巴西专利BR112014020156B1 DERIVATIVE BASED ON HYALURONIC ACID, METHOD OF PREPARING THE DERIVATIVE, HYDROGEL BASED ON THE CROSS

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专利摘要:
derivative based on hyaluronic acid, method of preparing the derivative, hydrogel based on the crosslinked derivative, hydrogel production method and use of the hydrogel the invention refers to a new hyaluronane derivative, according to the general formula (i) , to its method of preparation, to the hydrogel based on the new derivative, to the method of preparing the hydrogel and to the use of the hydrogel in the engineering of tissues, cosmetics, medicines or regenerative drugs, especially in the form of scaffolds for the treatment of joint cartilage or defects in bone tissue. (i)
公开号:BR112014020156B1
申请号:R112014020156-0
申请日:2013-02-26
公开日:2020-12-15
发明作者:Krzystof Niedoba；Lucie Wolfova；Marcela Foglarova；Martin Pravda；Miroslava Nemcova；Vladimir Velebny
申请人:Contipro A.S；
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FIELD OF THE INVENTION
[001] The invention relates to a new hyaluronic acid derivative, which is suitable for the preparation of hydrogels, and its respective method of preparation. It also refers to hydrogels based on this derivative, their properties, use and method of preparation. TECHNICAL STATUS
[002] Hyaluronan is a polysaccharide composed of disaccharide units containing D-glucuronic acid and D-N-acetylglucosamine, which are joined by alternating β-1,4 and β-1,3 glycosidic bonds. The average molecular weight (whenever the expression "molecular weight" is mentioned in the present, it will refer to the average molecular weight) in vivo is within the range of 3 kDa - 20 MDa. It is a polysaccharide easily soluble in aqueous medium, in which highly viscous solutions are formed, depending on their molecular weight and concentration.
[003] Hydrogels are materials that are formed in water by an insoluble network of polymers that are, at least partially, hydrophilic1. There are several ways to produce an insoluble network from an initially hydrophilic polymer. It is the hydrophobization2 of polymer or the use of a water-soluble polymer derivative that contains reactive functional groups, which may be present in subsequent chemical reactions, leading to the formation of a three-dimensional network of polymers3-5.
[004] The preparation of soluble hyaluronan derivatives and their consequent cross-links have been described by countless authors3-6. In the past, the use of phenolic derivative of hyaluronan for cross-linking reactions and the preparation of hydrogels has also been described. Calabro et. al. 4,7,8 describe the method of preparing phenolic hyaluronan derivatives by means of a reaction of carboxyls that are present within the structure of D-glucuronic hyaluronic acid with phenol aminoalkyl derivatives. This reaction produces hyaluronan amides. The fundamental characteristic for the process of this synthesis is the activation of the hyaluronan carboxyl for which the reaction with dehydrating agents of the carbodiimide type (such as EDC) is used. The most frequently used aminoalkyl phenol is tyramine6.
[005] In general, the cross-linking of phenolic derivatives of hyaluronan is initiated by adding a peroxidase (such as horseradish peroxidase - HRP) and a diluted hydrogen peroxide solution. Currently, horseradish peroxidase, HRP, E.C.1.11.1.7) is widely used as a catalyst for organic reactions and biotransformation9-13. It is characterized by a very broad substrate specificity and, therefore, is capable of oxidizing numerous organic and inorganic compounds13-15.
[006] It is an enzyme that has a sheath containing iron, like a prosthetic group. Iron has a degree of oxidation (III) in the non-activated state of the enzyme. The reaction with peroxides leads to the formation of an intermediate called HRP-I. The Fe (III) iron sheath is oxidized to the oxiferril group (Fe (IV) = O) and, at the same time, a cationic π- radical is formed in the porphyrin cycle. This activated enzyme is able to form complexes with the substrate molecules that, during this interaction, undergo oxidation.14,16-18
[007] The conversion of the oxidized enzyme to its initial form takes place in two stages. In the first, there is a reaction between the substrate molecule (S) and HRP-I, giving rise to the substrate radical (R ') and a partially reduced form of the HRP-II enzyme. HRP-II still conserves the oxiferril group (Fe (IV) = O), but it no longer contains the π-radical porphyrin. During the transition from an electron to the porphyrin radical, an H + is acquired by the protein at the same time. HRP-II undergoes the reaction with the substrate again, giving rise to the R ». The oxiferril group (Fe (IV) = O) is reduced to Fe (III) again during this reaction. This process is associated with the transfer of 2 H + to oxygen from the oxiferril group. A proton originates from the substrate (or solvent), the other form of the protein. This results in the formation of a water molecule (Equation I and Scheme I).
Equation I: Basic description of the catalysis mechanism of substrate oxidation by HRP
[008] The radicals resulting from the substrate are numerous times capable of interacting together, forming R-R dimers. This process is no longer affected by enzymatic means and is related to the stability and reactivity of the resulting radicals. 14.16-26
[009] Therefore, in the case of a cross-linked enzymatic reaction of the phenolic derivative of the polysaccharide, the substrate (phenol - reactive ligand attached to the polymer) is transformed into a reactive radical by means of an enzyme. This radical can then react with another phenolic radical, forming dithyramine. Assuming the free mobility of the molecules of the substrate (ligand) of the enzymatic reaction, and the reaction process following exactly Equation I, the enzyme should gradually transform (if a sufficient amount of peroxide is used) all the molecules of the substrate into radicals reactive, and these should all gradually undergo dimerization (or oligomerization), if sufficiently long reaction time is provided. In case of binding of the substrate (binder) to the polymer, the degree of cross-linking of the polymer must always reach the same value, although the time to obtain this value may vary, depending on the amount of enzyme used. In practice it is different, however. The literature27 describes in detail the relationship between the expected rate of intramolecular and intermolecular cross-links, as well as the molecular weight of the polymer segments between the cross-link sites (density of cross-links, distance between nodes in the network), while intramolecular interactions that lead to cross-links are indicated as elastically ineffective, if compared with intermolecular cross-links.
