Classifications

• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
  • B01D15/361—Ion-exchange
  • B01D15/362—Cation-exchange
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
  • B01D61/24—Dialysis ; Membrane extraction
  • B01D61/243—Dialysis
• • 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
  • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass

Definitions

• 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
• 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.
• 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 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) 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).
• 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.
• 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.
• 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.
• TEA Triethylamine
• a-D-Lactose Triethylamine
• 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 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.
• 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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• Chemical & Material Sciences (AREA)
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• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Biochemistry (AREA)
• Molecular Biology (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Organic Chemistry (AREA)
• Polymers & Plastics (AREA)
• Materials Engineering (AREA)
• Pharmacology & Pharmacy (AREA)
• Analytical Chemistry (AREA)
• Animal Behavior & Ethology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Sustainable Development (AREA)
• Epidemiology (AREA)
• Water Supply & Treatment (AREA)
• Urology & Nephrology (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Polysaccharides And Polysaccharide Derivatives (AREA)
• Materials For Medical Uses (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicinal Preparation (AREA)

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• 2022
  • 2022-09-12 IT IT102022000018585A patent/IT202200018585A1/it unknown
• 2023
  • 2023-09-08 JP JP2025538901A patent/JP2025528605A/ja active Pending
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  • 2023-09-08 CN CN202380065710.XA patent/CN119855838A/zh active Pending
  • 2023-09-08 WO PCT/IB2023/058903 patent/WO2024057154A1/en not_active Ceased
  • 2023-09-08 KR KR1020257008394A patent/KR20250067823A/ko active Pending
  • 2023-09-08 EP EP23783022.9A patent/EP4587481A1/de active Pending

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