Method for binding hyaluronic acid and hyaluronic acid conjugate obtained thereby

Abstract

The present invention describes a method for conjugating hyaluronic acid using a specific condensing agent under specific operating conditions, which allows for the efficient, rapid, and economical preparation of hyaluronic acid derivatives with disaccharides or oligosaccharides having the desired degree of conjugation. Hyaluronic acid conjugates with specific degrees of conjugation are also described, as well as their use for therapeutic purposes and as scaffolds for biomaterials.

Description

[Technical Field]

[0001] The present invention relates to a method for binding hyaluronic acid using a specific condensing agent under specific operating conditions, which allows for the efficient, rapid and economical production of hyaluronic acid derivatives with disaccharides or oligosaccharides having the desired degree of binding.

[0002] Therefore, the present invention also relates to hyaluronic acid conjugates having a specific degree of binding and uses thereof. [Background technology]

[0003] Hyaluronic acid, or simply "HA," is a non-sulfated linear glycosaminoglycan (GAG) composed of repeating disaccharide units consisting of glucuronic acid and N-acetylglucosamine residues linked by alternating β1→4 and β1→3 glycosidic bonds.

[0004] At physiological pH, the carboxyl groups of the glucuronic acid units are ionized, giving HA a high polarity and consequently good solubility in water. The molecular weight of HA is 10 3 ~10 7 Da, and this parameter influences its properties, allowing for a variety of applications.

[0005] HA is naturally present in both vertebrate organisms and bacteria. Its presence is primarily in the extracellular matrix (ECM) of epithelial cells and connective tissues. The biological properties of hyaluronan are due to its chemical and physical properties, such as viscosity and high water-holding capacity, which allow it to play a structural and hydration role. Currently, research on hyaluronan focuses on its role as a drug carrier through appropriate chemical functionalization. During enzymatic degradation of hyaluronan, cellular uptake by the CD44 receptor determines the intracellular release of drugs obtained by binding to HA or encapsulation within HA particles. In this regard, the possibility of functionalizing hyaluronan with modified lactose is particularly interesting, given its biological significance in terms of its interaction with galectins.

[0006] Indeed, since galectins are galactose-specific receptors, the addition of these galactose residues to the HA chains allows the polymer to interact not only with the CD44 receptor but also with galectins, creating a desirable synergistic effect.

[0007] A large number of derivatives of hyaluronic acid are known in the literature for the most diverse applications. However, it has been observed that there is still a need for efficient and economical functionalization processes that, above all, ensure the achievement of the desired degree of substitution, high yields, and a minimum of by-products. Summary of the Invention

[0008] The above object has been achieved by a method for binding hyaluronic acid using a specific condensing agent under specific operating conditions, as described in claim 1 of the present invention.

[0009] In another aspect, the present invention relates to a conjugate of hyaluronic acid or a pharmaceutically acceptable salt thereof with a disaccharide or oligosaccharide, wherein the average degree of substitution in the carboxyl group is 10 to 50%, more preferably 15 to 45%.

[0010] In a further aspect, the present invention relates to the use of said conjugates in the treatment of conditions resulting from altered galectin expression, including, but not limited to, non-alcoholic steatohepatitis, plaque psoriasis, rheumatoid arthritis, osteoarthritis, tumors, adhesions, and fibrotic skin, pulmonary, renal, and cardiovascular processes.

[0011] In a further aspect, the invention relates to the use of the conjugate in rheumatology, orthopedics, oncology, plastic / cosmetic surgery, hemodialysis, cardiology, angiology, ophthalmology, otolaryngology, pulmonology, dentistry, gynecology, urology, dermatology, oncology, and tissue repair.

[0012] The features and advantages of the present invention will become apparent from the following detailed description and from the embodiments provided as illustrative and non-limiting examples. DETAILED DESCRIPTION OF THE INVENTION

[0013] Thus, the present invention relates to a method for conjugating hyaluronic acid or a pharmaceutically acceptable salt thereof with a disaccharide or oligosaccharide, comprising the steps of: i) providing an aqueous solution of hyaluronic acid or a pharmaceutically acceptable salt thereof; ii) adding the triazine compound of formula (I) under stirring to form an ester adduct between the carboxyl group of hyaluronic acid or a pharmaceutically acceptable salt thereof and the triazine ring of the compound of formula (I); iii) waiting for at least 15 minutes under stirring, adding a primary amine of a disaccharide or oligosaccharide to form a conjugate between hyaluronic acid or a pharmaceutically acceptable salt thereof and said disaccharide or oligosaccharide via an amide bond; iv) adding a water-miscible organic solvent to promote precipitation of the resulting conjugate, thereby obtaining a crude conjugate, and optionally further washing the precipitated conjugate with an organic solvent optionally mixed with water; v) separating the loosely bound bodies, and optionally vi) purifying the crude conjugate by reprecipitation, ion exchange resin treatment, dialysis, or combinations and / or repetitions thereof; and vii) Obtaining the purified conjugate by drying. wherein the method is carried out at a temperature of 4 to 100°C and a pH of 5 to 8; The triazine compound has the formula (I): [ka]

[0014] wherein X is a halogen selected from I, Br, Cl and mixtures thereof, or a -NR1R2 group, which is selected from NH2, NMe2, NHPr, PhNH, 4-MeCH4NH, [ka]

[0015] and mixtures thereof.

[0016] As can be seen from the examples below, the method according to the invention makes it possible to conveniently and quickly obtain conjugates having the desired degree of substitution, as well as in high purity.

[0017] In step i), an aqueous solution of hyaluronic acid or a pharmaceutically acceptable salt thereof is provided.

[0018] Preferably, the hyaluronic acid in step i) has an average molecular weight of 50 to 5000 kDa, more preferably 50 to 500 kDa.

[0019] The term "pharmaceutically acceptable salts" of hyaluronic acid in step i) preferably means salts selected from sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, ammonium hyaluronate, tetrabutylammonium hyaluronate, and mixtures thereof.

[0020] Preferably, step i) is carried out at a pH of 5-7, more preferably 6-7, by addition of an acid, preferably 2-[N-morpholino]-ethanesulfonic acid (MES).