[010] Furthermore, it is known from the literature that in the case of the use of phenolic derivatives of HA, the amount of enzymes not only affects the speed of the cross-linking reaction, but also significantly affects the mechanical properties resulting from hydrogels4,6,7,28. The literature points out that, using rheological measures, it was found that the cut-off module (G ') is greater if a higher concentration of the enzyme is used. The reason for this phenomenon, according to the authors, is a higher cross-link density of hydrogels. If a hydrogel is prepared with maximum firmness, the cross-linking reaction should proceed with a relatively high concentration of peroxidase, and thus also faster. However, very rapid reaction processes can lead to the formation of a non-homogeneous cross-linked hydrogel. Locations can then appear in the samples without any cross-linking. In addition, very fast reaction processes can also cause problems when placing the gel at the site of their final application and others.
[011] The cause of this is a short distance from the reactive center of the basic polymer chain. The low mobility of the ligand decreases the likelihood of an effective collision of the ligand radicals to form dithyramine. Therefore, if there is a low concentration of the enzyme within the system, a small amount of reactive ligand forms can form in a unit of time. So, the slow cross-linking reaction is ineffective.
[012] Park et. al.29 tried to increase the reactivity of the ligands attached to the polymer by introducing a suitable spacer between the reactive ligand and the polymer chain. The document describes the insertion of a hydrophilic chain between the polysaccharide chain and the phenol or aniline ring to increase the reactivity of these substitutes. The main reason for the introduction of the hydrophilic chain in the polymer structure was to improve its solubility and improve the accessibility of the reactive centers (phenol or aniline ring). Facilitating the spatial accessibility of the reactive centers increases the likelihood of the reaction between the ligands. Most of the time, maintaining the same enzymatic activity, this step produces a higher degree of substitution, greater concentration and better homogeneity of the hydrogels cross-linking. In addition, according to the author, thanks to the introduction of this hydrophilic chain in the hydrogel structure, the biostability and mechanical properties of the hydrogel are increased. However, Park et al. use as a "spacer" a hydrophilic PEG polymer with a molecular weight of 3500 Da, and therefore, in the end it is more of a copolymer. However, such an intervention in the hydrogel structure, even in a low degree of substitution, produces significant changes in the physical properties of the original polymer. Furthermore, in the case of hyaluronan, a higher concentration of cross-links produces an increase in the hardness of the hydrogel, and at the same time, it also causes an increase in its fragility, which is undesirable for the intended use in tissue engineering. For example, when it comes to support material (scaffolds), for example, but not only, to support articular cartilage, the emphasis is placed on sufficient strength and resistance, while a more fragile material would be irreversibly deformed. with a higher charge, and, in the case of hydrogels, even total destruction can occur. SUMMARY OF THE INVENTION
[013] The aim of the invention is, therefore, to find a material that is strong enough and at the same time tenacious, and that does not show any significant changes in biological and physical properties compared to the original polymer. The resistance of the hyaluronan-based hydrogel in general can be increased by increasing the concentration of cross-links, such as by increasing the concentration of the polymer in the solution from which the hydrogel is formed, or by increasing the degree of polymer substitution. However, in the state of the art in the case of hyaluronan these two methods have also led to an increase in the fragility of the resulting hydrogel, which significantly limits the possible uses of the hydrogel.
[014] The solution that this invention presents to the problem is to find those derivatives that lead to an increase in the reactivity of the binders and an increase in the resistance of the hydrogel, while maintaining the physical and biological properties of the original polymer. Surprisingly, it was found that the introduction of a relatively small spacer (with a molecular weight of approximately 130 Da), according to the invention, between the reactive binder and HA, results in a significant increase in the toughness of the final hydrogels to a very low degree of substitution.
[015] Therefore, in one aspect the invention relates to an HA derivative containing reactive ligands joined by means of hydrophobic spacers, in order to increase the mobility of the ligands and thereby increase the likelihood of an effective collision of the same, even in the case of low concentration (low degree of substitution and low enzyme activity). It was found that despite the very low weight abundance of the spacer within the hydrogel, showing, for example, only 0.01 to 0.02%, a significant increase in the hydrogel's toughness and strength is obtained compared to a hydrogel based on an analogous HA derivative with no spacer inserted (i.e., identical concentration, molar weight and degree of substitution / cross-linking). Therefore, the invention relates to this new hyaluronan derivative suitable for the preparation of hydrogels and their respective method of preparation. It also refers to hydrogels based on this derivative, its use and its preparation method.
[016] The hydrogel is prepared using a method that uses crosslinked chains of the modified hyaluronan through a reaction that is catalyzed by horseradish peroxidase or its analogs. Suitable hyaluronan derivatives contain in their structure phenol or heteroaryl phenol rings covalently joined to the basic polysaccharide chain. The cross-linking procedure can be described as a cascade of consecutive chemical reactions that begin with the formation of reactive oxygen forms (ROS) within the system. These are added to the mixture or their formation is activated by the presence of chemical compounds that work as a "generator". The ROS activate the enzyme peroxidase or its analogs, which subsequently catalyzes the dimerization (or oligomerization) of the aromatic or heteroaromatic rings present within of the structure of the hyaluronan derivative. This leads to the formation of a three-dimensional polymer network.
[017] In accordance with the invention, hyaluranane modified by the joining of a linker containing aminoalkyl phenol or aminoalkyl heteroaryl phenol (eg, tyramine, 5-hydroxy tryptophan, serotonin) is used for the preparation of this hydrogel. The hyaluronan derivatives described in this invention contain a linker that is joined to the polysaccharide by means of a spacer. The presence of this spacer within the structure of the HA derivative leads, thanks to its flexibility, to an increase in elasticity and freedom of possibilities of conformational arrangement of the participating polymer segments, and thus also to the possibility of dissipating the deformation energy. The introduction of a spacer also increases the distance from the reactive aromatic center (phenol, heteroaryl phenol) to the basic polymer chain, improves its accessibility for an interaction with the enzyme and significantly affects the course of the cross-linking reaction and the properties of the resulting hydrogel.
