RU2792001C2 - Functionalized hyaluronic acid or its derivative for the treatment of inflammatory conditions - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: group of inventions relates to functionalized hyaluronic acid. Functionalized hyaluronic acid or its derivative of formula (I) is disclosed

(I),

where R₁, R₂, R₃, R₄ independently represent the H, SO_3-, acyl group of a carboxylic acid of the aliphatic, aromatic, arylaliphatic, cycloaliphatic or heterocyclic series, -CO-(CH₂)₂-COOY, where Y is a negative charge or H, and R is Z(1) or Z(2), and R₅ is -CO-CH₃, H, SO₃-, acyl group of carboxylic acid of the aliphatic, aromatic, arylaliphatic, cycloaliphatic or heterocyclic series, the acyl group of hyaluronic acid. A method for obtaining functionalized hyaluronic acid or its derivative and a pharmaceutical composition are also disclosed.

EFFECT: group of inventions provides treatment of pathologies caused by changes in the expression of galectins.

10 cl, 4 dwg, 1 tbl, 29 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to a functionalized hyaluronic acid or its derivative, as well as to a method for its preparation and its use as a biomaterial and as an ingredient in pharmaceutical compositions.

BACKGROUND OF THE INVENTION

Galectins are a family of proteins characterized by specific binding to β-galactosidic sugars such as N-acetyl-lactosamine, which can bind to proteins via N-glycosylation or O-glycosylation. In mammals, 15 galectins are known, which are encoded by the LGALS genes and numbered sequentially, while in humans only galectins -1, -2, -3, -4, -7, -8, -9, -10, -12 and -13.

They are either intracellular or extracellular. The latter enter into bivalent or multivalent interactions with glycans on the cell surface and induce various cellular responses, including the production of cytokines and other inflammatory mediators, cell adhesion, migration, and apoptosis. In addition, they can form spatial structures with membrane glycoprotein receptors and modulate the properties of these receptors. Intracellular galectins may be involved in signal transduction and alter biological responses, including apoptosis, cell differentiation, and cell motility. According to currently available data, galectins play an important role in acute and chronic inflammatory responses, as well as in a number of other pathological processes.

Recent studies have shown that some galectins are involved in the inflammatory response in several musculoskeletal disorders such as rheumatoid arthritis and osteoarthritis (D. Weinmann et al. Scientific Reports DOI: 10.1038/srep39112; Toegel S. et al Histochem Cell Biol 2014, 142 , 373; Toegel S. The Journal of Immunology 2016, 1910; Li S. et al. J. Clin. Cell Immunol 2013, 4(5), 1000164). Since many inflammatory pathologies are accompanied by overexpression of galectins, inhibition of metalloproteinase activity can, accordingly, lead to a marked decrease in the activity of processes in the inflammatory cascade.

In addition, galectins are known to play an active role in the development and progression of tumors. Therefore, studies of galectins are currently underway to improve both the diagnosis and treatment of neoplasms (Ebrahim AH, et al. Galectins in cancer: carcinogenesis, diagnosis and therapy. Ann Transl Med 2014;2(9):88. doi: 10.3978 /j.issn.2305-5839.2014.09.12).

Accordingly, it is an object of the present invention to provide a product that regulates the expression of these receptors for therapeutic effects on pathologies due to altered expression of galectins, while having suitable medical and pharmaceutical characteristics.

BRIEF DESCRIPTION OF THE INVENTION

This problem was solved using functionalized hyaluronic acid or its derivative according to claim 1 of the claims.

According to another aspect, the present invention relates to a method for producing functionalized hyaluronic acid or its derivative.

According to another aspect, the present invention relates to the use of the specified functionalized hyaluronic acid or its derivative for the treatment of pathologies due to changes in the expression of galectins. Non-limiting examples of pathologies due to over- or under-regulation of these receptors are non-alcoholic steatohepatitis, plaque psoriasis, rheumatoid arthritis, osteoarthritis, neoplasia, and fibrotic processes in the lungs, kidneys, and cardiovascular system.

According to another aspect, the present invention relates to the use of said functionalized hyaluronic acid or a derivative thereof as a biomaterial or scaffold for cell growth in the treatment of orthopedic diseases.

According to a further aspect, the present invention relates to the use of said functionalized hyaluronic acid or a derivative thereof as a biomaterial or scaffold for cell growth, in plastic/cosmetic surgery, hemodialysis, cardiology, angiology, ophthalmology, otolaryngology, odontology, gynecology, urology, dermatology, oncology and tissue restoration.

According to another aspect, the present invention relates to a pharmaceutical composition containing at least one functionalized hyaluronic acid or its derivative and at least one pharmacologically active substance and/or at least one biologically active substance.

According to a further aspect, the present invention relates to the use of said pharmaceutical composition for the treatment of pathologies due to altered expression of galectins. Non-limiting examples of pathologies due to over- or under-regulation of these receptors are non-alcoholic steatohepatitis, plaque psoriasis, rheumatoid arthritis, osteoarthritis, neoplasia, and fibrotic processes in the lungs, kidneys, and cardiovascular system.

According to another aspect, the present invention relates to the use of said pharmaceutical composition in plastic/cosmetic surgery, hemodialysis, cardiology, angiology, ophthalmology, otolaryngology, odontology, gynecology, urology, dermatology, oncology and tissue repair.

BRIEF DESCRIPTION OF GRAPHICS

The characteristics and advantages of the present invention will become apparent from the following detailed description, embodiments given by way of non-limiting examples, and the accompanying drawings, where:

- FIGURE 1 compares the IR spectra of lactose, deacetylated hyaluronic acid and a hyaluronic acid derivative, where R ₅ is Z(3) and Z3 is a galactose moiety.

- FIGURE 2 compares the IR spectra of hyaluronic acid, a reducing sugar amine derivative obtained by reductive amination, and a hyaluronic acid derivative, where R is Z(1) and Z3 is a galactose moiety.

