Process for conjugation of hyaluronic acid and conjugates of hyaluronic acid thus obtained

Abstract

The present invention describes a process for conjugating hyaluronic acid by using a specified condensing agent and under certain operating conditions, which process allows to obtain conjugates of hyaluronic acid with disaccharides or oligosaccharides with a desired degree of conjugation in an efficient, rapid and economical manner. Hyaluronic acid conjugates having a specified degree of conjugation are also described, as well as their use for therapeutic purposes and as scaffolds for biological materials.

Description

Field of the Invention

[0001] The present invention relates to a process for conjugating hyaluronic acid by using a specified condensing agent and under certain operating conditions, which allows obtaining a conjugate of hyaluronic acid with a disaccharide or oligosaccharide having a desired degree of conjugation in an efficient, rapid and economical manner.

[0002] Therefore, the present invention also relates to hyaluronic acid conjugates having a specified degree of conjugation and uses thereof. Background Art

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

[0004] At physiological pH, the carboxyl groups of the glucuronic acid units are ionized, which gives HA a high polarity and thus good solubility in water. The molecular weight of HA can be between 10 3 Da and 10 7 Da varies between Å and this parameter affects its properties, making different applications possible.

[0005] HA is naturally present in both vertebrate organisms and bacteria. It is present mainly in the extracellular matrix (ECM) of epithelial cells and connective tissue. The biological properties of hyaluronic acid can be attributed to its chemical and physical properties, such as viscosity and high water retention capacity, which allow the hyaluronic acid to achieve structural effects and hydration. Today, the study of hyaluronic acid involves its role as a drug carrier by appropriate chemical functionalization. During the enzymatic degradation of the hyaluronic acid, the cellular internalization of hyaluronic acid by means of CD44 receptors determines the intracellular release of the drug obtained by conjugating to HA or embedding in HA particles. Along this route, considering the biological significance in terms of interaction with galectins, the possibility of functionalizing hyaluronic acid with modified lactose is particularly interesting.

[0006] Indeed, galectins are galactose-specific receptors, and thus the addition of this residue to the HA chain allows the polymer to interact not only with the CD44 receptor, but also with galectins, resulting in the desired synergistic effect.

[0007] Many derivatives of hyaluronic acid are known in the literature for the most diverse applications. However, it has also been observed that there is still a need for functionalization processes that are efficient, economical and, above all, capable of guaranteeing the desired degree of substitution and high yields while also minimizing by-products. SUMMARY OF THE INVENTION

[0009] The object is achieved by the process for conjugating hyaluronic acid by means of a specified condensing agent and under certain operating conditions as described in claim 1.

[0010] In another aspect, the present invention relates to a conjugate of hyaluronic acid or a pharmaceutically acceptable salt thereof with a disaccharide or an oligosaccharide, the conjugate having an average degree of substitution in the carboxyl groups of 10% to 50%, more preferably 15% to 45%.

[0011] In a further aspect, the invention relates to the use of said conjugates in the treatment of conditions attributable to altered galectin expression. Non-limiting examples of such conditions are non-alcoholic steatohepatitis, plaque psoriasis, rheumatoid arthritis, osteoarthritis, neoplasms, adhesions and fibrotic processes of the skin, lungs, kidneys and cardiovascular.

[0012] In yet further aspects, the invention relates to the use of such conjugates in rheumatology, orthopedics, oncology, plastic / cosmetic surgery, hemodialysis, cardiology, vascular, ophthalmology, otolaryngology, pulmonology, dentistry, gynecology, urology, dermatology, oncology and tissue repair.

[0013] The characteristics and advantages of the invention will become apparent from the following detailed description and from the embodiments provided as illustrative, non-limiting examples. DETAILED DESCRIPTION OF THE INVENTION

[0015] Therefore, the present invention relates to a process for conjugating hyaluronic acid or a pharmaceutically acceptable salt thereof with a disaccharide or an oligosaccharide, the process comprising the following steps:

[0016] i) providing an aqueous solution of hyaluronic acid or a pharmaceutically acceptable salt thereof,

[0017] ii) adding a 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),

[0018] iii) waiting for at least 15 minutes and adding the primary amine of the disaccharide or oligosaccharide under stirring to form a conjugate of hyaluronic acid or a pharmaceutically acceptable salt thereof with the disaccharide or oligosaccharide via an amide bond,

[0019] iv) promoting the precipitation of said conjugate thus obtained, adding an organic solvent miscible with water, thereby obtaining a crude conjugate, and the precipitated conjugate may be washed with a further organic solvent, which may be miscible with water,

[0020] v) isolating the crude conjugate, and optionally

[0021] vi) purifying the crude conjugate by reprecipitation, by treatment with an ion exchange resin, by dialysis or by a combination thereof and / or repetition thereof, and

[0022] vii) obtaining a purified conjugate by drying,

[0023] The process is carried out at a temperature of 4°C-100°C and a pH of 5-8,

[0024] The triazine compound of formula (I) is:

[0025]

[0026] wherein X is a halogen selected from I, Br, Cl and mixtures thereof, or X is a group -NR1R2 selected from NH2, NMe2, NHPr, PhNH, 4-MeC6H4NH, and mixtures thereof.

[0027] As can also be seen from the following examples, the process according to the invention allows to conveniently and quickly obtain conjugates having the desired degree of substitution and also having a high purity.

[0028] In step i), an aqueous solution of hyaluronic acid or a pharmaceutically acceptable salt thereof is provided.

[0029] Preferably, the hyaluronic acid in step i) has an average molecular weight of 50 kDa-5000 kDa, more preferably 50 kDa-500 kDa.

[0030] In step i), the term "pharmaceutically acceptable salt" of hyaluronic acid means a salt preferably selected from sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, ammonium hyaluronate, tetrabutylammonium hyaluronate and mixtures thereof.

