Crosslinked hyaluronic acid butyrate or butyrate-formate derivatives and methods for crosslinking same

Abstract

The present invention provides cross-linked hyaluronic acid butyrate, which has increased chemical and biological stability of polysaccharides and at the same time has an improved rheological profile. [Solution] A method for preparing crosslinked hyaluronic acid butyrate, hyaluronic acid butyrate-formate, or a pharmaceutically acceptable salt thereof, by crosslinking in an organic solvent with a carboxylic acid activating reagent and a base, wherein the raw material hyaluronic acid butyrate, etc., is a mixture of a high-molecular-weight polysaccharide having a weight-average molecular weight of 1,000 kDa to 10,000 kDa and a low-molecular-weight polysaccharide having a weight-average molecular weight of 1 kDa to 900 kDa.

Description

[Technical Field]

[0001] The present invention relates to a process for the preparation of crosslinked hyaluronic acid butyrate or crosslinked hyaluronic acid butyrate-formate or salts thereof, to the products obtained by said process, and to their formulation for pharmaceutical or cosmetic use or as medical devices. [Background technology]

[0002] In particular, the present invention relates to the cross-linking of combinations of different molecular weight hyaluronic acid butyrate or hyaluronic acid butyrate-formate or salts thereof, which surprisingly results in polymers having different rheological profiles than polymers obtained by combining the same polymers that have been previously cross-linked.

[0003] The cross-linked butyrate-formate esters of hyaluronic acid prepared by the above method have better viscoelastic properties than gels obtained by combining pre-cross-linked polysaccharides with different molecular weights, and therefore can be advantageously used in the pharmaceutical and dermocosmetics fields and as medical devices, especially as injections.

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

[0005] WO98 / 23648 discloses the preparation of hyaluronic acid butyrate (SHB), in which the hydroxyl groups of hyaluronic acid are esterified with butyric acid residues.Hyaluronic acid butyrate has anti-inflammatory, anti-proliferative and skin-protecting properties as a skin elasticity agent and moisturizing agent.

[0006] WO2009 / 068215 discloses the preparation of mixed butyrate-formate esters of hyaluronic acid and their use in skin cosmetics with skin-protecting and anti-inflammatory activities. The mixed esters are prepared using butyric anhydride and formamide (FA) with N,N-dimethylaminopyridine (N,N-DMAP) as a basic catalyst.

[0007] EP341745 discloses the preparation of self-crosslinked hyaluronic acid, starting from hyaluronic acid or hyaluronic acid, in which the carboxyl groups are partially esterified with various types of alcohols. The carboxyl functional groups of hyaluronic acid (or its ester derivatives, defined as "external esters") participate in the formation of intramolecular or intermolecular esters with the alcohol hydroxyls of the repeating units of hyaluronic acid, resulting in crosslinks (defined as "self-crosslinks").

[0008] WO2008 / 081255 discloses the preparation of self-crosslinked hyaluronic acid characterized by the simultaneous presence of esters and non-polysaccharide carboxylic acids, including butyric acid-formic acid and esters between acid and alcohol groups of the starting polysaccharide, crosslinks between polysaccharide chains.

[0009] EP 2614090 discloses cooperative hybrid complexes between low and high molecular weight hyaluronic acid, in which the hyaluronic acid molecules in solution are characterized by cooperative interactions based on the formation of hydrophobic bonds and intra- and inter-chain hydrogen bonds, the extent of which depends on the molecular weight of the polysaccharide.

[0010] It has now been discovered that cross-linking hyaluronic acid butyrate or hyaluronic acid butyrate-formate with different molecular weights increases the chemical and biological stability of the polysaccharide and at the same time provides an improved rheological profile, which is particularly advantageous for applications in the pharmaceutical and dermocosmetic fields, as well as for medical devices, especially injectables. Summary of the Invention

[0011] The object of the present invention is a method for preparing crosslinked hyaluronic acid butyrate or crosslinked hyaluronic acid butyrate or an acceptable salt thereof, which method comprises reacting hyaluronic acid butyrate or hyaluronic acid butyrate-formate or a pharmaceutically acceptable salt thereof with a carboxyl group-activating reagent and a base in an organic solvent, wherein the hyaluronic acid butyrate or hyaluronic acid butyrate-formate or a pharmaceutically acceptable salt thereof is a mixture of a high molecular weight polysaccharide having a weight-average molecular weight in the range of 1,000 kDa to 10,000 kDa, preferably 1,000 kDa to 6,000 kDa, more preferably 1,000 kDa to 2,000 kDa, and a low molecular weight polysaccharide having a weight-average molecular weight in the range of 1 kDa to 900 kDa, preferably 10 kDa to 500 kDa, more preferably 50 kDa to 300 kDa.

[0012] The degree of substitution (DS), defined as the ratio between the number of butyric or butyric-formic acid residues per GlcNAc-GlcUA disaccharide unit of hyaluronic acid, can range, for example, from 0.1 to 2.2.

[0013] The high molecular weight polysaccharide and the low molecular weight polysaccharide are preferably used in a weight ratio ranging from 80:20 to 20:80.

[0014] By "acceptable salt" herein is meant a salt that is acceptable for pharmaceutical or cosmetic use or in medical devices, e.g., sodium, potassium, lithium or quaternary ammonium salts, e.g., tetrabutylammonium, preferably sodium salts.

[0015] The crosslinking reaction is preferably carried out in an organic solvent selected from polar aprotic organic solvents such as N,N-dimethylformamide, dimethylacetamide, dimethylsulfoxide, N-methylpyrrolidone or formamide. The preferred solvent is formamide.

[0016] The carboxyl group activating reagent is preferably selected from the group consisting of carbonyldiimidazole, 1,1'-carbonyl-di-(1,2,4-triazole), 1,1'-oxalylimidazole, 1,1'-thiocarbonylimidazole, 1,1'-carbonylbis(2-methylimidazole), N-hydroxysuccinimide, p-nitrophenol, p-nitrophenyl trifluoroacetate, 2-halo-N-alkylpyridine salts and acyl halides. More preferably, the carboxyl group activating reagent is carbonyldiimidazole.

[0017] The base used in the crosslinking reaction is preferably selected from inorganic bases such as carbonates, bicarbonates or hydroxides of alkali or alkaline earth metals, in particular sodium, potassium or magnesium, aromatic or aliphatic organic bases containing at least one trisubstituted nitrogen atom, such as pyridine or its homologues, such as collidine, basic amines, such as triethylamine, imidazole, N-methyl-piperazine or dimethylaminopyridine, or alkali or alkaline earth metal salts of organic acids, such as sodium or potassium acetate. More preferably, the base used is sodium carbonate or dimethylaminopyridine.

