Hydrogels based on zinc gluconate and hyaluronic acid esters

Abstract

Disclosed are hydrogels containing zinc gluconate and hyaluronic acid esters, methods for their preparation, compositions containing them, and uses of the hydrogels and compositions in the pharmaceutical and cosmetic fields or as topical or injectable medical devices.

Description

[Technical Field]

[0001] The present invention relates to a hydrogel containing zinc gluconate and a hyaluronic acid ester, a method for preparing the same, compositions containing the same, and the use of the hydrogel and the composition in the pharmaceutical and cosmetic fields or as a topical or injectable medical device. [Background technology]

[0002] Zinc is an essential trace element for the body. It is involved in proinflammatory cytokine regulation and exhibits radical scavenging activity against reactive oxygen species (ROS) [A.S.Rasad, Frontiers in Nutrition, 2014, Vol. 1, pp. 1-10]. It plays an important role in diseases such as osteoarthritis, which are accompanied by excessive production of free radicals, and low zinc levels have been observed in patients with these diseases [A.Mierzecki, Biol Trace Elem. Res., 2011, 143, pp. 854-862]. Zinc gluconate [WO 2016 / 141946] is used for its antibacterial properties and its ability to promote wound healing. Zinc is usually administered orally, absorbed in the intestine, and rapidly sequestered in plasma by proteins; it has a very rapid metabolic turnover and does not accumulate in the body [M.Jarosz, InflamMopharmacol, 2017, 25, pp. 11-24].

[0003] Hyaluronan is a glycosaminoglycan consisting of repeating units of glucuronic acid and N-acetylglucosamine, linked via glycosidic bonds β1→4 and β1→3. It is an essential element of connective tissue and is also present in synovial fluid, vitreous humor, and umbilical cord.

[0004] WO2009 / 098127 discloses a biocompatible injectable product for zinc delivery, which comprises zinc saccharin salts such as zinc hyaluronate or zinc gluconate dispersed in a matrix.This product can be used in medical devices or pharmaceutical or cosmetic preparations, such as for the treatment of wrinkles, arthritis and inflammation.

[0005] Hyaluronic acid lipoic acid ester, or hyaluronic acid lipoic acid / formate ester, is an ester derivative in which the hydroxyl groups of hyaluronic acid are esterified with lipoic acid or lipoic acid and formic acid residues at different degrees of substitution (DS). DS refers to the number of hydroxyls involved in ester bonds with lipoic acid or lipoic acid / formic acid residues per repeating disaccharide unit of hyaluronic acid. Hyaluronic acid lipoic acid / formate ester is known to have anti-inflammatory, antioxidant, and skin-protecting properties (WO2009080220), and its use in the field of trichology has also been reported (WO2012080223).

[0006] Lipoic acid (or thioctic acid) is a naturally occurring molecule isolated from mammalian liver. It serves as an essential cofactor for many enzymatic reactions, including the conversion of pyruvate to acetyl-CoA in the Krebs cycle. Lipoic acid in the body governs the production of antioxidant vitamins C and E and glutathione. It also exhibits radical scavenging activity in lipid tissues. It has a high affinity for metals, forming stable, water-insoluble complexes with them [J. Fuchs, Lipoic Acid in Health and Disease]. Lipoic acid's affinity for transition metals, especially zinc, allows for the preparation of systems that extend the metal's residence time in the body and enable its favorable biological activities, such as anti-inflammatory activity and free radical absorption. However, the poor water solubility of these complexes makes their use extremely difficult. Summary of the Invention [Problem to be solved by the invention]

[0007] The aim of the present invention is to provide a system that is particularly suitable for delivering zinc and that is characterized by optimal viscoelastic properties for topical and injectable (mesodermal, subcutaneous and intra-articular) applications that are useful in the cosmetic and pharmaceutical fields or in medical devices. [Means for solving the problem]

[0008] An object of the present invention is a hydrogel comprising: - hyaluronic acid esterified on the free hydroxyl group with lipoic acid, or lipoic acid and formic acid, or a pharmaceutically acceptable salt thereof; and - Zinc Gluconate. DETAILED DESCRIPTION OF THE INVENTION

[0009] The use of a strong hydrophilic carrier such as hyaluronic acid or its salt, which is modified by introducing lipoic acid residue and optionally formic acid residue through esterification reaction at the level of hydroxyl functional group, constitutes an ideal system for the formation of a complex between lipoic acid residue and zinc that is soluble in aqueous solvent.Because esterification is involved at the level of hyaluronic acid hydroxyl, the absence of carboxyl residue of lipoic acid, which is usually involved in the formation of complex with metal, is avoided by using the salt between zinc and gluconic acid.Because carboxyl group of hyaluronic acid is not involved, water solubility remains unchanged.

[0010] The esterified hyaluronic acid is preferably 1 kDa to 4 x 10 3 It has a molecular weight in the kDa range.

[0011] According to a preferred embodiment, the number of lipoic acid residues per GlcNAc-GlcUA disaccharide unit of hyaluronic acid is in the range of 0.01 to 0.5, and the number of formic acid residues per GlcNAc-GlcUA disaccharide unit of hyaluronic acid is in the range of 0 to 0.1.

[0012] According to a further preferred embodiment, the amount of zinc gluconate is 0.1 to 25 times by weight relative to the amount of sodium hyaluronate lipoate or sodium hyaluronate lipoic acid / formate.

