WO2024057154A1 - Procédé de conjugaison d'acide hyaluronique et conjugués d'acide hyaluronique ainsi obtenus - Google Patents

Procédé de conjugaison d'acide hyaluronique et conjugués d'acide hyaluronique ainsi obtenus Download PDF

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WO2024057154A1
WO2024057154A1 PCT/IB2023/058903 IB2023058903W WO2024057154A1 WO 2024057154 A1 WO2024057154 A1 WO 2024057154A1 IB 2023058903 W IB2023058903 W IB 2023058903W WO 2024057154 A1 WO2024057154 A1 WO 2024057154A1
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conjugate
hyaluronic acid
pharmaceutically acceptable
water
acceptable salt
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PCT/IB2023/058903
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English (en)
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Giulio Bianchini
Emiliano Esposito
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Glycocore Pharma S.R.L.
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates

Definitions

  • the present invention relates to a process for conjugating hyaluronic acid by using a specific condensing agent and under certain operating conditions which allow to obtain derivatives of hyaluronic acid with disaccharides or oligosaccharides having the desired degree of conjugation in an efficient, fast, and economical way.
  • the present invention therefore also relates to hyaluronic acid conjugates having specific degrees of conjugation and uses thereof.
  • Hyaluronic acid or HA' for brevity, is a non-sulfated linear glycosaminoglycan (GAG) consisting of repetitive disaccharide units consisting of a glucuronic acid residue and an N- Acetylglucosamine residue, linked together alternatively by 1— >4 and 01— >3 glycosidic bonds.
  • GAG non-sulfated linear glycosaminoglycan
  • the carboxyl groups of the glucuronic units are ionised, giving HA high polarity and consequently good solubility in water.
  • the molecular weight of HA can vary between 10 3 and 10 7 Da, and this parameter affects the properties thereof, thus making different applications possible.
  • HA is present naturally in both vertebrate organisms and bacteria. Its presence is mainly in the extracellular matrix (ECM) of epithelial cells and connective tissues.
  • ECM extracellular matrix
  • the biological properties of hyaluronic acid are attributable to the chemical and physical characteristics thereof, such as viscosity and high water retention capacity, which allow said hyaluronic acid to perform a structural and hydrating role.
  • Studies of hyaluronic acid concern its role as a drug carrier through appropriate chemical functionalisations.
  • the cellular internalisation of hyaluronic acid by means of CD44 receptors during enzymatic degradation of said hyaluronic acid determines the intracellular release of the drug obtained through conjugation with HA or entrapment in HA particles.
  • the possibility of functionalising hyaluronic acid with modified lactose is particularly interesting given the biological significance in terms of interaction with galectins.
  • galectins are galactose-specific receptors, so the addition of this residue to the HA chain allows the polymer to interact not only with the CD44 receptors but also with galectins, producing a desired synergistic effect.
  • the present invention concerns a conjugate of hyaluronic acid, or a pharmaceutically acceptable salt thereof, and a disaccharide or an oligosaccharide having an average degree of substitution in the carboxyl group of 10-50%, more preferably 15-45%.
  • the present invention relates to the use of said conjugate in the treatment of pathologies ascribable to altered galectin expression.
  • pathologies are non-alcoholic steatohepatitis, plaque psoriasis, rheumatoid arthritis, osteoarthritis, neoplasia, adhesions, and fibrotic dermal, pulmonary, renal, and cardiovascular processes.
  • the present invention relates to the use of such conjugate in rheumatology, orthopaedics, oncology, plastic/cosmetic surgery, haemodialysis, cardiology, angiology, ophthalmology, otolaryngology, pneumology, odontology, gynaecology, urology, dermatology, oncology, and tissue repair.
  • the invention therefore relates to 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 at least 15 minutes and adding a primary amine of a disaccharide or oligosaccharide, under stirring, to form a conjugate of hyaluronic acid, or a pharmaceutically acceptable salt thereof, with said disaccharide or oligosaccharide, via amide bond, iv) promoting precipitation of the conjugate thus obtained, adding a water-miscible organic solvent
  • the process according to the invention allows the conjugate to be obtained, conveniently and quickly, with the desired degree of substitution and likewise with high purity.
  • step i) an aqueous solution of hyaluronic acid or a pharmaceutically acceptable salt thereof is provided.
