Hydrogels based on zinc gluconate and hyaluronic acid esters

Abstract

Disclosed are hydrogels containing hyaluronic acid or pharmaceutically acceptable salts thereof esterified at its free hydroxyl groups with lipoic acid or with lipoic acid and formic acid and zinc gluconate, methods of preparing the hydrogels, compositions containing the hydrogels, and methods of preparing the hydrogels. And the use of said hydrogels and compositions thereof in the pharmaceutical and cosmetic field or as topical or injectable medical devices.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202080068524.8. The filing date of the original application is September 25, 2020, and the title is "Hydrogels Based on Zinc Gluconate and Hyaluronate Esters".

[0002] The present invention relates to hydrogels containing zinc gluconate and hyaluronate esters, a method for preparing the same, a composition containing the same, and the use of the hydrogels and the composition thereof in the fields of medicine and cosmetics or as local or injectable medical devices. Background Art

[0003] Zinc is an essential trace element for the human body. It participates in the regulatory mechanism of pro-inflammatory cytokines and exhibits free radical scavenger activity against reactive oxygen species (ROS) [A.S. Prasad, Frontiers in Nutrition, 2014, 1, pp. 1-10]. Zinc plays an important role in diseases such as osteoarthritis involving a large amount of free radical production, and low zinc levels are found in patients suffering from the above diseases [A. Mierzecki, Biol Trace Elem.Res., 2011, 143, pp. 854-862]. Zinc gluconate [WO2016 / 141946] is used due to its antibacterial properties and its role in accelerating the wound healing process. Zinc is usually administered orally, absorbed in the intestine, and rapidly sequestered by proteins in the plasma; its turnover rate is very fast and it does not accumulate in the body [M. Jarosz, InframmoPharmacol, 2017, 25, pp. 11-24].

[0004] Hyaluronic acid is a glycosaminoglycan composed of repeating units of glucuronic acid and N-acetylglucosamine, or bonded together by glycosidic bonds β1→4 and β1→3. It is an essential component of connective tissue and is also present in synovial fluid, vitreous humor, and umbilical cord.

[0005] WO2009 / 098127 discloses biocompatible injectable products for zinc delivery containing zinc sugar salts, such as zinc hyaluronate or zinc gluconate, dispersed in a matrix. The products can be used in medical devices or pharmaceutical or cosmetic formulations, for example, for the treatment of wrinkles, arthritis, and inflammation.

[0006] Hyaluronic acid thioester or thioester / formate ester is an ester derivative in which the hydroxyl groups of hyaluronic acid are esterified with lipoic acid groups or lipoic acid and formic acid with different degrees of substitution (DS). DS refers to the number of hydroxyl groups that form ester bonds with lipoic acid ester or lipoic acid ester / formate ester residues in each repeating disaccharide unit of hyaluronic acid. It is well known that hyaluronic acid thioester / formate ester has anti-inflammatory, antioxidant, and skin protection properties (WO2009080220), and its application in the field of trichology has also been reported (WO2012080223).

[0007] Lipoic acid (or thioctic acid) is a natural molecule isolated from mammalian livers and plays an essential cofactor role in many enzymatic reactions, including the conversion of pyruvate to acetyl coenzyme A in the Krebs cycle. Lipoic acid in the body controls the production of antioxidant vitamins C and E, as well as glutathione. It also exhibits free radical scavenging activity in lipid tissues. It has a high affinity for metals and forms stable water-insoluble complexes with metals [J.Fuchs “Lipoic acid in health and disease”]. The affinity of lipoic acid for transition metals, especially zinc, allows it to be formulated into systems that prolong the retention time of metals in the body and exhibit good biological activity in terms of anti-inflammatory activity and free radical absorption. However, the low water solubility of this complex makes the above uses very difficult.

[0008] The object of the present invention is to provide a system particularly suitable for delivering zinc, characterized by having optimal viscoelasticity and being suitable for topical and injectable (mesoderm, subcutaneous, and intra-articular) applications in the cosmetic and pharmaceutical fields or medical devices. Detailed Description of the Invention

[0010] The object of the present invention is a hydrogel, which comprises:

[0011] - Hyaluronic acid or a pharmaceutically acceptable salt thereof esterified with lipoic acid or lipoic acid and formic acid at free hydroxyl groups;

[0012] - Zinc gluconate.

[0013] Using a strongly hydrophilic carrier, such as hyaluronic acid or its salt, modified by introducing lipoic acid residues and optionally formic acid residues, and carrying out an esterification reaction at the level of hydroxyl functional groups, constitutes an ideal system for forming a complex soluble in aqueous solvents between lipoic acid residues and zinc. Since lipoic acid does not have carboxylic acid residues that usually participate in forming complexes with metals and participates in the esterification reaction at the level of hyaluronic acid hydroxyl groups, the absence of lipoic acid carboxyl residues usually related to forming complexes with metals can be avoided by using the salt between zinc and gluconic acid. Since the carboxyl groups of hyaluronic acid are not involved, the water solubility remains unchanged.

[0014] The esterified hyaluronic acid according to the present invention preferably has a molecular weight between 1 kDa - 4x10 3 kDa.

[0015] According to a preferred embodiment, the number of lipoic acid residues per GlcNAc-GlcUA disaccharide unit of hyaluronic acid is 0.01 - 0.5, while the number of formic acid residues per GlcNAc-GlcUA disaccharide unit is 0 - 0.1.

[0016] According to another preferred embodiment, the amount of zinc gluconate is in the range of 0.1 - 25% by weight, compared to thioctic acid hyaluronate or thioctic acid / formic acid hyaluronate sodium salt.

[0017] The esterified hyaluronic acid is preferably in the form of a pharmaceutically acceptable salt, more preferably the sodium salt.