[0013] The esterified hyaluronic acid is preferably in the form of a pharmacologically acceptable salt, more preferably a sodium salt.

[0014] The properties and preparation of hyaluronic acid lipoic acid and formate ester are described in WO2009 / 080220, which is fully incorporated herein by reference.For synthesis, please refer to page 5, line 20-7, line 5 and examples 1-5 of WO2009 / 080220 in particular.

[0015] Another aspect of the present invention relates to a method for producing a hydrogel, which method comprises mixing esterified hyaluronic acid or its salt with zinc gluconate in water to obtain a viscous solution, followed by standing for a period ranging from 1 to 48 hours, during which time a viscoelastic solution typical of a hydrogel is formed. The method may also include a hydrogel sterilization step. In a preferred embodiment, the product is mixed at a temperature ranging from 20°C to 30°C.

[0016] Another aspect of the present invention relates to a hydrogel obtainable by the above method.

[0017] A further aspect of the present invention relates to a pharmaceutical or cosmetic composition, supplement, or medical device comprising the hydrogel described herein, optionally in combination with a biologically active compound or substance, such as an anesthetic, particularly lidocaine.

[0018] The composition or device has a form or configuration suitable for topical, ophthalmic or injectable administration or application of the hydrogel, particularly intradermal, mesodermal or intra-articular administration. For topical application, the hydrogel is preferably formulated as an oil-in-water (O / W) or water-in-oil (W / O) emulsion, or as a gel, foam, or unfilled.

[0019] The use of hydrogels correlates with the presence of various biologically active components; the repair and maintenance activity typical of sodium hyaluronate is combined with (i) the antioxidant properties of lipoic acid and the various biological activities of zinc gluconate, with regard to their interaction with proteins, especially thionein (antioxidant-like properties of zinc in activated endothelial cells; Hennig B and McClain GJ, Journal of American College of Nutrition, 18(2):152-158, 1999), 5-α-reductase (Effect of a topical erythromycin-zinc formulation on sebum delivery, assessed by combined photometric - multi-step sampling with Sebutape, Pierard GE and Pierard-Franchimont C, Clinical and Experimental Dermatology, 18(5):410-413, 1993); (ii) anti-irritant activity and reduction of oxidative stress (antioxidant-like properties of zinc in activated endothelial cells, Hennig B and McClain GJ, Journal of American College of Nutrition, 18(2):152-158.1999).

[0020] In particular, the composition usable as a topical medical device can be used to treat skin disorders such as acne by reducing sebum production due to the regulatory activity of zinc gluconate on 5-alpha-reductase; rashes and burns due to the presence of a combination of anti-irritants such as hyaluronic acid lipoic acid sodium ester and zinc gluconate.

[0021] The hydrogels and compositions to which this patent relates may have ophthalmic applications due to the viscoelastic and wetting properties of hyaluronic acid and the antioxidant properties of lipoic acid.

[0022] The rheological properties of the hydrogel, particularly the reversibility of its viscoelastic structure after the application of force, combined with the antioxidant, lubricant, and regenerative properties of its components, make the hydrogel of the present invention highly useful in injectable medical devices as dermal fillers or for viscosity enhancement in joints. Thus, the hydrogel and corresponding compositions can be advantageously administered via injection into the joint capsule or dermis, where they exert a protective effect. Furthermore, the special molecular structure of the polysaccharide, which contains lipoate residues attached to the polymer chain via ester bonds, combined with the interaction of said residues with zinc gluconate to create a viscoelastic system, modifies the three-dimensional structure of the polymer, making it more resistant to enzymatic attack by hyaluronidase than crosslinks consisting of simple covalent interactions typical of currently available systems.

[0023] Furthermore, the hydrogels and corresponding compositions can be used in the cosmetic field due to the combination of the emollient and moisturizing properties of the polymeric components and the soothing and regenerating properties of the zinc and lipoic acid combination. In particular, cosmetic applications are intended for the treatment of skin stressed by aging (anti-aging agents) or external factors (anti-pollutants).

[0024] The hydrogels may also be used in cosmetic medicine applications or in mesotherapy.

[0025] For the applications and uses specified herein, the hydrogels may be used in combination with substances such as lidocaine, vitamins and amino acids.

[0026] The following examples illustrate the invention in more detail. [Example]

[0027] method Equipment used: -VARIAN VNMR 500MHz spectrometer equipped with a 5mM multinuclear inverse probe with z-gradient to measure the degree of substitution (DS); - Anton Paar MCR 301 rheometer equipped with parallel plates (25 mm diameter, satin finish) and thermostated to 25 °C.

[0028] Degree of substitution (DS) The degree of substitution in lipoester on the hyaluronic acid derivatives was determined by NMR spectroscopy. 1 H NMR spectra were performed in DO using a VARIAN VNMR 500 MHz spectrometer equipped with a 5 mm multinuclear reverse probe with z-gradients. The experiments were performed by thermostatting the measurement probe to 298°K. Quantitation of DS in lipoic acid esters was performed after exhaustive hydrolysis with NaOD directly in an NMR tube.

[0029] of hydrolysate 1 The H NMR spectrum allows integration of the signals due to lipoic acid (methylene and methane protons) and hyaluronic acid (two anomeric protons), the ratio of which determines the degree of substitution.

[0030] Measurement of elastic and viscous moduli by rheological testing Rheological testing of the gels was performed using an Anton Paar MCR 301 rheometer equipped with parallel plates (25 mm diameter, satin finish) thermostated at 25°C.