[018] In its first aspect, the invention relates to the derivative based on hyaluronic acid according to the general formula (I)
where Ar is phenyl and R1 is ethylene, ethylene, or Ar is indole and R1 is carboxyethylene, and where R2 is an alkyl having 3 to 7 carbons, and where n is within the range of 1 to 7500.
[019] In another aspect, the invention relates to the method of preparation of the derivative according to general formula (I), where first an aldehyde derivative of hyaluronic acid according to formula (II) is prepared,
where the aldehyde derivative is prepared using the 4-acetamido-TEMPO / NaClO oxidation system in a protic medium and has a substitution degree of 5 - 15% and molecular weight within the range of 10000 g / mol to 2000000 g / mol, then , separately, the compound according to general formula (III) is generally prepared <<DRAWCODE>> where Ar is phenyl and R1 is ethylene, or Ar is indole and R1 is ethylene, or Ar is indole and R1 is carboxyethylene, and where R2 is an alkyl having 3 to 7 carbons, and where n is within the range of 1 to 7500, where the compound according to the general formula (III) is prepared by a reaction of a spacer precursor according to the formula (IV) Z-NH — R2 — COOH (IV), where Z is a protecting group that is generally used to protect the primary amino group, with the linker according to formula (V)
in an aprotic medium at a temperature within the range of 40oC to 150oC for 1 to 24 hours in the presence of an activating agent for the carboxylic functional groups, forming the compound according to the general formula (VI) Z-NH — R2 — CO -NH-R1-Ar-OH (VI), from which the compound according to the general formula (III) is prepared by removing the protective group Z, and then the aldehyde derivative of hyaluronic acid according to the formula (II) is placed to react with the compound according to the general formula (III) at a pH within the range of 3 to 8 at room temperature for 1 to 72 hours in the presence of a picoline-borane complex, forming the derivative according to with formula (I).
[020] Therefore, the derivative according to the invention contains a linker capable of undergoing oligomerization by treatment with a suitable agent, and a flexible spacer that is inserted between the hyaluronan chain and the linker. The binder according to the general formula (V) according to the invention is preferably selected from the group containing tyramine, serotonin and 5-hydroxytryptophan. The compound according to the general formula (IV), that is, the spacer precursor is preferably selected from the group of amino acids including derivatives of w - [(tert butoxycarbonyl) amino] carboxylic acids where R2 is an alkyl with 3 to 7 carbons .
[021] In another embodiment of the method according to the invention, the reaction of the spacer precursor with the ligand takes place in THF or DMF at 50 ° C for 2 to 6 hours in the presence of 1,1'-carbodiimidazole.
[022] In addition, it is preferable that the removal of the protective group Z is done using trifluoroacetic acid or hydrochloric acid.
[023] For the purpose of this invention only, the intermediate spacer-linker is represented by the compounds according to the general formula: HO-Ar-R1-NH-CO-R2-NH2
[024] Preferably, the compound according to the general formula: -CO-R2-NH2 where R2 is an alkyl having 3 to 7, is used as a spacer.
[025] The method of preparing the derivative according to the invention can be characterized by Scheme I:
Scheme I: Example of a possible method of preparing the HA-spacer-ligand derivative according to the invention
[026] Furthermore, the invention relates to the hydrogel formed by cross-linking the derivative according to the general formula (I) and a method of preparing it. This method of preparing the hydrogel is that the derivative according to the general formula (I) is treated using a generator of reactive phenoxy radicals, preferably using a horseradish peroxidase system and a source of hydroxy radicals, which can be a solution of hydrogen peroxide in water, or an oxidase-oxygen-substrate system, for example, galactose oxidase - galactose or glucose oxidase - glucose, at pH within the range of 4 to 10.
[027] Therefore, for the oligomerization of the reactive ligands, agents capable of producing the formation of phenoxy radicals from the aromatic rings of the ligands are used. According to this invention, the peroxide / horseradish peroxidase system is preferably used. Peroxide can be introduced into the system as a diluted solution, or it is generated by chemical reaction in situ. Hydrogen peroxide can be generated in the mixture using various types of oxygen enzymes (oxidases), such as an electron receptor, and the respective electron donor in an oxidation-reduction reaction. Preferably, a combination of galactose oxidase or glucose oxidase and its substrates can be used: galactose and glucose can be used. Other agents capable of causing the formation of phenoxy radicals in the presence of a molecular oxygen are tyrosinase enzymes, lactase etc.
[028] As is well known, the properties of these hydrogels are affected by the chemical structure of the polymer and its concentration, as well as by the types of crosslinking agents chosen and the amount used. The physicochemical properties of the polymer (derived from HA) are mainly affected by the structure of the monomer, the conformation of the segments of the polymer chain, degree of cross-links and molecular weight. The mechanical properties of the polymer are also influenced by them. When the polymer is subjected to mechanical stress, its deformation occurs and a part of the absorbed deformation energy dissipates - it is consumed to change the conformation of the network nodes and segments of the polymer chain, and a part of the energy is inevitably transformed in heat. The amount of energy dissipated, and consequently also the possibility of adopting various conformation arrangements within the polymer structure, is associated with the rigidity of the macromolecular chains and reflects the degree of elastic resistance of the material to deformation. Polymer materials composed of rigid and inflexible chains and segments can then exhibit a low degree of elastic resistance to deformation and fragility.