[0018] The properties and preparation of thioctic acid hyaluronate and formic acid hyaluronate are described in WO2009 / 080220, the full text of which is incorporated herein by reference. For synthesis, reference is made in particular to page 5, lines 20 to 7, line 5 and Examples 1 - 5 of WO2009 / 080220.

[0019] Another aspect of the invention relates to a method for preparing a hydrogel, which comprises mixing an esterified hyaluronic acid or its salt and zinc gluconate in water until a viscous solution is obtained, and then leaving it to stand for 1 - 48 h to form a viscoelastic solution typical of a hydrogel. The method may also include a step of sterilizing the hydrogel. In a preferred embodiment, the product is mixed at a temperature of 20°C - 30°C.

[0020] Another aspect of the invention relates to a hydrogel obtained by the above method.

[0021] A further aspect of the invention relates to a pharmaceutical or cosmetic composition, supplement or medical device containing the hydrogel described herein, optionally in combination with a bioactive compound or substance (such as an anesthetic, especially lidocaine).

[0022] The composition or device has a form or structure suitable for topical, ophthalmic or injectable administration or application of the hydrogel, especially 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 a gel, foam or ointment.

[0023] The uses of the hydrogel are related to the presence of various bioactive ingredients; the typical restorative and maintenance activities of sodium hyaluronate are combined with the antioxidant properties of lipoic acid and the various bioactivities of zinc gluconate, manifested in (i) the interaction with proteins, especially thioproteins, (Antioxidant-like properties of Zinc In Activated Endothelial Cells, Hennig B and McClain GJ, Journal of the American College of Nutrition, 18(2):152-158. 1999) and enzymes, such as 5-α-reductase (Effect of a topical erythromycin-zinc formulation on sebum delivery. Evaluation by combined photometric-multi-step samplings 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 the American College of Nutrition, 18(2):152-158. 1999).

[0024] Specifically, the composition that can be used as a topical medical device reduces sebum production through the activity regulation of zinc gluconate on 5-α-reductase and can be used to treat skin diseases, such as acne; it can be used to treat rashes and burns due to anti-irritant combinations such as sodium hyaluronate lipoate and zinc gluconate.

[0025] Due to the viscoelastic and wetting properties of hyaluronic acid and the antioxidant properties of lipoic acid, the hydrogels and compositions involved in this patent can have ophthalmic applications.

[0026] The rheological properties of the hydrogel, especially 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 extremely useful as a dermal filler in injectable medical devices or for supplementing mucus at joints. Thus, the hydrogel and the corresponding compositions can be advantageously administered via injection into the joint capsule or dermis, where they exert a protective effect. In addition, the special molecular structure of the polysaccharide (containing lipoic acid residues bonded to the polymer chain via ester bonds) combined with the interaction of the residues with zinc gluconate creates a viscoelastic system that modifies the three-dimensional structure of the polymer, making it more resistant to enzymatic attack by hyaluronidase than crosslinks composed only of covalent interactions, which are common systems on the current market.

[0027] Furthermore, due to the combination of the emollient and moisturizing properties of the polymer components and the soothing and regenerative properties of the zinc and lipoic acid combination, the hydrogel and the corresponding compositions can be used in the cosmetic field. Specifically, the cosmetic use is intended to treat skin stressed by aging (anti-aging agent) or external factors (anti-pollution agent).

[0028] The hydrogel can also be used in aesthetic medicine applications or mesotherapy.

[0029] For the applications and uses specified herein, the hydrogel can be used in combination with substances such as lidocaine, vitamins, and amino acids.

[0030] The following examples illustrate the present invention in more detail. Examples

[0031] Method

[0032] Instruments used:

[0033] · Varian VNMR 500 MHz spectrometer, equipped with a 5 mm multinuclear inverse probe and a z-gradient for determining the degree of substitution (DS);

[0034] · Anton Paar MCR 301 rheometer, equipped with parallel plates (diameter 25 mm, satin finish) thermostated to 25 °C.

[0035] Degree of substitution (DS)

[0036] The degree of substitution of the lipoate ester of hyaluronic acid derivatives was quantified by NMR spectroscopy. 1H NMR spectra were recorded on a Varian VNMR 500 MHz spectrometer equipped with a 5 mm multinuclear inverse probe with z-gradient in D 2 2O 1 and the measurement probe was thermostated to 298 °K for the experiments.

[0037] The quantification of DS in lipoic acid esters was carried out directly in an NMR tube after complete hydrolysis with NaOD.

[0038] The 1 1H NMR spectrum of the hydrolysis product allowed the signal assignment of lipoic acid (methylene and methine protons) and hyaluronic acid (two heteropolymeric protons) separately; their ratio determined the degree of substitution.

[0039] The elastic modulus and viscous modulus were determined by rheological tests.

[0040] The rheological tests of the gels were carried out using an Anton Paar MCR 301 rheometer equipped with parallel plates (diameter 25 mm, satin) thermostated at 25 °C.

[0041] At a constant frequency of 1 Hz, the mechanical spectra of each gel were recorded in oscillatory mode (stress sweep) to determine the elastic modulus G′ and viscosity modulus G″ (measuring unit Pa); the flow curves of some gels were also recorded, which measured the viscosity η (measuring unit Pa·s) when the applied force changed.

[0042] Example 1: Synthesis of sodium hyaluronate lipoate / formate (molecular weight: 1500 kDa; DS lip : 0.4; DS for : 0.02)

[0043] 100 ml of formamide and 5 g of HANa with a molecular weight of 1500 kDa were introduced into a 1-liter reactor. The mixture was thermostated at 95 °C and stirred at a constant temperature for 1 h until the polymer was completely dissolved. The temperature was lowered to 25 °C, and the mixture was kept under stirring overnight.

[0044] The next day, the temperature was raised to 40 °C; then sodium carbonate (Na 2 CO 3 - 264 mg) was added, and after 0.5 h, a solution of fatty acyl imidazole in acetone (24.0 g; 20%) was added within about 1.5 h. The mixture was stirred at the same temperature for 0.5 h. The thermostat was turned off, 40 ml of acidic water was added to terminate the reaction, and the product was separated by precipitation with acetone followed by vacuum filtration.