- FIGURE 3 shows ¹ H-NMR spectra (400 MHz, D ₂ O, 343 K): 1) sodium hyaluronate; 2) sodium hyaluronate obtained according to Example 6; 3) amino derivatives of partially deacetylated hyaluronic acid (reductive amination using reducing sugars) obtained according to Example 13, and

- FIGURE 4 shows ¹ H-NMR spectra (400 MHz, D ₂ O, 298 K): 1) sodium hyaluronate; 2) amino reducing sugar obtained according to Example 3; 3) an amide derivative of partially deacetylated hyaluronic acid (amidation using amine derivatives of reducing sugars) obtained according to Example 13.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the present invention relates to a functionalized hyaluronic acid or a derivative thereof having the formula (I):

(I)

(I)

Where

R ₁ , R ₂ , R ₃ , R ₄ are independently H, SO ₃ ^- , an aliphatic, aromatic, arylaliphatic, cycloaliphatic or heterocyclic carboxylic acid acyl group, -CO-(CH ₂ ) ₂ -COOY, where Y is negative charge or H,

And

R is Z(1) or Z(2), and R ₅ is -CO-CH ₃ , H, SO ₃ ^- , an aliphatic, aromatic, arylaliphatic, cycloaliphatic or heterocyclic carboxylic acid acyl group, a hyaluronic acid acyl group,

where Z(1) is a fragment of formula (1):

(1)

(1)

where Z ₁ is -NR ₆ CH ₂ - and R ₆ is H or a substituted or unsubstituted aliphatic, aromatic, arylaliphatic, cycloaliphatic or heterocyclic group,

Z ₂ is -OH or -NHCOCH ₃ ,

Z ₃ is H, a monosaccharide, disaccharide or oligosaccharide,

or Z(2) is a fragment of formula (2):

(2)

(2)

where Z ₄ is -NR ₆ CH- and R ₆ is H or a substituted or unsubstituted aliphatic, aromatic, arylaliphatic, cycloaliphatic or heterocyclic group,

Z ₅ and Z ₆ are independently H, a monosaccharide, a disaccharide or an oligosaccharide,

or

R ₅ is Z(3) or Z(4), and R is NR ₆ R ₇ , or an aliphatic, aromatic, arylaliphatic, cycloaliphatic or heterocyclic alcohol group, OH, O ^- , an alcohol group of hyaluronic acid, an amino group of hyaluronic acid , and R ₆ , R ₇ independently represent H or a substituted or unsubstituted aliphatic, aromatic, arylaliphatic, cycloaliphatic or heterocyclic group,

where Z(3) is a fragment of formula (3):

(3)

(3)

where Z ₁ represents -CH ₂ - or -CO-,

Z ₂ is -OH or -NHCOCH ₃ ,

Z ₃ is H, a monosaccharide, disaccharide or oligosaccharide,

or Z(4) is a fragment of formula (4):

(4)

(4)

where Z ₄ represents -CH-,

Z ₅ and Z ₆ are independently H, a monosaccharide, a disaccharide or an oligosaccharide,

or

R is Z(1) or Z(2) and R ₅ is Z(3) or Z(4).

The term "aliphatic, aromatic, arylaliphatic, cycloaliphatic, heterocyclic" preferably means a linear, branched or cyclic moiety - saturated or unsaturated, aliphatic or aromatic, selected from: C1-C10 alkyl, substituted C1-C10 alkyl, C2-C10 alkenyl substituted with C2 -C10 alkenyl, C4-C10 dienyl, substituted C4-C10 dienyl, C2-C10 alkynyl, substituted C2-C10 alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, C1-C10 alkylthio substituted C1-C10 alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, carbonyl, substituted C1-C6 carbonyl, carboxyl, substituted C1-C6 carboxyl, amino, substituted C1-C6 amino, amide, substituted C1-C6 amide, sulfonyl, substituted C1-C6 sulfonyl, sulfonic acid, phosphonyl, substituted C1-C6 phosphonyl, polyaryl, substituted polyaryl, C3-C20 cycloalkyl, substituted C3-C20 cycloalkyl, C3-C20 substituted C3-C20 heterocycloalkyl heterocycloalkyl, C2-C10 cycloalkenyl, C2-C10 substituted cycloalkenyl, C4-C10 cyclodienyl, C4-C10 substituted cyclodienyl, or amino acid. The term "substituted" means "associated with at least one halogen, hydroxyl, C1-C4 alkyl, carboxyl, or combinations thereof."

Preferably Z ₃ , Z ₅ and Z ₆ are independently H, glucose, galactose, arabinose, xylose, mannose, lactose, trehalose, gentiobiose, cellobiose, cellotriose, maltose, maltotriose, chitobiose, chitotriose, mannobia, melibiose, fructose, N- acetylglucosamine, N-acetylgalactosamine fragment or a combination thereof.

More preferably Z ₃ is H, glucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine, or a combination thereof.

In particularly preferred embodiments, Z is a lactose or galactose moiety, where Z is any of Z(1), Z(2), Z(3) and Z(4).

As can be seen from the above structural formulas, hyaluronic acid or its derivative is functionalized by conjugation with a fragment of the formula Z - Z(1), Z(2), Z(3) or Z(4) - due to:

1) an amide bond between the carboxyl group of hyaluronic acid, or its derivative, and an amine, by reductive amination of the precursor Z with primary amines or ammonia sources,

2) an amine bond between the amino group of hyaluronic acid or its derivative, previously deacetylated, and the Z fragment, through reductive amination,

3) an amide bond between the amino group of hyaluronic acid or its derivative, previously deacetylated, and the carboxyl group of the Z fragment precursor.

Accordingly, according to another aspect, the present invention relates to a method for producing a functionalized hyaluronic acid or its derivative, comprising the following steps:

i) providing hyaluronic acid or a partially or fully deacetylated derivative thereof;

ii) providing an amino derivative of the monosaccharide, disaccharide or oligosaccharide by means of a reductive amination reaction;

iii) interactions:

a) said hyaluronic acid from step i) with an amino derivative from step ii) in the presence of carbodiimides and/or carboxyl group activators,

or

b) said partially or fully deacetylated derivative from step i) with a monosaccharide, disaccharide or oligosaccharide in the presence of an aminoborane;

or

c) a partially or completely deacetylated derivative from step i) with a carboxy derivative of a monosaccharide, disaccharide or oligosaccharide in the presence of carbodiimides and/or carboxyl group activators;

or

d) said derivative from step iii-b) with the amino derivative from step ii) in the presence of carbodiimides and/or carboxyl group activators;

or

e) said derivative from step iii-c) with an amino derivative from step ii) in the presence of carbodiimides and/or carboxyl group activators;

And

iv) precipitation of the formed functionalized hyaluronic acid or its derivative with an organic solvent.

Surprisingly, these aminoboranes have been found to exhibit significant selectivity for the reduction of the imino group over the carbonyl group and are compatible with aqueous media, thereby providing efficient amine reduction of reducing sugars in the presence of primary amines, ammonia sources and amine polysaccharide moieties. At the same time, the presence of carbodiimides and/or carboxyl group activators effectively promotes the formation of hyaluronic acid amide derivatives with excellent selectivity towards the formation of ester derivatives. Thus, this method generally provides the advantage of being able to combine monosaccharides, disaccharides and oligosaccharides with the main chain of hyaluronic acid without the need for the introduction of chemical spacers.

