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WO2024222981A1 - Ester conjugate of sphingolipid and hyaluronan, method of synthesis thereof, composition containing thereof, use of conjugate or composition - Google Patents

Ester conjugate of sphingolipid and hyaluronan, method of synthesis thereof, composition containing thereof, use of conjugate or composition Download PDF

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Publication number
WO2024222981A1
WO2024222981A1 PCT/CZ2024/050031 CZ2024050031W WO2024222981A1 WO 2024222981 A1 WO2024222981 A1 WO 2024222981A1 CZ 2024050031 W CZ2024050031 W CZ 2024050031W WO 2024222981 A1 WO2024222981 A1 WO 2024222981A1
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group
sphingolipid
carbon atoms
conjugate
alkyl group
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PCT/CZ2024/050031
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French (fr)
Inventor
Martin JUHASCIK
Gloria Huerta-Angeles
Katerina STARMANOVA
Petr Stepan
Andrea EXNEROVA
Kristina NESPOROVA
Andrej Kovacik
Martina BRANDEJSOVA
Petra VECEROVA
Jana POLASKOVA
Martina HERMANNOVA
Radovan Buffa
Hana VAGNEROVA
Vladimir Velebny
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Contipro A.S.
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Publication of WO2024222981A1 publication Critical patent/WO2024222981A1/en

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    • 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
    • A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • 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
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6903—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6907—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6955—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a plaster, a bandage, a dressing or a patch
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
    • A61K8/41—Amines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/68—Sphingolipids, e.g. ceramides, cerebrosides, gangliosides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions

Definitions

  • the present invention relates to an ester conjugate of sphingolipid and hyaluronan, a composition comprising thereof and its use. Furthermore, it relates to methods of the ester conjugate synthesis.
  • Sphingolipids are a class of lipids containing a backbone of sphingoid bases, a set of aliphatic amino alcohols.
  • Ceramides CERs
  • CERs Ceramides
  • CERs are epidermal sphingolipids necessary for the proper skin barrier function and are composed of fatty acid and a sphingoid base, varying the size and structure of their molecular chains.
  • CERs constituted up to 50% (M'/W) of the lipid matrix of the epidermis in the Stratum corneum (SC).
  • SC Stratum corneum
  • Newer formulations of skin-care products have incorporated CERs into their formulations, intending to exogenously apply CERs to help skin barrier function.
  • CERs support the skin’s natural protective layer renewal and form an effective epidermal barrier.
  • PEFA polyglycerol esters of fatty acids
  • an oil-in water nanoemulsion-based formulation containing ceramide IIIB using phase-inversion temperature was developed for transdermal delivery.
  • the composition consisted of ethanol, propylene glycol (PG), and glycerol in the cosmetic oil Tegosoft® G20 (octyldodecanol) and Tween® 80 (polysorbate 80).
  • the optimized composition showed an average droplet size from 11.2 to 40.1 nm, and polydispersity was estimated to be 0.12.
  • Tegosoft® G20 octyldodecanol
  • Tween® 80 polysorbate 80
  • Licciardi et al. synthesized a succinyl derivative of ceramide (Licciardi, Scialabba, Sardo, Cavallaro, & Giammona, 2014).
  • the authors reacted the ceramide AP with succinic anhydride in anhydrous tetrahydrofuran (THF).
  • THF anhydrous tetrahydrofuran
  • the resulting mixture was kept at 40 °C for 6 h followed by 16 h at 25 °C.
  • the authors reacted bis-(4-nitrophenyl)carbonate (BNPC) with inulin in a reaction under microwaves’ irradiation.
  • BNPC bis-(4-nitrophenyl)carbonate
  • a chitosan-ceramide (CS-CE) graft copolymer is suitable for forming polymeric nanoparticles as a carrier for hydrophobic drugs.
  • the conjugate CS-CE formed nanoparticles with a mucoadhesive chitosan corona and hydrophobically stabilized core by CER moieties, wherein hydrophobic drugs were encapsulated, providing an attractive oral drug delivery system.
  • Battogtokh and Tag Ko carried out the activation of the ceramide by means of N, -disuccinimidyl carbonate (DSC).
  • the activation reaction uses anhydrous solvents, wherein sodium salt of HA is insoluble.