[0045] The crude reaction product was purified by washing several times with acetone and methanol, and vacuum filtration was carried out after each washing. The precipitate was dried under vacuum at room temperature for about 17 h.

[0046] 10 mg of the sample was dissolved in 0.9 ml of deuterated water (D 2 2O) and transferred to an NMR tube.

[0047] After hydrolyzing lipoic acid ester and formic acid ester by adding NaOD (sodium deuteroxide), the NMR spectrum shows that the DS of lipoic acid is 0.4 and the DS of formic acid is 0.02.

[0048] Example 2: Synthesis of Sodium Hyaluronate Lipoic Acid Ester / Formic Acid Ester (Molecular Weight: 300 kDa; DS lip : 0.5; DS for : 0.03)

[0049] 100 ml of formamide and 10.05 g of HANa with a molecular weight of 300 kDa were introduced into a 1-liter reactor. The mixture was heated to 95 °C and stirred at a constant temperature for 1 h until the polymer was completely dissolved. The temperature was then lowered to 25 °C, and the mixture was kept stirring overnight.

[0050] The next day, the temperature was raised to 40 °C; sodium carbonate (Na 2 CO 3 - 528 mg) was added, and after 0.5 h, a solution of acyl imidazole in acetone (25.0 g; 20%) was added over about 1.5 h. The mixture was stirred at the same temperature for 0.5 h. The reaction was terminated by adding 80 ml of acidic water, and the product was separated by precipitation with acetone followed by vacuum filtration.

[0051] The crude reaction product was purified by washing several times with acetone and methanol, and vacuum filtration was carried out after each washing. The precipitate was dried in vacuo at room temperature for about 6 h.

[0052] 10 mg of the sample was dissolved in 0.9 ml of deuterated water (D 2 O) and transferred to an NMR tube.

[0053] After hydrolyzing lipoic acid ester and formic acid ester by adding NaOD (sodium deuteroxide), the NMR spectrum shows that the DS of lipoic acid is 0.5 and the DS of formic acid is 0.03.

[0054] Example 3: Synthesis of Sodium Hyaluronate Lipoic Acid Ester / Formic Acid Ester (Molecular Weight: 50 kDa; DS lip : 0.5; DS for : 0.03)

[0055] 100 ml of formamide and 10.0 g of HANa with a molecular weight of 50 kDa were introduced into a 1-liter reactor. The mixture was heated to 95 °C and stirred at a constant temperature for 1 h until the polymer was completely dissolved. The temperature was then lowered to 25 °C, and the mixture was kept stirring overnight.

[0056] The next day, the temperature was raised to 40 °C; then sodium carbonate (Na 2 CO 3-500 mg), after 0.5 h, a solution of acyl imidazole in dimethyl sulfoxide (20.0 g; 20%) was added within about 1.5 h. The mixture was stirred at the same temperature for 0.5 h. 40 ml of acidic water was added to stop the reaction, and the product was precipitated with acetone and then separated by vacuum filtration.

[0057] The crude reaction product was purified by washing several times with acetone and methanol, and vacuum filtration was carried out after each washing. The precipitate was dried under vacuum at room temperature for about 18 h.

[0058] 10 mg of the sample was dissolved in 0.9 ml of deuterated water (D 2 2O) and transferred to an NMR tube.

[0059] After adding NaOD (sodium deuteroxide) to hydrolyze lipoate and formate, the NMR spectrum showed that the DS of lipoic acid was 0.5 and the DS of formic acid was 0.03.

[0060] Example 4: Synthesis of sodium hyaluronate lipoate / formate (molecular weight: 1500 kDa; DS lip : 0.3; DS for : 0.02)

[0061] 200 ml of formamide and 10.0 g of HANa with a molecular weight of 1500 kDa were introduced into a 1-liter reactor. The mixture was heated to 95 °C and stirred at a constant temperature for 1 h until the polymer was completely dissolved. Then the temperature was lowered to 25 °C and the mixture was kept under stirring overnight.

[0062] The next day, the temperature was raised to 40 °C; then sodium carbonate (Na 2 2CO 3 3 - 527 mg) was added. After 0.5 h, a solution of acyl imidazole in acetone (47.2 g; 20%) was added within about 1.75 h. The mixture was stirred at the same temperature for 0.5 h. 80 ml of acidic water was added to terminate the reaction, and the product was precipitated with acetone and then separated by vacuum filtration.

[0063] The crude reaction product was purified by washing several times with acetone and methanol, and vacuum filtration was carried out after each washing. The precipitate was dried under vacuum at room temperature for about 18 h.

[0064] 10 mg of the sample was dissolved in 0.9 ml of deuterated water (D 2 2O) and transferred to an NMR tube.

[0065] After adding NaOD (sodium deuteroxide) to hydrolyze lipoate and formate, the NMR spectrum showed that the DS of lipoic acid was 0.3 and the DS of formic acid was 0.02.

[0066] Example 5: Synthesis of Sodium Hyaluronate Thioester / Formate (Molecular Weight: 300 kDa; DS lip : 0.3; DS for : 0.01)

[0067] 160 ml of formamide and 8 g of HANa with a molecular weight of 300 kDa were introduced into a 1-liter reactor. The mixture was heated to 95 °C and stirred at a constant temperature for 1.5 h until the polymer was completely dissolved. Then the temperature was lowered to 25 °C, and the mixture was kept under stirring overnight.

[0068] The next day, sodium carbonate (Na 2 CO 3 - 400 mg) was added. After 0.5 h, a solution of acyl imidazole in acetone (15 g; 20%) was added within about 1.75 h. The mixture was stirred at the same temperature for 1 h. 16 ml of acidic water was added to terminate the reaction, and the product was separated by precipitation with acetone followed by vacuum filtration.