Derivatives of hyaluronic acid that can be used to obtain functionalized derivatives according to the present invention are preferably:

- hyaluronic acid salts such as sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, ammonium hyaluronate, tetrabutylammonium hyaluronate and mixtures thereof,

- esters of hyaluronic acid, in which part of the carboxyl groups or all of the carboxyl groups are esterified with aliphatic, aromatic, arylaliphatic, cycloaliphatic or heterocyclic alcohols, as also described in EP 0216453 B1,

- self-crosslinked esters of hyaluronic acid, in which part of the carboxyl groups or all of the carboxyl groups are esterified with alcohol groups from the same polysaccharide chain or other chains, as also described in EP 0341745 B1,

- cross-linked hyaluronic acid compounds, in which part of the carboxyl groups or all of the carboxyl groups are esterified with aliphatic, aromatic, arylaliphatic, cycloaliphatic or heterocyclic polyalcohols, where the cross-link is formed by spacer chains, as also described in EP 0265116 B1,

- a half-ester of succinic acid or a heavy metal salt of succinic acid with hyaluronic acid or with partial or full esters of hyaluronic acid, as also described in WO 96/357207,

- O-sulfo derivatives, as also described in WO 95/25751, or N-sulfo derivatives, as also described in WO 1998/045335.

Said monosaccharides, disaccharides or oligosaccharides meet the definition given above for the Z moiety.

Said aminoborane is preferably 2-methyl-pyridineborane, 5-ethyl-2-methyl-pyridineborane, pyridineborane, trimethylamineborane, triethylamineborane, dimethylamineborane, t-butylamineborane or a mixture thereof. More preferably, said aminoborane is 2-methyl-pyridineborane, 5-ethyl-2-methyl-pyridineborane, or a mixture thereof.

Aminoboranes can be used without pre-treatment, or they can be pre-solubilized or dispersed in water-miscible organic solvents such as alcohols. Among such alcohols, methanol, ethanol, 2-propanol or a mixture thereof are most preferred.

The term "organic solvent" means a water-miscible organic solvent capable of lowering the dielectric constant of an aqueous reaction solution. Suitable organic solvents are acetone, methanol, ethanol, 2-propanol, or a mixture thereof, and preferably said organic solvent is ethanol or 2-propanol, or a mixture thereof.

The term "activator of the carboxyl group" means reagents that modify the hydroxy group of the specified group and thereby contribute to its elimination in substitution reactions. Carboxyl group activators include hydroxybenzotriazole, 1,1''-carbodiimidazole, p-nitrophenol, N-hydroxysulfosuccinimide sodium salt, N-hydroxysuccinimide, and mixtures thereof.

Suitable carbodiimides include dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride, 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide, N,N''-di-iso-propylcarbodiimide and their mixtures.

Optionally, the precipitate recovered in step iv) is washed with mixtures of water and an organic solvent where the water content is up to 30%, more preferably up to 10%.

Preferably in step iii), the molar ratio of monosaccharide, disaccharide or oligosaccharide to hyaluronic acid or derivative thereof is 0.5 to 30, more preferably 1 to 20, even more preferably 1 to 10.

According to another aspect, the present invention relates to the use of the specified functionalized hyaluronic acid or its derivative for the treatment of pathologies due to changes in the expression of galectins. Non-limiting examples of pathologies due to over- or under-regulation of these receptors are non-alcoholic steatohepatitis, plaque psoriasis, rheumatoid arthritis, osteoarthritis, neoplasia, and fibrotic processes in the lungs, kidneys, and cardiovascular system.

Examples of neoplasia and fibrotic processes include acute lymphoblastic leukemia, idiopathic pulmonary fibrosis, hepatic fibrosis, cardiac fibrosis, renal fibrosis, and tumors of the ovaries, prostate, lung, stomach, skin, thyroid, and pancreas.

According to another aspect, the present invention relates to the use of said functionalized hyaluronic acid or a derivative thereof as a biomaterial or scaffold for cell growth in the treatment of orthopedic diseases.

According to a further aspect, the present invention relates to the use of said functionalized hyaluronic acid or a derivative thereof as a biomaterial or scaffold for cell growth, in plastic/cosmetic surgery, hemodialysis, cardiology, angiology, ophthalmology, otolaryngology, odontology, gynecology, urology, dermatology, oncology and tissue restoration.

Functionalized hyaluronic acid or its derivative can also be used as a biomaterial for coating objects used in the field of medicine and other industries, giving the surface of such an object used as a medium, new biological characteristics.

Objects that can be coated include, for example, catheters, tubes, probes, heart valves, soft tissue prostheses, animal prostheses, artificial tendons, bone and vascular prostheses, contact lenses, blood oxygenators, artificial kidneys, heart , pancreas, liver, blood transfusion bags, syringes, surgical instruments, filtration systems, laboratory instruments, containers for culture storage and for cell and tissue recovery, media for peptides, proteins and antibodies.

Functionalized hyaluronic acid or its derivative can also be used in cosmetology and dermatology.

According to another aspect, the present invention relates to a pharmaceutical composition containing at least one functionalized hyaluronic acid or its derivative and at least one pharmacologically active substance and/or at least one biologically active substance.

Suitable pharmacologically active substances include antibiotics, anti-infective, anti-microbial, anti-viral, cytostatic, cytotoxic, anti-tumor and anti-inflammatory drugs, wound healing agents, anesthetics, analgesics, vasoconstrictors, cholinergic or andrenergic agonists and antagonists, antithrombotic agents, anticoagulants, hemostatic, fibrinolytic or thrombolytic agents. agents, proteins and fragments thereof, peptides, polynucleotides, growth factors, enzymes, vaccines, or combinations thereof.

Preferably said active 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, copolymer polylactic and polyglycolic acids, polycaprolactone, gelatin, albumin, polyglycolide-polycaprolactone copolymer, polyglycolide-polytrimethylene carbonate copolymer, hydroxyapatite, tricalcium phosphate, dicalcium phosphate, demineralized bone matrix, and mixtures thereof.

Preferably, said at least one functionalized hyaluronic acid or its derivative and said at least one biologically active substance are contained in a weight ratio of 100:1 to 1:150.

According to a further aspect, the present invention relates to the use of said pharmaceutical composition for the treatment of pathologies due to altered expression of galectins. Non-limiting examples of pathologies due to over- or under-regulation of these receptors are non-alcoholic steatohepatitis, plaque psoriasis, rheumatoid arthritis, osteoarthritis, neoplasia, and fibrotic processes in the lungs, kidneys, and cardiovascular system.

According to a further aspect, the present invention relates to the use of said pharmaceutical composition in plastic/cosmetic surgery, hemodialysis, cardiology, angiology, ophthalmology, otolaryngology, odontology, gynecology, urology, oncology, dermatology and tissue repair.

Preferably, the pharmaceutical composition according to the invention contains up to 10% of the mass. inclusive of the specified at least one functionalized hyaluronic acid or its derivative, based on the weight of the pharmaceutical composition, and more preferably, up to 5% of the mass. including said at least one functionalized hyaluronic acid or its derivative. Most preferred are pharmaceutical compositions in which the amount of said at least one functionalized hyaluronic acid or its derivative is from 0.5 to 5% of the mass. based on the weight of the specified pharmaceutical composition.