  • said hyaluronic acid in step i) has an average molecular weight of 50-5000 kDa, more preferably 50-500 kDa.
  • hyaluronic acid in step i) 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.
  • step i) takes place at a pH of 5-7, more preferably 6-7, upon addition of an acid, preferably 2-[N-morpholino]-ethanesulfonic acid (MES).
  • an acid preferably 2-[N-morpholino]-ethanesulfonic acid (MES).
  • 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: where “HACOO”” is the carboxyl group of hyaluronic acid, or a pharmaceutically acceptable salt thereof, which binds to the triazine ring and forms the ester adduct on the right, releasing group X.
  • said triazine compound of formula (I) is 4,6-Dimethoxy-l,3,5-triazin-2-yl)-4- methylmorpholinium chloride, ('DMTMM' for brevity), 2-Chloro-4,6-dimethoxy-l,3,5- triazine ('CDMT' for brevity), or a mixture thereof.
  • said triazine compound of formula (I) is DMTMM, and in step ii), the following reaction occurs:
  • DMTMM adduct methylmorpholine where “HACOO” is the carboxyl group of hyaluronic acid, or a pharmaceutically acceptable salt thereof, which binds to the triazine ring of DMTMM and forms the ester adduct on the right, releasing methylmorpholine.
  • the hyaluronic acid or pharmaceutically acceptable salt thereof and the triazine compound of formula (I), in step ii), are in a molar ratio of 3 : 1 to 1 :3.
  • the mixture that forms after the addition of the triazine compound of formula (I), is left to react for at least 15 minutes, preferably at least 30 minutes, after which the primary amine of a disaccharide or an oligosaccharide is added, under stirring, to form a conjugate of hyaluronic acid, or a pharmaceutically acceptable salt thereof, with said disaccharide or oligosaccharide, via amide bond, as per step iii).
  • primary amine refers not only to the free primary amine -NH2, but also to pharmaceutically acceptable salts thereof.
  • oligosaccharide means a saccharide polymer comprising 3 to 6 monosaccharides, i.e. from a trisaccharide to hexasaccharide.
  • said primary amine of a disaccharide or an oligosaccharide is a primary amine derivative of sucrose, maltose, lactose, trehalose, gentiobiose, cellobiose, maltotriose, raffinose, stachyose, maltotetraose, melezitose, isomaltose, panose, nystose, 1 -ketose, galactopinitol, galactosyllactose, or a mixture thereof.
  • a primary amine of a disaccharide is added which is a primary amine derivative of sucrose, maltose, lactose, trehalose, gentiobiose, cellobiose or a mixture thereof, more preferably a primary amine derivative of lactose.
  • the hyaluronic acid or pharmaceutically acceptable salt thereof and said primary amine, in step iii), are in a molar ratio of 3: 1 to 1:3.
  • the primary amine and the adduct are left to react at room temperature for at least 12 hours, preferably at least 24 hours, more preferably at least 48 hours.
  • step iii) after the at least 12 hours left for the reaction, a base is subsequently added, preferably NaOH 5M.
  • This basification step breaks the ester bonds that form - through secondary reactions of the activated carboxylic acid of HA - with the hydroxyl groups thereof or with lactosamine.
  • step iv) the precipitation of the conjugate thus obtained is promoted by adding an acid, preferably HC1 5M, and a water-miscible organic solvent, and obtaining a crude conjugate and possibly washing the precipitated conjugate with further organic solvent possibly mixed with water.
  • an acid preferably HC1 5M
  • a water-miscible organic solvent preferably a water-miscible organic solvent
  • the organic solvent serves to reduce the polarity of the water in order to increase the conjugate precipitation speed and yield.
  • said water-miscible organic solvent is acetone, acetonitrile, 1-4 dioxane, tetrahydrofuran or an alcohol, preferably with a number of carbon atoms of 1 to 5, more preferably selected from methanol, ethanol, 2-propanol, or a mixture thereof.
  • step v) the crude conjugate is separated from the solvent mixture.
  • this separation can be carried out by techniques such as decantation, filtration, evaporation, or centrifugation, or a combination thereof.
  • the crude conjugate thus separated undergoes a further purification procedure, which can be carried out by reprecipitation, ion-exchange resin treatment, or dialysis, to obtain a more purified conjugate.