[0031] Preferably, step i) is performed at a pH of 5-7, more preferably 6-7, after addition of the acid, preferably after addition of 2-[N-morpholino]-ethanesulfonic acid (MES).

[0032] In step ii), a 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:

[0033]

[0034] Wherein "HACOO-" is a carboxyl group of hyaluronic acid or a pharmaceutically acceptable salt thereof, which is combined with the triazine ring and forms an ester adduct on the right side, releasing the group X.

[0035] Preferably, the triazine compound of formula (I) is 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) or a mixture thereof.

[0036] In a particularly preferred embodiment, the triazine compound of formula (I) is DMTMM and in step ii), the following reaction occurs:

[0037]

[0038] Wherein "HACOO-" is a carboxyl group of hyaluronic acid or a pharmaceutically acceptable salt thereof, which combines with the triazine ring of DMTMM and forms an ester adduct on the right side, releasing methylmorpholine.

[0039] Preferably, in step ii), the hyaluronic acid or a pharmaceutically acceptable salt thereof and the triazine compound of formula (I) are in a molar ratio of 3:1 to 1:3.

[0040] The mixture formed after adding the triazine compound of formula (I) is allowed to react for at least 15 minutes, preferably at least 30 minutes, after which the primary amine of the disaccharide or oligosaccharide is added under stirring to form a conjugate of hyaluronic acid or a pharmaceutically acceptable salt thereof with the disaccharide or oligosaccharide via an amide bond, according to step iii).

[0041] The term "primary amine" refers not only to the free primary amine -NH2 but also to the pharmaceutically acceptable salts thereof.

[0042] Preferably, the term "oligosaccharide" means a sugar polymer comprising 3 to 6 monosaccharides, ie from trisaccharides to hexasaccharides.

[0043] 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, methyl inositol galactopinitol, galactosyl lactose or a mixture thereof.

[0044] In a preferred embodiment, in step iii), a primary amine of a disaccharide is added, which primary amine is a primary amine derivative of sucrose, maltose, lactose, trehalose, gentiobiose, cellobiose or a mixture thereof, more preferably a primary amine derivative of lactose. Adding the primary amine to the mixture containing the ester adduct described above, in addition to releasing 4,6-dimethoxy-1,3,5-triazine-2-ol, also leads to the formation of a conjugate, as shown below:

[0045]

[0046] wherein R-NH2 is the primary amine of a disaccharide or oligosaccharide, which for simplicity is represented as "R", and the resulting conjugate is represented schematically, highlighting the amide bond between the carboxyl group of hyaluronic acid or a pharmaceutically acceptable salt thereof and the amino group of the primary amine.

[0047] Preferably, in step iii), the molar ratio of hyaluronic acid or a pharmaceutically acceptable salt thereof to the primary amine is 3:1 to 1:3.

[0048] Preferably, in step iii), the primary amine and the adduct are allowed to react at room temperature for at least 12 hours, preferably at least 24 hours, more preferably at least 48 hours.

[0049] In a preferred embodiment, in step iii), after the reaction has lasted for at least 12 hours, a base, preferably 5 M NaOH, is subsequently added. This alkalization step disrupts the ester bonds formed by secondary reactions of the activated carboxylic acids of HA with their hydroxyl groups or with lactosamine.

[0050] In step iv), precipitation of the conjugate thus obtained is promoted by adding an acid, preferably 5M HCl, and an organic solvent miscible with water, and obtaining a crude conjugate, and the precipitated conjugate may be washed with a further organic solvent, which may be mixed with water.

[0051] Organic solvents are used to reduce the polarity of water in order to increase the conjugate precipitation rate and yield.

[0052] Preferably, the water-miscible organic solvent is acetone, acetonitrile, 1,4-dioxane, tetrahydrofuran or an alcohol, the alcohol preferably having a carbon number of 1 to 5, more preferably selected from methanol, ethanol, 2-propanol or a mixture thereof.

[0053] In step v), the crude conjugate is separated from the solvent mixture. Preferably, the separation may be performed by techniques such as decantation, filtration, evaporation or centrifugation or a combination thereof.

[0054] In optional step vi), the crude conjugate thus isolated is subjected to an additional purification procedure, which can be carried out by reprecipitation, ion exchange resin treatment or dialysis, to obtain a more purified conjugate. These three purification methods can also be carried out in combination with each other and can be repeated several times, for example reprecipitation, followed by ion exchange resin treatment, followed by reprecipitation.

[0055] Preferably, when the purification of the crude conjugate in step vi) is by treatment with an ion exchange resin, the following sub-steps are performed:

[0056] vi-a) dissolving the crude conjugate in water to obtain an aqueous solution,

[0057] vi-b) treating the aqueous solution with an ion exchange resin by using water as an eluent,

[0058] vi-c) separating the aqueous solution from the ion exchange resin,

[0059] vi-d) The separated solution is brought to pH neutral.

[0060] In a preferred embodiment, the cation exchange column is Amberlite TM IR120 (CAS No. 78922-04-0; IUPAC name: 3-[(3-chlorophenyl)sulfonylamino]benzoic acid).

[0061] In sub-step vi-d), the eluate can be brought to neutral pH by adding a salt such as NaHCO 3 .Also, the subsequent drying can be carried out by freeze drying.

[0062] Unreacted primary amine retained in the cation exchange column can then be recovered using NH3 as eluent.

[0063] Alternatively, when the purification of the crude conjugate in step vi) is carried out by dialysis, the following sub-steps are performed:

[0064] vi-i) dissolving the crude conjugate in water to obtain an aqueous solution,

[0065] vi-ii) The obtained solution is dialyzed against saline solution and / or water through a suitable membrane.

[0066] Alternatively and preferably, when the purification of the crude conjugate in step vi) is carried out by reprecipitation, the following sub-steps are performed:

[0067] vi-α) dissolving the crude conjugate in water to obtain an aqueous solution,

[0068] vi-β) adding a water-miscible organic solvent which may be the same as or different from the solvent of step iv) to promote reprecipitation of the conjugate,

[0069] vi-γ) washing the precipitated conjugate with additional organic solvent to obtain a purified conjugate.

