During ongoing hydrogel degradation were in predetermined time intervals withdrawn samples of the material, in which the viscosity at 25 °C and shear rate 0.1 s ¹ was measured. Total period of degradation was 3 h. Lrom Lig. 2 it is apparent that presence of ChS in prepared hydrogels (C - 5 mg/mL ChS; D - 10 mg/mL ChS) increases their resistance to the action of ROS. Example 15

Enzymatic degradation of hydrogels based on crosslinked derivative crossHA-TA

3 types of hydrogels were prepared for determining the influence of the presence of ChS on rate of degradation of hydrogels based on crossHA-TA by the action of bovine testicular hyaluronidase:

A - hydrogels without addition of chondroitin sulfate that were prepared according to the protocol of Example 2

B - hydrogels with addition of ChS at concentration 3.3 mg/mL that were prepared according to the protocol of Example 4 C - hydrogels with addition of ChS at concentration 10 mg/mL that were prepared according to the protocol of Example 5.

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

Hydrogels were immersed in degradation medium (solution of hyaluronidase BTH of activity 30 U/mg in solution of bovine serum albumin (BSA) in concentration 0.1 mg/mL in 0.01 mol/L acetate buffer (OP) pH 5.3). Degradation of hydrogels proceeded in incubator at 37 °C with stirring. After determined time intervals samples of degradation medium with products of hydrogel degradation were withdrawn. Concentration of disaccharide units HA in degradation medium was determined spectrophotometrically as concentration of N- acetylglucos amine. Fig. 3 represents increase of concentration of hydrogel degradation products in the medium in time. From the Fig. it is apparent that presence of ChS in hydrogels based on crossHA-TA leads to a decrease of the rate of degradation of the materials by the action of hyaluronidase.

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Claims

1. Hydrogel based on a crosslinked hydroxyphenyl derivative of hyaluronic acid, characterized in that, it contains 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.

2. Hydrogel according to claim 1, characterized in that, alkali salts or salts of alkali earth metal are selected from the group comprising of Na⁺, K⁺, Ca²⁺, Mg²⁺.

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 0.5 to 50 mg/mL hydrogel, preferably at concentration 1 to 20 mg/mL, more preferably 5 mg/mL.

4. Hydrogel according to any of claims 1 to 3, characterized in that, the content of crosslinked hydroxyphenyl derivative of hyaluronan is in the range 5 to 30 mg/mL, preferably 10 mg/mL hydrogel.

5. Hydrogel according to any of claims 1 to 4, characterized in that, it further contains hyaluronic acid or its pharmaceutically acceptable salt at concentration 1 to 20 mg/mL hydrogel, preferably 5 to 10 mg/mL hydrogel, more preferably 5 mg/mL hydrogel.