[0031] For each gel in oscillatory mode (stress sweep), a mechanical spectrum was recorded at a constant frequency of 1 Hz, which allowed the determination of the elastic modulus G' and the viscous modulus G" (measurement unit Pa); for some gels, flow curves were also recorded, measuring the viscosity η (measurement unit Pa*s) against variations in the applied force.

[0032] Example 1: Hyaluronic Acid Lipoic Acid / Sodium Formate (MW: 1500 kDa, DS lip :0.4, DS for :0.02) synthesis 100 mL of formamide and 5 g of HANa with a molecular weight of 1500 kDa are introduced into a 1-liter reactor. The mixture is heated to 95 °C and stirred at this temperature for 1 hour until the polymer is completely dissolved. The temperature is then lowered to 25 °C and the mixture is stirred overnight.

[0033] The next day, the temperature is raised to 40°C, followed by the addition of sodium carbonate (Na2CO3 - 264 mg), followed 0.5 hours later by the addition of lipoyl-imidazolide (24.0 g; 20%) in acetone over approximately 1.5 hours. The mixture is kept stirring at the same temperature for 0.5 hours. The thermostat is switched off and the reaction is stopped by adding 40 mL of acidified water, and the product is isolated by precipitation with acetone and subsequent vacuum filtration.

[0034] The crude reaction product is purified by washing with acetone and methanol several times and filtering under vacuum. The precipitate is dried under vacuum at room temperature for about 17 hours.

[0035] Dissolve 10 mg of sample in 0.9 mL of deuterium oxide (DO) and transfer to an NMR test tube. After hydrolysis of lipoic acid and formate esters by adding NaOD (deuterated sodium hydroxide), NMR spectra showed a DS of 0.4 in lipoic acid and 0.02 in formic acid.

[0036] Example 2: Hyaluronic Acid Lipoic Acid / Sodium Formate (MW: 300 kDa, DS lip :0.5, DS for :0.03) synthesis 100 mL of formamide and 10.05 g of HANa with a molecular weight of 300 kDa are introduced into a 1-liter reactor. The mixture is thermostated at 95 °C and stirred at this temperature for 1 hour until the polymer is completely dissolved. The temperature is then reduced to 25 °C and the mixture is stirred overnight.

[0037] The next day, the temperature is raised to 40°C, followed by the addition of sodium carbonate (Na2CO3 - 528 mg), followed by the addition of lipoyl-imidazolide (25.0 g; 20%) in acetone over approximately 1.5 hours. The mixture is stirred at the same temperature for 0.5 hours. The reaction is stopped by adding 80 mL of acidified water, and the product is precipitated with acetone and subsequently isolated by vacuum filtration.

[0038] The crude reaction product is purified by washing with acetone and methanol several times and filtering under vacuum. The precipitate is dried under vacuum at room temperature for about 6 hours.

[0039] Dissolve 10 mg of sample in 0.9 mL of deuterium oxide (DO) and transfer to an NMR test tube. After hydrolysis of lipoic acid and formate esters by adding NaOD (deuterated sodium hydroxide), NMR spectra showed a DS of 0.5 in lipoic acid and 0.03 in formic acid.

[0040] Example 3: Hyaluronic Acid Lipoic Acid / Sodium Formate (MW: 50 kDa; DS lip :0.5, DS for :0.03) synthesis 100 mL of formamide and 10.0 g of HANa with a molecular weight of 50 kDa are introduced into a 1-liter reactor. The mixture is thermostated at 95 °C and kept under stirring at this temperature for 1 hour until the polymer is completely dissolved. The temperature is then reduced to 25 °C and the mixture is kept under stirring overnight.

[0041] The next day, the temperature is raised to 40°C, followed by the addition of sodium carbonate (Na2CO3 - 500 mg), followed 0.5 hours later by the addition of a DMSO solution of lipoyl-imidazolide (20.0 g; 20%) over approximately 1.5 hours. The mixture is kept stirring at the same temperature for 0.5 hours. The reaction is stopped by adding 40 mL of acidified water, and the product is isolated by precipitation with acetone followed by vacuum filtration.

[0042] The crude reaction product is purified by washing with acetone and methanol several times and filtering under vacuum. The precipitate is dried under vacuum at room temperature for about 18 hours.

[0043] Dissolve 10 mg of sample in 0.9 mL of deuterium oxide (DO) and transfer to an NMR test tube.

[0044] After hydrolysis of lipoic acid and formate esters by adding NaOD (deuterated sodium hydroxide), NMR spectra showed a DS of 0.5 in lipoic acid and 0.03 in formic acid.

[0045] Example 4: Hyaluronic Acid Lipoic Acid / Sodium Formate (MW: 1500 kDa; DS lip :0.3, DS for :0.02) synthesis 200 mL of formamide and 10.0 g of HANa with a molecular weight of 1500 kDa are introduced into a 1-liter reactor. The mixture is thermostated at 95°C and stirred at this temperature for 1 hour until the polymer is completely dissolved. The temperature is then reduced to 25°C and the mixture is stirred overnight.

[0046] The next day, the temperature is raised to 40°C, followed by the addition of sodium carbonate (Na2CO3 - 527 mg), followed 0.5 hours later by the addition of lipoyl-imidazolide (47.2 g; 20%) in acetone over approximately 1.75 hours. The mixture is kept stirring at the same temperature for 0.5 hours. The reaction is stopped by adding 80 mL of acidified water, and the product is isolated by precipitation with acetone followed by vacuum filtration.