[0021] In step ii), the triazine compound of formula (I) is added under stirring to form an ester adduct between the carboxyl group of hyaluronic acid, or a pharmaceutically acceptable salt thereof, and the triazine ring, as shown below: [ka] (During the ceremony, “HACOO - " is the carboxyl group of hyaluronic acid that is attached to the triazine ring and releases the X group to form the ester adduct on the right, or a pharmaceutically acceptable salt thereof.

[0022] Preferably, the triazine compound of formula (I) is 4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (abbreviated as "DMTMM"), 2-chloro-4,6-dimethoxy-1,3,5-triazine (abbreviated as "CDMT"), or a mixture thereof.

[0023] In a particularly preferred embodiment, the triazine compound of formula (I) is DMTMM, and in step ii) the following reaction occurs: [ka] (During the ceremony, “HACOO - " is the carboxyl group of hyaluronic acid that binds to the triazine ring of DMTMM and releases methylmorpholine to form the ester adduct on the right, or a pharmaceutically acceptable salt thereof.

[0024] Preferably, the molar ratio of hyaluronic acid or a pharmaceutically acceptable salt thereof to the triazine compound of formula (I) in step ii) is 3:1 to 1:3.

[0025] The mixture formed after the addition of the triazine compound of formula (I) is allowed to react for at least 15 minutes, preferably at least 30 minutes, after which, according to step iii), a primary amine of a disaccharide or oligosaccharide is added under stirring to form a conjugate of hyaluronic acid, or a pharmaceutically acceptable salt thereof, with said disaccharide or oligosaccharide via an amide bond.

[0026] The term "primary amine" refers to the free primary amine -NH2 as well as pharmaceutically acceptable salts thereof.

[0027] Preferably, the term "oligosaccharide" refers to a glycopolymer containing 3 to 6 monosaccharides, ie, a trisaccharide to a hexasaccharide.

[0028] More preferably, the primary amine of the disaccharide or oligosaccharide is a primary amine derivative of sucrose, maltose, lactose, trehalose, gentiobiose, cellobiose, maltotriose, raffinose, stachyose, maltotetraose, melezitose, isomaltose, panose, nystose, 1-ketose, galactopinitol, galactosyllactose, or a mixture thereof.

[0029] In a preferred embodiment, in step iii), a primary amine of a disaccharide is added, which is a primary amine derivative of sucrose, maltose, lactose, trehalose, gentiobiose, cellobiose, or a mixture thereof, more preferably a primary amine derivative of lactose. The addition of said primary amine to the mixture containing the ester adduct leads to the formation of a conjugate in addition to the release of 4,6-dimethoxy-1,3,5-triazin-2-ol, as shown below: [ka] Here, R-NH2 is a primary amine of a disaccharide or oligosaccharide, abbreviated as "R", while the resulting conjugate is represented schematically, emphasizing the amide bond between the carboxyl group of hyaluronic acid, or a pharmaceutically acceptable salt thereof, and the amino group of the primary amine.

[0030] Preferably, the molar ratio of hyaluronic acid or a pharmaceutically acceptable salt thereof to the primary amine in step iii) is 3:1 to 1:3.

[0031] Preferably, in step iii) the primary amine and the adduct are reacted at room temperature for at least 12 hours, preferably at least 24 hours, more preferably at least 48 hours.

[0032] In a preferred embodiment, in step iii), after leaving the reaction for at least 12 hours, a base, preferably NaOH 5M, is subsequently added. This basification step cleaves the ester bond formed by secondary reaction of the activated carboxylic acid of HA with its hydroxyl group or lactosamine.

[0033] In step iv), the precipitation of the resulting conjugate is promoted by adding an acid, preferably HCl 5M, and a water-miscible organic solvent to obtain a crude conjugate, and optionally the precipitated conjugate is further washed with an organic solvent optionally mixed with water.

[0034] The organic solvent serves to reduce the polarity of water in order to increase the precipitation rate and yield of the conjugate.

[0035] Preferably, the water-miscible organic solvent is selected from acetone, acetonitrile, 1-4 dioxane, tetrahydrofuran, or an alcohol, preferably an alcohol having 1 to 5 carbon atoms, more preferably methanol, ethanol, 2-propanol, or a mixture thereof.

[0036] In step v), the crude conjugate is separated from the solvent mixture. Preferably, this separation can be achieved by techniques such as decantation, filtration, evaporation, or centrifugation, or a combination thereof.

[0037] In optional step vi), the crude conjugate thus isolated is subjected to a further purification procedure, which can be carried out by reprecipitation, ion exchange resin treatment, or dialysis, to obtain a more purified conjugate. The three purification methods can be performed in combination with each other or can be repeated multiple times, for example, reprecipitation followed by ion exchange resin treatment followed by reprecipitation.

[0038] Preferably, when the purification of the crude conjugate in step vi) is carried out by ion exchange resin treatment, the following substeps are carried out: vi-a) dissolving the crude binder in water to obtain an aqueous solution; vi-b) subjecting said aqueous solution to ion exchange resin treatment using water as an eluent; vi-c) separating the aqueous solution from the ion exchange resin; vi-d) Adjusting the separated solution to a neutral pH.

[0039] In a preferred embodiment, the cation exchange column is Amberlite™ IR120 (CAS number: 78922-04-0; IUPAC name: 3-[(3-chlorophenyl)sulfonylamino]benzoic acid).

[0040] In substep vi-d), the eluate can be brought to a neutral pH by adding a salt such as NaHCO3, while subsequent drying can be achieved by lyophilization.

[0041] The unreacted primary amines retained on the cation exchange column can then be recovered using NH3 as the eluent.

[0042] Alternatively, if the purification of the crude conjugate in step vi) is carried out by dialysis, the following substeps are performed: vi-i) dissolving the crude binder in water to obtain an aqueous solution; vi-ii) Dialyzing the resulting solution against saline and / or water through a suitable membrane.

[0043] Alternatively, and preferably, when the purification of the crude conjugate in step vi) is carried out by reprecipitation, the following substeps are carried out: vi-α) dissolving the crude conjugate in water to obtain an aqueous solution; vi-β) adding a water-miscible organic solvent, which may be the same as or different from the solvent of step iv), thereby promoting reprecipitation of the conjugate; vi-γ) Washing the precipitated conjugate with additional organic solvent, thereby obtaining a purified conjugate.