According to particularly preferred embodiments, the present invention relates to a pharmaceutical composition containing at least one functionalized hyaluronic acid or derivative thereof, as described above, and hydroxyapatite, tricalcium phosphate, or mixtures thereof. These compositions are successfully used in the field of orthopedics in relation to the skeletal system.

The pharmaceutical composition according to the invention can be administered orally, intramuscularly, intravenously, intra-articularly, transdermally, subcutaneously, or topically or orally, for example by surgical means.

Preferably, said pharmaceutical composition is administered intra-articularly, transdermally or topically.

Said pharmaceutical composition may further contain pharmaceutically acceptable excipients.

Suitable pharmaceutically acceptable excipients include, for example, pH adjusters, isotonicity adjusters, diluents, stabilizers, chelating agents, diluents, binders, disintegrants, lubricants, glidants, colorants, suspending agents, surfactants, cryoprotectants, preservatives, and antioxidants.

The present invention also relates to a biomaterial containing said functionalized hyaluronic acid or its derivative as described above, alone or in combination with at least one of the pharmacologically active and/or biologically active substances described above. Said biomaterial may be in the form of microspheres, nanospheres, membranes, sponges, wires, films, needles, guides, swabs, gels, hydrogels, tissues, nonwovens, cannulas, or combinations thereof.

It should be understood that all aspects designated as preferred and advantageous in relation to the specified hyaluronic acid or its derivative, should be considered as equally preferred and advantageous in relation to the method, compositions, biomaterials and applications described above.

It should also be understood that all possible combinations of the preferred aspects of the functionalized hyaluronic acid or derivative thereof, method of preparation, compositions, biomaterial and uses set forth above are equally preferred.

Below for illustrative purposes are examples of implementation of the present invention.

EXAMPLES

Example 1 Synthesis of primary amine derivatives of reducing sugars

(4-O-β-D-galactopyranosyl-1-amino-1-deoxy-D-glucitol hydrochloride)

A solution of lactose (6.25% w/v) and ammonium acetate (56% w/v) in methanol was treated at room temperature with stirring with an amount of 5-ethyl-2-methylpyridineborane complex equimolar to lactose. The resulting mixture was kept under the same conditions for 16 hours, after which the crude reaction product was mixed with an equal volume of isopropanol, and then acidified to pH 2-3 with 6N hydrochloric acid, resulting in the precipitation of the hydrochloride salt of the amine derivative of lactose. This precipitate was then isolated and washed with ethanol:water (9:1, 3x) and ethanol:6N sodium hydroxide (95:5) until pH 9, and then again with ethanol:water (9:1, 2x) and finally ethanol (1x). The solid thus obtained was dried under reduced pressure and used in the next synthetic steps without further purification. Said derivative was analyzed by IR spectroscopy. Reaction yield: 90%.

Example 2 Synthesis of benzylamine derivatives of reducing sugars

A solution of lactose (3% w/v), benzylamine (5% w/v) and 5-ethyl-2-methylpyridineborane (6% w/v) in water and methanol (3:1) was stirred at temperature of 55°C and left for the reaction to proceed for 20 hours. This mixture was then cooled, extracted with dichloromethane, and finally the aqueous phase was evaporated at low pressure to give a white crystalline solid, which was then washed with diethyl ether, after which it was isolated by decantation and dried under reduced pressure. The product was analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 90%.

Example 3 Synthesis of primary amine derivatives of reducing sugars

A solution of the derivative obtained according to Example 2 (4% w/v) in methanol and water (1:1) was stirred with a magnetic stirrer at room temperature. Pd on carbon (0.4% w/v) was then added and the system thus obtained was pressurized with hydrogen. After 48 hours the system was depressurized and an equal volume of water was added to the system and the decanted solid and solution filtered through celite. The solution thus obtained was then dried under reduced pressure to give a white solid. The resulting product was analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 96%.

Example 4 Synthesis of an acylating solution based on lactobionic acid imidazolamide

A solution of lactobionic acid (10% w/v) in dimethyl sulfoxide was mixed with 1,1-carbonyldiimidazole (1 eq) and stirred at room temperature for 2 hours. The resulting solution was used further without further purification.

Example 5 Synthesis of partially deacetylated sodium hyaluronate (48 hours)

A solution of hydrazine sulfate (1% w/v) in hydrazine monohydrate was mixed with sodium hyaluronate (2% w/v) and the system thus obtained was heated to 55° C. and left under stirring to react for 48 hours. Then the crude reaction product was cooled, precipitated with ethanol, isolated and washed with ethanol, and then dried for 24 hours under reduced pressure. After that, the resulting product (5% w/v) was dissolved in an aqueous solution of acetic acid (5% v/v), the solution was cooled to 4°C, and an aqueous solution of HIO ₃ (0.5 M, 60% vol. / vol.). The mixture was kept under the same conditions for 1 hour for the reaction to proceed, and then a solution of hydroiodic acid (57% w/v, 11% v/v in relation to the solution) was added, and the system was left for another 15 minutes for the course of the reaction. The solution was then extracted with ethyl ether until colorless, the pH of the aqueous phase was adjusted to 7-7.5 with NaOH (1N, 0.1N) and finally the product was precipitated with ethanol, washed with ethanol and dried. The product was analyzed by ¹ H-NMR and IR spectroscopy. Reaction yield: 83%, degree of deacetylation: 11%.

Example 6 Synthesis of partially deacetylated sodium hyaluronate (72 hours)

A solution of sodium hyaluronate (2% w/v) and hydrazine sulfate (1% w/v) in hydrazine hydrate was stirred with a magnetic stirrer at 55° C. for 72 hours. At the end of the reaction time, ethanol was added to precipitate the polymer, and the resulting solid was washed with more ethanol and dried under nitrogen flow. The resulting product was dissolved in an aqueous solution of acetic acid (6% w/v, 5% acetic acid), thermostated at 0-5°C and mixed with 0.8 eq. by volume of a solution of iodic acid in water (7.5% w/v). The resulting system was kept under stirring for 1 hour, then mixed with 0.11 eq. by volume of aqueous hydroiodic acid (57%) and left for another 15 minutes for the reaction to proceed. The pH was then adjusted to 9 with 1M NaOH aqueous solution and the solution was extracted with ethyl ether until colorless. After that, the product was precipitated with ethanol, washed with ethanol, dried under reduced pressure and analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 86%, degree of deacetylation: 20%.