[029] The increase in elasticity of these polymers is conducted according to the method of the invention, in which flexible segments are introduced into the polymer structure. These segments are characterized by a greater freedom of individual molecules on their connections through which they reach an increase in the possibilities of their conformational arrangement when subjected to the deformation energy, and in the possibilities of dissipating that energy. Therefore, the introduction of a suitable flexible spacer between the binder and the basic hyaluronan chain leads to greater elasticity, toughness and resistance of the final material, which is very beneficial for, for example, hydrogels intended for support (scaffolds) for the treatment of defects in certain tissues exposed to higher loads, such as articular cartilage and bones. As described above, the introduction of the flexible spacer between the binder and the basic hyaluronan chain can preferably be used also in the case where the mechanical properties of the hydrogels depend on the concentration of enzyme used as a catalyst for the cross-linking reaction. The introduction of the flexible spacer between the ligand and the basic hyaluronan chain provides sufficient steric accessibility of the reactive groups of the derivative for mutual dimerization, even after a partial cross-linking of the polymer.
[030] This solution results in the generation of a more effective cross-linking reaction, which causes greater homogeneity of the prepared hydrogels and thus leads to overcoming the technological problems associated with cross-linked hyaluronan modified by hydroxyphenyl or heteroaryl phenol (tyramine, serotonin etc.) in the event that the cross-linking agents are horseradish peroxidase and hydrogen peroxide (or another type of phenoxy radical generator).
[031] However, surprisingly, we also find that the introduction of our selected spacers between the binder and the basic hyaluronan chain leads, even in a very low degree of substitution, to a significant increase in the degree of elasticity, toughness and strength of the hydrogel final based on the mentioned HA derivative.
[032] In addition, the invention relates to the use of hydrogels based on derivatives according to the invention, especially in the field of tissue engineering, cosmetics, medicaments or regenerative medicaments. The use of the hydrogels described in the present application is especially intended for the basic material for forming support (scaffolds) in tissue engineering, mainly in the field of treatment of defects in joints and bones, such as coatings for wound healing, as a biodegradable barrier preventing the formation of post-surgical coalescences, for the increase of soft tissues and filling of tissue defects and others. When the hydrogel is used as a support material (scaffolds), the supoert (scaffolds) can be sown and not sown. If they are seeded, the type of cells to be incorporated in the support (scaffold) is selected according to the intended application site. BRIEF DESCRIPTION OF THE FIGURES
[033] Figure 1 represents the deformation properties ("stress-strain curves") obtained during the deformation determination of hydrogels based on derivatives prepared according to Examples VIII, IX, XI and XII in compression. PREFERRED EMBODIMENTS OF THE INVENTION 1. Example of the synthesis of derivatives
[034] The synthesis of hyaluronan derivatives was carried out in several steps (see Scheme 1). The first step is the preparation of an aldehyde derivative of hyaluronan (Example 1.7). Another step is the synthesis of several intermediate spacer-ligands (Examples 1.1 to 1.6) which were later joined to hyaluronan by the reductive amination process (Examples 1.9 - 1.14).
[035] Examples also include the synthesis of hyaluronan derivatives, in which the linker (tyramine, hydroxytryptophan) directly joins the polysaccharide without the need for a spacer (Examples VIII). These derivatives and the hydrogels prepared from them served to compare their properties with the properties of the derivatives described in the present application (HA-spacer-ligand derivatives - derivatives IX to XIV). Example 1.1: Synthesis of 6-amino-N- [2- (4-hydroxyphenyl) ethyl] hexanamide (spacer-intermediate linker (I))
[036] 6 - [(tert-butoxycarbonyl) amino] hexanoic acid (1.00 g, 4.3 mmol) was dissolved in 50 ml of tetrahydrofuran (THF). To that acid solution was added 1,1'-carbodiimidazole (0.70 g, 4.3 mmol). The mixture was heated to 50oC for sixty minutes, then the reaction vessel was washed with an inert gas. To the reaction mixture was added tyramine (0.59 g, 4.3 mmol). The mixture was again heated for another 2 hours. Then the THF was removed by means of reduced pressure distillation. The evaporation 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 dehydrated using a molecular sieve. Ethylacetate was removed by means of reduced pressure distillation. The evaporation residue was dissolved in 50 ml of MeOH and 2 ml of trifluoroacetic acid (TFA) was added. The solution was heated for 6 hours under reflux. The solvent was removed by means of reduced pressure distillation. The evaporation 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 dehydrated using a molecular sieve. Ethylacetate was removed by means of reduced pressure distillation. m = 0.75 g (70% theory) 1H NMR (D2O, ppm) δ: 1.17 (m, β H, Y-CH2-hexanoic acid); 1.48 (m, β H, p-CH2-hexanoic acid); 1.58 (m, β H, δ-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, aroma); 7.13 (d, 2 H, aroma). 13C NMR (D2O, ppm) δ: β4 (y-C-hexanoic acid); 26 (δ-C-hexanoic acid); YY (p-C-hexanoic acid); 35 (—C — CO—); 39 (—C — NH2); 40 (C — Ph); 63 (—C — NH — CO—); 115 (C3 aroma); 126 (C1 aroma); 130 (aromatic C2); 153 (C4 arom); 176 (-CO-). Example 1.2: Synthesis of 4-amino-N- [2- (4-hydroxyphenyl) ethyl] butanamide (spacer-intermediate linker (II))
[037] 4 - [(tert-butoxycarbonyl) amino] butanoic acid (0.50 g, 2.5 mmol) was dissolved in 25 mL of tetrahydrofuran (THF). To the acid solution, 1,1'-carbodiimidazole (0.40 g, 25 mmol) was added. The mixture was heated to 50 ° C for sixty minutes. Then the reaction vessel was washed with an inert gas. To the reaction mixture, tyramine (0.34 g, 25 mmol) was added. The mixture was again heated for another 2 hours. Then the THF was removed by means of reduced pressure distillation. The evaporation 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 dehydrated using a molecular sieve. Ethylacetate was removed by means of reduced pressure distillation. The evaporation residue was dissolved in 50 ml of MeOH and 2 ml of trifluoroacetic acid were added. The solution was heated for 6 hours under reflux. The solvent was removed by means of reduced pressure distillation. The evaporation 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 dehydrated using a molecular sieve. Ethylacetate was removed by means of reduced pressure distillation. m = 0.44 g (80% theory) 1H NMR (D2O, ppm) δ: 1.75 (m, β H, p-CH2-butanoic acid); 2.16 (t, 2 H, -CH2-CO-); 2.59 (m, 2 H, -CH2-In); 2.78 (m, 2 H, -CH2-NH2); 3.20 (m, 2 H, - CH2-NH-CO-); 6.69 (d, 2 H, aroma); 6.99 (d, 2 H, aroma). 13C NMR (D2O, ppm) δ: βY (p-C-butanoic acid); 25 (t, 2 H, -C-CO-); 32 (-C-NH2); 45 (CH2-Ar); 60 (-C- NH-CO-); 115 (C3 aroma); 117 (C1 aroma); 129 (aromatic C2); 155 (C4 aroma); 171 (-CO-). Example 1.3: Synthesis of 8-amino-N- [2- (4-hydroxyphenyl) ethyl] octanamide (spacer-intermediate linker (III))