[0047] The crude reaction product is purified by washing several times with acetone and methanol, followed by vacuum filtration, respectively.

[0048] The precipitate is dried under vacuum at room temperature for approximately 18 hours. 10 mg of the sample is dissolved in 0.9 mL of deuterium oxide (DO) and transferred to an NMR test tube.

[0049] After hydrolysis of lipoic acid and formate esters by adding NaOD (deuterated sodium hydroxide), NMR spectra showed a DS of 0.3 in lipoic acid and 0.02 in formic acid.

[0050] Example 5: Hyaluronic Acid Lipoic Acid / Sodium Formate (MW: 300 kDa, DS lip :0.3, DS for :0.01) synthesis 160 mL of formamide and 8 g of HANa with a molecular weight of 300 kDa are introduced into a 1-liter reactor. The mixture is thermostated at 95 °C and stirred at this temperature for 1.5 hours until the polymer is completely dissolved. The temperature is then lowered to 25 °C and the mixture is left stirring overnight.

[0051] The next day, sodium carbonate (Na2CO3 - 400 mg) is added, and after 0.5 hours, an aqueous solution of lipoyl-imidazolide in acetone (15 g; 20%) is added over approximately 1.75 hours. The mixture is kept stirring at the same temperature for 1 hour. The reaction is stopped by adding 16 mL of acidified water, and the product is precipitated with acetone and subsequently isolated by vacuum filtration.

[0052] The crude reaction product is purified by washing with acetone and methanol several times and filtering under vacuum. The precipitate is dried under vacuum at room temperature for about 18 hours.

[0053] Dissolve 10 mg of sample in 0.9 mL of deuterium oxide (DO) and transfer to an NMR test tube.

[0054] After hydrolysis of lipoic acid and formate esters by adding NaOD (deuterated sodium hydroxide), NMR spectra showed a DS of 0.3 in lipoic acid and 0.01 in formic acid.

[0055] Example 6: Hyaluronic Acid Lipoic Acid / Sodium Formate (MW: 50 kDa; DS lip :0.3, DS for :0.01) synthesis 30 mL of formamide and 3 g of HANa having a molecular weight of 50 kDa are introduced into a 500 mL three-necked flask. The mixture is adjusted to a temperature of 95°C and kept under stirring at a constant temperature for 1 hour until the polymer is completely dissolved. The temperature is then lowered to 25°C and the mixture is kept under stirring overnight.

[0056] The next day, the temperature is raised to 40°C, followed by the addition of sodium carbonate (Na2CO3 - 150 mg), followed 0.5 hours later by the addition of a DMSO solution of lipoyl-imidazolide (15.0 g; 20%) over approximately 1.5 hours. The mixture is stirred at the same temperature for 0.5 hours. The reaction is stopped by adding 10 mL of acidified water, and the product is isolated by precipitation with acetone followed by vacuum filtration.

[0057] The crude reaction product is purified by washing with acetone and methanol several times and filtering under vacuum. The precipitate is dried under vacuum at room temperature for about 18 hours.

[0058] Dissolve 10 mg of sample in 0.9 mL of deuterium oxide (DO) and transfer to an NMR test tube.

[0059] After hydrolysis of lipoic acid and formate esters by adding NaOD (deuterated sodium hydroxide), NMR spectra showed a DS of 0.3 in lipoic acid and 0.01 in formic acid.

[0060] Example 7: Hyaluronic Acid Lipoic Acid / Sodium Formate (80 MW 1500 kDa: 20 MW 300 kDa; DS lip :0.3, DS for :0.02) synthesis 100 mL of formamide is introduced into a 1-liter reactor, followed by 4 g of HANa with a molecular weight of 1500 kDa and 1 g of HANa with a molecular weight of 300 kDa. The mixture is heated to 90°C and stirred at this temperature for 1 hour until the polymer is completely dissolved. The temperature is then lowered to 25°C and the mixture is stirred overnight.

[0061] The next day, the temperature is raised to 40°C, followed by the addition of sodium carbonate (Na2CO3 - 264 mg), followed by the addition of lipoyl-imidazolide (31.5 g; 20%) in acetone over approximately 1.5 hours. The mixture is kept stirring at the same temperature for 0.5 hours. The reaction is stopped by adding 40 mL of acidified water, and the product is precipitated with acetone and subsequently isolated by vacuum filtration.

[0062] The crude reaction product is purified by washing with acetone and methanol several times and filtering under vacuum. The precipitate is dried under vacuum at room temperature for about 19 hours.

[0063] Dissolve 10 mg of sample in 0.9 mL of deuterium oxide (DO) and transfer to an NMR test tube.

[0064] After hydrolysis of lipoic acid and formate esters by adding NaOD (deuterated sodium hydroxide), NMR spectra showed a DS of 0.3 in lipoic acid and 0.02 in formic acid.

[0065] Example 8: Hyaluronic Acid Sodium Lipoate / Sodium Formate (20 MW 1500 kDa: 80 MW 300 kDa; DS lip :0.3, DS for :0.02) synthesis 100 mL of formamide is introduced into a 1-liter reactor, followed by 1 g of HANa with a molecular weight of 1500 kDa and 4 g of HANa with a molecular weight of 300 kDa. The mixture is heated to 95°C and stirred at this temperature for 1 hour until the polymer is completely dissolved. The temperature is then lowered to 25°C and the mixture is stirred overnight.