Example 7 Synthesis of partially deacetylated sodium hyaluronate (96 hours)

A solution of hydrazine sulfate (1% w/v) in hydrazine monohydrate was mixed with sodium hyaluronate (2% w/v) and the system thus obtained was heated to 55° C. and left under stirring to react for 96 hours. Then the crude reaction product was cooled, precipitated with ethanol, isolated and washed with ethanol, and then dried for 24 hours under reduced pressure. After that, the resulting product (5% w/v) was dissolved in an aqueous solution of acetic acid (5% v/v), the solution was cooled to 4°C, and an aqueous solution of HIO ₃ (0.5 M, 60% vol. / vol.). The mixture was kept under the same conditions for 1 hour for the reaction to proceed, and then a solution of hydroiodic acid (57% w/v, 11% v/v in relation to the solution) was added, and the system was left for another 15 minutes for the course of the reaction. The solution was then extracted with ethyl ether until colorless, the pH of the aqueous phase was adjusted to 7-7.5 with NaOH (1N, 0.1N) and finally the product was precipitated with ethanol, washed with ethanol and dried. The product was analyzed by ¹ H-NMR and IR spectroscopy. Reaction yield: 86%, degree of deacetylation: 21%.

Example 8 Synthesis of partially deacetylated sodium hyaluronate (120 h)

A solution of hydrazine sulfate (1% w/v) in hydrazine monohydrate was mixed with sodium hyaluronate (2% w/v) and the system thus obtained was heated to 55° C. and left under stirring to react for 120 hours. Then the crude reaction product was cooled, precipitated with ethanol, isolated and washed with ethanol, and then dried for 24 hours under reduced pressure. After that, the resulting product (5% w/v) was dissolved in an aqueous solution of acetic acid (5% v/v), the solution was cooled to 4°C, and an aqueous solution of HIO ₃ (0.5 M, 60% vol. / vol.). The mixture was kept under the same conditions for 1 hour for the reaction to proceed, and then a solution of hydroiodic acid (57% w/v, 11% v/v in relation to the solution) was added, and the system was left for another 15 minutes for the course of the reaction. The solution was then extracted with ethyl ether until colorless, the pH of the aqueous phase was adjusted to 7-7.5 with NaOH (1N, 0.1N) and finally the product was precipitated with ethanol, washed with ethanol and dried. The product was analyzed by ¹ H-NMR and IR spectroscopy. Reaction yield: 89%, degree of deacetylation: 26%.

Example 9 Synthesis of partially deacetylated sodium hyaluronate (24 hours)

A solution of sodium hyaluronate (2% w/v) and ammonium iodide (0.7% w/v) in hydrazine hydrate was stirred with a magnetic stirrer at 60°C for 24 hours. At the end of the reaction time, ethanol was added to precipitate the polymer, and the resulting solid was washed with ethanol and dried under nitrogen flow. The resulting product was dissolved in an aqueous solution of acetic acid (6% w/v, 5% acetic acid), thermostated at 0-5°C and mixed with 0.8 eq. by volume of a solution of iodic acid in water (7.5% w/v). The resulting system was kept under stirring for 1 hour, then mixed with 0.11 eq. by volume of aqueous hydroiodic acid (57%) and left for another 15 minutes for the reaction to proceed. The pH was then adjusted to 9 with 1M NaOH aqueous solution and the solution was extracted with ethyl ether until colorless. Thereafter, the product was precipitated with ethanol, washed with ethanol and dried under reduced pressure. The solid thus obtained was analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 88%, degree of deacetylation: 15%.

Example 10 Preparation of Tetrabutylammonium Salt of Hyaluronic Acid

An aqueous solution of sodium hyaluronate (1.6% w/v) was passed through a column packed with sulfosmol in the form of tetrabutylammonium salt (50% v/v with respect to the solution), previously activated with a solution of tetrabutylammonium (40% w/v .). The eluted solution was then lyophilized.

Example 11 Preparation of Partially Deacetylated Hyaluronic Acid Tetrabutylammonium Salt

An aqueous solution of partially deacetylated sodium hyaluronate (1.6% w/v) was passed through a column filled with sulfosmol in the form of tetrabutylammonium salt (50% v/v with respect to the solution), previously activated with a solution of tetrabutylammonium (40% wt. /about.). The eluted solution was then lyophilized.

Example 12 Hyaluronic acid amide derivatives (amidation using amine derivatives of reducing sugars)

A solution of sodium hyaluronate (0.25% w/v) in water was mixed with the amine derivative prepared in Example 1 (30 eq) and the resulting solution was adjusted to pH 6.8 with the appropriate amount of sodium hydroxide (1N, 0. 1N) or hydrochloric acid (1N, 0.1N). Then, a solution of (3-dimethylaminopropyl)-N"-ethylcarbodiimide hydrochloride (5 eq., 11% w/v) and hydroxybenzotriazole (3.5 eq., 6% w/v) previously solubilized in mixtures of water:dimethyl sulfoxide (1.1:1). The pH of the resulting solution was adjusted to 6.8 with an appropriate amount of sodium hydroxide (1N, 0.1N) and the resulting crude product was left at room temperature to react for 16 hours. The pH was then adjusted to 7 with sodium hydroxide/hydrochloric acid (0.1N) and the resulting solution repeatedly dialyzed with water (cutoff values: 12-14000). After that, sodium chloride was added to the solution until a titer of 5% w/v was reached, and the target product was precipitated with ethanol, dried and analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 88%, amidation using a reducing sugar amine derivative: 88%.

Example 13 Hyaluronic acid amide derivatives (amidation using amine derivatives of reducing sugars)

Aqueous solution containing sodium hyaluronate (3% w/v), hydroxybenzotriazole (0.4% w/v), N-ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.6% w/v) vol.) and lactose amine derivative obtained according to Example 3 (2% wt./vol.), kept under stirring for 22 hours, maintaining the pH equal to 6.8 by adding aqueous solutions of 0.1 M NaOH or 0.1 M HCl. NaCl (5 g/100 ml) was then added and the product precipitated with methanol. The solid thus obtained was isolated by decantation, washed with methanol and water (4:1), pure methanol and finally dried under reduced pressure. The product was analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 86%, amidation using a reducing sugar amine derivative: 27%.

Example 14 Hyaluronic acid amide derivatives (amidation using amine derivatives of reducing sugars)

A solution of water and dioxane (1:1) containing sodium hyaluronate (0.5% w/v), N-hydroxysuccinimide (1.3% w/v), N-ethyl-N-(3- dimethylaminopropyl)carbodiimide (1.0% w/v) and the lactose amine derivative prepared according to Example 3 (2.1% w/v) were stirred at room temperature for 12 hours. After the reaction time was added sodium bicarbonate, brought the pH to about 9-10 and left the solution under stirring for another 3 hours. The resulting mixture was adjusted to pH 7 by adding acetic acid (50%, v/v), then sodium chloride (5 g/100 ml) was added and the product was precipitated with ethanol, washed with ethanol and finally dried under reduced pressure. The product was analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 85%, amidation using an amine reducing sugar: 21%.