[0070] In sub-step vi-β), preferably, a saline solution is also added, more preferably a 0.5 M NaCl solution.

[0071] In sub-step vi-γ), the act of washing the conjugate with additional organic solvent may be repeated several times in order to increase the final purity of the conjugate.

[0072] Alternatively and preferably, when the purification of the crude conjugate in step vi) is carried out by dialysis, the following sub-steps are performed:

[0073] vi-●) dissolving the crude conjugate in water to obtain an aqueous solution,

[0074] vi-●●) transferred to a 6 kDa-8 kDa membrane and dialyzed against NaCl saline solution for at least 24 hours,

[0075] vi-●●●) The retentate was collected and freeze-dried.

[0076] 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.

[0077] The primary amines described above and also the relevant pharmaceutically acceptable salts are commercially available or, alternatively, they can be prepared by commonly known methods for the amination of disaccharides or oligosaccharides or even by a process comprising the following steps:

[0078] - reacting the disaccharide or oligosaccharide with ammonium acetate in the presence of ammonia and NaCNBH3, under stirring and at a temperature of at least 60°C,

[0079] - the product thus obtained is dried and placed on a cation exchange column, eluted first with deionized water in order to desalt the product, and then with ammonia,

[0080] - The eluate coming out of the column is collected, brought to neutral pH, and then dried to obtain the purified primary amine.

[0081] In addition, the primary amine can be prepared by a process comprising the following steps:

[0082] - reacting the disaccharide or oligosaccharide with benzylamine in the presence of borane, under stirring and at a temperature between 5° C. and 60° C.,

[0083] - isolating the benzylamine derivative obtained by the reductive amination reaction,

[0084] - reacting the benzylamine derivative with a hydrogen source in the presence of a catalyst for the hydrogenolysis reaction, under stirring and at a temperature between 4° C. and 100° C.,

[0085] - Isolating the benzylamine derivative and purifying the benzylamine derivative by passing through an ion exchange column to desalt the product and then dry it.

[0086] 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, cetyltrimethylammonium borohydride, borane tetrahydrofuran complex, picoline-borane complex, complex), 5-ethyl-2-methylpyridine-borane complex, dimethyl sulfide-borane complex, pyridine-borane complex, trimethylamine-borane complex, triethylamine-borane complex, morpholine-borane complex, tert-butylamine-borane complex, ammonia-borane complex, diphenylphosphine-borane complex, 4-methylmorpholine-borane complex, borane ethylenediamine complex, or a mixture thereof.

[0087] The term "catalyst" preferably refers to a Raney nickel catalyst of metal group 8-11, a carbon-supported metal group 8-11, silica, alumina, aluminosilicate, zirconia, or a mixture thereof.

[0088] The term "hydrogen source" refers to H2 or a compound capable of releasing H+ ions under process conditions.

[0089] 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, the conjugate having an average degree of substitution of the carboxyl groups of the amide bonds of the disaccharide or oligosaccharide with 10%-50%, more preferably 15%-45%, which can be obtained from the process described above, in particular when:

[0090] - in step ii), the molar ratio of hyaluronic acid or a pharmaceutically acceptable salt thereof to the triazine compound of formula (I) is 3:1 to 1:3, and / or

[0091] - In step iii), the molar ratio of hyaluronic acid or a pharmaceutically acceptable salt thereof to the primary amine is 3:1 to 1:3.

[0092] Indeed, it has surprisingly been observed that by adjusting the ratios between the reagents as described above, it is possible to obtain conjugates with the desired degree of substitution in an efficient and quantitative manner.

[0093] The conjugate can be administered by inhalation, oral, nasal, ocular, urogynaecological, intraarticular, intramuscular, intravenous, intradermal, transdermal, subcutaneous or external or internal topical routes such as by surgery.

[0094] Preferably, the conjugate is administered by the oral route, the nasal route, the ocular route, the urogynecological route, the intra-articular route, the intradermal route or the inhalation route.

[0095] In certain embodiments, the conjugate is in an injectable form suitable for hard or soft body tissues such as organs, adipose tissue, mucosa, and gums, preferably by intradermal, subcutaneous, intramuscular or intraarticular routes.

[0096] The conjugate may be in the form of a soft gel capsule or in a solid form such as a tablet, mini-tablet, micro-tablet, granule, microparticle, pellet, multiparticulate or micronized granule or powder, or in the form of a solution, emulsion, gel, ointment, drops, nebuliser solution or spray.

[0097] In a preferred embodiment, the conjugate is in the form of a powder, solution, emulsion, gel, ointment, drops, aerosol solution, spray or injectable form for administration by nasal or inhalation or intra-articular or intradermal or ocular or urogynecological route.

[0098] In yet another aspect, the invention relates to the use of the conjugate in the treatment of a condition attributable to altered galectin expression. Non-limiting examples of such conditions are non-alcoholic steatohepatitis, plaque psoriasis, rheumatoid arthritis, osteoarthritis, vegetations, adhesions, and fibrotic processes of the skin, lungs, kidneys, and cardiovascular. Examples of neoplasia 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.

[0099] In a further aspect, the present invention relates to the use of the conjugate as a biomaterial or scaffold for cell growth in the treatment of orthopedic diseases.

[0100] In a further preferred aspect, the invention relates to the use of said conjugate in tissue repair or reconstruction, preferably in the production or replacement of biological tissue, or in the filling of biological tissue such as filling of skin, recesses, bone cartilage or joints.