  • the three purification methods can also be carried out in reciprocal combination and can be repeated several times, e.g. reprecipitation, followed by ion-exchange resin treatment, followed by reprecipitation.
  • step vi) when the purification of the crude conjugate in step vi) is by ion-exchange resin treatment, the following sub-steps are carried out: vi-a) dissolving the crude conjugate in water to obtain an aqueous solution, vi-b) ion-exchange resin treating said aqueous solution, by using water as an eluting agent, vi-c) isolating the aqueous solution from the ion exchange resin, vi-d) bringing the isolated solution to pH neutral.
  • said cation exchange column is an AmberliteTM IR120 (CAS n.78922-04-0; IUPAC name: 3-[(3-chlorophenyl)sulfonylamino]benzoic acid).
  • the eluate can be brought to neutral pH by adding a salt such as NaHCOi.
  • a salt such as NaHCOi.
  • the unreacted primary amine retained in the cation exchange column can subsequently be recovered using NH3 as an eluting agent.
  • step vi) when the crude conjugate purification in step vi) is performed by dialysis, the following sub-steps are carried out: vi-i) dissolving the crude conjugate in water to obtain an aqueous solution, vi-ii) dialysing the solution obtained against aqueous saline solution and/or water through an appropriate membrane.
  • step vi) when the crude conjugate purification in step vi) is performed by reprecipitation, the following sub-steps are carried out: vi-a) dissolving the crude conjugate in water to obtain an aqueous solution, vi-
  • a saline solution is preferably furthermore added, more preferably a 0.5 M NaCl solution.
  • the action of washing the conjugate with further organic solvent can be repeated several times in order to increase the final purity of said conjugate.
  • step vi) when the crude conjugate purification in step vi) is performed by dialysis, the following sub-steps are carried out: vi-*) dissolving the crude conjugate in water to obtain an aqueous solution, vi-**) transferring to a 6-8 kDa membrane and dialysing for at least 24 hours against an NaCl saline solution, vi-***) collecting and freeze-drying the retentate.
  • the process according to the invention is conducted at a temperature of 15 -75 °C and a pH of 6-11.
  • the primary amines described above, and likewise the related pharmaceutically acceptable salts, are commercially available, or alternatively they can be prepared with commonly known methods of amination of disaccharides or oligosaccharides or even by a process comprising the following steps:
  • said primary amines can be prepared by a process comprising the following steps:
  • 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)borohydrate, potassium tri(l-pyrazolyl) borohydride, cetyltrimethylammonium borohydride, borane tetrahydrofuran complex, picoline-borane complex, 5-ethyl-2-methylpyridine-borane complex, dimethyl sulfide-borane complex, pyridine-borane complex, trimethylamine-borane complex, triethylamine-borane complex, morpholine-borane complex, t-butyl
  • catalyst preferably refers to Raney catalyst of metal groups 8-11, carbon-supported metal groups 8-11, silica, alumina, aluminosilicates, zirconia, or a mixture thereof.
  • hydrogen source refers to H2 or a compound capable of releasing H+ ions under the conditions of the process.
  • the present invention concerns a conjugate of hyaluronic acid, or a pharmaceutically acceptable salt thereof, and a disaccharide or an oligosaccharide having an average degree of substitution of the carboxyl group of 10-50%, more preferably 15-45% with the amide bond to said disaccharide or oligosaccharide, which can be obtained from the process described above, in particular when:
  • step ii) the hyaluronic acid or pharmaceutically acceptable salt thereof and the triazine compound of formula (I), in step ii), are in a molar ratio of 3 : 1 to 1 :3, and/or
  • step iii) the hyaluronic acid or pharmaceutically acceptable salt thereof and said primary amine, in step iii), are in a molar ratio of 3 : 1 to 1 :3.
  • Said conjugate can be administered by inhalation, oral, nasal, ocular, urogynaecological, intra-articular, intramuscular, intravenous, intradermal, transdermal, subcutaneous, or external or internal topical route, for example by surgical route.
  • the conjugate is administered by oral, nasal, ocular, urogynaecological, intraarticular, intradermal, or inhalation route.
  • the conjugate is in an injectable form suitable for hard or soft body tissues, such as organs, adipose tissues, mucous membranes, and gums, preferably by intradermal, subcutaneous, intramuscular, or intra-articular route.