Example 15 Amido derivatives of hyaluronic acid (amidation using amine derivatives of reducing sugars) in an organic medium

A solution of tetrabutylammonium salt of hyaluronic acid (2% w/v) in dimethyl sulfoxide was treated with aqueous hydrochloric acid to bring the pH to 3, and then 1,1-carbonyldiimidazole (1.5 eq) was added and allowed to react for 12 hours . The solution was then filtered through a Gooch crucible to remove solids, the amine derivative prepared in Example 1 (2 eq.) was added, and the resulting mixture was left to react for 48 hours. After that, a saturated solution of sodium chloride was added in an amount sufficient to obtain a final titer of 5% wt./about. by sodium chloride. The mixture was stirred for 1 hour and finally precipitated the product by adding acetone. The resulting solid was isolated and dried. The product was analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 80%, amidation using an amine reducing sugar: 10%.

Example 16 Amido derivatives of hyaluronic acid (amidation using amine derivatives of reducing sugars) in an organic medium

A solution of sodium hyaluronate (2% w/v) in dimethylformamide was mixed with 1,1-carbonyldiimidazole (1 eq.). The resulting solution was allowed to react for 6 hours, then the amine derivative obtained in Example 1 (5 eq.) was added and the system was left to react for another 36 hours. The product was then precipitated with acetone, isolated, washed with acetone and dried under reduced pressure. The product was analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 80%, amidation using a reducing sugar amine derivative: 57%.

Example 17 Amino derivatives of partially deacetylated hyaluronic acid (reductive amination using reducing sugars)

Lactose (10 eq.) was added to an aqueous solution of partially deacetylated sodium hyaluronate prepared according to Example 7 (1.5 w/v) and the pH was adjusted to about 5.5 with acetic acid (100%). The system thus obtained was heated to 60°C, a solution of 2-methylpyridineborane (10 eq., 10% w/v) in methanol was added and left under the same conditions for 2 hours for the reaction to proceed. The pH of the solution was then adjusted to about 2-3 with aqueous hydrochloric acid (4N) and the system was kept under these conditions for 15 minutes. The system was then cooled, adjusted to pH 7-7.5 with NaOH (1N) and the resulting solution repeatedly dialyzed with water (cutoff values: 12-14000). Finally, sodium chloride was added to the solution until a titer of 5% w/v was reached, and the desired product was precipitated with ethanol, dried and analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 80%, amination with reducing sugar: 21%.

Example 18 Amino Derivatives of Partially Deacetylated Hyaluronic Acid (Reductive Amination Using Reducing Sugars)

Lactose (10 eq.) was added to an aqueous solution of partially deacetylated sodium hyaluronate prepared according to Example 7 (1.5 w/v) and the pH was adjusted to about 5.5 with acetic acid (100%). The system thus obtained was heated to 60°C, a solution of 2-methylpyridineborane (10 eq., 10% w/v) in methanol was added and left under the same conditions for 2 hours for the reaction to proceed. The pH of the solution was then adjusted to about 2-3 with aqueous hydrochloric acid (4N) and the system was kept under these conditions for 15 minutes. After that, the system was cooled, the pH was adjusted to 7-7.5 with NaOH (1N) and sodium chloride was added to achieve a titer of 5% wt./about. The target product was precipitated with ethanol, dried, and analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 84%, amination with reducing sugar: 21%.

Example 19 Amino derivatives of partially deacetylated hyaluronic acid (reductive amination using reducing sugars)

Lactose (10 eq.) was added to an aqueous solution of partially deacetylated sodium hyaluronate prepared according to Example 5 (1.5 w/v) and the pH was adjusted to about 5.5 with acetic acid (100%). The system thus obtained was heated to 60°C, a solution of 2-methylpyridineborane (10 eq., 10% w/v) in methanol was added and left under the same conditions for 2 hours for the reaction to proceed. The pH of the solution was then adjusted to about 2-3 with aqueous hydrochloric acid (4N) and the system was kept under these conditions for 15 minutes. The system was then cooled, adjusted to pH 7-7.5 with NaOH (1N) and the resulting solution repeatedly dialyzed with water (cutoff values: 12-14000). Finally, sodium chloride was added to the solution until a titer of 5% w/v was reached, and the desired product was precipitated with ethanol, dried and analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 78%, amination with reducing sugar: 11%.

Example 20 Amino derivatives of partially deacetylated hyaluronic acid (reductive amination using reducing sugars)

To an aqueous solution of partially deacetylated sodium hyaluronate prepared according to Example 6 (2 w/v) was added lactose (3 eq) and adjusted the pH to about 5.5 with acetic acid (100%). The system thus obtained was heated to 60°C, a solution of 2-methylpyridineborane (1 eq., 10% w/v) in isopropanol was added and left under the same conditions for 3 hours for the reaction to proceed. The pH of the reaction mixture was then adjusted to about 2-3 with aqueous hydrochloric acid (4N) and the system was kept under these conditions for 15 minutes. The system was then cooled and the product precipitated by adding isopropanol, washed with isopropanol:water (80:20 and 90:10) and dried under reduced pressure. The product was analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 95%, amination with reducing sugar: 20%.

Example 21 Amino derivatives of partially deacetylated hyaluronic acid (reductive amination using reducing sugars)

To an aqueous solution of partially deacetylated sodium hyaluronate prepared according to Example 9 (2 w/v) was added lactose (3 eq) and adjusted the pH to about 5.5 with acetic acid (100%). The system thus obtained was heated to 60°C, a solution of 2-methylpyridineborane (1 eq., 10% w/v) in isopropanol was added and left under the same conditions for 3 hours for the reaction to proceed. The pH of the reaction mixture was then adjusted to about 2-3 with aqueous hydrochloric acid (4N) and the system was kept under these conditions for 15 minutes. The system was then cooled and the product precipitated by adding isopropanol, washed with isopropanol:water (80:20 and 90:10) and dried under reduced pressure. The product was analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 95%, amination with reducing sugar: 15%.

Example 22 Amido derivatives of the compounds obtained according to Examples 17-21 (amidation of derivatives obtained by reductive amination of hyaluronic acid with reducing sugars)

A solution of the hyaluronic acid amine derivative prepared according to Example 17 (0.25% w/v) in water was mixed with the amine derivative obtained in Example 1 (30 eq.) and the pH of the resulting solution was adjusted to 6.8 with the appropriate amount of sodium hydroxide (1N, 0.1N) or hydrochloric acid (1N, 0.1N). Then, a solution of (3-dimethylaminopropyl)-N"-ethylcarbodiimide hydrochloride (5 eq., 11% w/v) and hydroxybenzotriazole (3.5 eq., 6% w/v) previously solubilized in mixtures of water:dimethyl sulfoxide (1.1:1). The pH of the resulting solution was adjusted to 6.8 with an appropriate amount of sodium hydroxide (1N, 0.1N) and the resulting crude product was left at room temperature to react for 16 hours. The pH was then adjusted to 7 with sodium hydroxide/hydrochloric acid (0.1N) and the resulting solution repeatedly dialyzed with water (cutoff values: 12-14000). After that, sodium chloride was added to the solution until a titer of 5% w/v was reached, and the target product was precipitated with ethanol, dried and analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 90%, amidation using an amine reducing sugar: 90%.