[0101] In yet another aspect, the present invention relates to the use of the conjugate in rheumatology, orthopedics, oncology, plastic / cosmetic surgery, hemodialysis, cardiology, vascular, ophthalmology, otolaryngology, pulmonology, dentistry, gynecology, urology, dermatology, oncology and tissue repair; furthermore, the present invention relates to the use of the conjugate in traumatic and / or post-surgical tissue processes and / or chronic fibrotic processes associated with autoimmune diseases, in traumatic and post-surgical sequelae involving dermal and abdominal tissues, or in post-surgical sequelae of intranasal surgery, in post-surgical sequelae of tendon and / or cartilage tissues.

[0102] Particularly preferred is the use of the conjugates according to the invention for the treatment of asthma, COPD, IPF, tonsillitis, laryngitis, pharyngitis, nasopharyngitis, sinusitis, rhinitis, tracheitis, hoarseness and inflammation of the vocal cords with or without dysphonia.

[0103] The conjugates can also be used in the field of cosmetics and dermatology, in dermatological products or cosmetic products, or as biomedical products, preferably as bioresorbable implants.

[0104] In yet a further aspect, the invention relates to the use of such conjugates in psoriasis and psoriatic osteoarthritis.

[0105] In another aspect, the present invention relates to a composition comprising the conjugate described above and at least one pharmacologically active substance and / or at least one substance having an optional biological function.

[0106] Suitable pharmacologically active substances include: antibiotics, anti-infectives, antimicrobials, antivirals, cytostatics, cytotoxic agents, anti-tumor drugs, anti-inflammatory drugs, cicatrizing agents, anesthetics, analgesics, vasoconstrictors, cholinergic or adrenergic agonists and antagonists, antithrombotic agents, anticoagulants, hemostatics, fibrinolytics, thrombolytics, proteins and fragments thereof, peptides, polynucleotides, growth factors, enzymes, vaccines or combinations thereof.

[0107] Preferably, the substance having an optional biological function is selected from the group consisting of 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.

[0108] The composition according to the invention has advantageous application in the same uses as listed above for the conjugates.

[0109] It is also to be understood that all aspects identified as preferred and advantageous with respect to the conjugation procedure are also to be considered as being likewise preferred and advantageous with respect to the above-mentioned conjugates obtainable therefrom and with respect to the compositions and uses of the conjugates.

[0110] Furthermore, it is to be understood that all possible combinations of the preferred aspects of the invention described above are considered to be equally described and preferred.

[0111] Working examples of the present invention are provided below for illustrative and non-limiting purposes. Example

[0112] Material

[0113] As substrates, 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.

[0114] The main reagents used for HA functionalization testing and for the synthesis of lactosamine are:

[0115] EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride),

[0116] NHS (N-hydroxysuccinimide),

[0117] HCTU (O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate),

[0118] DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride),

[0119] TEA (triethylamine),

[0120] α-D-lactose,

[0121] NH3 28%-30% (ammonium hydroxide solution),

[0122] NaCNBH3 (sodium cyanoborohydride),

[0123] NH4OAc (ammonium acetate),

[0124] IR-120 (ion exchanger),

[0125] SEC Pephadex G-10 size exclusion column.

[0126] Example 1.

[0127] Lactosamine Synthesis Procedure

[0128] 360 mg (1 mmol) of α-D-lactose was dissolved in 20 mL of NH4OAc / EtOH saturated solution ([c] = 0.05 M), and the mixture was kept at 40°C for about 30 min to complete the dissolution. 8 mL of 28%-30% aqueous ammonia (2.24 g; 131 mmol) was added to the mixture, and then 190 mg of NaCNBH3 (3 mmol) was added, and the resulting mixture was stirred at 90°C. After 8 hours, the solvent and excess ammonia were evaporated under reduced pressure, and the white solid was first treated with water and then with separate EtOH several times before loading onto an Amberlite IR-120 (H+) column (20 equivalents (eqs); ) and undergo elution with deionized water until the acidic conditions disappear (desalting). The amine product anchored on the sulfonic acid resin in the first step is recovered by subsequent elution with 50 mL of 10% aqueous ammonia solution and another 50 mL of water until complete elution of the amine product detected on the TLC plate with 10% sulfuric acid. All eluted fractions containing the product are evaporated and the residue is treated first with water and then with EtOH until the weight remains constant. 312 mg of white product is obtained with a yield of 90%.

[0129] Example 2.

[0130] Lactosamine Synthesis Procedure

[0131] 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 placed under stirring at a temperature of 55°C and allowed to react for 20 hours. Subsequently, the mixture was cooled, extracted with dichloromethane and, finally, the aqueous phase was evaporated at low pressure to obtain a crystalline white solid which was then washed with ether and finally recovered by decantation and dried under reduced pressure. The product was characterized by means of IR and 1 The product was characterized by H-NMR spectroscopy. Reaction yield: 90%.

[0132] A solution of the derivative thus obtained in methanol and water (1:1) (4% w / v) was placed under magnetic stirring at room temperature. Subsequently, Pd on charcoal (0.4% w / v) was added and the resulting system was pressurized with hydrogen. After 48 hours, the system was decompressed, mixed with an equal volume of water, the solid was decanted and the solution was filtered on celite. The solution thus obtained was dried under reduced pressure to afford a white solid. The product thus obtained was characterized by means of IR and 1 The product was characterized by H-NMR spectroscopy. Reaction yield: 96%.

[0133] Example 3.

[0134] a) Preparation of hyaluronic acid lactosylate

[0135] 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was stirred under magnetic force for 2 h. 4.12 g (14.90 mmol; 0.5 equivalent) of condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride) was added to the homogeneous solution, and 100 mL of an aqueous solution containing 5.12 g (14.9 mmol; 0.5 equivalent) of lactosamine was added after 1 h, and the mixture was kept at pH 6-7 at 25 ° C for 48 h. The product was then isolated by precipitation with 2.5 L of isopropanol at 4 ° C (75%), enriching the suspension with 3 mL of saturated NaCl solution (about 1 g of salt) to improve the precipitation step, and then recovered by centrifuging the suspension at 15000 rpm for 10 min. The obtained 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 then purified according to steps b1, b2 and c, the remaining 225 mL solution (9 / 10 of the total amount) was diluted to 350 mL and transferred to a 6 kDa-8 kDa membrane, and first dialyzed against 24 liters (12 l + 12 l) of 0.1 M NaCl saline solution for 2 days, and then dialyzed against deionized water for 3 days. The entire retentate (about 1 liter) was then concentrated under reduced pressure to about 1 / 3 of the initial volume, and freeze-dried to obtain a white solid.