[0044] In substep vi-β), preferably an additional aqueous salt solution is added, more preferably a 0.5 M NaCl solution.

[0045] In substep vi-γ), the washing operation of the conjugate with additional organic solvents can be repeated several times to increase the final purity of said conjugate.

[0046] Alternatively, and preferably, when the purification of the crude conjugate in step vi) is carried out by dialysis, the following substeps are carried out: vi-●) dissolving the crude conjugate in water to obtain an aqueous solution; vi) Transfer to a 6-8 kDa membrane and dialyze against NaCl salt solution for at least 24 hours; vi-●●●) A step in which the retentate is collected and freeze-dried.

[0047] In a preferred embodiment, the process according to the invention is carried out at a temperature of 15-75°C and a pH of 6-11.

[0048] The above primary amines, and likewise the related pharmaceutically acceptable salts, are either commercially available or can be prepared by commonly known methods of amination of di- or oligosaccharides or by a method comprising the following steps: - reacting said disaccharide or oligosaccharide with ammonium acetate under stirring in the presence of ammonia and NaCNBH3 at a temperature of at least 60°C, - drying the product thus obtained and placing it on a cation exchange column, eluting first with deionized water to desalt the product and then with ammonia, - collecting the eluate coming out of the column, bringing said eluate to a neutral pH and then drying to obtain the purified primary amine.

[0049] Additionally, the primary amine can be prepared by a method comprising the steps of: - reacting said disaccharide or oligosaccharide with benzylamine in the presence of borane at a temperature between 5 and 60°C under stirring, - isolating the benzylamine derivative obtained from the reductive amination reaction, - reacting the benzylamine derivative with a hydrogen source under stirring in the presence of a catalyst for hydrogenolysis at a temperature between 4 and 100°C; - isolating the benzylamine derivative, purifying it by passing it through an ion exchange column, desalting it, and then drying the product.

[0050] The term "borane" includes sodium borohydride, sodium cyanoborohydride, sodium acetate borohydride, sodium triacetoxyborohydride, lithium borohydride, potassium borohydride, tetrabutylammonium borohydride, calcium borohydride, magnesium borohydride, tetraethylammonium borohydride, methyltrioctylammonium borohydride, bis(triphenylphosphine)copper(I) borohydride, potassium tri(1-pyrazolyl)borohydride, water Examples of suitable borane complexes include cetyltrimethylammonium boron hydride, borane-tetrahydrofuran complex, picoline-borane complex, 5-ethyl-2-methylpyridine-borane complex, dimethylsulfide-borane complex, pyridine-borane complex, trimethylamine-borane complex, triethylamine-borane complex, morpholine-borane complex, t-butylamine-borane complex, ammonia-borane complex, diphenylphosphine-borane complex, 4-methylmorpholine-borane complex, borane-ethylenediamine complex, or mixtures thereof.

[0051] The term "catalyst" preferably refers to a Raney catalyst using a metal from Groups 8 to 11, a metal from Groups 8 to 11 supported on carbon, silica, alumina, aluminosilicate, zirconia, or mixtures thereof.

[0052] The term "hydrogen source" refers to a compound capable of releasing H2 or H+ ions under the conditions of the process.

[0053] In a further aspect, the present invention relates to a conjugate of hyaluronic acid or a pharmaceutically acceptable salt thereof with a disaccharide or oligosaccharide, wherein the average degree of substitution of carboxyl groups by amide bonds with said disaccharide or oligosaccharide is 10 to 50%, more preferably 15 to 45%, and which is obtained by the above-mentioned method, in particular when: - in step ii) the molar ratio of hyaluronic acid or a pharmaceutically acceptable salt thereof to the triazine compound of formula (I) is between 3:1 and 1:3, and / or - in step iii), the molar ratio of hyaluronic acid or its pharmaceutically acceptable salt to the primary amine is 3:1 to 1:3.

[0054] In fact, it has been surprisingly observed that by adjusting the ratio between the reagents as described above, conjugates having the desired degree of substitution can be obtained effectively and quantitatively.

[0055] The conjugates can be administered by inhalation, oral, nasal, ophthalmic, urological, intraarticular, intramuscular, intravenous, intradermal, transdermal, subcutaneous, or topical or internal route, e.g., surgical route.

[0056] Preferably, the conjugate is administered by oral, nasal, ophthalmic, urological, intraarticular, intradermal, or inhalation routes.

[0057] In certain embodiments, the conjugate is in an injectable form suitable for injection into hard or soft tissues, such as organs, adipose tissue, mucosa and gums, preferably by intradermal, subcutaneous, intramuscular, or intraarticular route.

[0058] The conjugate can be in the form of a softgel capsule, or in a solid form such as a tablet, mini-tablet, micro-tablet, granule, micro-granule, pellet, multi- or micronized particle, or powder, or in the form of a solution, emulsion, gel, ointment, eye drop, nebulizer solution, or spray.

[0059] In a preferred embodiment, the conjugate is in the form of a powder, solution, emulsion, gel, ointment, eye drops, nebulizer solution, spray, or injectable form, for administration via nasal or inhaled or intra-articular or intradermal or ophthalmic or urological routes.

[0060] In a further aspect, the present invention relates to said conjugates in the treatment of conditions resulting from altered galectin expression. Non-limiting examples of such conditions include non-alcoholic steatohepatitis, plaque psoriasis, rheumatoid arthritis, osteoarthritis, tumors, adhesions, and fibrotic skin, lung, kidney, and cardiovascular processes. Examples of tumors and fibrotic processes include acute lymphoblastic leukemia, idiopathic pulmonary fibrosis, liver fibrosis, cardiac fibrosis, kidney fibrosis, and tumors of the ovary, prostate, lung, stomach, skin, thyroid, and pancreas.

[0061] In a further aspect, the present invention relates to the use of said conjugate as a biomaterial or scaffold for cell growth in the treatment of orthopedic diseases.

[0062] In a further preferred embodiment, the present invention relates to the use of said conjugates in tissue repair or reconstruction, preferably in creating or replacing biological tissue or filling biological tissue such as skin, cavities, bone cartilage or joints.