[0066] The next day, the temperature is raised to 40°C, followed by the addition of sodium carbonate (NaCO - 264 mg), followed 0.5 hours later by the addition of lipoyl-imidazolide (31.5 g; 20%) in acetone over approximately 2.5 hours. The reaction is stopped by the addition of 40 mL of acidified water, and the product is isolated by precipitation with acetone followed by vacuum filtration.

[0067] The crude reaction product is purified by washing with acetone and methanol several times and filtering under vacuum. The precipitate is dried under vacuum at room temperature for about 19 hours.

[0068] Dissolve 10 mg of sample in 0.9 mL of deuterium oxide (DO) and transfer to an NMR test tube.

[0069] After hydrolysis of lipoic acid and formate esters by adding NaOD (deuterated sodium hydroxide), NMR spectra showed a DS of 0.3 in lipoic acid and 0.02 in formic acid.

[0070] Example 9: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (2 w / V; MW: 1500 kDa; DS lip :0.4, DS for Preparation of hydrogels containing ZnO (0.02 mM; Zn 0.1 mM) 100 mL of water for injection, 4.6 mg of zinc gluconate and 0.9 g of NaCl are introduced into a 200 mL flask and 2 g of the sample of Example 1 are added to the mixture. The system is mixed with an Ultra-turrax.

[0071] After centrifugation, the mixture is dispensed into syringes. A gel is obtained with the following properties: max =68000Pa*s;G':399.6Pa;G":88.7Pa.

[0072] Some syringes are subjected to a sterilization cycle in an autoclave (121°C; 15 min). A gel is obtained with the following properties: max =42000Pa*s;G':98.1Pa;G":29.7Pa.

[0073] Example 10: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (2 w / V; MW: 300 kDa; DS lip :0.5;DS for Preparation of hydrogels containing ZnO (0.03 mM; Zn 0.1 mM) 100 mL of water for injection, 4.5 mg of zinc gluconate and 0.9 g of NaCl are introduced into a 200 mL flask and 2 g of the sample of Example 2 is added to the mixture. The system is mixed with an Ultra-turrax.

[0074] After centrifugation, the mixture is dispensed into syringes. A gel is obtained with the following properties: G': 117.9 Pa; G": 33.1 Pa.

[0075] Some syringes are subjected to a sterilization cycle in an autoclave (121°C; 15 min). A gel is obtained with the following properties: max =200000Pa*s;G':134.3Pa;G":7.7Pa.

[0076] Example 11: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (2 w / V; MW: 50 kDa; DS lip :0.5;DS for Preparation of hydrogels containing ZnO (0.03 mM; Zn 0.1 mM) 100 mL of water for injection, 4.5 mg of zinc gluconate and 0.9 g of NaCl are introduced into a 200 mL flask and 2 g of the sample of Example 3 are added to the mixture. The system is mixed with an Ultra-turrax.

[0077] After centrifugation, the mixture is dispensed into syringes.

[0078] Example 12: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (2 w / V; MW: 1500 kDa; DS lip :0.4;DS for Preparation of hydrogels containing ZnO (0.02 mM; Zn 0.2 mM) 100 mL of water for injection, 9.1 mg of zinc gluconate and 0.9 g of NaCl are introduced into a 200 mL flask and 2 g of the sample of Example 1 are added to the mixture. The system is mixed with an Ultra-turrax.

[0079] After centrifugation, the mixture is dispensed into syringes.

[0080] Some syringes are subjected to a sterilization cycle in an autoclave (121°C; 15 min). A gel is obtained with the following properties: max =40000Pa*s;G':46.8Pa;G":12.6Pa.

[0081] Example 13: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (2 w / V; MW: 300 kDa; DS lip :0.5;DS for Preparation of hydrogels containing ZnO (0.03 mM; Zn 0.2 mM) 100 mL of water for injection, 9.1 mg of zinc gluconate and 0.9 g of NaCl are introduced into a 200 mL flask and 2 g of the sample of Example 2 is added to the mixture. The system is mixed with an Ultra-turrax.

[0082] After centrifugation, the mixture is dispensed into syringes.

[0083] Example 14: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (MW: 50 kDa; DS lip :0.5;DS for Preparation of hydrogels containing ZnO (Na = 0.03; Zn = 0.2 mM) 100 mL of water for injection, 9.2 mg of zinc gluconate and 0.9 g of NaCl are introduced into a 200 mL flask and 2 g of the sample of Example 3 are added to the mixture. The system is mixed with an Ultra-turrax.

[0084] After centrifugation, the mixture is dispensed into syringes.

[0085] Example 15: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (2 w / V; MW: 1500 kDa; DS lip :0.4;DS FOR Preparation of hydrogels containing ZnO (0.02; Zn 0.15 mM) 100 mL of water for injection, 6.8 mg of zinc gluconate and 0.9 g of NaCl are introduced into a 200 mL flask and 2 g of the sample of Example 1 are added to the mixture. The system is mixed with an Ultra-turrax.

[0086] After centrifugation, the mixture is dispensed into syringes.

[0087] Some syringes are subjected to a sterilization cycle in an autoclave (121°C; 15 min). A gel is obtained with the following properties: max =16500Pa*s;G':17.9Pa;G":6.3Pa.