  • the conjugate can be in the form of soft gel capsules or in solid form, such as a tablet, a minitablet, a micro-tablet, a granule, a micro-granule, pellet, multiparticulate or micronised particulate, or powder or in the form of a solution, emulsion, gel, ointment, drops, nebuliser solution, or spray.
  • the conjugate is in the form of a powder, solution, emulsion, gel, ointment, drops, nebuliser solution, spray, or injectable form, for administration by nasal or inhalation or intra-articular or intradermal or ophthalmic or urogynaecological route.
  • the present invention relates to said conjugate in the treatment of pathologies ascribable to altered galectin expression.
  • pathologies are non-alcoholic steatohepatitis, plaque psoriasis, rheumatoid arthritis, osteoarthritis, neoplasia, adhesions, and fibrotic dermal, pulmonary, renal, and cardiovascular processes.
  • neoplasia and fibrotic processes include acute lymphoblastic leukaemia, idiopathic pulmonary fibrosis, hepatic fibrosis, cardiac fibrosis, renal fibrosis, and ovarian, prostate, lung, stomach, skin, thyroid, and pancreas tumours.
  • the present invention concerns the use of said conjugate as a biomaterial or a scaffold for cell growth, in the treatment of orthopaedic diseases.
  • the present invention concerns the use of said conjugate in tissue repair or reconstruction, preferably in the creation or replacement of biological tissues or in filling biological tissues, such as filling skin, depressions, bone cartilage, or joints.
  • the present invention concerns the use of said conjugate in rheumatology, orthopaedics, oncology, plastic/cosmetic surgery, haemodialysis, cardiology, angiology, ophthalmology, otolaryngology, pneumology, odontology, gynaecology, urology, dermatology, oncology, and tissue repair; furthermore, the present invention concerns the use of said 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 the dermis and abdominal tissues, or in post-surgical sequelae of endonasal procedures, in post-surgical sequelae of tendon and/or cartilaginous tissues.
  • conjugate according to the invention in the treatment of asthma, COPD, IPF, tonsillitis, laryngitis, pharyngitis, nasopharyngitis, sinusitis, rhinitis, tracheitis, hoarseness, and inflammation of the vocal cords with or without dysphonia.
  • the conjugate can also be used in the cosmetic and dermatological field, in dermatological or cosmetic products, or as a biomedical product, preferably as a bio-absorbable implant.
  • the present invention relates to the use of such conjugate in psoriasis and psoriatic osteoarthritis.
  • the present invention concerns a composition
  • a composition comprising the conjugate described above and at least one pharmacologically active substance and/or at least one substance with an optionally biological function.
  • Suitable pharmacologically active substances include: antibiotics, anti-infectives, antimicrobials, antivirals, cytostatics, cytotoxics, anti-tumour drugs, anti-inflammatory drugs, cicatrizants, anaesthetics, analgesics, vasoconstrictors, cholinergic or adrenergic agonists and antagonists, antithrombotics, anticoagulants, haemostatics, fibrinolytics, thrombolytics, proteins and fragments thereof, peptides, polynucleotides, growth factors, enzymes, vaccines, or combinations thereof.
  • said substance with an optionally biological function is chosen from collagen, fibrinogen, fibrin, alginic acid, sodium alginate, potassium alginate, magnesium alginate, cellulose, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, eparan 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, demineralised bone matrix, and mixtures thereof.
  • composition according to the invention finds advantageous application in the same uses as those listed above for the conjugate.
  • Lactosamine As a substrate, two samples of HA with a molecular weight of 82 kDa and 300 kDa respectively, and an amine derivative of lactose, hereinafter referred to as Lactosamine (Lat- NH2), were used.
  • HCTU O-(lH-6-Chlorobenzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate
  • TEA Triethylamine
  • a-D-Lactose Triethylamine
  • the product was then isolated by precipitation with 2.5 L isopropanol at 4°C (75%) enriching the suspension with 3 mL saturated NaCl solution (approximately 1 g salt) to improve the precipitation step and subsequently recovered by centrifugation of the suspension at 15000 rpm for 10 min.
  • the white solid obtained was washed several times with isopropanol, solubilised in 250 mL deionised water.