[0018] The reaction mixture is preferably maintained at a temperature in the range of 20°C to 30°C for 4 to 24 hours.

[0019] Once the reaction is complete, the mixture is diluted with water and the product is recovered by precipitation in a suitable solvent. The product thus obtained is then purified, for example, by successive washings with a suitable solvent and filtration.

[0020] The mixture of high and low molecular weight derivatives of hyaluronic acid (hyaluronic acid butyrate or hyaluronic acid butyrate salts or salts thereof) used in the crosslinking reaction can be prepared by mixing a high molecular weight hyaluronic acid derivative with a low molecular weight derivative, or by derivatizing a mixture of high and low molecular weight hyaluronic acid with butyric acid or butyric acid and formamide.

[0021] A further object of the present invention is the cross-linked hyaluronic acid butyrate or cross-linked hyaluronic acid butyrate-formate or pharmaceutically acceptable salts thereof obtainable by the above process.

[0022] The cross-linked polysaccharides obtained by the method have a different rheological profile than those obtained by combining the same starting polysaccharides that have been previously cross-linked.

[0023] The rheological profile of the cross-linked polysaccharides according to the invention is characterized by high viscosity and high elastic modulus (G') and viscosity (G") values.

[0024] In particular, cross-linked polysaccharides according to the present invention obtained by cross-linking a mixture of high and low molecular weight polysaccharides have a higher viscosity and a higher modulus (G') and viscosity (G") than a mixture of cross-linked high and low molecular weight polysaccharides.

[0025] Furthermore, the presence of polysaccharides with different molecular weights not only results in the typical biological properties of high molecular weight sodium hyaluronate, such as proliferative activity in the cells that make up the extracellular matrix and anti-inflammatory activity, but also in the typical biological properties of low molecular weight sodium hyaluronate, such as angiogenic activity and modulation of inflammatory processes, thus conferring a unique profile of biological activity while still maintaining the biocompatibility typical of hyaluronic acid.

[0026] Finally, the cross-linked polysaccharides obtained by the method according to the invention exhibit improved resistance to enzymatic degradation, which promotes long-lasting activity in vivo.

[0027] A further object of the present invention is a pharmaceutical or cosmetic preparation or a medical device comprising crosslinked hyaluronic acid butyrate or crosslinked hyaluronic acid butyrate-formate or a pharmaceutically acceptable salt thereof obtainable by the above-described method, and at least one pharmaceutically or cosmetically acceptable excipient and / or carrier.

[0028] The pharmaceutical or cosmetic preparation or medical device may also contain a local anesthetic, such as lidocaine.

[0029] The cosmetic preparations can be used for dermocosmetic anti-aging or rejuvenating treatments and in mesotherapy applications.

[0030] The pharmaceutical preparation or medical device can be used for the topical treatment of skin lesions and rashes and eye lesions.

[0031] A further object of the invention is a medical device which can be used as an adjuvant for ophthalmic applications, for example in eye surgery or in the treatment of dry eye, or as an adjuvant in the treatment of osteoarthritis, or as a dermal filler.

[0032] Hyaluronic acid butyrate-formate esters are prepared as disclosed in WO2009 / 068215, and hyaluronic acid butyrate esters are prepared as disclosed in WO2016 / 113192.

[0033] Cross-linked hyaluronic acid butyrate or cross-linked hyaluronic acid butyrate-formate or their salts obtained by the above-mentioned method have considerable anti-irritant, anti-inflammatory and antioxidant activity, which influences acute inflammatory responses. Due to these characteristics, the cross-linked polysaccharides according to the invention are particularly indicated for injectable, dermocosmetic or intra-articular applications, when acute inflammation caused by subcutaneous injection or inflammation associated with osteoarthritis constitutes a problematic target. [Example]

[0034] method Equipment used: VARIAN VNMR 500MHz spectrometer equipped with a 5mm multinuclear inverse probe with z-gradient to measure degree of substitution (DS); An Anton Paar MCR301 rheometer was equipped with parallel plates (25 mm diameter, satin finish) and thermostated at 25°C.

[0035] Determination of degree of substitution (DS) The degree of substitution of butyrate esters 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-gradient. The experiments were performed by thermostatting the measurement probe to 298°K.

[0036] Quantitation of DS in butyrate esters was performed after exhaustive hydrolysis with NaOD directly in the NMR tube.

[0037] of hydrolysate 1 The H NMR spectrum allows integration of signals due to butyric acid (vicinal methyl and methylene protons) and hyaluronic acid (sugar protons, excluding the two anomeric protons), and their ratio determines the degree of substitution.

[0038] Rheological testing to measure elastic and viscous moduli.

[0039] Rheological testing of the gels was performed using an Anton Paar MCR01 rheometer equipped with parallel plates (25 mm diameter, satin finish) thermostated at 25°C. Measurements were performed on samples swollen in ultrapure water at a concentration of 1% w / v 24 hours after mixing.

[0040] For each gel in oscillatory mode (stress sweep), a mechanical spectrum was recorded at a constant frequency of 1 Hz, allowing the determination of the elastic modulus G' and the viscous modulus G" (measured in units of Pa); for some gels, flow curves were also recorded, measuring the viscosity η (measured in units of Pa*s) against variations in the applied force.

[0041] Example 1: Synthesis of hyaluronic acid butyrate-sodium formate (MW: 1500kDa; DS but :0.3;DS for :0.01) 100 ml of formamide was introduced into a 1 L reactor, followed by 5.0 g of sodium hyaluronate with a molecular weight of 1500 kDa. The mixture was adjusted to a temperature of 95°C and stirred at a constant temperature for 1.5 hours until the polymer was completely dissolved. The temperature was then lowered to 25°C, and the mixture was kept under stirring overnight.

[0042] Sodium carbonate (NaCO-262.0 mg) was then added, followed 0.5 hours later by butyryl-imidazolide (1.1 g). The mixture was stirred at 25°C for 1.5 hours. The reaction was quenched with 50 ml of acidified water, and the product was isolated by precipitation with acetone and subsequent filtration.

[0043] The crude reaction product was purified by washing with acetone and methanol several times, followed by vacuum filtration. The precipitate was dried under vacuum at a temperature of ≦60° C. for approximately 24 hours.

[0044] 10 mg of sample was dissolved in 0.9 ml of deuterium oxide (D2O) and transferred to an NMR test tube.

[0045] After hydrolysis of the butyrate-formate esters by adding NaOD (deuterated sodium hydroxide), the NMR spectra showed a DS of 0.3 in butyrate and a DS of 0.01 in formate.