[0063] In a further aspect, the present invention relates to the use of said conjugates in rheumatology, orthopedics, oncology, plastic / cosmetic surgery, hemodialysis, cardiology, angiology, ophthalmology, otorhinolaryngology, pulmonology, dentistry, gynecology, urology, dermatology, oncology, and tissue repair. Furthermore, the present invention relates to the use of said conjugates in traumatic and / or post-operative tissue processes and / or chronic fibrotic processes associated with autoimmune diseases, traumatic and post-operative sequelae involving the dermis and abdominal tissues, or post-operative sequelae of intranasal surgery, post-operative sequelae of tendon and / or cartilage tissue.

[0064] Particularly preferred is the use of the conjugates according to the invention in the treatment of asthma, COPD, IPF, tonsillitis, laryngitis, pharyngitis, nasopharyngitis, sinusitis, rhinitis, tracheitis, hoarseness and inflammation of the vocal cords with or without dysphonia.

[0065] The conjugates can also be used in the cosmetic and dermatological fields, in dermatological or cosmetic products or in biomedical products, preferably as bioabsorbable implants.

[0066] In a further aspect, the invention relates to the use of this conjugate in psoriasis and psoriatic osteoarthritis.

[0067] In another embodiment, the present invention relates to a composition comprising the above conjugate and at least one pharmacologically active substance and / or at least one substance having any biological function.

[0068] Suitable pharmacologically active substances include antibiotics, anti-infectives, antibacterial agents, antivirals, cell growth inhibitors, cytotoxic agents, anti-tumor agents, anti-inflammatory agents, scar-forming agents, anesthetics, analgesics, vasoconstrictors, cholinergic or adrenergic agonists and antagonists, antithrombotic agents, anticoagulants, hemostatic agents, fibrinolytic agents, thrombolytic agents, proteins and fragments thereof, peptides, polynucleotides, growth factors, enzymes, vaccines, or combinations thereof.

[0069] Preferably, the optional biologically functional substance is selected from collagen, fibrinogen, fibrin, alginic acid, sodium alginate, potassium alginate, magnesium alginate, cellulose, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, laminin, fibronectin, elastin, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), polycaprolactone, gelatin, albumin, poly(glycolide-co-caprolactone), poly(glycolide-co-trimethylene carbonate), hydroxyapatite, tricalcium phosphate, dicalcium phosphate, demineralized bone matrix, and mixtures thereof.

[0070] The compositions according to the invention find advantageous application in the same uses as those listed for the conjugates above.

[0071] It should also be understood that all aspects identified as preferred and advantageous for the conjugation method should be considered as equally preferred and advantageous for the above conjugates resulting therefrom, as well as for the compositions and uses thereof.

[0072] Furthermore, it is to be understood that all possible combinations of the preferred aspects of the invention described above are likewise described and considered to be preferred.

[0073] Examples of the present invention are provided below for illustrative and non-limiting purposes. [Example]

[0074] (material) Two HA samples with molecular weights of 82 kDa and 300 kDa, respectively, and an amine derivative of lactose (hereinafter referred to as lactosamine (Lat-NH2)) were used as substrates.

[0075] The main reagents used for the functionalization of HA and the synthesis of lactosamine were as follows: EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride), NHS (N-hydroxysuccinimide), HCTU (O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride), TEA (triethylamine), α-D-lactose, NH328-30% (ammonium hydroxide solution), NaCNBH3 (sodium cyanoborohydride), NH4OAc (ammonium acetate), Amberlite™ IR-120 (ion exchanger), SEC Pephadex G-10 exclusion column.

[0076] Example 1 (Lactosamine synthesis procedure) 360 mg (1 mmol) of α-D-lactose was dissolved in 20 mL of saturated NHOAc / EtOH solution ([c] = 0.05 M), and the mixture was kept at 40 °C for approximately 30 min to complete dissolution. 8 mL of 28-30% aqueous ammonia (2.24 g; 131 mmol) was added to the mixture, followed by 190 mg of NaCNBH (3 mmol), and the resulting mixture was left stirring at 90 °C. After 8 h, the solvent and excess ammonia were evaporated under reduced pressure, and the white solid was treated several times, first with water and then with EtOH alone, before being loaded onto an Amberlite IR-120 (H+) column (20 eq.; 15 mL, φ = 1 h = 20 cm) and eluted with deionized water until the acidity disappeared (desalting). The amine product immobilized on the sulfonic acid resin in the first step was recovered by elution with the next 50 mL of 10% ammonia solution and an additional 50 mL of water until the amine product was completely eluted as detected by 10% sulfuric acid on a TLC plate. All eluted fractions containing the product were evaporated, and the residue was treated first with water and then with EtOH until the weight was constant. 312 mg of white product was obtained, a yield of 90%.

[0077] Example 2 (Lactosamine synthesis procedure) A solution of lactose (3% w / v), benzylamine (5% w / v) and 5-ethyl-2-methylpyridine borane (6% w / v) in water and methanol (3:1) was stirred at a temperature of 55°C and allowed to react for 20 hours. The mixture was then cooled, extracted with dichloromethane and finally the aqueous phase was evaporated at low pressure to give a crystalline white solid. This was then washed with ethyl ether and finally recovered by decantation and dried under reduced pressure. The product was analyzed by IR and NMR. 1 Characterized by H-NMR spectroscopy. Reaction yield: 90%.

[0078] A solution (4% w / v) of the derivative thus obtained in methanol and water (1:1) was placed under magnetic stirring at room temperature. Pd on carbon (0.4% w / v) was then added and the resulting system was pressurized with hydrogen. After 48 hours, the system was decompressed, an equal volume of water was mixed, the solid was decanted and the solution was filtered over Celite. The solution thus obtained was dried under reduced pressure to give a white solid. The product thus obtained had IR and 1 Characterized by H-NMR spectroscopy. Reaction yield: 96%.