Example 23 Amido derivatives of partially deacetylated hyaluronic acid (acylation with carboxyl derivatives of reducing sugars)

To a solution of deacetylated sodium hyaluronate prepared according to Example 7 (0.30% w/v) in water was added lactobionic acid (30 eq.) and the resulting solution was adjusted to pH 6.8 with an appropriate amount of sodium hydroxide (1N, 0.1N) or hydrochloric acid (1N, 0.1N). Then, a solution of (3-dimethylaminopropyl)-N''-ethylcarbodiimide hydrochloride (5 eq., 11% w/v) and hydroxybenzotriazole (3.5 eq., 6% w/v), previously solubilized, was added dropwise. in a mixture of water: dimethyl sulfoxide (1.1: 1). The pH of the resulting solution was adjusted to 6.8 with an appropriate amount of sodium hydroxide (1N, 0.1N) and the resulting crude product was left at room temperature to react for 16 hours. The pH was then adjusted to 7 with sodium hydroxide/hydrochloric acid (0.1N) and the resulting solution repeatedly dialyzed with water (cutoff values: 12-14000). After that, sodium chloride was added to the solution until a titer of 5% w/v was reached, and the target product was precipitated with ethanol, dried and analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 79%, acylation with lactobionic acid: 5%.

Example 24 Amido derivatives of partially deacetylated hyaluronic acid (acylation with carboxyl derivatives of reducing sugars)

To a solution of deacetylated sodium hyaluronate prepared according to Example 7 (0.5 eq., 0.30% w/v) in water was added a solution of lactobionic acid prepared according to Example 4 and the resulting crude product was left at room temperature for 16 hours for the reaction to proceed. The pH was then adjusted to 7 with sodium hydroxide/hydrochloric acid (0.1N) and the resulting solution repeatedly dialyzed with water (cutoff values: 12-14000). After that, sodium chloride was added to the solution until a titer of 5% w/v was reached, and the target product was precipitated with ethanol, dried and analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 87%, acylation with lactobionic acid: 16%.

Example 25 Amido derivatives of partially deacetylated hyaluronic acid (acylation with carboxyl derivatives of reducing sugars)

To a solution of deacetylated sodium hyaluronate prepared according to Example 7 (0.5 eq., 30% w/v) in water was added a solution of lactobionic acid prepared according to Example 4, and the resulting crude product was left at room temperature for 16 hours for the course of the reaction. Then was added a saturated solution of sodium chloride in an amount sufficient to obtain a final titer of 5% wt./about. by sodium chloride. The mixture was stirred for 1 hour and finally precipitated the product by adding acetone. The resulting solid was isolated and dried. The product was analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 85%, acylation with lactobionic acid: 16%.

Example 26. Amido derivatives of partially deacetylated hyaluronic acid (acylation with carboxyl derivatives of reducing sugars) in an organic medium

To a solution of deacetylated tetrabutylammonium hyaluronate obtained according to Example 11 (0.5 eq., 2% w/v) in water was added a solution of lactobionic acid obtained according to Example 4, and the resulting crude product was left at room temperature for 16 hours for the course of the reaction. Then was added a saturated solution of sodium chloride in an amount sufficient to obtain a final titer of 5% wt./about. by sodium chloride. The mixture was stirred for 1 hour and finally precipitated the product by adding acetone. The resulting solid was isolated and dried. The product was analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 80%, acylation with lactobionic acid: 10%.

Example 27 Amido derivatives of partially deacetylated hyaluronic acid (acylation with carboxyl derivatives of reducing sugars) in an organic medium

To a solution of deacetylated sodium hyaluronate prepared according to Example 7 (0.5 eq., 2% w/v) in dimethylformamide was added a solution of lactobionic acid prepared according to Example 4, and the resulting crude product was left at room temperature for 16 hours for the course of the reaction. Then was added a saturated solution of sodium chloride in an amount sufficient to obtain a final titer of 5% wt./about. by sodium chloride. The mixture was stirred for 1 hour and finally precipitated the product by adding acetone. The resulting solid was isolated and dried. The product was analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 88%, acylation with lactobionic acid: 19%.

Example 28 Amido derivatives of the compounds obtained according to Examples 23-27 (amidation of derivatives obtained by acylation of hyaluronic acid with amino derivatives of reducing sugars)

A solution of the hyaluronic acid amide derivative prepared according to Example 24 (0.25% w/v) in water was mixed with the amine derivative obtained in Example 1 (30 eq.) and the pH of the resulting solution was adjusted to 6.8 with the appropriate amount of sodium hydroxide (1N, 0.1N) or hydrochloric acid (1N, 0.1N). Then, a solution of (3-dimethylaminopropyl)-N"-ethylcarbodiimide hydrochloride (5 eq., 11% w/v) and hydroxybenzotriazole (3.5 eq., 6% w/v) previously solubilized in mixtures of water:dimethyl sulfoxide (1.1:1). The pH of the resulting solution was adjusted to 6.8 with an appropriate amount of sodium hydroxide (1N, 0.1N) and the resulting crude product was left at room temperature to react for 16 hours. The pH was then adjusted to 7 with sodium hydroxide/hydrochloric acid (0.1N) and the resulting solution repeatedly dialyzed with water (cutoff values: 12-14000). After that, sodium chloride was added to the solution until a titer of 5% w/v was reached, and the target product was precipitated with ethanol, dried and analyzed by IR and ¹ H-NMR spectroscopy. Reaction yield: 84%, amidation using a reducing sugar amine derivative: 93%.

Example 29 Decreased Markers of Inflammation

NIH-3T3 mouse fibroblasts were grown in Dulbecco's Modified Eagle's Medium (DMEM) in the presence of 10% fetal bovine serum PBS and treated with IL1β 1 ng/ml for 24 hours. Then, some of these cultures were incubated with the hyaluronic acid amide derivative prepared according to Example 13 at a concentration of 1.25 mg/ml. Cellular RNA was isolated 6, 12, and 24 hours after treatment for subsequent analysis of the expression of the pro-inflammatory cytokine TGF-β1 by qPCR. This assay was performed using the RotorGene Q series, which allows for the quantification of gene expression during the amplification reaction. The qPCR reaction mixture contained a fluorescent molecule that binds to the minor groove in the double-stranded DNA molecule. A negative control sample (reaction mixture without cDNA) was run for each reaction. Experiments were performed in duplicate, and statistical analysis was performed using a two-tailed Student's t-test. At a Student's t-test <0.05, the discrepancies were considered significant. The value shown in the table corresponds to 2^-ΔΔCt obtained by comparative quantification according to the 2^-ΔΔCt method of Pfaffl (Pfaffl M.V. Nucleic Acid Research 2001, 29 (9): e45):

ΔCt = Housekeeping gene Ct - Target gene Ct

ΔΔCt = Δ∆Ct sample - ΔCt control

2^-ΔΔCt

Time watch) Raw control Control treated with IL1β Treated with IL1β and hyaluronic acid amide TGF-β1 TGF-β1 TGF-β1 6 1,000 2.101 1.289 12 1,000 1.112 0.946 24 1,000 0.778 0.462

Example 29 demonstrates the ability to regulate galectin-mediated pathological processes, where the hyaluronic acid derivatives prepared according to the present invention show a significant ability to reduce the expression of TGF-β1, a cytokine downstream of the inflammatory cascade in galectin-3-mediated processes such as fibrosis.