[0069] The crude reaction product was purified by washing several times with acetone and methanol, and vacuum filtration was carried out after each washing. The precipitate was dried under vacuum at room temperature for about 18 h.

[0070] 10 mg of the sample was dissolved in 0.9 ml of deuterated water (D 2 O) and transferred to an NMR tube.

[0071] After hydrolyzing the thioester and formate by adding NaOD (deuterated sodium hydroxide), the NMR spectrum showed that the DS of lipoic acid was 0.3 and the DS of formic acid was 0.01.

[0072] Example 6: Synthesis of Sodium Hyaluronate Thioester / Formate (Molecular Weight: 50 kDa; DS lip : 0.3; DS for : 0.01)

[0073] 30 ml of formamide and 3 g of HANa with a molecular weight of 50 kDa were introduced into a 500-ml three-necked flask. The mixture was heated to 95 °C and stirred at a constant temperature for 1 h until the polymer was completely dissolved. The temperature was lowered to 25 °C, and the mixture was kept under stirring overnight.

[0074] The next day, the temperature was raised to 40 °C; sodium carbonate (N a 2CO 3 - 150 mg) was added. After 0.5 h, a solution of acyl imidazole in dimethyl sulfoxide (15.0 g; 20%) was added within about 1.5 h. The mixture was stirred at the same temperature for 0.5 h. 10 ml of acidic water was added to terminate the reaction, and the product was separated by precipitation with acetone followed by vacuum filtration.

[0075] The crude reaction product was purified by washing several times with acetone and methanol, and vacuum filtration was carried out after each washing. The precipitate was dried in vacuo at room temperature for about 18 h.

[0076] 10 mg of the sample was dissolved in 0.9 ml of deuterated water (D 2 2O) and transferred to an NMR tube.

[0077] After adding NaOD (sodium deuteroxide) to hydrolyze the lipoate and formate, the NMR spectrum showed a DS of 0.3 for lipoic acid and a DS of 0.01 for formic acid.

[0078] Example 7: Synthesis of sodium hyaluronate lipoate / formate (80 MW 1500 kDa: 20 MW 300 kDa; DS lip : 0.3; DS for : 0.02)

[0079] 100 ml of formamide, and then 4 g of HA-Na with a molecular weight of 1500 kDa and 1 g of HA-Na with a molecular weight of 300 kDa were introduced into a 1 L reactor. The mixture was heated to 90 °C and stirred at a constant temperature for 1 h until the polymer was completely dissolved. The temperature was then lowered to 25 °C, and the mixture was kept under stirring overnight.

[0080] The next day, the temperature was raised to 40 °C; then sodium carbonate (Na 2 2CO 3 3 - 264 mg) was added. After 0.5 h, a solution of acyl imidazole in acetone (31.5 g; 20%) was added within about 1.5 h. The mixture was stirred at the same temperature for 0.5 h. 40 ml of acidic water was added to stop the reaction, and the product was separated by precipitation with acetone followed by vacuum filtration.

[0081] The crude reaction product was purified by washing several times with acetone and methanol, and vacuum filtration was carried out after each washing. The precipitate was dried in vacuo at room temperature for about 19 h.

[0082] 10 mg of the sample was dissolved in 0.9 ml of deuterated water (D 2 2O) and transferred to an NMR tube.

[0083] After adding NaOD (sodium deuteroxide) to hydrolyze the lipoate and formate, the NMR spectrum showed a DS of 0.3 for lipoic acid and a DS of 0.02 for formic acid.

[0084] Example 8: Synthesis of sodium hyaluronate lipoate / formate (20 MW 1500 kDa: 80 MW 300 kDa; DS lip : 0.3; DS for : 0.02)

[0085] Introduce 100 ml of formamide and 1 g of HANa with a molecular weight of 1500 kDa and 4 g of HANa with a molecular weight of 300 kDa into a 1-liter reactor. Keep the mixture at a constant temperature of 95 °C and stir it at a constant temperature for 1 h until the polymer is completely dissolved. Lower the temperature to 25 °C and keep the mixture under stirring overnight.

[0086] The next day, raise the temperature to 40 °C; add sodium carbonate (Na 2 CO 3 - 264 mg), and after 0.5 h, add a solution of fatty acyl imidazole in acetone (31.5 g; 20%) within about 2.5 h. Add 40 ml of acidic water to stop the reaction and precipitate with acetone, and then separate the product by vacuum filtration.

[0087] The crude reaction product is purified by washing several times with acetone and methanol, and vacuum filtration is carried out after each washing. The precipitate is vacuum dried at room temperature for about 19 h.

[0088] Dissolve 10 mg of the sample in 0.9 ml of deuterated water (D 2 O) and transfer it to an NMR tube.

[0089] After adding NaOD (deuterated sodium hydroxide) to hydrolyze lipoic acid esters and formic acid esters, the NMR spectrum shows that the DS of lipoic acid is 0.3 and the DS of formic acid is 0.02.

[0090] Example 9: Preparation of a hydrogel containing sodium hyaluronate lipoic acid ester / formic acid ester zinc gluconate complex (2 w / v; MW: 1500 kDa; DS lip : 0.4; DS for : 0.02; Zn 0.1 mM)

[0091] Introduce 100 ml of injectable water, 4.6 mg of zinc gluconate, and 0.9 g of NaCl into a 200-ml flask, and add 2 g of the sample of Example 1 to the mixture. The system is mixed with an Ultra-turrax.

[0092] After centrifugation, distribute the mixture into syringes. A gel with the following properties is obtained: η max = 68000 Pa*s; G′: 399.6 Pa; G″: 88.7 Pa.

[0093] Some syringes are subjected to a sterilization cycle (121 °C; 15 min) in an autoclave. A gel with the following properties is obtained: η max = 42000 Pa*s; G′: 98.1 Pa; G″: 29.7 Pa.