[0079] Example 3 a) Preparation of lactosylated hyaluronic acid 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was left under magnetic stirring for 2 hours. To the homogeneous solution, 4.12 g (14.90 mmol; 0.5 eq.) of the condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) was added. After 1 hour, 100 mL of an aqueous solution containing 5.12 g (14.9 mmol; 0.5 eq.) of lactosamine was added, and the mixture was left at pH 6-7 for 48 hours at 25 °C. The product was then isolated by precipitation with 2.5 L of isopropanol (75%) at 4 °C. To improve the precipitation step, the suspension was concentrated with 3 mL of saturated NaCl solution (approximately 1 g of salt), followed by centrifugation at 15,000 rpm for 10 minutes. The resulting white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. A 25 mL aliquot (1 / 10; 1.14 g) was lyophilized and subsequently purified according to steps b1, b2, and c. The remaining 225 mL solution (9 / 10 of the total) was diluted to 350 mL, transferred to a 6-8 kDa membrane, and dialyzed first against 24 liters (12 + 12 L) of 0.1 M NaCl aqueous solution for 2 days, then against deionized water for 3 days. The entire retentate (approximately 1 L) was then concentrated under reduced pressure to approximately 1 / 3 of its initial volume and lyophilized to obtain a white solid.

[0080] b1) Purification by continuous reprecipitation The 570 mg sample obtained after the initial precipitation and freeze-drying was dissolved in 40 mL of deionized water at a concentration of 15 mg / mL and reprecipitated with 60 mL of isopropanol (4 °C, 60% v / v). To improve the precipitation step, the mixture was concentrated with a few drops of saturated NaCl solution. The suspension was centrifuged at 15,000 rpm, and the resulting solid was washed several times with isopropanol, then recovered with deionized water and freeze-dried to obtain a white solid.

[0081] b2) Purification by ion exchange resin treatment The 570 mg sample obtained after the initial precipitation and lyophilization was dissolved in 40 mL of deionized water ([c] = 15 mg / mL) and then eluted with an additional 60 mL of deionized water on an Amberlite cation column IR-120 (H+) (30 eq.; 15 mL, φ = 1 h = 20 cm) until the acidic solution was completely eluted. The entire eluted mixture was then neutralized with a stoichiometric amount of NaHCO3 (106 mg). The mixture was kept under stirring in a vacuum system for approximately 1 h to promote acid exchange of the sodium form of glucuronic acid. The mixture was then concentrated under reduced pressure to approximately one-third of its initial volume and lyophilized to obtain a white solid.

[0082] c) Recovery of lactosamine from samples purified by ion exchange resin treatment The previously used Amberlite IR-120 (H+) column (30 eq.; 15 mL, φ=1 h=20 cm) was eluted with 40 mL of 9% aqueous NH3, followed by 80 mL of water alone until neutral. The entire eluate was dried under reduced pressure, and the residue was treated several times, first with water (5 x 10 mL) and finally with EtOH, until the weight was constant, yielding a sample weighing 56 mg.

[0083] Example 4 a) Preparation of lactosylated hyaluronic acid 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was left under magnetic stirring for 2 hours. To the homogeneous solution, 8.24 g (29.83 mmol; 1.0 eq.) of the condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) was added. After 1 hour, 100 mL of an aqueous solution containing 10.24 g (29.83 mmol; 1.0 eq.) of lactosamine was added, and the mixture was left at pH 6-7 for 48 hours at 25 °C. The product was then isolated by precipitation with 2.5 L of isopropanol (75%) at 4 °C. To improve the precipitation step, the suspension was concentrated with 3 mL of saturated NaCl solution (approximately 1 g of salt), followed by centrifugation of the suspension at 15,000 rpm for 10 minutes. The resulting white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL, transferred to a 6-8 kDa membrane, and dialyzed first against 24 liters (12 + 12 L) of 0.1 M NaCl aqueous solution for 2 days, then against deionized water for 3 days. The total retentate (approximately 1 L) was then concentrated under reduced pressure to approximately one-third of its original volume and lyophilized to yield a white solid.

[0084] Alternatively, the white solid obtained after the precipitation and washing steps was further purified according to the techniques described in Example 3, b1 and b2.

[0085] Example 5 a) Preparation of lactosylated hyaluronic acid 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was left under magnetic stirring for 2 hours. To the homogeneous solution, 12.36 g (44.75 mmol; 1.5 eq.) of the condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) was added. After 1 hour, 100 mL of an aqueous solution containing 15.36 g (44.75 mmol; 1.5 eq.) of lactosamine was added, and the mixture was left at pH 6-7 for 48 hours at 25 °C. The product was then isolated by precipitation with 2.5 L of isopropanol (75%) at 4 °C. To improve the precipitation step, the suspension was concentrated with 3 mL of saturated NaCl solution (approximately 1 g of salt), followed by centrifugation at 15,000 rpm for 10 minutes. The resulting white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL, transferred to a 6-8 kDa membrane, and dialyzed first against 24 liters (12 + 12 L) of 0.1 M NaCl aqueous solution for 2 days, then against deionized water for 3 days. The total retentate (approximately 1 L) was then concentrated under reduced pressure to approximately one-third of its original volume and lyophilized to yield a white solid.

[0086] Alternatively, the white solid obtained after the precipitation and washing steps was further purified according to the techniques described in Example 3, b1 and b2.

[0087] Example 6 a) Preparation of lactosylated hyaluronic acid 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was left under magnetic stirring for 2 hours. To the homogeneous solution, 12.36 g (44.75 mmol; 1.5 eq.) of the condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) was added. After 1 hour, 100 mL of an aqueous solution containing 15.36 g (44.75 mmol; 1.5 eq.) of lactosamine was added, and the mixture was left at pH 6-7 for 24 hours at 25 °C. The product was then isolated by precipitation with 2.5 L of isopropanol (75%) at 4 °C. To improve the precipitation step, the suspension was concentrated with 3 mL of saturated NaCl solution (approximately 1 g of salt), followed by centrifugation of the suspension at 15,000 rpm for 10 minutes. The resulting white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL, transferred to a 6-8 kDa membrane, and dialyzed first against 24 liters (12 + 12 L) of 0.1 M NaCl aqueous solution for 2 days, then against deionized water for 3 days. The total retentate (approximately 1 L) was then concentrated under reduced pressure to approximately one-third of its original volume and lyophilized to yield a white solid.

[0088] Alternatively, the white solid obtained after the precipitation and washing steps was further purified according to the techniques described in Example 3, b1 and b2.