[0088] Example 16: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (2 w / V; 50:50; MW: 1500 kDa; DS lip :0.3;DS for :0.02;+MW:300kDa;DS lip :0.5;DS for Preparation of hydrogels containing ZnO (0.03 mM; Zn 0.1 mM) 100 mL of water for injection, 4.6 mg of zinc gluconate, 0.9 g of NaCl and 0.3 g of lidocaine are introduced into a 200 mL flask, and 1 g of the sample of Example 4 and 1 g of the sample of Example 2 are added to the mixture.

[0089] The system is mixed with an Ultra-turrax.

[0090] After centrifugation, the mixture is dispensed into syringes, which are subjected to a sterilization cycle in an autoclave (121°C; 15 min). A gel is obtained with the following properties: max =2400Pa*s;G':19.6Pa;G":5.5Pa.

[0091] Example 17: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (2 w / V; 20:80; MW: 1500 kDa; DS lip :0.3;DS for :0.02;+MW:300kDa;DS lip :0.5;DS for Preparation of hydrogels containing ZnO (0.03 mM; Zn 0.1 mM) 100 mL of water for injection, 4.6 mg of zinc gluconate, 0.9 g of NaCl and 0.3 g of lidocaine are introduced into a 200 mL flask. 0.4 g of the sample from Example 4 and 1.6 g of the sample from Example 2 are added to the mixture. The system is mixed with an Ultra-turrax.

[0092] After centrifugation, the mixture is dispensed into syringes, which are subjected to a sterilization cycle in an autoclave (121°C; 15 min). A gel is obtained with the following properties: max =57,500Pa*s;G':53.4Pa;G":5.7Pa.

[0093] Example 18: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (2 w / V; 50:50; MW: 1500 kDa; DS lip :0.3;DS for :0.02;+MW:300kDa; DS lip :0.5;DS for Preparation of hydrogels containing ZnO (0.03 mM; Zn 0.2 mM) 100 mL of water for injection, 9.2 mg of zinc gluconate and 0.9 g of NaCl are introduced into a 200 mL flask and 1 g of sample from Example 4 and 1 g of sample from Example 2 are added to the mixture. The system is mixed with an Ultra-turrax.

[0094] After centrifugation, the mixture is dispensed into syringes, which are subjected to a sterilization cycle in an autoclave.

[0095] Example 19: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (2 w / V; DS lip :0.3;DS for Preparation of hydrogels containing ZnO (0.02 mM; Zn 0.2 mM) 100 mL of water for injection, 9.1 mg of zinc gluconate, 0.9 g of NaCl and 0.3 g of lidocaine are introduced into a 200 mL flask. 2 g of the sample of Example 8 is added to the mixture. The system is mixed with an Ultra-turrax.

[0096] After centrifugation, the mixture is dispensed into syringes, which are subjected to a sterilization cycle in an autoclave (121°C; 15 min). A gel is obtained with the following properties: max :1800Pa*s;G':6.0Pa;G":5.2Pa.

[0097] Example 20: Sodium Hyaluronate Lipoate / Zinc Formate Gluconate Complex (80:20 MW:1500 kDa; DS lip :0.3;DS for :0.02;+MW:300kDa; DS lip :0.5;DS for Preparation of hydrogels containing ZnO (0.03 mM; Zn 0.1 mM) 100 mL of water for injection, 4.5 mg of zinc gluconate, 0.9 g of NaCl and 0.3 g of lidocaine are introduced into a 200 mL flask. 1.6 g of the sample from Example 4 and 0.4 g of the sample from Example 2 are added to the mixture. The system is mixed with an Ultra-Turrax.

[0098] After centrifugation, the mixture is dispensed into syringes.

[0099] Example 21: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (2 w / V; DS lip :0.3;DS for Preparation of hydrogels containing ZnO (0.02 mM; Zn 0.2 mM) 100 mL of water for injection, 9.2 mg of zinc gluconate, 0.9 g of NaCl and 0.3 g of lidocaine are introduced into a 200 mL flask. 2 g of the sample of Example 7 is added to the mixture. The system is mixed with an Ultra-turrax.

[0100] Example 22: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (4 w / V; DS lip :0.3;DS for Preparation of hydrogels containing ZnO (0.02 mM; Zn 0.2 mM) 100 mL of water for injection, 9.2 mg of zinc gluconate, 0.9 g of NaCl and 0.3 g of lidocaine are introduced into a 200 mL flask. 4 g of the sample of Example 7 is added to the mixture. The system is mixed with an Ultra-turrax.

[0101] After centrifugation, the mixture is dispensed into syringes, which are subjected to a sterilization cycle in an autoclave (121°C; 15 min). A gel is obtained with the following properties: max =58800Pa*s;G':118.1Pa;G":40.0Pa.

[0102] Example 23: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (6 w / V; DS lip :0.3;DS for Preparation of hydrogels containing ZnO (0.02 mM; Zn 0.2 mM) 100 mL of water for injection, 9.2 mg of zinc gluconate, 0.9 g of NaCl and 0.3 g of lidocaine are introduced into a 200 mL flask. 6 g of the sample of Example 7 is added to the mixture. The system is mixed with an Ultra-turrax.

[0103] After centrifugation, the mixture is dispensed into syringes, which are subjected to a sterilization cycle in an autoclave (121°C; 15 min). A gel is obtained with the following properties: max =60000Pa*s;G':184.2Pa;G":81.2Pa.

[0104] Example 24: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (4 w / V; DS lip :0.3;DS for Preparation of hydrogels containing ZnO (0.02 mM; Zn 0.2 mM) 100 mL of water for injection, 9.2 mg of zinc gluconate, 0.9 g of NaCl and 0.3 g of lidocaine are introduced into a 200 mL flask. 4 g of the sample of Example 8 is added to the mixture. The system is mixed with an Ultra-turrax.