[0046] Example 2: Synthesis of hyaluronic acid butyrate-formate sodium salt (MW: 1500kDa; DS but :0.95;DS for :0.01) 150 ml of formamide was introduced into a 1 L reactor, followed by 7.5 g of sodium hyaluronate with a molecular weight of 1500 kDa. The mixture was adjusted to a temperature of 95°C and stirred at a constant temperature for 1.5 hours until the polymer was completely dissolved. The temperature was then lowered to 25°C, and the mixture was kept under stirring overnight.

[0047] Sodium carbonate (NaCO - 600 mg) was then added, followed 0.5 hours later by butyryl-imidazolide (6.7 g). The mixture was stirred at 25 °C for 1.5 hours. The reaction was quenched with 65 ml of acidified water, and the product was isolated by precipitation with acetone and subsequent filtration.

[0048] The crude reaction product was purified by washing with acetone and methanol several times, followed by vacuum filtration. The precipitate was dried under vacuum at a temperature of ≦60° C. for approximately 24 hours.

[0049] 10 mg of sample was dissolved in 0.9 ml of deuterium oxide (D2O) and transferred to an NMR test tube.

[0050] After hydrolysis of the butyrate-formate ester by adding NaOD (deuterated sodium hydroxide), the NMR spectrum showed a DS of 0.95 in butyrate and 0.01 in formate.

[0051] Example 3: Synthesis of hyaluronic acid butyrate-formate sodium salt (MW: 1500kDa; DS but :1.6;DS for :0.03) 200 ml of formamide was introduced into a 1 L reactor, followed by 10.15 g of HANa with a molecular weight of 1500 kDa. The mixture was heated to 95°C and maintained at this temperature for 1 hour under stirring until the polymer was completely dissolved. The temperature was then reduced to 25°C, and the mixture was maintained under stirring overnight.

[0052] Sodium carbonate (NaCO - 2.7 g) was then added, followed 0.5 hours later by butyryl-imidazolide (26.2 g). The mixture was stirred at 25 °C for 1.5 hours. The reaction was quenched with 200 ml of acidified water, and the product was isolated by precipitation with acetone followed by decantation.

[0053] The crude reaction product was purified by washing with acetone and methanol several times, followed by vacuum filtration. The precipitate was dried under vacuum at a temperature of ≦60° C. for approximately 16 hours.

[0054] 10 mg of sample was dissolved in 0.9 ml of deuterium oxide (D2O) and transferred to an NMR test tube.

[0055] After hydrolysis of the butyrate-formate esters by adding NaOD (deuterated sodium hydroxide), the NMR spectra showed a DS of 1.6 for butyrate and 0.03 for formate.

[0056] Example 4: Synthesis of hyaluronic acid butyrate-formate sodium salt (MW: 300 kDa; DS but :0.3;DS for :0.02) 2.67 L of formamide was introduced into a 15 L reactor, followed by 200.5 g of sodium hyaluronate with a molecular weight of 300 kDa. The mixture was adjusted to a temperature of 95°C and stirred at a constant temperature for 1.5 hours until the polymer was completely dissolved. The temperature was then lowered to 25°C, and the mixture was kept under stirring overnight.

[0057] Sodium carbonate (NaCO, 10.57 g) was then added, followed 0.5 hours later by butyryl-imidazolide (36.81 g). The mixture was stirred at 25°C for 1.5 hours. The reaction was quenched with 1.38 L of acidified water, and the product was isolated by precipitation with acetone followed by decantation.

[0058] The crude reaction product was purified by washing with acetone and methanol several times, followed by vacuum filtration. The precipitate was dried under vacuum at a temperature of ≦60° C. for approximately 16 hours.

[0059] 10 mg of sample was dissolved in 0.9 ml of deuterium oxide (D2O) and transferred to an NMR test tube.

[0060] After hydrolysis of the butyrate-formate ester by adding NaOD (deuterated sodium hydroxide), the NMR spectrum showed a DS of 0.3 in butyrate and 0.02 in formate.

[0061] Example 5: Synthesis of hyaluronic acid butyrate-formate sodium salt (MW: 300 kDa; DS but :0.95;DS for :0.01) 80 ml of formamide was added to a 1 L reactor, followed by 6 g of sodium hyaluronate with a molecular weight of 300 kDa. The mixture was then adjusted to 95°C and stirred at this temperature for 1.5 hours until the polymer was completely dissolved. The temperature was then lowered to 25°C.

[0062] Sodium carbonate (NaCO - 480 mg) was then added, followed 0.5 hours later by butyryl-imidazolide (4.2 g). The mixture was stirred at 25 °C for 1.5 hours. The reaction was quenched with 40 ml of acidified water, and the product was isolated by precipitation with acetone and subsequent filtration.

[0063] The crude reaction product was purified by washing with acetone and methanol several times, followed by vacuum filtration. The precipitate was dried under vacuum at a temperature of ≦60° C. for approximately 24 hours.

[0064] 10 mg of sample was dissolved in 0.9 ml of deuterium oxide (D2O) and transferred to an NMR test tube.

[0065] After hydrolysis of the butyrate-formate ester by adding NaOD (deuterated sodium hydroxide), the NMR spectrum showed a DS of 0.95 in butyrate and 0.01 in formate.

[0066] Example 6: Synthesis of hyaluronic acid butyrate-formate sodium salt (MW: 300 kDa; DS but :2.16;DS for :0.02) 50 ml of formamide was added to a 0.5 L flask, followed by 5 g of sodium hyaluronate with a molecular weight of 300 kDa. The mixture was adjusted to a temperature of 95°C and stirred at a constant temperature for 1.5 hours until the polymer was completely dissolved. The temperature was then lowered to 25°C, and the system was kept under stirring overnight.

[0067] Sodium carbonate (NaCO - 1.34 g) was then added, followed 0.5 hours later by butyryl-imidazolide (18.41 g). The mixture was stirred at 25 °C for 1.5 hours. The reaction was quenched with 70 ml of acidified water, and the product was isolated by precipitation with acetone followed by filtration.

[0068] The crude reaction product was purified by washing with acetone and methanol several times, followed by vacuum filtration. The precipitate was dried under vacuum at a temperature of ≦60° C. for approximately 24 hours.

[0069] 10 mg of sample was dissolved in 0.9 ml of deuterium oxide (D2O) and transferred to an NMR test tube.

[0070] After hydrolysis of the butyrate-formate esters by adding NaOD (deuterated sodium hydroxide), the NMR spectra showed a DS of 2.16 for butyrate and 0.02 for formate.

[0071] Example 7: Synthesis of sodium hyaluronate butyrate (MW: 25kDa, DS but :0.35) 25 ml of water was introduced into a 0.5 L flask, followed by 5 g of sodium hyaluronate with a molecular weight of 25 kDa. The mixture was thermostated at 25°C and stirred at constant temperature for 1.5 hours until the polymer was completely dissolved.