[0089] Example 7 a) Preparation of lactosylated hyaluronic acid 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was left under magnetic stirring for 2 hours. To the homogeneous solution, 12.36 g (44.75 mmol; 1.5 eq.) of the condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) was added. After 1 hour, 100 mL of an aqueous solution containing 15.36 g (44.75 mmol; 1.5 eq.) of lactosamine was added, and the mixture was left at pH 6-7 for 48 hours at 25 °C. The product was then isolated by precipitation with 2.5 L of isopropanol (75%) at 4 °C. To improve the precipitation step, the suspension was concentrated with 3 mL of saturated NaCl solution (approximately 1 g of salt), followed by centrifugation at 15,000 rpm for 10 minutes. The resulting white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL, transferred to a 6-8 kDa membrane, and dialyzed first against 24 liters (12 + 12 L) of 0.1 M NaCl aqueous solution for 2 days, then against deionized water for 3 days. The total retentate (approximately 1 L) was then concentrated under reduced pressure to approximately one-third of its original volume and lyophilized to yield a white solid.

[0090] Alternatively, the white solid obtained after the precipitation and washing steps was further purified according to the techniques described in Example 3, b1 and b2.

[0091] Example 8 a) Preparation of lactosylated hyaluronic acid 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was left under magnetic stirring for 2 hours. To the homogeneous solution, 20.60 g (74.58 mmol; 2.5 eq.) of the condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) was added. After 1 hour, 100 mL of an aqueous solution containing 25.60 g (74.58 mmol; 2.5 eq.) of lactosamine was added, and the mixture was left at pH 6-7 for 48 hours at 25 °C. The product was then isolated by precipitation with 2.5 L of isopropanol (75%) at 4 °C. To improve the precipitation step, the suspension was concentrated with 3 mL of saturated NaCl solution (approximately 1 g of salt), followed by centrifugation of the suspension at 15,000 rpm for 10 minutes. The resulting white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL, transferred to a 6-8 kDa membrane, and dialyzed first against 24 liters (12 + 12 L) of 0.1 M NaCl aqueous solution for 2 days, then against deionized water for 3 days. The total retentate (approximately 1 L) was then concentrated under reduced pressure to approximately one-third of its original volume and lyophilized to yield a white solid.

[0092] Alternatively, the white solid obtained after the precipitation and washing steps was further purified according to the techniques described in Example 3, b1 and b2.

[0093] Example 9 a) Preparation of lactosylated hyaluronic acid 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was left under magnetic stirring for 2 hours. To the homogeneous solution, 12.36 g (44.75 mmol; 1.5 eq.) of the condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) was added. After 1 hour, 100 mL of an aqueous solution containing 15.36 g (44.75 mmol; 1.5 eq.) of lactosamine was added, and the mixture was left at pH 6-7 for 48 hours at 25 °C. The product was then isolated by precipitation with 2.5 L of isopropanol (75%) at 4 °C. To improve the precipitation step, the suspension was concentrated with 3 mL of saturated NaCl solution (approximately 1 g of salt), followed by centrifugation at 15,000 rpm for 10 minutes. The resulting white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL, transferred to a 6-8 kDa membrane, and dialyzed first against 24 liters (12 + 12 L) of 0.1 M NaCl aqueous solution for 2 days, then against deionized water for 3 days. The total retentate (approximately 1 L) was then concentrated under reduced pressure to approximately one-third of its original volume and lyophilized to yield a white solid.

[0094] Alternatively, the white solid obtained after the precipitation and washing steps was further purified according to the techniques described in Example 3, b1 and b2.

[0095] Example 10 a) Preparation of lactosylated hyaluronic acid 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was left under magnetic stirring for 2 hours. To the homogeneous solution, 8.24 g (29.83 mmol; 1.0 eq.) of the condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) was added. After 1 hour, 100 mL of an aqueous solution containing 10.24 g (29.83 mmol; 1.0 eq.) of lactosamine was added, and the mixture was left at 25 °C for 12 hours at pH 5-8. The product was then isolated by precipitation with 2.5 L of isopropanol (75%) at 4 °C. To improve the precipitation step, the suspension was concentrated with 3 mL of saturated NaCl solution (approximately 1 g of salt), followed by centrifugation of the suspension at 15,000 rpm for 10 minutes. The resulting white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL, transferred to a 6-8 kDa membrane, and dialyzed first against 24 liters (12 + 12 L) of 0.1 M NaCl aqueous solution for 2 days, then against deionized water for 3 days. The total retentate (approximately 1 L) was then concentrated under reduced pressure to approximately one-third of its original volume and lyophilized to yield a white solid.

[0096] Alternatively, the white solid obtained after the precipitation and washing steps was further purified according to the techniques described in Example 3, b1 and b2.

[0097] Example 11 a) Preparation of lactosylated hyaluronic acid 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was left under magnetic stirring for 2 hours. To the homogeneous solution, 12.36 g (44.75 mmol; 1.5 eq.) of the condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) was added. After 1 hour, 100 mL of an aqueous solution containing 15.36 g (44.75 mmol; 1.5 eq.) of lactosamine was added, and the mixture was left at pH 6-7 for 48 hours at 25 °C. The product was then isolated by precipitation with 2.5 L of isopropanol (75%) at 4 °C. To improve the precipitation step, the suspension was concentrated with 3 mL of saturated NaCl solution (approximately 1 g of salt), followed by centrifugation at 15,000 rpm for 10 minutes. The resulting white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL, transferred to a 6-8 kDa membrane, and dialyzed first against 24 liters (12 + 12 L) of 0.1 M NaCl aqueous solution for 2 days, then against deionized water for 3 days. The total retentate (approximately 1 L) was then concentrated under reduced pressure to approximately one-third of its original volume and lyophilized to yield a white solid.

[0098] Alternatively, the white solid obtained after the precipitation and washing steps was further purified according to the techniques described in Example 3, b1 and b2.

[0099] Example 12 a) Preparation of lactosylated hyaluronic acid 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was left under magnetic stirring for 2 hours. To the homogeneous solution, 12.36 g (44.75 mmol; 1.5 eq.) of the condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) was added. After 1 hour, 100 mL of an aqueous solution containing 15.36 g (44.75 mmol; 1.5 eq.) of lactosamine was added, and the mixture was left at pH 6-7 for 24 hours at 25 °C. The product was then isolated by precipitation with 2.5 L of isopropanol (75%) at 4 °C. To improve the precipitation step, the suspension was concentrated with 3 mL of saturated NaCl solution (approximately 1 g of salt), followed by centrifugation of the suspension at 15,000 rpm for 10 minutes. The resulting white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL, transferred to a 6-8 kDa membrane, and dialyzed first against 24 liters (12 + 12 L) of 0.1 M NaCl aqueous solution for 2 days, then against deionized water for 3 days. The total retentate (approximately 1 L) was then concentrated under reduced pressure to approximately one-third of its original volume and lyophilized to yield a white solid.