[0094] Example 10: Preparation of a hydrogel containing sodium hyaluronate lipoic acid ester / formic acid ester zinc gluconate complex (2 w / v; MW: 300 kDa; DSlip : 0.5; DS for : 0.03; Preparation of zinc gluconate (0.1 mM) hydrogel

[0095] 100 ml of injectable water, 4.5 mg of zinc gluconate and 0.9 g of NaCl were introduced into a 200 ml flask, and 2 g of the sample of Example 2 was added to the mixture. The system was mixed with an Ultra - turrax.

[0096] After centrifugation, the mixture was dispensed into syringes. Gels with the following characteristics were obtained: G′: 117.9 Pa; G″: 33.1 Pa.

[0097] Some syringes were subjected to a sterilization cycle in an autoclave (121 °C; 15 min). Gels with the following properties were obtained: η max = 200000 Pa*s; G′: 134.3 Pa; G″: 7.7 Pa.

[0098] Example 11: Preparation of a hydrogel containing sodium hyaluronate thioester / formate zinc gluconate complex (2 w / v; MW: 50 kDa; DS lip : 0.5; DS for : 0.03; Zn 0.1 mM)

[0099] 100 ml of injectable water, 4.5 mg of zinc gluconate and 0.9 g of NaCl were introduced into a 200 ml flask, and 2 g of the sample of Example 3 was added to the mixture. The system was mixed with an Ultra - turrax.

[0100] After centrifugation, the mixture was dispensed into syringes.

[0101] Example 12: Preparation of a hydrogel containing sodium hyaluronate thioester / formate zinc gluconate complex (2 w / v; MW: 1500 kDa; DS lip : 0.4; DS for : 0.02; Zn 0.2 mM)

[0102] 100 ml of injectable water, 9.1 mg of zinc gluconate and 0.9 g of NaCl were introduced into a 200 ml flask, and 2 g of the sample of Example 1 was added to the mixture. The system was mixed with an Ultra - turrax.

[0103] After centrifugation, the mixture was dispensed into syringes.

[0104] Some syringes were subjected to a sterilization cycle in an autoclave (121 °C; 15 min). Gels with the following properties were obtained: η max= 40000 Pa*s; G': 46.8 Pa; G": 12.6 Pa.

[0105] Example 13: Preparation of a hydrogel containing sodium hyaluronate thioester / formate zinc gluconate complex (2 w / v; MW: 300 kDa; DS lip : 0.5; DS for : 0.03; Zn 0.2 mM)

[0106] 100 ml of injectable water, 9.1 mg of zinc gluconate and 0.9 g of NaCl were introduced into a 200 ml flask, and 2 g of the sample of Example 2 was added to the mixture. The system was mixed with an Ultra-turrax.

[0107] After centrifugation, the mixture was dispensed into syringes.

[0108] Example 14: Preparation of a hydrogel containing sodium hyaluronate thioester / formate zinc gluconate complex (MW: 50 kDa; DS lip : 0.5; DS for : 0.03; Zn 0.2 mM)

[0109] 100 ml of injectable water, 9.2 mg of zinc gluconate and 0.9 g of NaCl were introduced into a 200 ml flask, and 2 g of the sample of Example 3 was added to the mixture. The system was mixed with an Ultra-turrax.

[0110] After centrifugation, the mixture was dispensed into syringes.

[0111] Example 15: Preparation of a hydrogel containing sodium hyaluronate thioester / formate zinc gluconate complex (2 w / v; MW: 1500 kDa; DS lip : 0.4; DS for : 0.02; Zn 0.15 mM)

[0112] 100 ml of injectable water, 6.8 mg of zinc gluconate and 0.9 g of NaCl were introduced into a 200 ml flask, and 2 g of the sample of Example 1 was added to the mixture. The system was mixed with an Ultra-turrax.

[0113] After centrifugation, the mixture was dispensed into syringes.

[0114] Some syringes were subjected to a sterilization cycle in an autoclave (121 °C; 15 min). Gels with the following properties were obtained: η max = 16500 Pa*s; G': 17.9 Pa; G": 6.3 Pa.

[0115] Example 16: Preparation of a hydrogel containing a complex of sodium hyaluronate thioester / formate zinc gluconate (2 w / v; 50:50; MW: 1500 kDa; DS lip : 0.3; DS for : 0.02; +MW: 300 kDa; DS lip : 0.5; DS for : 0.03; Zn 0.1 mM)

[0116] 100 ml of water for injection, 4.6 mg of zinc gluconate, 0.9 g of sodium chloride and 0.3 g of lidocaine were injected into a 200 ml flask. 1 g of the sample of Example 4 and 1 g of the sample of Example 2 were added to the mixture. The system was mixed with an Ultra-turrax.

[0117] After centrifugation, the mixture was dispensed into syringes, and the syringes were subjected to a sterilization cycle in an autoclave (121 °C; 15 min). A gel with the following properties was obtained: η max = 2400 Pa*s; G′: 19.6 Pa; G″: 5.5 Pa.

[0118] Example 17: Preparation of a hydrogel containing a complex of sodium hyaluronate thioester / formate zinc gluconate (2 w / v; 20:80; MW: 1500 kDa; DS lip : 0.3; DS for : 0.02; +MW: 300 kDa; DS lip : 0.5; DS for : 0.03; Zn 0.1 mM)

[0119] 100 ml of water for injection, 4.6 mg of zinc gluconate, 0.9 g of sodium chloride and 0.3 g of lidocaine were injected into a 200 ml flask. 0.4 g of the sample of Example 4 and 1.6 g of the sample of Example 2 were added to the mixture. The system was mixed with an Ultra-turrax.

[0120] After centrifugation, the mixture was dispensed into syringes, and the syringes were subjected to a sterilization cycle in an autoclave (121 °C; 15 min). A gel with the following properties was obtained: η max = 57500 Pa*s; G′: 53.4 Pa; G″: 5.7 Pa.