[0072] Sodium carbonate (NaCO - 0.8 g) was then added, followed 0.5 hours later by butyryl-imidazolide (0.9 g). The mixture was stirred at 25 °C for 1.0 hour. The reaction was quenched with 5 ml of acidified water, and the product was isolated by precipitation with isopropanol and subsequent filtration.

[0073] The crude reaction product was purified by washing with isopropanol and water several times, followed by vacuum filtration. The precipitate was dried under vacuum at 25° C. for about 48 hours.

[0074] 10 mg of sample was dissolved in 0.9 ml of deuterium oxide (D2O) and transferred to an NMR test tube.

[0075] After hydrolysis of the butyrate ester by adding NaOD (deuterated sodium hydroxide), the NMR spectrum showed a DS of 0.35 in butyric acid.

[0076] Example 8: Synthesis of sodium hyaluronate butyrate (MW: 25kDa, DS but :0.9) 25 ml of water was introduced into a 0.5 L flask, followed by 5 g of sodium hyaluronate with a molecular weight of 25 kDa. The mixture was thermostated at 25°C and stirred at constant temperature for 1.5 hours until the polymer was completely dissolved.

[0077] Sodium carbonate (NaCO - 4.0 g) was then added, followed 0.5 hours later by butyryl-imidazolide (2.5 g). The mixture was stirred at 25 °C for 1.0 hour. The reaction was quenched with 20 ml of acidified water, and the product was isolated by precipitation with isopropanol and subsequent filtration.

[0078] The crude reaction product was purified by washing with isopropanol and water several times, followed by vacuum filtration. The precipitate was dried under vacuum at 25° C. for about 48 hours.

[0079] 10 mg of sample was dissolved in 0.9 ml of deuterium oxide (D2O) and transferred to an NMR test tube.

[0080] After hydrolysis of the butyrate ester by adding NaOD (deuterated sodium hydroxide), the NMR spectrum showed a DS of 0.9 in butyric acid.

[0081] Example 9: Synthesis of sodium hyaluronate butyrate (MW: 25kDa, DS but :1.6 35 ml of water was introduced into a 0.5 L flask, followed by 5 g of sodium hyaluronate with a molecular weight of 25 kDa. The mixture was thermostated at 25°C and kept under stirring at constant temperature for 1.5 hours until the polymer was completely dissolved.

[0082] Sodium carbonate (NaCO - 6.61 g) was then added, followed 0.5 hours later by butyryl-imidazolide (11.9 g). The mixture was stirred at 25 °C for 1.0 hour. The reaction was quenched with 20 ml of acidified water, and the product was isolated by precipitation with isopropanol and subsequent filtration.

[0083] The crude reaction product was purified by washing with isopropanol and water several times, followed by vacuum filtration. The precipitate was dried under vacuum at 25° C. for about 48 hours.

[0084] 10 mg of sample was dissolved in 0.9 ml of deuterium oxide (D2O) and transferred to an NMR test tube.

[0085] After hydrolysis of the butyrate ester by adding NaOD (deuterated sodium hydroxide), the NMR spectrum showed a DS of 1.6 in butyric acid.

[0086] Example 10: Synthesis of cross-linked hyaluronic acid butyrate-formate sodium salt (80:20; MW: 1500 kDa; DS but :1.6;DS for :0.03:MW:300kDa;DS but :0.3;DS for :0.02) 20 ml of formamide was introduced into a 100 ml three-necked flask, followed by 0.8 g of the product obtained in Example 3 and 0.2 g of the product obtained in Example 4. The mixture was thermostated at 95° C. and kept under constant stirring for 2.5 hours until the polymer was completely dissolved.

[0087] The temperature was then reduced to 25°C and the system was kept under stirring overnight.

[0088] Sodium carbonate (Na2CO3 - 60 mg) was added, followed 0.5 h later by CDI (300 mg dissolved in 1.2 ml DMSO). The mixture was left under stirring at 25°C for 24 h.

[0089] The reaction was quenched by adding 40 ml of water and the product was isolated by precipitation in acetone followed by decantation.

[0090] The crude reaction product was purified by washing with acetone and methanol several times and then vacuum filtering. The precipitate was dried under vacuum at room temperature for about 16 hours.

[0091] 50 mg of sample was added to 5 ml of ultrapure water (concentration 1% w / v); 24 hours after mixing, the resulting gel gave G'=1550 Pa and G"=186 Pa in rheological tests.

[0092] Example 11: Synthesis of cross-linked hyaluronic acid butyrate-formate sodium salt (50:50; MW: 1500 kDa; DS but :1.6;DS for :0.03:MW:300kDa;DS but :0.3;DS for :0.02) 20 ml of formamide was introduced into a 100 ml three-necked flask, followed by 0.5 g of the product obtained in Example 3 and 0.5 g of the product obtained in Example 4. The mixture was thermostated at 95° C. and kept under constant stirring for 2.5 hours until the polymer was completely dissolved.

[0093] The temperature was then reduced to 25°C and the system was kept under stirring overnight.

[0094] Sodium carbonate (Na2CO3 - 60 mg) was added, followed 0.5 h later by CDI (300 mg dissolved in 1.2 ml DMSO). The mixture was left under stirring at 25°C for 24 h.

[0095] The reaction was quenched by adding 40 ml of water and the product was isolated by precipitation in acetone followed by decantation.

[0096] The crude reaction product was purified by washing with acetone and methanol several times and then vacuum filtering. The precipitate was dried under vacuum at room temperature for about 16 hours.

[0097] 50 mg of sample was added to 5 ml of ultrapure water (concentration 1% w / v); 24 hours after mixing, the resulting gel gave G'=73 Pa and G"=16 Pa in rheological tests.

[0098] Example 12: Synthesis of cross-linked hyaluronic acid butyrate-formate sodium salt (20:80;;MW:1500kDa;DS but :1.6;DS for :0.03:MW:300kDa;DS but :0.3;DS for :0.02) 20 ml of formamide was introduced into a 100 ml three-necked flask, followed by 0.2 g of the product obtained in Example 3 and 0.8 g of the product obtained in Example 4. The mixture was thermostated at 95° C. and kept under constant stirring for 2.5 hours until the polymer was completely dissolved.

[0099] The temperature was then reduced to 25°C and the system was kept under stirring overnight.

[0100] Sodium carbonate (Na2CO3 - 73 mg) was added, followed 0.5 hours later by CDI (345 mg dissolved in 1.4 ml DMSO). The mixture was left under stirring at 25°C for 24 hours.

[0101] The reaction was quenched by adding 40 ml of water and the product was isolated by precipitation in acetone followed by decantation.