[0100] Alternatively, the white solid obtained after the precipitation and washing steps was further purified according to the techniques described in Example 3, b1 and b2.

[0101] Example 13 a) Preparation of lactosylated hyaluronic acid 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was left under magnetic stirring for 2 hours. To the homogeneous solution, 12.36 g (44.75 mmol; 1.5 eq.) of the condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) was added. After 1 hour, 100 mL of an aqueous solution containing 20.48 g (59.67 mmol; 1.5 eq.) of lactosamine was added, and the mixture was left at pH 6-7 for 48 hours at 25 °C. The product was then isolated by precipitation with 2.5 L of isopropanol (75%) at 4 °C. To improve the precipitation step, the suspension was concentrated with 3 mL of saturated NaCl solution (approximately 1 g of salt), followed by centrifugation at 15,000 rpm for 10 minutes. The resulting white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL, transferred to a 6-8 kDa membrane, and dialyzed first against 24 liters (12 + 12 L) of 0.1 M NaCl aqueous solution for 2 days, then against deionized water for 3 days. The total retentate (approximately 1 L) was then concentrated under reduced pressure to approximately one-third of its original volume and lyophilized to yield a white solid.

[0102] Alternatively, the white solid obtained after the precipitation and washing steps was further purified according to the techniques described in Example 3, b1 and b2.

[0103] Example 14 a) Preparation of lactosylated hyaluronic acid 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was left under magnetic stirring for 2 hours. To the homogeneous solution, 12.36 g (44.75 mmol; 1.5 eq.) of the condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) was added. After 1 hour, 100 mL of an aqueous solution containing 20.48 g (59.67 mmol; 1.5 eq.) of lactosamine was added, and the mixture was left at pH 5-8 and 60 °C for 5 hours. The product was then isolated by precipitation with 2.5 L of isopropanol (75%) at 4 °C. To improve the precipitation step, the suspension was concentrated with 3 mL of saturated NaCl solution (approximately 1 g of salt), followed by centrifugation of the suspension at 15,000 rpm for 10 minutes. The resulting white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL, transferred to a 6-8 kDa membrane, and dialyzed first against 24 liters (12 + 12 L) of 0.1 M NaCl aqueous solution for 2 days, then against deionized water for 3 days. The total retentate (approximately 1 L) was then concentrated under reduced pressure to approximately one-third of its original volume and lyophilized to yield a white solid.

[0104] Alternatively, the white solid obtained after the precipitation and washing steps was further purified according to the techniques described in Example 3, b1 and b2.

[0105] Examples 15-26 The procedure of Examples 3-14 was repeated using 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) in place of DMTMM to similarly afford the desired product.

[0106] Example 27 Samples from Examples 3-14 were used to measure molecular weight (MW) and degree of substitution (DS).

[0107] The molecular weight was measured by size exclusion chromatography (HP-SEC / TDA).

[0108] The degree of substitution was measured by two-dimensional heterocorrelation (1H, 13 C) HSQC-DEPT nuclear magnetic resonance (NMR) techniques were used.

[0109] The results are shown in the table below.

[0110] [Table 1]

[0111] Example 28 Samples 14, 13 and 6 were tested in a cell model to evaluate their anti-inflammatory properties.

[0112] The anti-inflammatory activity of molecules 14, 13, and 6 was compared to that of a negative control (CTRL, human bronchial fibroblasts from smokers, untreated) and a positive control (TC, human bronchial fibroblasts from smokers incubated with a culture of U937 human monocytes activated to macrophages with PMA (50 μg / ml, 24 hours) and LPS (1 μg / ml, 1 hour)) by qPCR analysis of IL-1β gene expression. The table below shows the expression of the genes analyzed after 4 hours of incubation with compounds 14, 13, and 6.

[0113] [Table 2]

[0114] All compounds showed a significant reduction of IL-1β in TC.

[0115] Example 29 Samples 14, 13 and 6 were tested in a cell model to evaluate their anti-fibrotic properties.

[0116] The antifibrotic activity of molecules 14, 13, and 6 was compared with that of a negative control (CTRL, human peripheral lung fibroblasts, untreated, NHLF line) and a positive control (TGF-β, human peripheral lung fibroblasts (NHLF) incubated with 5 ng / ml TGF-β for 24 hours) by qPCR analysis of COL-1 gene expression. The table below shows the expression of the genes analyzed after 4 hours of incubation with compounds 14, 13, and 6.

[0117] [Table 3]

[0118] All compounds showed a significant decrease in COL-1 in response to TGF-β.

Claims

1. 1. A method for conjugating hyaluronic acid or a pharmaceutically acceptable salt thereof with a disaccharide or oligosaccharide, comprising the steps of: i) providing an aqueous solution of hyaluronic acid or a pharmaceutically acceptable salt thereof; ii) adding the triazine compound of formula (I) under stirring to form an ester adduct between the carboxyl group of hyaluronic acid or a pharmaceutically acceptable salt thereof and the triazine ring of the compound of formula (I); iii) waiting for at least 15 minutes under stirring, adding a primary amine of a disaccharide or oligosaccharide to form a conjugate between hyaluronic acid or a pharmaceutically acceptable salt thereof and said disaccharide or oligosaccharide via an amide bond; iv) adding a water-miscible organic solvent to promote precipitation of the resulting conjugate, thereby obtaining a crude conjugate, and optionally further washing the precipitated conjugate with an organic solvent optionally mixed with water; v) separating the loosely bound bodies, and optionally vi) purifying the crude conjugate by reprecipitation, ion exchange resin treatment, dialysis, or combinations and / or repetitions thereof; and vii) obtaining the purified conjugate by drying; The process is carried out at a temperature of 4 to 100°C and a pH of 5 to 8; The triazine compound has the formula (I): 【Chemical 1】 wherein X is a halogen selected from I, Br, Cl and mixtures thereof, or —NR １ R ２ is a group, and this group is NH ２ , NMe ２ , NHPr, PhNH, 4-MeC ６ H ４ N.H., 【Chemistry 2】 and mixtures thereof.) That's the method.