  • the product was then isolated by precipitation with 2.5 L isopropanol at 4°C (75%) enriching the suspension with 3 mL saturated NaCl solution (approximately 1 g salt) to improve the precipitation step and subsequently recovered by centrifugation of the suspension at 15000 rpm for 10 min.
  • the white solid obtained was washed several times with isopropanol, solubilised in 250 mL deionised water.
  • the solution was diluted to 400 mL and transferred to a 6-8 kDa membrane and dialysed first for 2 days against 24 litres (12+12 1) 0.1M NaCl saline solution and subsequently for 3 days against deionised water.
  • the entire retentate (approximately 1 litre) was then concentrated under reduced pressure to approximately 1/3 of the initial volume and freeze-dried to obtain a white solid.
  • the white solid obtained after the precipitation and washing step was further purified according to the techniques described in bl and b2 of Example 3.
  • the product was then isolated by precipitation with 2.5 L isopropanol at 4°C (75%) enriching the suspension with 3 mL saturated NaCl solution (approximately 1 g salt) to improve the precipitation step and subsequently recovered by centrifugation of the suspension at 15000 rpm for 10 min.
  • the white solid obtained was washed several times with isopropanol, solubilised in 250 mL deionised water.
  • the solution was diluted to 400 mL and transferred to a 6-8 kDa membrane and dialysed first for 2 days against 24 litres (12+12 1) 0.1M NaCl saline solution and subsequently for 3 days against deionised water.
  • the entire retentate (approximately 1 litre) was then concentrated under reduced pressure to approximately 1/3 of the initial volume and freeze-dried to obtain a white solid.
  • the white solid obtained after the precipitation and washing step was further purified according to the techniques described in bl and b2 of Example 3.
  • the product was then isolated by precipitation with 2.5 L isopropanol at 4°C (75%) enriching the suspension with 3 mL saturated NaCl solution (approximately 1 g salt) to improve the precipitation step and subsequently recovered by centrifugation of the suspension at 15000 rpm for 10 min.
  • the white solid obtained was washed several times with isopropanol, solubilised in 250 mL deionised water.
  • the solution was diluted to 400 mL and transferred to a 6-8 kDa membrane and dialysed first for 2 days against 24 litres (12+12 1) 0.1M NaCl saline solution and subsequently for 3 days against deionised water.
  • the entire retentate (approximately 1 litre) was then concentrated under reduced pressure to approximately 1/3 of the initial volume and freeze-dried to obtain a white solid.
  • the white solid obtained after the precipitation and washing step was further purified according to the techniques described in bl and b2 of Example 3.
  • Example 7 a) Preparation of hyaluronic acid lactosylate 12.02 g (29.83 mmol) sodium hyaluronate was solubilised in 0.7 L deionised water, leaving the mixture under magnetic stirring for 2 h.
  • the product was then isolated by precipitation with 2.5 L isopropanol at 4°C (75%) enriching the suspension with 3 mL saturated NaCl solution (approximately 1 g salt) to improve the precipitation step and subsequently recovered by centrifugation of the suspension at 15000 rpm for 10 min.
  • the white solid obtained was washed several times with isopropanol, solubilised in 250 mL deionised water.
  • the solution was diluted to 400 mL and transferred to a 6-8 kDa membrane and dialysed first for 2 days against 24 litres (12+12 1) 0.1M NaCl saline solution and subsequently for 3 days against deionised water.
  • the entire retentate (approximately 1 litre) was then concentrated under reduced pressure to approximately 1/3 of the initial volume and freeze-dried to obtain a white solid.
  • the white solid obtained after the precipitation and washing step was further purified according to the techniques described in bl and b2 of Example 3.
  • the product was then isolated by precipitation with 2.5 L isopropanol at 4°C (75%) enriching the suspension with 3 mL saturated NaCl solution (approximately 1 g salt) to improve the precipitation step and subsequently recovered by centrifugation of the suspension at 15000 rpm for 10 min.
  • the white solid obtained was washed several times with isopropanol, solubilised in 250 mL deionised water.
  • the solution was diluted to 400 mL and transferred to a 6-8 kDa membrane and dialysed first for 2 days against 24 litres (12+12 1) 0.1M NaCl saline solution and subsequently for 3 days against deionised water.
  • the entire retentate (approximately 1 litre) was then concentrated under reduced pressure to approximately 1/3 of the initial volume and freeze-dried to obtain a white solid.