[0102] The crude reaction product was purified by washing with acetone and methanol several times and filtering under vacuum, respectively. The precipitate was dried under vacuum at room temperature for about 5 hours.

[0103] Example 13: Synthesis of cross-linked hyaluronic acid butyrate-formate sodium salt (80:20; MW: 1500 kDa; DS but :0.95;DS for :0.01:MW:300kDa;DS but :0.95;DS for :0.01) 20 ml of formamide was introduced into a 100 ml three-necked flask, followed by 0.8 g of the product obtained in Example 2 and 0.2 g of the product obtained in Example 5. The mixture was thermostated at 95°C and stirred at a constant temperature for 2.5 hours until the polymer was completely dissolved. The temperature was then lowered to 25°C, and the system was kept under stirring overnight.

[0104] Sodium carbonate (Na2CO3 - 60 mg) was then added, followed 0.5 hours later by CDI (340 mg dissolved in 1.4 ml DMSO). The mixture was left under stirring at 25°C for 24 hours.

[0105] The reaction was quenched by adding 40 ml of water and the product was isolated by precipitation in acetone followed by decantation.

[0106] The crude reaction product was purified by washing with acetone and methanol several times and then vacuum filtering. The precipitate was dried under vacuum at room temperature for about 16 hours.

[0107] Example 14: Synthesis of cross-linked hyaluronic acid butyrate-formate sodium salt (80:20; MW:25kDa; DS but :0.9:MW:1500kDa;DS but :0.95;DS for :0.01) 20 ml of formamide was introduced into a 100 ml three-necked flask, followed by 0.8 g of the product obtained in Example 8 and 0.2 g of the product obtained in Example 2. The mixture was thermostated at 95°C and kept stirring at a constant temperature for 2.5 hours until the polymer was completely dissolved. The temperature was then lowered to 25°C and the system was kept under stirring overnight.

[0108] Sodium carbonate (Na2CO3 - 65 mg) was then added, followed 0.5 hours later by CDI (345 mg dissolved in 1.4 ml DMSO). The mixture was left under stirring at 25°C for 24 hours.

[0109] The reaction was quenched by adding 40 ml of water and the product was isolated by precipitation in acetone followed by decantation.

[0110] The crude reaction product was purified by washing with acetone and methanol several times and then vacuum filtering. The precipitate was dried under vacuum at room temperature for about 16 hours.

[0111] Example 15: Synthesis of cross-linked hyaluronic acid butyrate-formate sodium salt (20:80;;MW:25kDa;DS but :0.9:MW:1500kDa;DS but :0.95;DS for :0.01) 20 ml of formamide was introduced into a 100 ml three-necked flask, followed by 0.2 g of the product obtained in Example 8 and 0.8 g of the product obtained in Example 2. The mixture was thermostated at 95°C and kept stirring at a constant temperature for 2.5 hours until the polymer was completely dissolved. The temperature was then lowered to 25°C, and the system was kept under stirring overnight.

[0112] Sodium carbonate (Na2CO3 - 70 mg) was then added, followed 0.5 hours later by CDI (348 mg dissolved in 1.4 ml DMSO). The mixture was left under stirring at 25°C for 24 hours.

[0113] The reaction was quenched by adding 40 ml of water and the product was isolated by precipitation in acetone followed by decantation.

[0114] The crude reaction product was purified by washing with acetone and methanol several times and then vacuum filtering. The precipitate was dried under vacuum at room temperature for about 16 hours.

[0115] Example 16: Synthesis of cross-linked hyaluronic acid butyrate-formate sodium salt (80:20; MW: 25 kDa; DS but :0.35:MW:1500 Da;DS but :1.6;DS for :0.03) 20 ml of formamide was introduced into a 100 ml three-necked flask, followed by 0.8 g of the product obtained in Example 7 and 0.2 g of the product obtained in Example 3. The mixture was thermostated at 95°C and stirred at a constant temperature for 2.5 hours until the polymer was completely dissolved. The temperature was then lowered to 25°C, and the system was kept under stirring overnight.

[0116] Sodium carbonate (Na2CO3 - 78 mg) was then added, followed 0.5 hours later by CDI (360 mg dissolved in 1.3 ml DMSO). The mixture was left under stirring at 25°C for 24 hours.

[0117] The reaction was quenched by adding 40 ml of water and the product was isolated by precipitation in acetone followed by decantation.

[0118] The crude reaction product was purified by washing with acetone and methanol several times, followed by vacuum filtration, and the precipitate was dried under vacuum at room temperature for approximately 18 hours.

[0119] Example 17: Synthesis of cross-linked hyaluronic acid butyrate-formate sodium salt (20:80; MW: 25kDa; DS but :0.35:MW:1500kDa;DS but :1.6;DS for :0.03) 20 ml of formamide was introduced into a 100 ml three-necked flask, followed by 0.2 g of the product obtained in Example 7 and 0.8 g of the product obtained in Example 3. The mixture was thermostated at 95°C and stirred at a constant temperature for 2.5 hours until the polymer was completely dissolved. The temperature was then lowered to 25°C, and the system was kept under stirring overnight.

[0120] Sodium carbonate (Na2CO3 - 68 mg) was then added, followed 0.5 hours later by CDI (340 mg dissolved in 1.4 ml DMSO). The mixture was left under stirring at 25°C for 24 hours.

[0121] The reaction was quenched by adding 40 ml of water and the product was isolated by precipitation in acetone followed by decantation.

[0122] The crude reaction product was purified by washing with acetone and methanol several times and then vacuum filtering. The precipitate was dried under vacuum at room temperature for about 20 hours.

[0123] Example 18: Synthesis of cross-linked hyaluronate sodium butyrate-formate (80:20; MW: 25 kDa; DS but :1.6:MW:1500kDa;DS but :0.3;DS for:0.01) 20 ml of formamide was introduced into a 100 ml three-necked flask, followed by 0.8 g of the product obtained in Example 9 and 0.2 g of the product obtained in Example 1. The mixture was thermostated at 95° C. and stirred at a constant temperature for 2.5 hours until the polymer was completely dissolved. The temperature was then lowered to 25° C. and the system was kept under stirring overnight.

[0124] Sodium carbonate (Na2CO3 - 75 mg) was then added, followed 0.5 hours later by CDI (372 mg dissolved in 1.4 ml DMSO). The mixture was left under stirring at 25°C for 24 hours.

[0125] The reaction was quenched by adding 40 ml of water and the product was isolated by precipitation in acetone followed by decantation.

[0126] The crude reaction product was purified by washing with acetone and methanol several times and then vacuum filtering. The precipitate was dried under vacuum at room temperature for about 16 hours.