[038] 8 - [(tert-butoxycarbonyl) amino] octanoic acid (0.50 g, 1.9 mmol) was dissolved in 25 ml of tetrahydrofuran (THF). To the acid solution, 1,1'-carbodiimidazole (0.31 g, 1.9 mmol) was added. The mixture was heated to 50 ° C for sixty minutes. Then the reaction vessel was washed with an inert gas. To the reaction mixture, tyramine (0.26 g, 1.9 mmol) was added. The mixture was again heated for another 2 hours. Then the THF was removed by means of reduced pressure distillation. The evaporation 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 dehydrated using a molecular sieve. Ethylacetate was removed by means of reduced pressure distillation. The evaporation residue was dissolved in 50 ml of MeOH and 2 ml of trifluoroacetic acid were added. The solution was heated for 6 hours under reflux. The solvent was removed by means of reduced pressure distillation. The evaporation 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 dehydrated using a molecular sieve. Ethylacetate was removed by means of reduced pressure distillation. m = 0.40 g (75% theory) 1H NMR (CDCl3, ppm) δ: 1.16-1.34 (m, 6 H, C4 az C6 -CH2-octanoic acid); 1.56 - 1.44 (m, 4 H, C3 to C7 octanoic acid); 2.58 (m, 2 H, -CH2-Ar); 2.78 (m, 2 H, -CH2-NH2); 3.19 (m, 2 H, -CH2-NH-CO-); 6.68 (d, 2 H, aroma); 6.98 (d, 2 H, aroma). 13C NMR (CDCl3, ppm) δ: 21 (C7 octanoic acid); 24 (C4 octanoic acid); 26 (C6-octanoic acid); 28 (C5-octanoic acid); 33 (C3-octanoic acid); 35 (-C-CO-); 39 (-C-NH2); 40 (C-Ph); 63 (-C-NH-CO-); 115 (C3 aroma); 126 (C1 aroma); 130 (aromatic C2); 153 (C4 arom); 176 (-CO-). Example 1.4: Synthesis of 4-amino-N- [2- (5-hydroxy-1H-indol-3-yl) ethyl] butanamide (spacer-intermediate linker (IV))
[039] 4 - [(tert-butoxycarbonyl) amino] butanoic acid (0.50 g, 2.5 mmol) was dissolved in 25 ml of N, N-dimethylformamide (DMF). To the acid solution, 1,1'-carbodiimidazole (0.40 g, 2.5 mmol) was added. The mixture was heated to 50 ° C for sixty minutes. Then the reaction vessel was washed with an inert gas. To the reaction mixture, a solution of 5-hydroxytryptamine hydrochloride (0.52 g, 2.5 mmol) and triethylamine (0.68 ml; 4.9 mmol) in 25 ml DMF was added. The mixture was again heated for a further 2 hours. The mixture was diluted by adding ethyl acetate (100 ml). The resulting solution was washed with 300 ml of purified water (divided into three parts). The organic layer was dehydrated using a molecular sieve . Ethylacetate was removed by means of reduced pressure distillation. The evaporation residue was dissolved in 50 ml of MeOH and 2 ml of trifluoroacetic acid were added. The solution was heated for 6 hours under reflux. The solvent was removed by means of reduced pressure distillation. The evaporation 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 dehydrated using a molecular sieve. Ethylacetate was removed by means of reduced pressure distillation. m = 0.43 g (65% theory) 1H NεR: (DεSO, ppm) δ: 1.77 (m, β H, 3-CH2-butanoic acid); 2.20 (t, 2 H, -CH2-CO-); 2.73 (m, 2 H, -CH2-In); 2.81 (m, 2 H, -CH2-NH2); 3.30 (m, 2 H, - CH2-NH-CO-); 6.60 (d, 1 H, C6-aroma); 6.82 (s, 1 H, C4-aroma); 7.03 (s, 1 H, C2-aroma); 7.13 (d, 1 H, C7-arom). 13C NMR (DMSO, ppm) δ: 23 (p-C-butanoic acid); 25 (t, 2 H, -C-CO-); 32 (-C-NH2); 39 (CH2-In); 60 (-C- NH-CO-); 102 (C4 arom); 110 (C6 aroma); 111 (aromatic C7); 111 (aromatic C3); 123 (C2 aroma); 127 (C7 - C-NH-arom); 131 (C4-C-C3-arom); 150 (C5 aroma); 171 (-CO-). Example 1.5: Synthesis of 6-amino-N- [2- (5-hydroxy-1H-indol-3-yl) ethyl] hexanamide (spacer-intermediate linker (V))
[040] 6 - [(tert-butoxycarbonyl) amino] hexanoic acid (1.00 g, 4.3 mmol) was dissolved in 50 ml of N, N-dimethylformamide (DMF). To the acid solution, 1,1'-carbodiimidazole (0.70 g, 4.3 mmol) was added. The mixture was heated to 50 ° C for sixty minutes. Then the reaction vessel was washed with an inert gas. To the reaction mixture, a solution of 5-hydroxytryptamine hydrochloride (0.91 g, 4.3 mmol) and triethylamine (0.68 ml; 49 mmol) in 25 ml DMF was added. The mixture was again heated for a further 2 hours. The mixture was diluted by adding ethyl acetate (100 ml). The resulting solution was washed with 300 ml of purified water (divided into three parts). The organic layer was dehydrated using a molecular sieve . Ethylacetate was removed by means of reduced pressure distillation. The evaporation residue was dissolved in 50 ml of MeOH and 2 ml of trifluoroacetic acid were added. The solution was heated for 6 hours under reflux. The solvent was removed by means of reduced pressure distillation. The evaporation 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 dehydrated using a molecular sieve. Ethylacetate was removed by means of reduced pressure distillation. m = 0.75 g (60% theory) 1H NεR: (DεSO, ppm) δ: 1.17 (m, β H, Y-CH2-hexanoic acid); 1.48 (m, β H, p-CH2-hexanoic acid); 1.58 (m, β H, δ-CH2-hexanoic acid); 2.17 (t, 2 H, -CH2-CO-); 2.73 (m, 2 H, -CH2-In); 2.91 (m, 2 H, - CH2-NH2); 3.42 (m, 2 H, -CH2-NH-CO-); 6.60 (d, 1 H, C6-aroma); 6.82 (s, 1 H, C4-aroma); 7.03 (s, 1 H, C2-aroma); 7.13 (d, 1 H, C7-arom). 13C NεR (DεSO, ppm) δ: β4 (Y-C-hexanoic acid); 26 (δ-C-hexanoic acid); YY (p-C-hexanoic acid); 35 (-C-CO-); 39 (-C-NH2); 40 (C-In); 63 (-C-NH-CO-); 102 (C4 arom); 110 (C6 aroma); 111 (aromatic C7); 111 (aromatic C3); 123 (C2 aroma); 127 (C7 - C-NH-arom); 131 (C4-C-C3-arom); 150 (C5 aroma); 171 (-CO-). Example 1.6: Preparation of 2 - [(6-aminohexanoyl) amino] -3- (5-hydroxy-1H-indol-3-yl) -propanoic acid (spacer-intermediate linker VI)
[041] 6 - [(tert-butoxycarbonyl) amino] hexanoic acid (0.50 g, 2.2 mmol) was dissolved in 50 ml of tetrahydrofuran (THF). To that acid solution was added 1,1'-carbodiimidazole (0.35 g, 2.2 mmol). The mixture was heated to 50 ° C for sixty minutes. Then the reaction vessel was washed with an inert gas. To the reaction mixture, 5-hydroxytryptophan (0.48 g, 2.2 mmol) was added. The mixture was again heated for a further 2 hours. The mixture was diluted by adding ethyl acetate (100 ml). The resulting solution was washed with 300 ml of purified water (divided into three parts). The organic layer was dehydrated using a molecular sieve . Ethylacetate was removed by means of reduced pressure distillation. The evaporation residue was dissolved in 50 ml of MeOH and 2 ml of trifluoroacetic acid were added. The solution was heated for 6 hours under reflux. The solvent was removed by means of reduced pressure distillation. The evaporation 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 dehydrated using a molecular sieve. Ethylacetate was removed by means of reduced pressure distillation. m = 0.62 g (85% theory) 1H NεR: (DεSO, ppm) δ: 1.17 (m, β H, Y-CH2-hexanoic acid); 1.48 (m, β H, p-CH2-hexanoic acid); 1.58 (m, β H, δ-CH2-hexanoic acid); 2.19 (t, 2 H, -CH2-CO-); 2.51 (m, 2 H, -CH2-In); 2.90 (m, 2 H, - CH2-NH2); 3.81 (m, 2 H, -CH2-NH-CO-); (m, 2 H, -CH2-NH-CO-); 6.61 (d, 1 H, C6-aroma); 6.95 (s, 1 H, C4-aroma); 7.02 (s, 1 H, C2-aroma); 7.13 (d, 1 H, C7-arom). 13C NεR (DεSO, ppm) δ: β4 (y-C-hexanoic acid); 26 (δ-C-hexanoic acid); YY (p-C-hexanoic acid); 35 (—C — CO—); 39 (—C — NH2); 40 (C — Ph); 55 (—C — NH — CO—); 102 (C4 arom); 110 (C6 aroma); 111 (aromatic C7); 111 (aromatic C3); 123 (C2 aroma); 127 (C7 - C-NH-arom); 131 (C4-C-C3-arom); 150 (C5 aroma); 171 (-CO-). Example 1.7: Preparation of aldehyde derivative (HA-CHO) - general procedure (VII)