  • the white solid obtained after the precipitation and washing step was further purified according to the techniques described in bl and b2 of Example 3.
  • the product was then isolated by precipitation with 2.5 L isopropanol at 4°C (75%) enriching the suspension with 3 mL saturated NaCl solution (approximately 1 g salt) to improve the precipitation step and subsequently recovered by centrifugation of the suspension at 15000 rpm for 10 min.
  • the white solid obtained was washed several times with isopropanol, solubilised in 250 mL deionised water.
  • the solution was diluted to 400 mL and transferred to a 6-8 kDa membrane and dialysed first for 2 days against 24 litres (12+12 1) of 0.1M NaCl saline solution and subsequently for 3 days against deionised water.
  • the entire retentate (approximately 1 litre) was then concentrated under reduced pressure to approximately 1/3 of the initial volume and freeze-dried to obtain a white solid.
  • the white solid obtained after the precipitation and washing step was further purified according to the techniques described in bl and b2 of Example 3.
  • the product was then isolated by precipitation with 2.5 L isopropanol at 4°C (75%) enriching the suspension with 3 mL saturated NaCl solution (approximately 1 g salt) to improve the precipitation step and subsequently recovered by centrifugation of the suspension at 15000 rpm for 10 min.
  • the white solid obtained was washed several times with isopropanol, solubilised in 250 mL deionised water.
  • the solution was diluted to 400 mL and transferred to a 6-8 kDa membrane and dialysed first for 2 days against 24 litres (12+12 1) of 0.1M NaCl saline solution and subsequently for 3 days against deionised water.
  • the entire retentate (approximately 1 litre) was then concentrated under reduced pressure to approximately 1/3 of the initial volume and freeze-dried to obtain a white solid.
  • the white solid obtained after the precipitation and washing step was further purified according to the techniques described in bl and b2 of Example 3.
  • the product was then isolated by precipitation with 2.5 L isopropanol at 4°C (75%) enriching the suspension with 3 mL saturated NaCl solution (approximately 1 g salt) to improve the precipitation step and subsequently recovered by centrifugation of the suspension at 15000 rpm for 10 min.
  • the white solid obtained was washed several times with isopropanol, solubilised in 250 mL deionised water.
  • the solution was diluted to 400 mL and transferred to a 6-8 kDa membrane and dialysed first for 2 days against 24 litres (12+12 1) of 0.1M NaCl saline solution and subsequently for 3 days against deionised water.
  • the entire retentate (approximately 1 litre) was then concentrated under reduced pressure to approximately 1/3 of the initial volume and freeze-dried to obtain a white solid.
  • the white solid obtained after the precipitation and washing step was further purified according to the techniques described in bl and b2 of Example 3.
  • the product was then isolated by precipitation with 2.5 L isopropanol at 4°C (75%) enriching the suspension with 3 mL saturated NaCl solution (approximately 1 g salt) to improve the precipitation step and subsequently recovered by centrifugation of the suspension at 15000 rpm for 10 min.
  • the white solid obtained was washed several times with isopropanol, solubilised in 250 mL deionised water.
  • the solution was diluted to 400 mL and transferred to a 6-8 kDa membrane and dialysed first for 2 days against 24 litres (12+12 1) of 0.1M NaCl saline solution and subsequently for 3 days against deionised water.
  • the entire retentate (approximately 1 litre) was then concentrated under reduced pressure to approximately 1/3 of the initial volume and freeze-dried to obtain a white solid.
  • the white solid obtained after the precipitation and washing step was further purified according to the techniques described in bl and b2 of Example 3.
  • the product was then isolated by precipitation with 2.5 L isopropanol at 4°C (75%) enriching the suspension with 3 mL saturated NaCl solution (approximately 1 g salt) to improve the precipitation step and subsequently recovered by centrifugation of the suspension at 15000 rpm for 10 min.
  • the white solid obtained was washed several times with isopropanol, solubilised in 250 mL deionised water.
  • the solution was diluted to 400 mL and transferred to a 6-8 kDa membrane and dialysed first for 2 days against 24 litres (12+12 1) of 0.1M NaCl saline solution and subsequently for 3 days against deionised water.
  • the entire retentate (approximately 1 litre) was then concentrated under reduced pressure to approximately 1/3 of the initial volume and freeze-dried to obtain a white solid.