2. 2. The method according to claim 1, wherein the hyaluronic acid in step i) has an average molecular weight of 50 to 5000 kDa, preferably 50 to 500 kDa.

3. 3. The method according to claim 1 or 2, wherein the pharmaceutically acceptable salt of hyaluronic acid in step i) is selected from sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, ammonium hyaluronate, tetrabutylammonium hyaluronate and mixtures thereof.

4. 4. The method according to claim 1, wherein the primary amine of a disaccharide or a tri- to hexasaccharide oligosaccharide is a primary amino derivative of sucrose, maltose, lactose, trehalose, gentiobiose, cellobiose, maltotriose, raffinose, stachyose, maltotetraose, melezitose, isomaltose, panose, nystose, 1-kestose, galactopinitol, galactosyllactose, or a mixture thereof.

5. 5. The method according to any one of claims 1 to 4, wherein in step iii) a primary amine of a disaccharide is added, which is a primary amino derivative of sucrose, maltose, lactose, trehalose, gentiobiose, cellobiose or a mixture thereof, preferably a primary amino derivative of lactose.

6. 6. The method according to any one of claims 1 to 5, wherein the triazine compound of formula (I) is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), or a mixture thereof, preferably DMTMM.

7. In step vi), if the purification of the crude conjugate is by treatment with an ion exchange resin, the following sub-steps: vi-a) dissolving the crude conjugate in water to obtain an aqueous solution; vi-b) subjecting said aqueous solution to ion exchange resin treatment using water as eluent; vi-c) separating the aqueous solution from the ion exchange resin; vid-d) adjusting the separated solution to neutral pH; The method according to any one of claims 1 to 6, wherein the following is carried out:

8. In step vi), if the purification of the crude conjugate is by dialysis, the following sub-steps: vi-i) dissolving the crude conjugate in water to obtain an aqueous solution; vi-ii) dialyzing the resulting solution against saline and / or water through a suitable membrane; The method according to any one of claims 1 to 6, wherein the following is carried out:

9. In step vi), if the purification of the crude conjugate is by reprecipitation, the following sub-steps: vi-α) dissolving the crude conjugate in water to obtain an aqueous solution; vi-β) adding a water-miscible organic solvent, which may be the same as or different from the solvent of step iv), thereby facilitating reprecipitation of the conjugate; vi-γ) washing the precipitated conjugate with additional organic solvent, thereby obtaining a purified conjugate; The method according to any one of claims 1 to 6, wherein the following is carried out:

10. The process according to any one of claims 1 to 9, wherein the process is carried out at a temperature of 15 to 75°C and a pH of 6 to 7.

11. The method according to any one of claims 1 to 10, wherein in step ii), the hyaluronic acid or its pharmaceutically acceptable salt and the triazine compound of formula (I) are in a molar ratio of 3:1 to 1:

3.

12. The method according to claim 11, wherein in step iii), the hyaluronic acid or its pharmaceutically acceptable salt and the primary amine are in a molar ratio of 3:1 to 1:

3.

13. A conjugate of hyaluronic acid or a pharmaceutically acceptable salt thereof with a disaccharide or oligosaccharide, wherein the average substitution degree of carboxyl groups with amide bonds to the disaccharide or oligosaccharide is 10 to 50%, preferably 15 to 45%, and the conjugate is obtainable by the method according to claim 11 or 12.

14. 14. A composition comprising the conjugate of claim 13 and at least one pharmacologically active substance and / or at least one substance having any biological function, - the pharmacologically active substance is selected from antibiotics, anti-infectives, antibacterial agents, antivirals, cell growth inhibitors, cytotoxic agents, antitumor agents, anti-inflammatory agents, healing agents, anesthetics, analgesics, vasoconstrictors, cholinergic or adrenergic agonists and antagonists, antithrombotic agents, anticoagulants, hemostatic agents and fragments thereof, peptides, polynucleotides, growth factors, enzymes, vaccines, and combinations thereof; and the optionally biologically functional substance is selected from collagen, fibrinogen, fibrin, alginic acid, sodium alginate, potassium alginate, magnesium alginate, cellulose, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, laminin, fibronectin, elastin, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), polycaprolactone, gelatin, albumin, poly(glycolide-co-caprolactone), poly(glycolide-co-trimethylene carbonate), hydroxyapatite, tricalcium phosphate, dicalcium phosphate, demineralized bone matrix, and mixtures thereof; composition.

15. 15. The conjugate of claim 13 or the composition of claim 14 in the form of a powder, solution, emulsion, gel, ointment, eye drop, aerosol, spray, or injectable, for administration by nasal, inhalation, intra-articular, intradermal, ophthalmic, or urological route.

16. The conjugate of claim 13 or the composition of claim 14 for use in: - for use in the treatment of diseases caused by altered galectin expression, said diseases including non-alcoholic steatohepatitis, plaque psoriasis, rheumatoid arthritis, osteoarthritis, tumors, adhesions, and fibrotic processes of the skin, lungs, kidneys, and cardiovascular systems; or - as a biomaterial or scaffold for cell growth; or - for use in tissue repair or reconstruction, preferably for creating or replacing biological tissue or for filling biological tissue such as skin, cavities, bone cartilage or joints; or - for use in dermatological or cosmetic products or as biomedical products, preferably bioresorbable implants; or - for use in rheumatology, orthopedics, oncology, cosmetic and plastic surgery, hemodialysis, cardiology, angiology, ophthalmology, otorhinolaryngology, pulmonology, dentistry, gynecology, urology, dermatology, oncology, and tissue repair; or - for use in traumatic and / or postoperative tissue processes and / or chronic fibrotic processes associated with autoimmune diseases; or - for use in traumatic and postoperative sequelae involving the dermis and abdominal tissues, or in postoperative sequelae of intranasal surgery, postoperative sequelae of tendon and / or cartilage tissue; or - Use in psoriasis and psoriatic osteoarthritis.

Images