[0121] Example 18: Preparation of a hydrogel containing a complex of sodium hyaluronate thioester / formate zinc gluconate (2 W / v; 50:50; MW: 1500 kDa; DS lip : 0.3; DS for : 0.02; +MW: 300 kDa; DS lip: 0.5; DS for : 0.03; Preparation of zinc gluconate (2W / V; DS

[0122] Inject 100 ml of water for injection, 9.2 mg of zinc gluconate and 0.9 g of sodium chloride into a 200 ml flask. Add 1 g of the sample of Example 4 and 1 g of the sample of Example 2 to the mixture. The system is mixed with an Ultra - turrax.

[0123] After centrifugation, the mixture is dispensed into syringes, and the syringes are subjected to a sterilization cycle in an autoclave.

[0124] Example 19: Containing sodium hyaluronate thioester / formate zinc gluconate complex (2W / V; DS lip : 0.3; DS for : 0.02; Preparation of zinc gluconate (2W / V; DS

[0125] Inject 100 ml of water for injection, 9.1 mg of zinc gluconate, 0.9 g of sodium chloride and 0.3 g of lidocaine into a 200 ml flask. Add 2 g of the sample of Example 8 to the mixture. The system is mixed with an Ultra - turrax.

[0126] After centrifugation, the mixture is dispensed into syringes, and the syringes are subjected to a sterilization cycle (121 °C; 15 min) in an autoclave. A gel with the following properties is obtained: η max : 1800 Pa*s; G′: 6.0 Pa; G″: 5.2 Pa.

[0127] Example 20: Containing sodium hyaluronate thioester / formate zinc gluconate complex (80:20 MW: 1500 kDa; DS lip : 0.3; DS for : 0.02; +MW: 300 kDa; DS lip : 0.5; DS for : 0.03; Preparation of zinc gluconate (2W / V; DS

[0128] Add 100 ml of water for injection, 4.5 mg of zinc gluconate, 0.9 g of sodium chloride and 0.3 g of lidocaine to a 200 ml flask. Add 1.6 g of the sample of Example 4 and 0.4 g of the sample of Example 2 to the mixture. The system is mixed with an Ultra - turrax.

[0129] After centrifugation, the mixture is dispensed into syringes.

[0130] Example 21: Containing sodium hyaluronate thioester / formate zinc gluconate complex (2w / V; DS lip: 0.3; DS for : 0.02; Preparation of zinc gluconate hydrogel with Zn 0.2 mM

[0131] Add 100 ml of water for injection, 9.2 mg of zinc gluconate, 0.9 g of sodium chloride and 0.3 g of lidocaine to a 200 ml flask. Add 2 g of the sample of Example 7 to the mixture. The system is mixed with an Ultra - turrax.

[0132] After centrifugation, distribute the mixture into syringes.

[0133] Example 22: Preparation of a hydrogel containing sodium hyaluronate thioester / formate zinc gluconate complex (4 w / V; DS lip : 0.3; DS for : 0.02; Preparation of zinc gluconate hydrogel with Zn 0.2 mM

[0134] Add 100 ml of water for injection, 9.2 mg of zinc gluconate, 0.9 g of sodium chloride and 0.3 g of lidocaine to a 200 ml flask. Add 4 g of the sample of Example 7 to the mixture. The system is mixed with an Ultra - turrax.

[0135] After centrifugation, distribute the mixture into syringes, and the syringes are subjected to a sterilization cycle (121 °C; 15 min) in an autoclave. A gel with the following properties is obtained: η max = 58800 Pa*s; G′: 118.1 Pa; G″: 40.0 Pa.

[0136] Example 23: Preparation of a hydrogel containing sodium hyaluronate thioester / formate zinc gluconate complex (6 w / V; DS lip : 0.3; DS for : 0.02; Preparation of a hydrogel with Zn 0.2 mM

[0137] Add 100 ml of water for injection, 9.2 mg of zinc gluconate, 0.9 g of sodium chloride and 0.3 g of lidocaine to a 200 ml flask. Add 6 g of the sample of Example 7 to the mixture. The system is mixed with an Ultra - turrax.

[0138] After centrifugation, distribute the mixture into syringes, and the syringes are subjected to a sterilization cycle (121 °C; 15 min) in an autoclave. A gel with the following properties is obtained: η max = 60000 Pa*s; G′: 184.2 Pa; G″: 81.2 Pa.

[0139] Example 24: Preparation of a hydrogel containing sodium hyaluronate thioester / formate zinc gluconate complex (4 w / V; DS lip: 0.3; DS for : 0.02; Preparation of hydrogel with Zn 0.2 mM

[0140] 100 ml of water for injection, 9.2 mg of zinc gluconate, 0.9 g of sodium chloride and 0.3 g of lidocaine were added to a 200 ml flask. 4 g of the sample of Example 8 was added to the mixture. The system was mixed with an Ultra-turrax.

[0141] After centrifugation, the mixture was dispensed into syringes, and the syringes were subjected to a sterilization cycle in an autoclave (121 °C; 15 min). A gel with the following properties was obtained: η max = 57000 Pa*s; G′: 122.9 Pa; G″: 35.4 Pa.

[0142] Example 25: Preparation of a hydrogel containing a complex of sodium hyaluronate thioester / formate zinc gluconate (6 w / V; DS lip : 0.3; DS for : 0.02; Zn 0.2 mM)

[0143] 100 ml of water for injection, 9.2 mg of zinc gluconate, 0.9 g of sodium chloride and 0.3 g of lidocaine were added to a 200 ml flask. 6 g of the sample of Example 8 was added to the mixture. The system was mixed with an Ultra-turrax.

[0144] After centrifugation, the mixture was dispensed into syringes, and the syringes were subjected to a sterilization cycle in an autoclave (121 °C; 15 min). A gel with the following properties was obtained: η max = 170000 Pa*s; G′: 283.0 Pa; G″: 84.3 Pa.