[0136] b1) Purification by successive reprecipitation

[0137] The 570 mg sample obtained after the first precipitation and freeze drying was dissolved in 40 mL of deionized water at a concentration of 15 mg / ml, and subjected to reprecipitation with 60 mL of isopropanol (4° C., 60% v / v), the mixture was enriched with a few drops of saturated NaCl solution to improve the precipitation step. The suspension was centrifuged at 15000 rpm, and the solid obtained was washed several times with isopropanol, then recovered with deionized water and freeze dried to obtain a white solid.

[0138] b2) Purification by treatment with ion exchange resin

[0139] 570 mg of the sample obtained after the first precipitation and freeze-drying was dissolved in 40 mL of deionized water ([c] = 15 mg / ml) and precipitated on an Amberlite cation column IR-120 (H + ) (30 eq.; ) with other 60mL deionized water wash-out, until acid solution wash-out completely.Then use stoichiometric NaHCO (106mg) neutralize the mixture of whole wash-out, the mixture is remained on in vacuum system under agitation and continues about 1h, so as to promote the acid exchange of the glucuronic acid in sodium form.Then the mixture is concentrated to about 1 / 3 of the initial volume and freeze-dried under reduced pressure, to obtain a white solid.

[0140] c) Recovery of lactosamine from samples purified by ion exchange resin treatment

[0141] From the previously used Amberlite column IR-120 (H+) (30 equiv.; ), 40 mL 9% NH3 aqueous solution was loaded and eluted, followed by another separate 80 mL water, until neutral conditions were obtained. The entire eluate was dried under reduced pressure, and the obtained residue was first treated with water (5×10 ml) and finally with EtOH several times until the weight was constant, obtaining a sample weighing 56 mg.

[0142] Example 4.

[0143] a) Preparation of hyaluronic acid lactobionate

[0144] 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was stirred under magnetic force for 2 h. 8.24 g (29.83 mmol; 1.0 equivalent) of condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride) was added to the homogeneous solution, and 100 mL of an aqueous solution containing 10.24 g (29.83 mmol; 1.0 equivalent) of lactosamine was added after 1 h, and the mixture was kept at pH 6-7 at 25 ° C for 48 h. The product was then isolated by precipitation with 2.5 L of isopropanol at 4 ° C (75%), enriching the suspension with 3 mL of saturated NaCl solution (about 1 g of salt) to improve the precipitation step, and then recovered by centrifuging the suspension at 15000 rpm for 10 min. The obtained white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL and transferred to a 6 kDa-8 kDa membrane and first dialyzed against 24 liters (12 l + 12 l) of 0.1 M NaCl saline solution for 2 days and then dialyzed against deionized water for 3 days. The entire retentate (about 1 liter) was then concentrated under reduced pressure to about 1 / 3 of the initial volume and freeze-dried to obtain a white solid.

[0145] Alternatively, the white solid obtained after the precipitation and washing steps is further purified according to the techniques described in Example 3, b1 and b2.

[0146] Example 5.

[0147] a) Preparation of hyaluronic acid lactobionate

[0148] 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was stirred under magnetic force for 2 h. 12.36 g (44.75 mmol; 1.5 equivalents) of condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride) was added to the homogeneous solution, and 100 mL of an aqueous solution containing 15.36 g (44.75 mmol; 1.5 equivalents) of lactosamine was added after 1 h, and the mixture was kept at pH 6-7 at 25 ° C for 48 h. The product was then isolated by precipitation with 2.5 L of isopropanol at 4 ° C (75%), enriching the suspension with 3 mL of saturated NaCl solution (about 1 g of salt) to improve the precipitation step, and then recovered by centrifuging the suspension at 15000 rpm for 10 min. The obtained white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL and transferred to a 6 kDa-8 kDa membrane and first dialyzed against 24 liters (12 l + 12 l) of 0.1 M NaCl saline solution for 2 days and then dialyzed against deionized water for 3 days. The entire retentate (about 1 liter) was then concentrated under reduced pressure to about 1 / 3 of the initial volume and freeze-dried to obtain a white solid.

[0149] Alternatively, the white solid obtained after the precipitation and washing steps is further purified according to the techniques described in Example 3, b1 and b2.

[0150] Example 6.

[0151] a) Preparation of hyaluronic acid lactobionate

[0152] 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was stirred under magnetic force for 2 h. 12.36 g (44.75 mmol; 1.5 equivalents) of condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride) was added to the homogeneous solution, and 100 mL of an aqueous solution containing 15.36 g (44.75 mmol; 1.5 equivalents) of lactosamine was added after 1 h, and the mixture was kept at pH 6-7 at 25 ° C for 24 h. The product was then isolated by precipitation with 2.5 L of isopropanol at 4 ° C (75%), enriching the suspension with 3 mL of saturated NaCl solution (about 1 g of salt) to improve the precipitation step, and then recovered by centrifuging the suspension at 15000 rpm for 10 min. The obtained white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL and transferred to a 6 kDa-8 kDa membrane and first dialyzed against 24 liters (12 l + 12 l) of 0.1 M NaCl saline solution for 2 days and then dialyzed against deionized water for 3 days. The entire retentate (about 1 liter) was then concentrated under reduced pressure to about 1 / 3 of the initial volume and freeze-dried to obtain a white solid.

[0153] Alternatively, the white solid obtained after the precipitation and washing steps is further purified according to the techniques described in Example 3, b1 and b2.