[0105] After centrifugation, the mixture is dispensed into syringes, which are subjected to a sterilization cycle in an autoclave (121°C; 15 min). A gel is obtained with the following properties: max =57000Pa*s;G':122.9Pa;G”:35.4Pa.

[0106] Example 25: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (6 w / V; DS lip :0.3;DS for Preparation of hydrogels containing ZnO (0.02 mM; Zn 0.2 mM) 100 mL of water for injection, 9.2 mg of zinc gluconate, 0.9 g of NaCl and 0.3 g of lidocaine are introduced into a 200 mL flask. 6 g of the sample of Example 8 is added to the mixture. The system is mixed with an Ultra-turrax.

[0107] After centrifugation, the mixture is dispensed into syringes, which are subjected to a sterilization cycle in an autoclave (121°C; 15 min). A gel is obtained with the following properties: max =170000Pa*s;G':283.0Pa;G”:84.3Pa.

[0108] Example 26: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (2 w / V; 80:20 MW: 1500 kDa; DS lip :0.3;DS for :0.02;+MW:300kDa;DS lip :0.5;DS for Preparation of hydrogels containing ZnO (0.03; Zn 0.5 mM) 100 mL of water for injection, 22.5 mg of zinc gluconate, 0.9 g of NaCl and 0.3 g of lidocaine are introduced into a 200 mL flask. 1.6 g of the sample from Example 4 and 0.4 g of the sample from Example 2 are added to the mixture. The system is mixed with an Ultra-Turrax.

[0109] After centrifugation, the mixture is dispensed into syringes.

[0110] Example 27: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (2 w / V; MW: 300 kDa; DS lip :0.5;DS for Preparation of hydrogels containing ZnO (0.03; Zn 10 mM) 100 mL of water for injection, 455 mg of zinc gluconate and 0.9 g of NaCl are introduced into a 200 mL flask. 2 g of the sample of Example 2 is added to the mixture. The system is mixed with an Ultra-turrax.

[0111] After centrifugation, the mixture is dispensed into syringes. A gel is obtained with the following properties: max :200000Pa*s;G':314Pa;G":29Pa.

[0112] Some syringes are subjected to a sterilization cycle in an autoclave (121°C; 15 min). A gel is obtained with the following properties: max =130000Pa*s;G':106.5Pa;G":9.9Pa.

[0113] Example 28: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (2 w / V; MW: 1500 kDa; DS lip :0.3;DS for Preparation of hydrogels containing ZnO (0.02; Zn 0.5 mM) 100 mL of water for injection, 22.5 mg of zinc gluconate and 0.9 g of NaCl are introduced into a 200 mL flask. 2 g of the sample of Example 4 is added to the mixture. The system is mixed with an Ultra-turrax.

[0114] After centrifugation, the mixture is dispensed into syringes.

[0115] Example 29: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (2 w / V; MW: 1500 kDa; DS lip 0.4;DS for Preparation of hydrogels containing ZnO (0.02; Zn 0.5 mM) 100 mL of water for injection, 22.5 mg of zinc gluconate and 0.9 g of NaCl are introduced into a 200 mL flask. 2 g of the sample of Example 1 is added to the mixture. The system is mixed with an Ultra-turrax.

[0116] After centrifugation, the mixture is dispensed into syringes.

[0117] Example 30: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (2 w / V; MW: 50 kDa; DS lip :0.3;DS for Preparation of hydrogels containing ZnO (0.01 mM; Zn 0.1 mM) 100 mL of water for injection, 4.5 mg of zinc gluconate and 0.9 g of NaCl are introduced into a 200 mL flask. 2 g of the sample of Example 6 is added to the mixture. The system is mixed with an Ultra-turrax.

[0118] After centrifugation, the mixture is dispensed into syringes.

[0119] Example 31: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (2 w / V; MW: 1500 kDa; DS lip :0.3;DS for Preparation of hydrogels containing ZnO (0.02 mM; Zn 0.1 mM) 100 mL of water for injection, 4.5 mg of zinc gluconate and 0.9 g of NaCl are introduced into a 200 mL flask. 2 g of the sample of Example 4 is added to the mixture. The system is mixed with an Ultra-turrax.

[0120] After centrifugation, the mixture is dispensed into syringes.

[0121] Example 32: Hyaluronic Acid Alcohol (MW: 1500 kDa; DS lip :0.45;DS for :0.0) synthesis 200 mL of formamide and 5 g of HANa with a molecular weight of 1500 kDa are introduced into a 1-liter reactor. The mixture is heated to 75 °C and stirred at this temperature for 1 hour until the polymer is completely dissolved. The temperature is then lowered to 25 °C and the mixture is stirred overnight.

[0122] The next day, the temperature is raised to 40°C, followed by the addition of sodium carbonate (Na2CO3 - 660 mg), followed by the addition of lipoyl-imidazolide (46.9 g; 20%) in acetone over approximately 1.5 hours. The mixture is kept stirred at the same temperature for 0.5 hours. The reaction is stopped by adding 40 mL of acidified water, and the product is precipitated with acetone and subsequently isolated by vacuum filtration.

[0123] The crude reaction product is purified by washing with acetone and methanol several times and filtering under vacuum. The precipitate is dried under vacuum at room temperature for about 19 hours.