[042] Hyaluronan (10.00 g, Mw. = 2 MDa) was dissolved in 750 mL of 2.5% (w / w) Na2HPO4 solution. 12 H2O. The solution was cooled to 5 ° C. To the resulting solution, 2.60 g of NaBr and 0.05 g of 4-acetamide-2,2,6,6-tetramethylpiperidine-1-oxyl were added. After a complete homogenization of the solution, 3 mL of the NaClO solution (10-15% available Cl2) was added to the reaction mixture. The reaction proceeded for 15 min and was continuously stirred. The reaction was stopped with the addition of 100 ml of 40% propane-2-ol solution. The product was purified by ultrafiltration and isolated by propane-2-ol precipitation. IR (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, YH, CH3-), 3.37 - 3.93 (m, hyaluronan body), 4.46 (s, 1H, anomer), 4.54 (s, 1H anomer., -O-CH ( OH) -), 5.27 (geminal glycol -CH- (OH) 2). Example 1.8: Tyramine derivative synthesis (VIII)
[043] Aldehyde HA derivative (VII) (5.00 g) was dissolved in 500 ml of demineralized water. The pH of the solution was adjusted to 3 using acetic acid. Then, tyramine as a solution in 100 ml of 40% propane-2-ol was added to the reaction mixture (1.70 g). The mixture was stirred for an additional 1 hour at room temperature. Then a solution of the picoline-borane complex (0.50 g) in 50 ml of 40% propane-2-ol was added to the mixture. The reaction mixture was stirred for an additional 12 hours at room temperature. Low molecular weight ballast substances were removed from the product by ultrafiltration. The product was obtained by precipitation with propane-2-ol. Moisture and residual propane-2-ol were removed from the precipitate by drying in a hot air dryer (40 ° C, 3 days). IR (KBr) :: 3400, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm-1. 1H NMR (D2O) δ: 2.01 (s, YH, CH3-), 2.66 - 2.77 (m, 4 H, -CH2-CH2-NH-), 3.00 (s, 1H, H-CH-NH-), 3.37 - 3.93 (m, hyaluronan body), 4.46 (s, 1H, anomer), 4.54 (s, 1H anomer., -O-CH (OH) -), 6.59 (d, 2H, aroma), 7.04 (d , 2H. Arom). Example 1.9: Preparation of the tyramine HA derivative with a C6 (IX) spacer
[044] Aldehyde HA derivative (VII) (5.00 g) was dissolved in 500 ml of demineralized water. The pH of the solution was adjusted to 3 using acetic acid. Then, 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. Then the picoline-borane complex (0.270 g, 2.5 mmol) was 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 material retained by propane-2-ol precipitation. Moisture and residual propane-2-ol were removed from the precipitate by drying in a hot air dryer (40 ° C, 3 days). IR (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, Y -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-), 2.7Y (m, 2H, e-CH2- aminohexanoic acid), 3.37 - 3.93 (m, hyaluronan body), 4.46 (s, 1H, anomer), 4.54 (s, 1H anomer., -O-CH (OH) -), 6.59 (d, 2H, aroma), 7.01 (d, 2H. Aroma). Example 1.10: Preparation of the HA derivative with a C4 spacer and 5-hydroxy tryptamine (X)
[045] Derived from aldehyde HA (VII) (3.00 g) and Na2HPO4. 12 H2O (7.50 g) was dissolved in 300 ml of demineralized water. Then 4-amino-N- [2- (5-hydroxy-1H-indol-3-yl) ethyl] amide butane (0.40 g, 1.5 mmol) - (intermediate (IV)) was added to the solution of HA-CHO. The mixture was stirred for 2 hours at room temperature. Then the picoline-borane complex (0.16 g, 1.5 mmol) was 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 material retained by propane-2-ol precipitation. Moisture and residual propane-2-ol were removed from the precipitate by drying in a hot air dryer (40 ° C, 3 days). IR (KBr) :: 3400, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm-1. 1H NMR (D2O) δ: 1.73 (m, β H, β -CH2- aminobutanoic acid), 2.01 (s, 3 H, CH3), β.60 (m, 2H, Y -CH2- aminobutanoic acid), 2.93 ( m, 2H, Ind-CH2), 3.37 - 3.93 (m, hyaluronan body), 4.46 (s, 1H, anomer), 4.54 (s, 1H anomer., -O-CH (OH) -), 6.85 (d , 1H, aroma), 7.09 (s, 1H, aroma), 7.21 (s, 1H, aroma), 7.40 (s, 1H, aroma). Example 1.11: Preparation of the HA derivative tainted with a C4 spacer (XI)
[046] Aldehyde HA derivative (VII) (3.50 g) was dissolved in 350 ml of demineralized water. The pH of the solution was adjusted to 3 using acetic acid. Then, 4-amino-N- [2- (4-hydroxyphenyl) ethyl] amide butane (0.40 g, 1.8 mmol) - (intermediate (II)) was added to the HA-CHO solution. The mixture was stirred for 2 hours at room temperature. Then the picoline-borane complex (0.19 g, 1.8 mmol) was 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 material retained by propane-2-ol precipitation. Moisture and residual propane-2-ol were removed from the precipitate by drying in a hot air dryer (40 ° C, 3 days). IR (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, Y -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-), 2.7Y (m, 2H, e-CH2- aminohexanoic acid), 3.37 - 3.93 (m, hyaluronan body), 4.46 (s, 1H, anomer), 4.54 (s, 1H anomer., -O-CH (OH) -), 6.59 (d, 2H, aroma), 7.01 (d, 2H. Aroma). Example 1.12: Preparation of the HA derivative tainted with a C8 spacer (XII)
[047] Aldehyde HA derivative (VII) (2.90 g) was dissolved in 300 ml of demineralized water. The pH of the solution was adjusted to 3 using acetic acid. Then, 8-amino-N- [2- (4-hydroxyphenyl) ethyl] amide octane (0.40 g, 1.4 mmol) - (intermediate (III)) was added to the HA-CHO solution. The mixture was stirred for 2 hours at room temperature. Then the picoline-borane complex (0.15 g, 1.4 mmol) was 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 material retained by propane-2-ol precipitation. Moisture and residual propane-2-ol were removed from the precipitate by drying in a hot air dryer (40 ° C, 3 days). IR (KBr) :: 3425, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm-1. 1H NMR (D2O) δ: 1.16-1.34 (m, 6 H, C4 to C6 - CH2 - octanoic acid); 1.56 - 1.44 (m, 4 H, C3 to C7 octanoic acid); 2.01 (s, 3 H, CH 3 -), 2.58 (m, 2 H, -CH 2 -Ar); 2.78 (m, 2 H, -CH2-NH-), 3.37 - 3.93 (m, hyaluronan body), 4.46 (s, 1H, anomer), 4.54 (s, 1H anomer., -O-CH (OH) - ), 6.59 (d, 2H, aroma), 7.01 (d, 2H, aroma). Example 1.13: Preparation of the HA derivative with a C6 spacer and 5-hydroxy tryptamine (XIII)
[048] Derivative of aldehyde HA (VII) (5.00 g) and Na2HPO4. 12 H2O (12.5 g) were dissolved in 500 mL of demineralized water. Then, 6-amino-N- [2- (5-hydroxy-1H-indol-3-yl) ethyl] amide hexane (0.73 g, 2.5 mmol) - (intermediate (V)) was added to the HA-CHO solution. The mixture was stirred for 2 hours at room temperature. Then the picoline-borane complex (0.27 g, 2.5 mmol) was 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 material retained by propane-2-ol precipitation. Moisture and residual propane-2-ol were removed from the precipitate by drying in a hot air dryer (40 ° C, 3 days). IR (KBr) :: 3400, 2893, 2148, 1660, 1620, 1549, 1412, 1378, 1323, 1236, 1204, 1154, 1078, 1038, 945, 893 cm-1. 1H NMR (D2O) δ: 1.25 (t, β H, Y - 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-), 2.7Y (m, 2H, ε-CH2- aminohexanoic acid), 3.37 - 3.93 (m, hyaluronan body), 4.46 (s, 1H, anomer), 4.54 (s, 1H anomer., -O-CH (OH) -), 6.85 (d, 1H, aroma), 7.09 (s, 1H. Aroma), 7.21 (s, 1H. Arom), 7.40 (s, 1H. Arom). Example 1.14: Preparation of the HA derivative with a C6 spacer and 5-hydroxy tryptophan (XIV)