[0145] Example 26: Preparation of a hydrogel containing a complex of sodium hyaluronate thioester / formate zinc gluconate (2 w / V; 80:20 MW: 1500 kDa; DS lip : 0.3; DS for : 0.02; +MW: 300 kDa; DS lip : 0.5; DS for : 0.03; Zn 0.5 mM)

[0146] 100 ml of water for injection, 22.5 mg of zinc gluconate, 0.9 g of sodium chloride and 0.3 g of lidocaine were injected into a 200 ml flask. 1.6 g of the sample of Example 4 and 0.4 g of the sample of Example 2 were added to the mixture. The system was mixed with an Ultra-turrax.

[0147] After centrifugation, the mixture is dispensed into a syringe.

[0148] Example 27: Preparation of a hydrogel containing a complex of sodium hyaluronate thioester / formate zinc gluconate (2 w / v; MW: 300 kDa; DS lip : 0.5; DS for : 0.03; Zn 10 mM)

[0149] 100 ml of water for injection, 455 mg of zinc gluconate and 0.9 g of sodium chloride were injected into a 200 ml flask. 2 g of the sample of Example 2 was added to the mixture. The system was mixed with an Ultra - turrax.

[0150] After centrifugation, the mixture was dispensed into a syringe. A gel with the following properties was obtained: η max : 200000 Pa*s; G′: 314 Pa; G″: 29 Pa.

[0151] Some syringes were subjected to a sterilization cycle in an autoclave (121 °C; 15 min). A gel with the following properties was obtained: η max = 130000 Pa*s; G′: 106.5 Pa; G″: 9.9 Pa.

[0152] Example 28: Preparation of a hydrogel containing a complex of sodium hyaluronate thioester / formate zinc gluconate (2 w / v; MW: 1500 kDa; DS lip : 0.3; DS for : 0.02; Zn 0.5 mM)

[0153] 100 ml of water for injection, 22.5 mg of zinc gluconate and 0.9 g of sodium chloride were injected into a 200 ml flask. 2 g of the sample of Example 4 was added to the mixture. The system was mixed with an Ultra - turrax.

[0154] After centrifugation, the mixture was dispensed into a syringe.

[0155] Example 29: Preparation of a hydrogel containing a complex of sodium hyaluronate thioester / formate zinc gluconate (2 w / v; MW: 1500 kDa; DS lip : 0.4; DS for : 0.02; Zn 0.5 mM)

[0156] 100 ml of water for injection, 22.5 mg of zinc gluconate and 0.9 g of sodium chloride were injected into a 200 ml flask. 2 g of the sample of Example 1 was added to the mixture. The system was mixed with an Ultra - turrax.

[0157] After centrifugation, the mixture was dispensed into syringes.

[0158] Example 30: Preparation of a hydrogel containing a complex of sodium hyaluronate thioester / formate zinc gluconate (2 w / v; MW: 50 kDa; DS lip : 0.3; DS for : 0.01; Zn 0.1 mM)

[0159] 100 ml of water for injection, 4.5 mg of zinc gluconate and 0.9 g of sodium chloride were injected into a 200 ml flask. 2 g of the sample of Example 6 was added to the mixture. The system was mixed with an Ultra - turrax.

[0160] After centrifugation, the mixture was dispensed into syringes. Example 31: Preparation of a hydrogel containing a complex of sodium hyaluronate thioester / formate zinc gluconate (2 w / v; MW: 1500 kDa; DS lip : 0.3; DS for : 0.02; Zn 0.1 mM) 100 ml of water for injection, 4.5 mg of zinc gluconate and 0.9 g of sodium chloride were injected into a 200 ml flask. 2 g of the sample of Example 4 was added to the mixture. The system was mixed with an Ultra - turrax.

[0161] After centrifugation, the mixture was dispensed into syringes.

[0162] Example 32: Synthesis of sodium hyaluronate thioester (molecular weight: 1500 kDa; DS lip : 0.45; DS for : 0.0)

[0163] 200 ml of formamide and 5 g of HANa with a molecular weight of 1500 kDa were introduced into a 1 - liter reactor. The mixture was heated to 75 °C and stirred at a constant temperature for 1 h until the polymer was completely dissolved. Then the temperature was lowered to 25 °C and the mixture was kept stirring overnight.

[0164] The next day, the temperature was raised to 40 °C; sodium carbonate (Na 2 CO 3 - 660 mg) was added. After 0.5 h, a solution of acyl imidazole in acetone (46.9 g; 20%) was added within about 1.5 h. The mixture was stirred at the same temperature for 0.5 h. 40 ml of acidic water was added to stop the reaction, and the product was precipitated by acetone and then separated by vacuum filtration.

[0165] The crude reaction product was purified by washing several times with acetone and methanol, and vacuum filtration was carried out after each washing. The precipitate was dried under vacuum at room temperature for about 19 h.

[0166] Dissolve 10 mg of the sample in 0.9 ml of deuterium oxide (D 2 2O) and transfer it to an NMR tube.

[0167] After adding NaOD (sodium deuteroxide) to hydrolyze lipoate and formate, the NMR spectrum shows that the DS of lipoic acid is 0.45 and the DS of formic acid is 0.0.

[0168] Example 33: Preparation of a hydrogel containing sodium hyaluronate lipoate / formate zinc gluconate complex (2 w / v; DS lip : 0.3; DS for : 0.0; Zn 0.2 mM)

[0169] Add 100 ml of water for injection, 9.2 mg of zinc gluconate, 0.9 g of sodium chloride and 0.3 g of lidocaine to a 200 ml flask. Add 2 g of the sample of Example 32 to the mixture. The system is mixed with an Ultra-turrax.

[0170] After centrifugation, distribute the mixture into syringes.