[0154] Example 7.

[0155] a) Preparation of hyaluronic acid lactobionate

[0156] 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was stirred under magnetic force for 2 h. 12.36 g (44.75 mmol; 1.5 equivalents) of condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride) was added to the homogeneous solution, and 100 mL of an aqueous solution containing 15.36 g (44.75 mmol; 1.5 equivalents) of lactosamine was added after 1 h, and the mixture was kept at pH 6-7 at 25 ° C for 48 h. The product was then isolated by precipitation with 2.5 L of isopropanol at 4 ° C (75%), enriching the suspension with 3 mL of saturated NaCl solution (about 1 g of salt) to improve the precipitation step, and then recovered by centrifuging the suspension at 15000 rpm for 10 min. The obtained white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL and transferred to a 6 kDa-8 kDa membrane and first dialyzed against 24 liters (12 l + 12 l) of 0.1 M NaCl saline solution for 2 days and then dialyzed against deionized water for 3 days. The entire retentate (about 1 liter) was then concentrated under reduced pressure to about 1 / 3 of the initial volume and freeze-dried to obtain a white solid.

[0157] Alternatively, the white solid obtained after the precipitation and washing steps is further purified according to the techniques described in Example 3, b1 and b2.

[0158] Example 8.

[0159] a) Preparation of hyaluronic acid lactobionate

[0160] 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was stirred under magnetic force for 2 h. 20.60 g (74.58 mmol; 2.5 equivalents) of condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride) was added to the homogeneous solution, and 100 mL of an aqueous solution containing 25.60 g (74.58 mmol; 2.5 equivalents) of lactosamine was added after 1 h, and the mixture was kept at pH 6-7 at 25 ° C for 48 h. The product was then isolated by precipitation with 2.5 L of isopropanol at 4 ° C (75%), enriching the suspension with 3 mL of saturated NaCl solution (about 1 g of salt) to improve the precipitation step, and then recovered by centrifuging the suspension at 15000 rpm for 10 min. The obtained white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL and transferred to a 6 kDa-8 kDa membrane and first dialyzed against 24 liters (12 l + 12 l) of 0.1 M NaCl saline solution for 2 days and then dialyzed against deionized water for 3 days. The entire retentate (about 1 liter) was then concentrated under reduced pressure to about 1 / 3 of the initial volume and freeze-dried to obtain a white solid.

[0161] Alternatively, the white solid obtained after the precipitation and washing steps is further purified according to the techniques described in Example 3, b1 and b2.

[0162] Example 9.

[0163] a) Preparation of hyaluronic acid lactobionate

[0164] 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was stirred under magnetic force for 2 h. 12.36 g (44.75 mmol; 1.5 equivalents) of condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride) was added to the homogeneous solution, and 100 mL of an aqueous solution containing 15.36 g (44.75 mmol; 1.5 equivalents) of lactosamine was added after 1 h, and the mixture was kept at pH 6-7 at 25 ° C for 48 h. The product was then isolated by precipitation with 2.5 L of isopropanol at 4 ° C (75%), enriching the suspension with 3 mL of saturated NaCl solution (about 1 g of salt) to improve the precipitation step, and then recovered by centrifuging the suspension at 15000 rpm for 10 min. The obtained white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL and transferred to a 6 kDa-8 kDa membrane and first dialyzed against 24 liters (12 l + 12 l) of 0.1 M NaCl saline solution for 2 days and then dialyzed against deionized water for 3 days. The entire retentate (about 1 liter) was then concentrated under reduced pressure to about 1 / 3 of the initial volume and freeze-dried to obtain a white solid.

[0165] Alternatively, the white solid obtained after the precipitation and washing steps is further purified according to the techniques described in Example 3, b1 and b2.

[0166] Example 10.

[0167] a) Preparation of hyaluronic acid lactobionate

[0168] 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was stirred under magnetic force for 2 h. 8.24 g (29.83 mmol; 1.0 equivalent) of condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride) was added to the homogeneous solution, and 100 mL of an aqueous solution containing 10.24 g (29.83 mmol; 1.0 equivalent) of lactosamine was added after 1 h, and the mixture was kept at pH 5-8 at 25 ° C for 12 h. The product was then isolated by precipitation with 2.5 L of isopropanol at 4 ° C (75%), enriching the suspension with 3 mL of saturated NaCl solution (about 1 g of salt) to improve the precipitation step, and then recovered by centrifuging the suspension at 15000 rpm for 10 min. The obtained white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL and transferred to a 6 kDa-8 kDa membrane and first dialyzed against 24 liters (12 l + 12 l) of 0.1 M NaCl saline solution for 2 days and then dialyzed against deionized water for 3 days. The entire retentate (about 1 liter) was then concentrated under reduced pressure to about 1 / 3 of the initial volume and freeze-dried to obtain a white solid.

[0169] Alternatively, the white solid obtained after the precipitation and washing steps is further purified according to the techniques described in Example 3, b1 and b2.

[0170] Example 11.

[0171] a) Preparation of hyaluronic acid lactobionate

[0172] 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was stirred under magnetic force for 2 h. 12.36 g (44.75 mmol; 1.5 equivalents) of condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride) was added to the homogeneous solution, and 100 mL of an aqueous solution containing 15.36 g (44.75 mmol; 1.5 equivalents) of lactosamine was added after 1 h, and the mixture was kept at pH 6-7 at 25 ° C for 48 h. The product was then isolated by precipitation with 2.5 L of isopropanol at 4 ° C (75%), enriching the suspension with 3 mL of saturated NaCl solution (about 1 g of salt) to improve the precipitation step, and then recovered by centrifuging the suspension at 15000 rpm for 10 min. The obtained white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL and transferred to a 6 kDa-8 kDa membrane and first dialyzed against 24 liters (12 l + 12 l) of 0.1 M NaCl saline solution for 2 days and then dialyzed against deionized water for 3 days. The entire retentate (about 1 liter) was then concentrated under reduced pressure to about 1 / 3 of the initial volume and freeze-dried to obtain a white solid.