[0124] Dissolve 10 mg of sample in 0.9 mL of deuterium oxide (DO) and transfer to an NMR test tube.

[0125] After hydrolysis of lipoic acid and formate esters by adding NaOD (deuterated sodium hydroxide), NMR spectra showed a DS of 0.45 in lipoic acid and a DS of 0.0 in formic acid.

[0126] Example 33: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (2 w / V; DS lip :0.3;DS for Preparation of hydrogels containing ZnO (0.0 mM; Zn 0.2 mM) 100 mL of water for injection, 9.2 mg of zinc gluconate, 0.9 g of NaCl and 0.3 g of lidocaine are introduced into a 200 mL flask. 2 g of the sample of Example 32 is added to the mixture. The system is mixed with an Ultra-turrax.

[0127] After centrifugation, the mixture is dispensed into syringes.

[0128] Example 34: Hyaluronic Acid Lipoic Acid / Sodium Formate Zinc Gluconate Complex (2 w / V; MW: 1500 kDa; DS lip :0.4;DS for Enzymatic degradation of hydrogels containing Zn(II) 0.02; Zn(II) 0.1 mM) and commercial hydrogels based on sodium hyaluronate cross-linked with BDDE Hydrogel prepared as shown in Example 9 (2 mL of gel 2 w / v; MW: 1500 kDa; DS sterilized by a heat treatment cycle at 121°C for 15 minutes) lip :0.4DS for The resistance of the gel (a syringe containing 0.1 mM Zn:0.02; Zn:0.1 mM) to enzymatic degradation was assessed according to degradation kinetics by measuring the decrease in elastic modulus (G') over time. 0.5 mL of gel was extruded directly from the syringe onto the lower plate of the rheometer, and the elastic modulus G' was assessed at a temperature of 25 °C at a constant frequency (1 Hz) and force (1 Pa). Degradation kinetics was performed by adding 50 μl of a solution of bovine testicular hyaluronidase in 30 mM acetate buffer, pH 5.5 (activity 1500 U / mL) to 0.5 mL of gel. The decrease in elastic modulus G' over time, indicating polymer chain scission, was evaluated, and the fracture kinetics were carried out under the same experimental conditions using a commercial hydrogel crosslinked with BDDE (1,4-butanediol diglycidyl ether) at a concentration of 2% in phosphate-buffered saline (pH 7) in a 1 mL syringe for comparison purposes. 60 The modulus of elasticity 60 minutes after addition of hyaluronidase, denoted as G', was used as a comparison parameter. Table 1 below shows the measured values ​​of G' and identifies the resistance to enzymatic degradation and residual modulus after 60 minutes compared to the initial modulus.

[0129] [Table 1]

Claims

1. hyaluronic acid esterified on its free hydroxyl by lipoic acid or by lipoic acid and formic acid, or a pharmaceutically acceptable salt thereof, and - zinc gluconate, A process comprising the steps of: (a) mixing zinc gluconate and esterified hyaluronic acid or a salt thereof in water until a viscous solution is obtained; (b) leaving the viscous solution obtained in (a) for 1 to 48 hours; A hydrogel obtained by

2. The hyaluronic acid is 1 kDa to 4 x 10 ３ 10. The hydrogel of claim 1, having a molecular weight in the kDa range.

3. 3. The hydrogel according to claim 1, wherein the number of lipoic acid residues per GlcNAc-GlcUA disaccharide unit of hyaluronic acid is in the range of 0.01 to 0.

5.

4. 4. The hydrogel according to claim 1, wherein the number of formic acid residues per GlcNAc-GlcUA disaccharide unit of hyaluronic acid is in the range of 0 to 0.

1.

5. 5. The hydrogel according to claim 1, wherein the esterified hyaluronate is a sodium salt.

6. 6. The hydrogel according to any one of claims 1 to 5, wherein the amount of zinc gluconate ranges from 0.1% to 25% by weight of the hyaluronic acid sodium lipoic acid, or hyaluronic acid lipoic acid and sodium formate.

7. The following process: (a) mixing zinc gluconate and esterified hyaluronic acid or a salt thereof in water until a viscous solution is obtained; and 7. A method for preparing a hydrogel according to claim 1, comprising: (b) leaving the viscous solution obtained in (a) for 1 to 48 hours.

8. The method of claim 7 further comprising sterilizing the hydrogel.

9. 8. The method of claim 7, wherein the mixing in water is carried out at a temperature in the range of 20°C to 30°C.

10. A pharmaceutical or cosmetic composition or a medical device comprising a hydrogel according to any one of claims 1 to 6 in combination with a biologically active compound.

11. 11. The composition or device of claim 10, wherein the compound is selected from lidocaine, vitamins, and amino acids.

12. The composition according to claims 10-11, which is in a form suitable for local administration selected from ocular application; injectable administration; and ocular administration.

13. 13. The composition of claim 12, wherein the injectable administration is selected from intradermal, intramesodermal, intraarticular or intraocular injection.

14. A hydrogel according to any one of claims 1 to 6 or a composition according to any one of claims 10 to 13 for use in the treatment of skin diseases or in the treatment of joint inflammation.

15. 15. The hydrogel or composition of claim 14, wherein the treatment of skin diseases is the treatment of acne, erythema and burns.

16. Use of a hydrogel according to any one of claims 1 to 6 or a composition according to any one of claims 10 to 13 in the manufacture of a cosmetic composition for treating skin wounds, or an anti-aging or anti-pollution agent.

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