[0127] Example 19: Synthesis of cross-linked hyaluronic acid butyrate-formate sodium salt (20:80; MW:25kDa; DS but :1.6:MW:1500kDa;DS but :0.3;DS for :0.01) 20 ml of formamide was introduced into a 100 ml three-necked flask, followed by 0.2 g of the product obtained in Example 9 and 0.8 g of the product obtained in Example 1. The mixture was thermostated at 95° C. and kept stirring at a constant temperature for 2.5 hours until the polymer was completely dissolved. The temperature was then lowered to 25° C. and the system was kept under stirring overnight.

[0128] Sodium carbonate (Na2CO3 - 70 mg) was then added, followed 0.5 hours later by CDI (3348 mg dissolved in 1.4 ml DMSO). The mixture was left under stirring at 25°C for 24 hours.

[0129] The reaction was quenched by adding 40 ml of water and the product was isolated by precipitation in acetone followed by decantation.

[0130] The crude reaction product was purified by washing with acetone and methanol several times and then vacuum filtering. The precipitate was dried under vacuum at room temperature for about 20 hours.

[0131] Example 20: Synthesis of hyaluronic acid butyrate-formate sodium salt (MW:1500:300kDa 80:20;DS but :0.9;DS for :0.02) 150 ml of formamide was introduced into a 1 L reactor, followed by 6 g of sodium hyaluronate with a molecular weight of 1500 kDa and 1.5 g of sodium hyaluronate with a molecular weight of 300 kDa. The mixture was thermostated at 95°C and stirred at a constant temperature for 1.5 hours until the polymer was completely dissolved. The temperature was then lowered to 25°C, and the system was kept under stirring overnight.

[0132] Sodium carbonate (Na2CO3 - 0.6 g) was then added, followed 0.5 hours later by butyryl-imidazolide (5.7 g). The mixture was stirred at 25°C for 1.5 hours.

[0133] The reaction was quenched with 65 ml of acidified water and the product was isolated by precipitation with acetone followed by decantation.

[0134] The crude reaction product was purified by washing with acetone and methanol several times, followed by vacuum filtration, and the precipitate was dried under vacuum at room temperature for approximately 18 hours.

[0135] 10 mg of sample was dissolved in 0.8 ml of deuterium oxide (D2O) and transferred to an NMR tube.

[0136] After hydrolysis of the butyric and formic acid esters by adding NaOD (deuterated sodium hydroxide), the NMR spectra showed a DS of 0.9 in butyric acid and 0.02 in formic acid.

[0137] Example 21: Synthesis of cross-linked sodium hyaluronate butyrate from Example 20 + carbonyldiimidazole (CDI) 20 ml of formamide and 2 g of the product obtained in Example 20 were introduced into two different 100 mL three-neck flasks (designated A and B). The mixture was thermostated at 95°C and kept stirring at a constant temperature for 1 hour until the product was completely dissolved. The temperature was then lowered to 25°C and the system was kept under stirring overnight.

[0138] Dimethylaminopyridine (DMAP - 263 mg, solubilized in 1.5 ml of formamide) was added to flask A, followed one hour later by CDI (350 mg dissolved in 1.5 ml of DMSO). The mixture was left under stirring at 25°C for 2 hours.

[0139] Dimethylaminopyridine (DMAP-263 mg, solubilized in 1.5 ml of FA) was added to flask B and the mixture was left under stirring at 25° C. for 2 hours.

[0140] Mixture A was added to Mixture B, and the system was reacted under stirring for about 4 hours. The reaction was quenched with 70 ml of water, and the pH was adjusted to 10-7.5 by adding 10 ml of 0.5 M HCl.

[0141] The crude product was isolated by precipitation with acetone, washed several times with methanol, and purified by vacuum filtration after each wash. The precipitate was dried under vacuum at room temperature for approximately 16 hours.

[0142] 50 mg of sample was added to 5 ml of ultrapure water (concentration 1% w / v); 24 hours after mixing, the resulting gel gave G'=480 Pa and G"=70 Pa in rheological tests.

[0143] Example 22: Synthesis of hyaluronic acid butyrate-formate sodium salt (MW: 1500: 300kDa 20: 80; DS but :0.9;DS for :0.02)+Crosslink 150 ml of formamide was introduced into a 1 L reactor, followed by 1.5 g of sodium hyaluronate with a molecular weight of 1500 kDa and 6 g of sodium hyaluronate with a molecular weight of 300 kDa. The mixture was adjusted to a temperature of 95°C and stirred at a constant temperature for 1.5 hours until the polymer was completely dissolved. The temperature was then lowered to 25°C, and the system was kept under stirring overnight.

[0144] Sodium carbonate (NaCO -0.6 g) was then added, followed 0.5 hours later by butyryl-imidazolide (5.7 g). The mixture was stirred at 25°C for 1.5 hours. 547.5 mg of sodium carbonate was then added, and the mixture was left stirring for 0.5 hours, after which 2.6 g of CDI dissolved in 11 ml of dimethyl sulfoxide was added.

[0145] After 24 hours, the product was precipitated by adding acetone to the crude reaction product.

[0146] The isolated crude product was purified by successively washing with acetone and methanol, followed by vacuum filtration. The precipitate was dried under vacuum at room temperature for approximately 10 hours.

[0147] 10 mg of sample was dissolved in 0.8 ml of deuterium oxide (D2O) and transferred to an NMR tube.

[0148] After hydrolysis of the butyric and formic acid esters by adding NaOD (deuterated sodium hydroxide), the NMR spectra showed a DS of 0.9 in butyric acid and 0.02 in formic acid.

[0149] Example 23: Synthesis of hyaluronic acid butyrate-formate sodium salt (MW: 1500: 25kDa 20: 80; DS but :0.9;DS for :0.02) 150 ml of formamide was introduced into a 1 L reactor, followed by 1.5 g of sodium hyaluronate with a molecular weight of 1500 kDa and 6 g of sodium hyaluronate with a molecular weight of 25 kDa. The mixture was adjusted to 95°C and stirred at a constant temperature for 1.5 hours until the polymer was completely dissolved. The temperature was then lowered to 25°C, and the system was kept under stirring overnight.

[0150] Sodium carbonate (Na2CO3 - 0.6 g) was then added, followed 0.5 hours later by butyryl-imidazolide (5.7 g). The mixture was stirred at 25°C for 1.5 hours.

[0151] The reaction was quenched with 65 ml of acidified water and the product was isolated by precipitation with acetone followed by decantation.

[0152] The crude reaction product was purified by washing with acetone and methanol several times, followed by vacuum filtration, and the precipitate was dried under vacuum at room temperature for approximately 18 hours.