[0173] Alternatively, the white solid obtained after the precipitation and washing steps is further purified according to the techniques described in Example 3, b1 and b2.

[0174] Example 12.

[0175] a) Preparation of hyaluronic acid lactobionate

[0176] 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was stirred under magnetic force for 2 h. 12.36 g (44.75 mmol; 1.5 equivalents) of condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride) was added to the homogeneous solution, and 100 mL of an aqueous solution containing 15.36 g (44.75 mmol; 1.5 equivalents) of lactosamine was added after 1 h, and the mixture was kept at pH 6-7 at 25 ° C for 24 h. The product was then isolated by precipitation with 2.5 L of isopropanol at 4 ° C (75%), enriching the suspension with 3 mL of saturated NaCl solution (about 1 g of salt) to improve the precipitation step, and then recovered by centrifuging the suspension at 15000 rpm for 10 min. The obtained white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL and transferred to a 6 kDa-8 kDa membrane and first dialyzed against 24 liters (12 l + 12 l) of 0.1 M NaCl saline solution for 2 days and then dialyzed against deionized water for 3 days. The entire retentate (about 1 liter) was then concentrated under reduced pressure to about 1 / 3 of the initial volume and freeze-dried to obtain a white solid.

[0177] Alternatively, the white solid obtained after the precipitation and washing steps is further purified according to the techniques described in Example 3, b1 and b2.

[0178] Example 13.

[0179] a) Preparation of hyaluronic acid lactobionate

[0180] 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was stirred under magnetic force for 2 h. 12.36 g (44.75 mmol; 1.5 equivalents) of condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride) was added to the homogeneous solution, and 100 mL of an aqueous solution containing 20.48 g (59.67 mmol; 1.5 equivalents) of lactosamine was added after 1 h, and the mixture was kept at pH 6-7 at 25 ° C for 48 h. The product was then isolated by precipitation with 2.5 L of isopropanol at 4 ° C (75%), enriching the suspension with 3 mL of saturated NaCl solution (about 1 g of salt) to improve the precipitation step, and then recovered by centrifuging the suspension at 15000 rpm for 10 min. The obtained white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL and transferred to a 6 kDa-8 kDa membrane and first dialyzed against 24 liters (12 l + 12 l) of 0.1 M NaCl saline solution for 2 days and then dialyzed against deionized water for 3 days. The entire retentate (about 1 liter) was then concentrated under reduced pressure to about 1 / 3 of the initial volume and freeze-dried to obtain a white solid.

[0181] Alternatively, the white solid obtained after the precipitation and washing steps is further purified according to the techniques described in Example 3, b1 and b2.

[0182] Example 14.

[0183] a) Preparation of hyaluronic acid lactobionate

[0184] 12.02 g (29.83 mmol) of sodium hyaluronate was dissolved in 0.7 L of deionized water, and the mixture was stirred under magnetic force for 2 h. 12.36 g (44.75 mmol; 1.5 equivalents) of condensing agent DMTMM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride) was added to the homogeneous solution, and 100 mL of an aqueous solution containing 20.48 g (59.67 mmol; 1.5 equivalents) of lactosamine was added after 1 h, and the mixture was kept at pH 5-8 at 60 ° C for 5 h. The product was then isolated by precipitation with 2.5 L of isopropanol at 4 ° C (75%), enriching the suspension with 3 mL of saturated NaCl solution (about 1 g of salt) to improve the precipitation step, and then recovered by centrifuging the suspension at 15000 rpm for 10 min. The obtained white solid was washed several times with isopropanol and dissolved in 250 mL of deionized water. The solution was diluted to 400 mL and transferred to a 6 kDa-8 kDa membrane and first dialyzed against 24 liters (12 l + 12 l) of 0.1 M NaCl saline solution for 2 days and then dialyzed against deionized water for 3 days. The entire retentate (about 1 liter) was then concentrated under reduced pressure to about 1 / 3 of the initial volume and freeze-dried to obtain a white solid.

[0185] Alternatively, the white solid obtained after the precipitation and washing steps is further purified according to the techniques described in Example 3, b1 and b2.

[0186] Example 15-Example 26

[0187] The procedures in Example 3 to Example 14 were repeated using 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) instead of DMTMM to obtain the desired product.

[0188] Example 27.

[0189] Samples from Examples 3-14 were used to determine the molecular weight (MW) and degree of substitution (DS).

[0190] Molecular weight determination was performed by size exclusion chromatography (HP-SEC / TDA).

[0191] After enzymatic hydrolysis of the samples with hyaluronidase, two-dimensional hetero-correlated (1H, 13 C) HSQC-DEPT nuclear magnetic resonance (NMR) technique was used to determine the degree of substitution.

[0192] The results are shown in the table below:

[0193]

[0194]

[0195] Example 28.

[0196] Sample 14, Sample 13 and Sample 6 were tested on a cell model to evaluate their anti-inflammatory properties.

[0197] By qPCR analysis of IL-1β gene expression, the anti-inflammatory activity of molecule 14, molecule 13 and molecule 6 was compared with that of negative control (CTRL, human bronchial fibroblasts from smokers, untreated) and positive control (TC, human bronchial fibroblasts from smokers, incubated with cultures of U937 human monocytes activated into macrophages with PMA (50 μg / ml, 24 h) and LPS (1 μg / ml, 1 h). The following table shows the expression of the genes analyzed after 4 hours of incubation with compound 14, compound 13 and compound 6:

[0198] Mean (fold change) Standard Deviation CTRL 1 ±0 TC 58.12 ±10.18 #14 31.85 ±12.67 #6 41.51 ±9.181 #13 34.99 ±8.040

[0199] All compounds showed significant reduction of IL-1β relative to TC.

[0200] Example 29.