  • the white solid obtained after the precipitation and washing step was further purified according to the techniques described in bl and b2 of Example 3.
  • the product was then isolated by precipitation with 2.5 L isopropanol at 4°C (75%) enriching the suspension with 3 mL saturated NaCl solution (approximately 1 g salt) to improve the precipitation step and subsequently recovered by centrifugation of the suspension at 15000 rpm for 10 min.
  • the white solid obtained was washed several times with isopropanol, solubilised in 250 mL deionised water.
  • the solution was diluted to 400 mL and transferred to a 6-8 kDa membrane and dialysed first for 2 days against 24 litres (12+12 1) of 0.1M NaCl saline solution and subsequently for 3 days against deionised water.
  • the entire retentate (approximately 1 litre) was then concentrated under reduced pressure to approximately 1/3 of the initial volume and freeze-dried to obtain a white solid.
  • the white solid obtained after the precipitation and washing step was further purified according to the techniques described in bl and b2 of Example 3.
  • the samples from Examples 3-14 were used to determine the molecular weight (MW) and the degree of substitution (DS).
  • the determination of the degree of substitution was carried out using a two-dimensional hetero-correlated (IH, 13 C) HSQC-DEPT nuclear magnetic resonance (NMR) technique following enzymatic hydrolysis of the sample with hyaluronidase.
  • IH, 13 C HSQC-DEPT nuclear magnetic resonance
  • Samples 14, 13 and 6 were tested on a cell model to evaluate their anti-inflammatory properties.
  • the anti-inflammatory activity of molecules 14, 13, and 6 was compared with that of the negative control (CTRL, smoker's human bronchial fibroblasts, untreated) and with that of the positive control (TC, smoker's human bronchial fibroblasts incubated with culture of U937 human monocytes activated to macrophages with PMA (50 ug/ml, 24h) and LPS (1 ug/ml, Ih) by qPCR analysis of IL-ip gene expression.
  • CTR negative control
  • TC smoker's human bronchial fibroblasts incubated with culture of U937 human monocytes activated to macrophages with PMA (50 ug/ml, 24h) and LPS (1 ug/ml, Ih) by qPCR analysis of IL-ip gene expression.
  • TC smoker's human bronchial fibroblasts incubated with culture of U937 human monocytes activated to macrophages with PMA (50 ug
  • Samples 14, 13 and 6 were tested on a cell model to evaluate their anti-fibrotic properties.
  • the anti-fibrotic activity of molecules 14, 13, and 6 was compared with that of the negative control (CTRL, human peripheral lung fibroblasts, untreated, NHLF line) and with that of the positive control (TGF-P, human peripheral lung fibroblasts (NHLF) incubated with 5 ng/ml TGF-P for 24h) by qPCR analysis of COL-1 gene expression.
  • CTRL human peripheral lung fibroblasts
  • TGF-P human peripheral lung fibroblasts

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Abstract

La présente invention décrit un procédé de conjugaison d'acide hyaluronique à l'aide d'un agent de condensation spécifique et dans certaines conditions de fonctionnement qui permettent d'obtenir des dérivés d'acide hyaluronique avec des disaccharides ou des oligosaccharides présentant le degré de conjugaison souhaité de manière efficace, rapide et économique. L'invention concerne également des conjugués d'acide hyaluronique présentant des degrés de conjugaison spécifiques et, de manière analogue, leurs utilisations à des fins thérapeutiques et en tant qu'échafaudages pour des biomatériaux.
PCT/IB2023/058903 2022-09-12 2023-09-08 Procédé de conjugaison d'acide hyaluronique et conjugués d'acide hyaluronique ainsi obtenus WO2024057154A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017114867A1 (fr) * 2015-12-29 2017-07-06 Galderma S.A. Réticulateur de glucides
WO2020129028A1 (fr) * 2018-12-21 2020-06-25 Nestlé Skin Health S.A. Compositions d'hydrogel encapsulant des particules solides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017114867A1 (fr) * 2015-12-29 2017-07-06 Galderma S.A. Réticulateur de glucides
WO2020129028A1 (fr) * 2018-12-21 2020-06-25 Nestlé Skin Health S.A. Compositions d'hydrogel encapsulant des particules solides

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