  • DSC reaction is also known to activate secondary alcohols (Z. Li et al., 2003). Thus, it might act as a crosslinking agent of HA.
  • the modified ceramide was conjugated to the carboxyl moiety of HA-TBA via ester bond formation in a reaction performed in a mixture of THF/acetonitrile (4:1, v/v).
  • the degree of substitution (DS) was reported as 2.38%.
  • the authors reported that DS-Y30 ceramide (8.59 mmol) was added to 12.21 mmol of HA, which means the reaction produced a low conversion and needed organic solvents making the process expensive for upscale.
  • the decomposition products of the aromatic spacer are not known, and the polysaccharide was modified on HA's carboxyl moiety, and therefore limiting the ability of the conjugate to interact with the CD44 receptor. Similar conditions were reported by Jin et al. for physicochemical characterization. Critical micellar concentration was reported as 0.042 mg. ml/ 1 for HA-CE and was determined by fluorescence (Jin et al., 2012).
  • Turley etal. chemically modified HA with phospholipids or sphingolipids selected from the group consisting of phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, sphingosines, ceramides, and ceramide-based lipids.
  • phospholipids or sphingolipids selected from the group consisting of phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, sphingosines, ceramides, and ceramide-based lipids.
  • the most preferred is the use of phosphatidylethanolamines.
  • the modification efficiency would be decreased if the reaction is performed in water (US20150368373-A1).
  • the subject-matter of the invention concerns an ester conjugate of sphingolipid and hyaluronan of a general formula I:
  • R 5 is H + or pharmaceutically acceptable salt
  • R 4 is -H or linked sphingolipid substituent of a general formula II
  • R 1 is a straight chain or branched alkyl group having 10 to 22 carbon atoms which may optionally contain one or more double bonds, any of H bonded to C atom of said alkyl group is optionally substituted with one or more hydroxyl groups, preferably is a straight chain alkyl group having 12 to 18 carbon atoms, more preferably is a straight chain alkyl group having 13 carbon atoms,
  • R 6 is H or COR 2 , wherein
  • R 2 is a straight chain or branched alkyl group having 13 to 55 carbon atoms, preferably 15 to 50 carbon atoms, more preferably 17 to 23 carbon atoms, that may optionally contain one or more double bonds; the alkyl chain may contain an internal ester group; or H of the alkyl group may optionally be substituted with one or more hydroxyl groups,
  • At least one linked sphingolipid substituent of the Formula II that it is stated above as possible group R 4 binds to hyaluronic acid via carbon of the end group -OC- of R 3 substituent, forming an ester bonds between them and to form the ester conjugate of sphingolipid and hyaluronan of the general Formula I according to the present invention.
  • the weight average molecular weight of the conjugate of the formula I according to the present invention is in the range of 3,000 g/mol to 1,600,000 g/mol, preferably in the range from 3,000 to 20,000 g/mol, more preferably 5,000 to 7,000 g/mol.
  • the degree of substitution in the conjugate of hyaluronan according to the present invention is in the range from 0.4 to 15 %, preferably the degree of substitution is in the range of 5.5 to 10.5%, more preferably the degree of substitution is in the range of 6.5 to 8.5 %.
  • the pharmaceutically acceptable salt of the conjugate of hyaluronan according to the present invention wherein is selected from a group comprising any of ions of alkali metals or ions of alkaline-earth metals, preferably Na + , K + or Mg 2+ .