[0153] 10 mg of sample was dissolved in 0.8 ml of deuterium oxide (D2O) and transferred to an NMR tube.

[0154] After hydrolysis of the butyric and formic acid esters by adding NaOD (deuterated sodium hydroxide), the NMR spectra showed a DS of 0.9 in butyric acid and 0.02 in formic acid.

[0155] Example 24: Synthesis of cross-linked sodium hyaluronate butyrate from Example 23 + carbonyldiimidazole (CDI) 20 ml of formamide and 2 g of the product obtained in Example 23 were introduced into two different 100 ml three-necked flasks (designated A and B). The mixture was thermostated at 95°C and kept stirring at a constant temperature for 1 hour until the product was completely dissolved. The temperature was then lowered to 25°C and the system was kept under stirring overnight.

[0156] Sodium carbonate (Na2CO3 - 114.4 mg) was added to flask A, followed one hour later by CDI (350 mg dissolved in 1.5 ml of DMSO). The mixture was left under stirring at 25°C for 2 hours.

[0157] Sodium carbonate (Na2CO3-114.4 mg) was added to flask B, and the mixture was stirred at 25°C for 2 hours.

[0158] Mixture A was added to Mixture B, and the system was reacted under stirring for about 4 hours. The reaction was quenched with 70 ml of water, and the pH was adjusted from 10 to 7.5 by adding 0.5 M HCl.

[0159] The crude product was isolated by precipitation with acetone, washed several times with acetone and methanol, and purified by vacuum filtration. The precipitate was dried under vacuum at room temperature for approximately 16 hours.

[0160] Example 25: Synthesis of hyaluronic acid butyrate-formate sodium salt (MW: 1500: 25kDa 80: 20; DS but :0.9;DS for :0.02)+Crosslink 150 ml of formamide was introduced into a 1 L reactor, followed by 6.0 g of sodium hyaluronate with a molecular weight of 1500 kDa and 1.5 g of sodium hyaluronate with a molecular weight of 25 kDa. The mixture was thermostated at 95°C and stirred at a constant temperature for 1.5 hours until the polymer was completely dissolved. The temperature was then lowered to 25°C, and the system was kept under stirring overnight.

[0161] Sodium carbonate (Na2CO3 - 0.65 g) was then added, followed 0.5 hours later by butyryl-imidazolide (5.9 g). The mixture was stirred at 25°C for 1.5 hours.

[0162] Then 550.0 mg of sodium carbonate were added and the mixture was left under stirring for 0.5 hours, after which 2.7 g of CDI dissolved in 11 ml of dimethyl sulfoxide were added.

[0163] After 24 hours, the crude reaction product was precipitated by adding acetone.

[0164] The isolated crude product was purified by successively washing with acetone and methanol, followed by vacuum filtration. The precipitate was dried under vacuum at room temperature for approximately 10 hours.

[0165] 10 mg of sample was dissolved in 0.8 ml of deuterium oxide (D2O) and transferred to an NMR tube.

[0166] After hydrolysis of the butyric and formic acid esters by adding NaOD (deuterated sodium hydroxide), the NMR spectra showed a DS of 0.9 in butyric acid and 0.02 in formic acid.

[0167] Example 26: Synthesis of cross-linked hyaluronic acid butyrate-formate sodium salt (80:20; MW: 1500 kDa; DS but :1.6DS for : 0.03, MW: 300kDa; DS but :2.16;DS for :0.02) 40 ml of formamide was introduced into a 250 ml three-necked flask, followed by 1.6 g of the product obtained in Example 3 and 0.4 g of the product obtained in Example 6. The mixture was heat-cured to 95°C and maintained at a constant temperature under stirring for 1 hour until the polymer was completely dissolved. The temperature was then reduced to 25°C and the system was maintained under stirring overnight.

[0168] Sodium carbonate (Na2CO3 - 122 mg) was then added, followed 0.75 hours later by CDI (252 mg dissolved in 1 ml DMSO). The mixture was left under stirring at 25°C for 24 hours.

[0169] The reaction was quenched by adding 80 ml of water and the product was isolated by precipitation in acetone followed by filtration.

[0170] The crude reaction product was purified by successively washing with acetone and methanol, followed by vacuum filtration. The precipitate was dried under vacuum at room temperature for approximately 16 hours.

[0171] The swelling test was carried out twice: (1) 100 mg of polymer was dissolved in 10 mM PBS (pH 7.2) at a concentration of 2%. The resulting sample was then sterilized in an autoclave at 121 °C for 15 minutes. After leaving it for 24 hours, rheological measurements were performed. (2) 100 mg of polymer was dissolved in 10 ml of water (concentration: 1%). After 24 hours, the gel was manually homogenized and rheological measurements were performed.

[0172] The results are shown in Table 1 below.

[0173] [Table 1]

[0174] Example 27: Synthesis of cross-linked hyaluronic acid butyrate-formate sodium salt (MW: 1500kDa; DS but :1.6DS for :0.03) 40 ml of formamide was introduced into a 250 ml three-necked flask, followed by 2 g of the product obtained in Example 3. The mixture was heat-cured to 95°C and maintained at a constant temperature under stirring for 1 hour until the polymer was completely dissolved. The temperature was then reduced to 25°C, and the system was maintained under stirring overnight.

[0175] Sodium carbonate (Na2CO3 - 124 mg) was then added, followed 0.75 hours later by CDI (258 mg dissolved in 1 ml DMSO). The mixture was left under stirring at 25°C for 24 hours.

[0176] The reaction was quenched by adding 80 ml of water and the product was isolated by precipitation in acetone followed by filtration.

[0177] The crude reaction product was purified by successively washing with acetone and methanol, followed by vacuum filtration. The precipitate was dried under vacuum at room temperature for approximately 16 hours.

[0178] Example 28: Synthesis of cross-linked hyaluronic acid butyrate sodium-formate (molecular weight: 300 kDa; DS but :2.16;DS for :0.02) 40 ml of formamide was introduced into a 250 ml three-necked flask, followed by 2 g of the product obtained in Example 6. The mixture was heat-cured to 95°C and maintained at a constant temperature under stirring for 1 hour until the polymer was completely dissolved. The temperature was then reduced to 25°C and the system was maintained under stirring for 3 hours.

[0179] Sodium carbonate (Na2CO3 - 116 mg) was then added, followed 0.75 hours later by CDI (240 mg dissolved in 1 ml DMSO). The mixture was left under stirring at 25°C for 24 hours.

[0180] The reaction was stopped by adding 80 ml of water, and the product was purified by dialysis and recovered by lyophilization.