[049] Derivative of aldehyde HA (VII) (3.50 g) and Na2HPO4. 12 H2O (8.75 g) were dissolved in 350 mL of demineralized water. Then, 2- [(6-aminohexanoyl) amino] -3- (5-hydroxy-1H-indol-3-yl) propanoic acid (0.60 g, 1.8 mmol) - (intermediate (VI) ) to the HA-CHO solution. The mixture was mixed for 2 hours at room temperature. Then the picoline-borane complex (0.19 g, 1.8 mmol) was 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 material retained by propane-2-ol precipitation. Moisture and residual propane-2-ol were removed from the precipitate by drying in a hot air dryer (40 ° C, 3 days). IR (KBr) :: 3400, 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, Y -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-), 2.7Y (m, 2H, e-CH2- aminohexanoic acid), 3.37 - 3.93 (m, hyaluronan body), 4.46 (s, 1H, anomer), 4.54 (s, 1H anomer., -O-CH (OH) -), 6.85 (d, 1H, aroma), 7.09 (s, 1H. Aroma), 7.21 (s, 1H. Arom), 7.40 (s, 1H. Arom). Example 1.15: General procedure for the preparation of the hydrogel based on the HA derivative with a spacer and 5-hydroxytryptophan and based on a tyramine derivative
[050] The selected HA derivative is dissolved in 0.1 M PBS pH 7.4. The amount of derivative is chosen according to the desired concentration. The desired amount of enzyme is added to the derivative solution. After complete homogenization, a diluted hydrogen peroxide solution is added. The mixture is homogenized again and a clear gel is formed. Example 1.16: Preparation of hydrogel based on tyramine derivative
[051] 40 to 60 mg (according to the desired concentration of the polymer solution) of the HA derivative prepared according to example 1.8 (VIII) are dissolved in 2 ml of 0.1 M PBS with pH 7.4. Then, 20 µL of the HRP enzyme solution (24 mg of the HRP enzyme dissolved in 1 mL of 0.1 M PBS with pH 7.4) was added to the derivative solution. After complete homogenization, 100 μl of H2O2 solution (33 μl 30% H2O2 dissolved in 10 mL of 0.1 M PBS with pH 7.4) was added. The mixture is homogenized and a clear gel is formed. Example 1.17: Preparation of the hydrogel based on the HA tyramine derivative with a spacer
[052] 40 - 60 mg (according to the desired concentration of the polymer solution) of the HA derivative prepared according to example 1.9 (IX), 1.11 (XI) or 1.12 (XII) are dissolved in 2 ml of 0.1 M PBS with pH 7.4. To the derivative solution, 10 μL of the HRP enzyme solution is added (2.4 mg of the HRP enzyme dissolved in 1 ml of 0.1 M PBS with pH 7.4). After complete homogenization, 100 μl of H2O2 solution (33 μL 30% H2O2 dissolved in 10 mL of 0.1 M PBS with pH 7.4) was added. The mixture is homogenized and a clear gel is formed. 2. Differences in the properties of hydrogels Example 2.1: Difference in the mechanical properties of hydrogels depending on the type of HA derivative used and the amount of enzyme added
[053] Hydrogel samples obtained from derivatives VIII (tyramine, without built-in spacer), IX, XI and XII (with built-in spacer) were prepared according to examples 1.16 or 1.17, depending on the type of derivative used. After complete homogenization, the samples were allowed to mature for 120 minutes at room temperature. The derivative analogs used for the preparation of the comparison hydrogels have always had comparable molecular weight and degree of substitution. All samples had the same dimensions and were tested under constant laboratory conditions (temperature, pressure, humidity).
[054] Young's modulus of elasticity, in compression, toughness, compression force and corresponding deformation of the sample, was measured for each sample; and for the viscoelastic properties of the samples, the cutting module and the loss angle were measured.
[055] The data obtained clearly indicate that the introduction of the flexible spacer between the binder and the basic hyaluronan chain leads to greater elasticity, toughness and resistance of hydrogels based on the aforementioned derivatives, compared to hydrogels based on the hyaluronan analog derivatives without the use of a spacer.
[056] TABLE 1 shows the comparison of the mechanical properties of the hydrogel depending on the type of derivative used in its preparation. The concentration (%) means the concentration of the polymer in the solution from which the hydrogel was prepared, the degree of substitution (%) indicates the degree of substitution by the reactive ligand / crosslink, that is, the number of ligands joined for each 100 structural units of the polymer, where in the case of HA the structural unit of the polymer is disaccharide (or dimer) glucosamine + glucuronic acid.
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权利要求:
Claims (10)
[0001]
1. DERIVATIVE BASED ON HYALURONIC ACID characterized by the form of the general formula (I)
[0002]
2. DERIVATIVE PREPARATION METHOD defined by the general formula (I), characterized by the fact that a first aldehyde derivative of hyaluronic acid according to formula (II) is prepared
[0003]
3. PREPARATION METHOD according to claim 2, characterized in that the ligand, according to the general formula (V), is selected from the group containing tyramine, serotonin and 5-hydroxytryptophane.
[0004]
4. PREPARATION METHOD according to claims 2 to 3, characterized in that the compound, according to the general formula (IV), that is, the spacer precursor, is selected from the group of amino acids including derivatives of carboxylic acids w - [(tert butoxycarbonyl) amino] where R2 is an alkyl with 3 to 7 carbon atoms.
[0005]
5. PREPARATION METHOD according to claims 2 to 4, characterized in that the reaction of the spacer precursor with the binder occurs in THF or DMF at a temperature of 50 ° C for 2 to 6 hours in the presence of 1.1 ' - carbodiimidazole. . , ......
[0006]
6. PREPARATION METHOD according to claims 2 to 5, characterized in that the removal of the protective group Z is carried out using trifluoroacetic acid or hydrochloric acid.
[0007]
7. HYDROGEL BASED ON THE CROSS-LINK DERIVATIVE characterized by the fact that it presents a general formula (I)
[0008]
8. HYDROGEL PRODUCTION METHOD defined in claim 7, characterized by the fact that the derivative, according to the general formula (I), is treated with a generator of reactive phenoxy radicals with pH within the range of 4 to 10.
[0009]
9. PRODUCTION METHOD, according to claim 8, characterized by the fact that the generator of reactive phenoxy radicals is selected from the group containing the horseradish peroxidase system and a source of hydroxy radicals, where the source of hydroxy radicals can be a solution of hydrogen peroxide in water, or an oxidase-oxygen-substrate system.
[0010]
10. USE OF THE HYDROGEL defined in claim 7, characterized by the production of cosmetic preparations, medications or regenerative medications.