  • R 1 is a straight chain or branched alkyl group having 10 to 22 carbon atoms which may optionally contain one or more double bonds, any of H bonded to C atom of said alkyl group is optionally substituted with one or more hydroxyl groups, preferably is a straight chain alkyl group having 12 to 18 carbon atoms, more preferably is a straight chain alkyl group having 13 carbon atoms,
  • R 6 is H or COR 2 , wherein
  • R 2 is a straight chain or branched alkyl group having 13 to 55 carbon atoms, preferably 15 to 50 carbon atoms, more preferably 17 to 23 carbon atoms; the alkyl chain may contain an internal ester group; may optionally contain one or more double bonds, and may optionally be substituted with one or more hydroxyl groups; is attached to a linker that is a rest of succinic acid or a rest of maleic acid by adding of succinic anhydride or maleic anhydride in a polar solvent in the presence of an organic base or mixtures thereof to form a linked sphingolipid of a general formula IV wherein
  • R 1 is a straight chain or branched alkyl group having 10 to 22 carbon atoms which may optionally contain one or more double bonds, any of H bonded to C atom of said alkyl group is optionally substituted with one or more hydroxyl groups, preferably is a straight chain alkyl group having 12 to 18 carbon atoms, more preferably is a straight chain alkyl group having 13 carbon atoms, R 6 is H or COR 2 , wherein
  • R 2 is a straight chain or branched alkyl group having 13 to 55 carbon atoms, preferably 15 to 50 carbon atoms, more preferably 17 to 23 carbon atoms; the alkyl chain may contain an internal ester group; may optionally contain one or more double bonds, and may optionally be substituted with one or more hydroxyl groups;
  • R 1 is a straight chain or branched alkyl group having 10 to 22 carbon atoms which may optionally contain one or more double bonds, any of H bonded to C atom of said alkyl group is optionally substituted with one or more hydroxyl groups, preferably is a straight chain alkyl group having 12 to 18 carbon atoms, more preferably is a straight chain alkyl group having 13 carbon atoms, R 6 is H or COR 2 , wherein
  • R 2 is a straight chain or branched alkyl group having 13 to 55 carbon atoms, preferably 15 to 50 carbon atoms, more preferably 17 to 23 carbon atoms; the alkyl chain may contain an internal ester group; may optionally contain one or more double bonds, and may optionally be substituted with one or more hydroxyl groups;
  • R 9 is substituent selected from a group of substituents comprising a substituent of a general formula VI or a substituent of formula VII or a substituent of formula VIII, wherein R 8 is one or more substituents selected from a group comprising H, -NO2, -COOH, halides, Ci-Ce alkyloxy, preferably halides, methoxy or ethoxy, more preferably Cl, that reacts with hyaluronic acid or a pharmaceutically acceptable salt thereof in the presence of a mixture of water and water-miscible organic solvent or in the presence of an organic solvent, preferably dimethyl sulfoxide.
  • the concentration of the sphingolipid of the general formula III is preferably ranging from 0.88 % to 2.5 % (w/v) and the molar equivalent of succinic anhydride or maleic anhydride is ranging from 1.0 to 2.0, preferably 1.5 equivalents with respect to sphingolipid of the general formula III.
  • the attachment of the linker is preferably earned out in the range of temperatures from 0 °C to 50°C for 4 to 20 hours, more preferably for 6 to 8 hours in the range of temperatures from 0 °C to 40°C, followed by stirring at a temperature in the range of 0 °C to 29 °C, preferably at room temperature for 5 to 24 hours, more preferably for 16 to 20 hours.
  • the organic base is preferably selected from the group comprising a secondary or tertiary amine having a linear or branched or cyclic or aromatic, saturated or unsaturated C3- C30 alkyl group
  • the polar solvent is selected from the group comprising tetrahydrofurane (THF), dioxane, dimethyl sulfoxide, dichloromethane and chloroform or mixtures thereof.
  • the organic base is preferably selected from the group comprising pyridine, triethylamine, 7V-methylmorpholine, dimethylaminopyridine or mixtures thereof, and the polar solvent is preferably dichloromethane or tetrahydrofurane (THF) or their mixture.
  • the activation agent is preferably benzoyl chloride that is a substituted or nonsubstituted benzoyl chloride or its derivatives of the general formula IX wherein R 8 is one or more substituents selected from a group comprising H, -NO2, -COOH, halides, Ci-Ce alkyloxy, preferably halides, methoxy or ethoxy, more preferably Cl.
  • the forming of the activated linked sphingolipid of the general formula V is preferably carried out at the temperature in the range of 5 °C to 60 °C, more preferably 40 °C, for 0.5 to 24 hours.
  • the molar amount of the activation agent is preferably in the range of 0.03 to 2 molar equivalents, more preferably 1 molar equivalent to a linked sphingolipid of a general formula IV.
  • the organic solvent is preferably selected from the group comprising isopropanol, tert- butanol, dioxane, dimethyl sulfoxide, acetonitrile and tetrahydrofuran.
  • concentration of the activated linked sphingolipid of the general formula V that reacts with hyaluronic acid or a pharmaceutically acceptable salt thereof is preferably in the range of 0.25 % to 7.0 % (w/v) in the solution.