Claims (52)

1. Functionalized hyaluronic acid or its derivative of formula (I)

(I) Where R ₁ , R ₂ , R ₃ , R ₄ are independently H, SO ₃ ^- , an aliphatic, aromatic, arylaliphatic, cycloaliphatic or heterocyclic carboxylic acid acyl group, -CO-(CH ₂ ) ₂ -COOY, where Y is negative charge or H, And R is Z(1) or Z(2), and R ₅ is -CO-CH ₃ , H, SO ₃ ^- , an aliphatic, aromatic, arylaliphatic, cycloaliphatic or heterocyclic carboxylic acid acyl group, a hyaluronic acid acyl group, where Z(1) is a fragment of formula (1):

(1), where Z ₁ is -NR ₆ CH ₂ - and R ₆ is H or a substituted or unsubstituted aliphatic, aromatic, arylaliphatic, cycloaliphatic or heterocyclic group, Z ₂ is -OH or -NHCOCH ₃ , Z ₃ is H, a monosaccharide, disaccharide or oligosaccharide, or Z(2) is a fragment of formula (2):

(2) where Z ₄ is -NR ₆ CH- and R ₆ is H or a substituted or unsubstituted aliphatic, aromatic, arylaliphatic, cycloaliphatic or heterocyclic group, Z ₅ and Z ₆ are independently H, a monosaccharide, a disaccharide or an oligosaccharide, or R ₅ is Z(3) or Z(4), and R is NR ₆ R ₇ , or an aliphatic, aromatic, arylaliphatic, cycloaliphatic or heterocyclic alcohol group, OH, O ^- , an alcohol group of hyaluronic acid, an amino group of hyaluronic acid , and R ₆ , R ₇ independently represent H or a substituted or unsubstituted aliphatic, aromatic, arylaliphatic, cycloaliphatic or heterocyclic group, where Z(3) is a fragment of formula (3):

(3) where Z ₁ represents -CH ₂ - or -CO-, Z ₂ is -OH or -NHCOCH ₃ , Z ₃ is H, a monosaccharide, disaccharide or oligosaccharide, or Z(4) is a fragment of formula (4):

(4), where Z ₄ represents -CH-, Z ₅ and Z ₆ are independently H, a monosaccharide, a disaccharide or an oligosaccharide, or R is Z(1) or Z(2) and R ₅ is Z(3) or Z(4). 2. Functionalized hyaluronic acid or its derivative according to claim 1, in which Z ₃ , Z ₅ and Z ₆ independently represent H, glucose, galactose, arabinose, xylose, mannose, lactose, trehalose, gentiobiose, cellobiose, cellotriose, maltose, maltotriose, chitobiose, chitotriose, mannobiose, melibiose, fructose, N-acetylglucosamine, N-acetylgalactosamine moiety or a combination thereof. 3. The functionalized hyaluronic acid or derivative thereof according to claim 1 or 2, wherein Z ₃ is H, glucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine, or a combination thereof. 4. Functionalized hyaluronic acid or its derivative according to any one of paragraphs. 1-3, in which any of Z(1), Z(2), Z(3) and Z(4) is a lactose or galactose moiety. 5. A method for producing a functionalized hyaluronic acid or its derivative according to claim 1, including the steps: i) providing hyaluronic acid or a partially or fully deacetylated derivative thereof; ii) interactions: a) said hyaluronic acid from step (i) with an amine derivative of a monosaccharide, disaccharide or oligosaccharide obtained by a reductive amination reaction in the presence of carbodiimides and/or carboxyl group activators, or b) said partially or fully deacetylated derivative from step (i) with a monosaccharide, disaccharide or oligosaccharide in the presence of an aminoborane; or c) a partially or completely deacetylated derivative from step (i) with a carboxy derivative of a monosaccharide, disaccharide or oligosaccharide in the presence of carbodiimides and/or carboxyl group activators; or d) said derivative from step (ii)-(b) with an amino derivative of a monosaccharide, disaccharide or oligosaccharide obtained by a reductive amination reaction in the presence of carbodiimides and/or carboxyl group activators; or e) said derivative from step (ii)-(c) with an amino derivative of a monosaccharide, disaccharide or oligosaccharide obtained by a reductive amination reaction in the presence of carbodiimides and/or carboxyl group activators; And iii) precipitating the resulting functionalized hyaluronic acid or its derivative with an organic solvent. 6. The method according to claim 5, wherein the molar ratio of monosaccharide, disaccharide or oligosaccharide from step (ii) to hyaluronic acid or its derivative is from 0.5 to 30, preferably from 1 to 20, more preferably from 1 to 10. 7. Functionalized hyaluronic acid or its derivative according to claim 1 for use in the treatment of pathologies due to changes in the expression of galectins, where these pathologies include non-alcoholic steatohepatitis, plaque psoriasis, rheumatoid arthritis, osteoarthritis, neoplasia and fibrous processes in the lungs, kidneys and cardiovascular system. 8. Functionalized hyaluronic acid or its derivative according to claim 1 for use as a biomaterial or scaffold for cell growth in the treatment of orthopedic pathologies. 9. A pharmaceutical composition containing at least one functionalized hyaluronic acid or its derivative according to claim 1 and at least one pharmacologically active substance and / or at least one biologically active substance, where: said pharmacologically active substance is selected from antibiotics, anti-infective, antimicrobial, antiviral, cytostatic, cytotoxic, antitumor or anti-inflammatory agents, wound healing agents, anesthetics, analgesics, vasoconstrictors, cholinergic or andrenergic agonists and antagonists, antithrombotic agents, anticoagulants, hemostatic, fibrinolytic or thrombolytic agents, proteins and fragments thereof, peptides, polynucleotides, growth factors, enzymes, vaccines and combinations thereof, and - said biologically active 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, copolymer polylactic and polyglycolic acids, polycaprolactone, gelatin, albumin, polyglycolide-polycaprolactone copolymer, polyglycolide-polytrimethylene carbonate copolymer, hydroxyapatite, tricalcium phosphate, dicalcium phosphate, demineralized bone matrix, and mixtures thereof. 10. Pharmaceutical composition according to claim 9 for use in the treatment of pathologies due to changes in the expression of galectins, where these pathologies include non-alcoholic steatohepatitis, plaque psoriasis, rheumatoid arthritis, osteoarthritis, neoplasia and fibrotic processes in the lungs, kidneys and cardiovascular system.

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