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同族专利:

公开号 | 公开日

WO2013127374A1|2013-09-06|

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KR101953709B1|2019-03-04|

EP2820051B1|2015-09-09|

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RU2014138544A|2016-04-20|

EP2820051A1|2015-01-07|

JP2015508118A|2015-03-16|

CZ303879B6|2013-06-05|

RU2586931C2|2016-06-10|

US9492586B2|2016-11-15|

PL2820051T3|2016-02-29|

JP6247645B2|2017-12-13|

US20150000561A1|2015-01-01|

KR20140127286A|2014-11-03|

ES2549668T3|2015-10-30|

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law|

2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: C08B 37/08 (2006.01), A61L 27/52 (2006.01), A61K 8 |

2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

2018-04-03| B25A| Requested transfer of rights approved|Owner name: CONTIPRO PHARMA A.S. (CZ) |

2018-04-24| B25D| Requested change of name of applicant approved|Owner name: CONTIPRO A.S. (CZ) |

2019-04-16| B07E| Notice of approval relating to section 229 industrial property law|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

2020-04-22| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|

2020-08-18| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application according art. 36 industrial patent law|

2020-10-20| B09A| Decision: intention to grant|

2020-12-15| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 26/02/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

申请号 | 申请日 | 专利标题

CZ20120136A|CZ303879B6|2012-02-28|2012-02-28|Derivatives based on hyaluronic acid capable of forming hydrogels, process of their preparation, hydrogels based on these derivatives, process of their preparation and use|

CZPV2012-136|2012-02-28|

PCT/CZ2013/000023|WO2013127374A1|2012-02-28|2013-02-26|Derivates based on hyaluronic acid, capable of forming hydrogels, method of preparation thereof, hydrogels based on said derivatives, method of preparation thereof and use|

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