[0171] Example 34: Enzymatic degradation of a hydrogel containing sodium hyaluronate lipoate / formate zinc gluconate complex (2 w / v; MW: 1500 kDa; DS lip : 0.4; DS for : 0.02; Zn 0.1 mM) and a commercially available hydrogel based on sodium hyaluronate crosslinked with BDDE

[0172] The resistance of the hydrogel prepared as shown in Example 9 (syringe containing 2 ml of gel 2 w / v; MW: 1500 kDa; DS lip : 0.4; DS for : 0.02; Zn 0.1 mM, sterilized by thermal cycling at 121 °C for 15 min) to enzymatic degradation was evaluated according to the degradation kinetics by measuring the decrease in elastic modulus (G′) over time. Extrude 0.5 ml of the gel directly from the syringe onto the lower plate of the rheometer and evaluate the elastic modulus G′ at a constant frequency (1 Hz) and force (1 Pa) at a temperature of 25 °C. Add 50 μl of bovine testicular hyaluronidase solution to 0.5 mL of the gel in 30 mM acetate buffer pH 5.5 (activity 1500 U / ml) for decomposition kinetics determination. Evaluate the decrease in elastic modulus G′ over time indicating polymer chain breakage. For comparison purposes, the degradation kinetics determination was carried out under the same test conditions using a commercially available general hydrogel crosslinked with BDDE (1,4-butanediol diglycidyl ether) at a 2% concentration in pH 7 phosphate buffered saline in a 1 ml syringe. The elastic modulus after adding hyaluronidase for 60 min (expressed as G'60 ) is used as a comparison parameter. Table 1 below shows the measured values of G′, specifying the enzymatic degradation resistance and the residual elastic modulus after 60 min compared to the initial modulus.

[0173] Table 1

[0174] Hydrogel 2 <![CDATA[G’ 0 [Pa]]]> <![CDATA[G’ 60 [Pa]]]> Residual G’ Example 9 95 38 41 Commercially available product crosslinked with BDDE 79 7 9

Claims

1. A hydrogel, comprising: - a hyaluronic acid ester or a pharmaceutically acceptable salt thereof esterified with lipoic acid or lipoic acid and formic acid on its free hydroxyl groups, and - 0.1% - 25% by weight of zinc gluconate based on the weight of sodium hyaluronate lipoate or sodium hyaluronate lipoate and formate, wherein the hyaluronic acid has a molecular weight of 1 kDa - 4x10 3 kDa, the number of lipoic acid residues per GlcNAc GlcUA disaccharide unit of the hyaluronic acid is 0.01 - 0.5, the number of formic acid residues per GlcNAc GlcUA disaccharide unit of the hyaluronic acid is 0 - 0.1, the esterified hyaluronate is a sodium salt, and The polysaccharide contains a special molecular structure in which lipoic acid residues are bonded to the polymer chain through ester bonds, combined with the interaction of the residues with zinc gluconate, creating a viscoelastic system that changes the three-dimensional structure of the polymer.

2. The hydrogel according to claim 1, wherein the viscosity η max , the values of the elastic modulus G' and the viscous modulus G" are selected from: - 42000 Pa*s, 98.1 Pa, 29.7 Pa; - 200000 Pa*s, 134.3 Pa, 7.7 Pa; - 40000 Pa*s, 46.8 Pa, 12.6 Pa; - 16500 Pa*s, 17.9 Pa, 6.3 Pa; - 2400 Pa*s, 19.6 Pa, 5.5 Pa; - 57,500 Pa*s, 53.4 Pa, 5.7 Pa; - 1800 Pa*s, 6.0 Pa, 5.2 Pa; - 58800 Pa*s, 118.1 Pa, 40.0 Pa; - 60000 Pa*s, 184.2 Pa, 81.2 Pa; - 57000 Pa*s, 122.9 Pa, 35.4 Pa; - 170000 Pa*s, 283.0 Pa, 84.3 Pa; - 130000 Pa*s, 106.5 Pa, 9.9 Pa.

3. The hydrogel according to claim 2, wherein the viscosity η max , the values of the elastic modulus G' and the viscosity modulus G" are selected from: - 42000 Pa*s, 98.1 Pa, 29.7 Pa; - 200000 Pa*s, 134.3 Pa, 7.7 Pa.

4. The hydrogel according to claim 2 or 3, wherein the values of the viscosity η max , elastic modulus G’, and viscous modulus G” are 42000 Pa*s, 98.1 Pa, and 29.7 Pa, respectively.

5. A method for preparing the hydrogel according to claims 1 - 4, which comprises the following steps: (a) Mix zinc gluconate and the esterified hyaluronic acid or its salt in water until a viscous solution is obtained; (b) Let the viscous solution obtained in (a) stand for 1 - 48 hours.

6. The method according to claim 5, which further comprises sterilization of the hydrogel.

7. The method according to claim 5, wherein the mixing is carried out in water at 20°C - 30°C.

8. A hydrogel obtained by the method according to claims 5 - 7.

9. A pharmaceutical or cosmetic composition or a medical device containing the hydrogel according to claims 1 - 4 and 8, optionally in combination with a bioactive compound.

10. The composition or device according to claim 9, wherein the compound is selected from lidocaine, vitamins, and amino acids.

11. The composition according to claims 9 - 10, which is in a form suitable for topical administration, and the topical administration is selected from ophthalmic applications; injectable administration, especially intradermal, mesoderm, intra-articular, or intra-ocular injection; and ophthalmic administration.

12. The hydrogel according to claims 1 - 4 and 8 or the composition according to claims 9 - 11, which is used for the treatment of skin disorders selected from acne, erythema, and burns, or for the treatment of joint inflammation.

13. Use of the hydrogel according to claims 1 - 4 and 8 or the composition according to claims 9 - 11 for the preparation of a drug for treating skin blemishes or as an anti-aging agent or an anti-pollution agent.