[0201] Sample 14, Sample 13 and Sample 6 were tested on a cell model to evaluate their anti-fibrotic properties.

[0202] By qPCR analysis of COL-1 gene expression, the anti-fibrotic activity of molecule 14, molecule 13 and molecule 6 was compared with the anti-fibrotic activity of negative control (CTRL, human peripheral lung fibroblasts, untreated, NHLF line) and positive control (TGF-β, human peripheral lung fibroblasts (NHLF) incubated with 5ng / ml TGF-β for 24h). The following table shows the expression of the genes analyzed after 4 hours of incubation with compound 14, compound 13 and compound 6:

[0203]

[0204]

[0205] All compounds demonstrated significant reduction in COL-1 relative to TGF-β.

Claims

1. A process for conjugating hyaluronic acid or a pharmaceutically acceptable salt thereof with a disaccharide or an oligosaccharide, comprising the following steps: i) providing an aqueous solution of hyaluronic acid or a pharmaceutically acceptable salt thereof, ii) adding a 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 and adding the primary amine of the disaccharide or oligosaccharide under stirring to form a conjugate of hyaluronic acid or a pharmaceutically acceptable salt thereof with the disaccharide or oligosaccharide via an amide bond, iv) promoting the precipitation of said conjugate thus obtained, adding an organic solvent miscible with water, thereby obtaining a crude conjugate, and the precipitated conjugate may be washed with a further organic solvent, which may be miscible with water, v) isolating the crude conjugate, and optionally vi) purifying the crude conjugate by reprecipitation, by treatment with an ion exchange resin, by dialysis or by a combination thereof and / or repetition thereof, and vii) obtaining a purified conjugate by drying, The process is carried out at a temperature of 4°C-100°C and a pH of 5-8, The triazine compound of formula (I) is: wherein X is a halogen selected from I, Br, Cl and mixtures thereof, or X is a group -NR1R2 selected from NH2, NMe2, NHPr, PhNH, 4-MeC6H4NH, and mixtures thereof.

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

3. The process 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. The process according to any one of claims 1 to 3, wherein the primary amine of a disaccharide or an oligosaccharide from a trisaccharide to a hexasaccharide is a primary amine derivative of sucrose, maltose, lactose, trehalose, gentiobiose, cellobiose, maltotriose, raffinose, stachyose, maltotetraose, melezitose, isomaltose, panose, nesitose, 1-ketose, methyl inositol galactopyranose, galactosyl lactose or a mixture thereof.

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

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

7. The process according to any one of claims 1 to 6, wherein When the purification of the crude conjugate in step vi) is by treatment with an ion exchange resin, the following sub-steps are performed: vi-a) dissolving the crude conjugate in water to obtain an aqueous solution, vi-b) treating the aqueous solution with an ion exchange resin using water as eluent, vi-c) separating the aqueous solution from the ion exchange resin, vi-d) The separated solution is adjusted to neutral pH.

8. The process according to any one of claims 1 to 6, wherein When the purification of the crude conjugate in step vi) is by dialysis, the following sub-steps are performed: vi-i) dissolving the crude conjugate in water to obtain an aqueous solution, vi-ii) The obtained solution is dialyzed against saline solution and / or water through a suitable membrane.

9. The process according to any one of claims 1 to 6, wherein When the purification of the crude conjugate in step vi) is by reprecipitation, the following sub-steps are performed: vi-α) dissolving the crude conjugate in water to obtain an aqueous solution, vi-β) adding a water-miscible organic solvent which is the same as or different from the organic solvent in step iv) to promote reprecipitation of the conjugate, vi-γ) washing the precipitated conjugate with additional organic solvent to obtain a purified conjugate.

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

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

3.

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

3.

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

14. A composition comprising the conjugate according to claim 13 and at least one pharmacologically active substance and / or at least one substance optionally having a biological function, wherein: - the pharmacologically active substance is selected from the group consisting of antibiotics, anti-infectives, antimicrobials, antivirals, cytostatics, cytotoxics, anti-tumor agents, anti-inflammatory agents, healing agents, anesthetics, analgesics, vasoconstrictors, cholinergic or adrenergic agonists and antagonists, antithrombotic agents, anticoagulants, hemostatics and fragments thereof, peptides, polynucleotides, growth factors, enzymes, vaccines and combinations thereof, and - the optionally biologically functional substance is selected from the group consisting of 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.

15. The conjugate according to claim 13 or the composition according to claim 14 in the form of a powder, solution, emulsion, gel, ointment, drops, aerosol, spray or in an injectable form for administration via the nasal, inhalation, intra-articular, intradermal, ocular or urogynecological route.

16. The conjugate according to claim 13 or the composition according to claim 14, for use in: - for use in the treatment of diseases attributable to altered expression of galectins, including nonalcoholic steatohepatitis, plaque psoriasis, rheumatoid arthritis, osteoarthritis, neoplasms, adhesions, and fibrotic processes of the skin, lungs, kidneys, and cardiovascular, or - Use as a biomaterial or scaffold for cell growth, or - for use in tissue repair or reconstruction, preferably in the production or replacement of biological tissue or in the filling of biological tissue, such as in the filling of skin, recesses, bone cartilage or joints, or - for use in dermatological or cosmetic products, or as a biomedical product, preferably as a bioresorbable implant, or - For use in rheumatology, orthopedics, oncology, plastic and cosmetic surgery, hemodialysis, cardiology, vascular, ophthalmology, otolaryngology, pulmonology, dentistry, gynecology, urology, dermatology, oncology and tissue repair, or - Use in traumatic and / or post-surgical tissue processes and / or chronic fibrotic processes associated with autoimmune diseases, or - Use in traumatic and post-operative sequelae involving the dermis and abdominal tissues, or in post-operative sequelae of intranasal surgery, in post-operative sequelae of tendon and / or cartilage tissues, or -For use in psoriasis and psoriatic osteoarthritis.

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