[0181] Example 29 (Comparative): Crosslinked hyaluronic acid butyrate-formate sodium salt (MW: 1500 kDa; DS but :1.6DS for :0.03) and cross-linked hyaluronic acid butyrate-formate sodium salt (MW: 300 kDa; DS but :2.1;DS for :0.02) (80:20) (1) 80 mg of the product from Example 27 and 20 mg of that from Example 28 were dissolved in 10 mM PBS, pH 7.2, at a concentration of 2%. The resulting samples were then sterilized in an autoclave at 121°C for 15 minutes. After leaving for 24 hours, rheological measurements were performed. (2) 80 mg of the product from Example 27 and 20 mg from Example 28 were dissolved in 10 ml of water (1% concentration). After 24 hours, the gel was manually homogenized and rheological measurements were performed. The results are shown in Table 2 below.

[0182] [Table 2]

[0183] Example 30: Preparation of a medical device in the form of a syringe containing 2.0 ml of a 2% w / v hydrogel of the crosslinked product obtained according to Example 26 and 0.3% w / v lidocaine 40 mg of the cross-linked esterified polymer in powder form obtained according to Example 26 was weighed into a sterile 2.5 ml syringe together with 6 mg of lidocaine hydrochloride; 2.0 ml of an aqueous solution of 10 mM PBS buffer (pH 7.2) was introduced into the syringe. The polymer was allowed to swell at room temperature for 24 hours. The syringe was then steam sterilized in an autoclave at 121°C for 15 minutes according to a standard cycle. 24 hours after sterilization, the resulting gel was extruded from the syringe and subjected to rheological characterization. The results are shown in Table 3 below.

[0184] [Table 3]

[0185] Example 31: Preparation of a 2% w / v hydrogel of the cross-linked product obtained according to Example 26 containing an amino acid 107 mg of the cross-linked esterified polymer in powder form obtained according to Example 26 was weighed into a sterile 10.0 ml glass bottle; 5.0 ml of an aqueous solution of lactated Ringer's (pH 7.0) containing 50 mM histidine was poured into the glass bottle. The polymer was allowed to swell at room temperature for 24 hours. The sealed glass bottle was then steam sterilized in an autoclave at 121°C for 15 minutes according to a standard cycle. 30 days after sterilization, the resulting gel was subjected to rheological characterization. The results are shown in Table 4 below.

[0186] [Table 4]

[0187] Example 32: Ophthalmic preparation starting from the crosslinked product obtained according to Example 26 5 ml of water was added to a 10 ml glass bottle, and 0.045 g of sodium chloride was dissolved therein. 0.025 g of the cross-linked esterified polymer in powder form obtained according to Example 26 was then added. The polymer was allowed to swell at room temperature for 24 hours. The sealed glass bottle was then steam sterilized in an autoclave at 121 °C for 15 minutes according to the standard cycle.

[0188] Example 33: Topical formulation starting from the crosslinked product obtained according to Example 26

[0189] [Table 5]

Claims

1. A method for preparing crosslinked hyaluronic acid butyrate or crosslinked hyaluronic acid butyrate-formate or an acceptable salt thereof, the method comprising crosslinking hyaluronic acid butyrate or hyaluronic acid butyrate-formate or a pharmaceutically acceptable salt thereof in an organic solvent with a carboxyl group-activating reagent and a base, wherein the hyaluronic acid butyrate or hyaluronic acid butyrate-formate or a pharmaceutically acceptable salt thereof is a mixture of a high molecular weight polysaccharide having a weight-average molecular weight in the range of 1,000 kDa to 10,000 kDa and a low molecular weight polysaccharide having a weight-average molecular weight in the range of 1 kDa to 900 kDa.

2. 2. The method of claim 1, wherein the low molecular weight polysaccharide has a weight average molecular weight in the range of 1 kDa to 500 kDa and the high molecular weight polysaccharide has a weight average molecular weight in the range of 1000 kDa to 6000 kDa.

3. 3. The method of claim 2, wherein the low molecular weight polysaccharide has a weight average molecular weight in the range of 50 kDa to 300 kDa and the high molecular weight polysaccharide has a weight average molecular weight in the range of 1000 kDa to 2000 kDa.

4. 4. The method of claim 1, wherein the high molecular weight polysaccharide and the low molecular weight polysaccharide are present in a weight ratio of 80:20 to 20:

80.

5. The method according to any one of claims 1 to 4, wherein the hyaluronic acid butyrate or hyaluronic acid butyrate-formate or a pharmaceutically acceptable salt thereof has a degree of substitution ranging from 0.1 to 2.

2.

6. The method according to any one of claims 1 to 5, wherein the acceptable salt of hyaluronic acid butyrate or hyaluronic acid butyrate-formate is a sodium, potassium or lithium salt, or a quaternary ammonium salt.

7. 7. The method of claim 6, wherein the acceptable salt is a sodium salt.

8. The method according to any one of claims 1 to 7, wherein the organic solvent is selected from basic polar aprotic solvents.

9. 9. The method of claim 8, wherein the organic solvent is formamide.

10. The method according to any one of claims 1 to 9, wherein the carboxyl group activating reagent is selected from the group consisting of carbonyldiimidazole, 1,1'-carbonyl-di-(1,2,4-triazole), 1,1'-oxalylimidazole, 1,1'-thiocarbonylimidazole, 1,1'-carbonylbis(2-methylimidazole), N-hydroxysuccinimide, p-nitrophenol, p-nitrophenyl trifluoroacetate, 2-halo-N-alkylpyridine salts, and acyl halides.

11. The method of claim 10, wherein the carboxyl group activating reagent is carbonyldiimidazole.

12. 12. The method according to any one of claims 1 to 11, wherein the base is selected from inorganic bases selected from alkali metal or alkaline earth metal carbonates, bicarbonates and hydroxides, aromatic or aliphatic organic bases containing at least one trisubstituted nitrogen atom, or basic amines selected from triethylamine, N-methyl-piperazine and dimethylaminopyridine, or alkali or alkaline earth metal salts of organic acids.

13. 13. The method of claim 12, wherein the base is sodium carbonate or dimethylaminopyridine.

14. A process according to any one of claims 1 to 13, wherein the reaction mixture is maintained at a temperature in the range of from 20 to 30°C for a period of from 4 to 24 hours.

15. A pharmaceutical or cosmetic formulation or a medical device comprising crosslinked hyaluronic acid butyrate or crosslinked hyaluronic acid butyrate-formate or a pharmaceutically acceptable salt thereof obtainable by the method according to claim 14, and at least one pharmaceutically acceptable excipient and / or carrier.

16. 16. A pharmaceutical or cosmetic preparation or medical device according to claim 15, containing a local anesthetic.

17. 16. A pharmaceutical formulation or medical device according to claim 15 for use in the topical treatment of skin lesions, rashes and eye lesions.

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