  • the reaction of the activated linked sphingolipid of the formula V and hyaluronic acid or the pharmaceutically acceptable salt thereof is preferably carried out in the range of temperatures 5 °C to 37 °C, for 1 to 72 hours, more preferably at 25°C, for 2 hours.
  • the water-miscible organic solvent is preferably selected from a group comprising isopropanol, /e/7-butanol, dioxane, dimethylsulfoxide and tetrahydrofuran.
  • the molar amount of the activated linked sphingolipid of the formula V is 0.01 to 2.0 equivalents, preferably 0.03 to 0.5 equivalents with respect to a dimer of hyaluronic acid.
  • a composition comprises the ester conjugate according to the present invention, preferably the ester conjugate is in a form of a self-assembled polymeric particle.
  • the concentration of the ester conjugate of the general Formula I in the composition according to the present invention is ranging from 0.001 to 50 % (w/w), preferably from 0.001 to 5 % (w/w), more preferably from 0.001 to 1 % (w/w).
  • composition according to the present invention is preferably in the form selected from a group comprising emulsion, serum, hydrogel or buccal patch.
  • composition according to the present invention is in the form of emulsion, while the concentration of the ester conjugate according to the general formula I is ranging from 0.001 to 5 % (w/w), preferably from 0.001 to 1 % (w/w).
  • composition according to the present invention preferably contains water and at least one cosmetic or pharmaceutical excipient selected from a group containing oil, wax, butter, emulsifier, auxiliary active ingredient, thickener and preservative.
  • the oil is preferably selected from a group comprising coconut, olive, avocado, sesame, almond, castor, sunflower, hemp, jojoba, argan, apricot, borage, marula, cottonseed, evening primrose, grapeseed, hazelnut, linseed, meadowfoam, moringa, plum, poppy, rice, rosehip, safflower, wheat germ, macadamia oils and squalene; butter is selected from a group comprising cocoa butter, illipe butter, kokum butter, murumuru butter, mango butter, cupuacu butter, avocado butter and shea butter; wax is selected from a group comprising lanolin, beeswax, carnauba wax, candelilla wax and petroleum jelly.
  • the emulsifier is preferably selected from a group comprising glyceryl stearate, glyceryl caprylate, behenyl alcohol, glyceryl behenate, cetearyl glucoside, methyl glucose sequistearate, glyceryl stearate citrate, polyglyceryl-3 stearate, cetearyl olivate, lecithin, stearyl alcohol, sorbitan oleate, polysorbates, stearic acid, cetyl alcohol and cetearyl alcohol, sodium acrylate, sodium acryloyldimethyltaurate copolymer or mixtures of thererof.
  • the auxiliary active ingredient is preferably selected from a group comprising vitamins A, D, E, K, C and B group vitamins; curcumin, coenzyme Q10, allantoin, bisabolol, lactic acid, amino acids, a-hydroxy and ⁇ -hydroxy acids, ceramides, peptides, preferably acetyl hexapeptide- 8, palmitoyl tripeptide- 1, palmitoyl tetrapeptide-7, copper tripeptide- 1, palmitoyl pentapeptide-4, Saccharomyces peptides, hexapeptide- 1 and protein, preferably rice protein, soy protein, quinoa protein or wheat protein; polysaccharides, hyaluronic acid or a pharmaceutically and cosmetically acceptable salt thereof, carboxymethyl glucan, schizophyllan, glucomannan; panthenol; urea; glycerin; pentylene glycol; plant extracts, preferably aloe vera extract
  • the preservative is preferably selected from a group comprising aromatic acids and their derivatives, preferably benzoic acid, salicylic acid, dehydroacetic acid, potassium sorbate, parabens; alcohols preferably ethanol, isopropanol, benzyl alcohol, phenoxyethanol, phenethyl alcohol; imidazole derivatives preferably hydantoin, imidazolidinyl urea; cationic surfactants preferably benzalkonium chloride.
  • aromatic acids and their derivatives preferably benzoic acid, salicylic acid, dehydroacetic acid, potassium sorbate, parabens
  • alcohols preferably ethanol, isopropanol, benzyl alcohol, phenoxyethanol, phenethyl alcohol
  • imidazole derivatives preferably hydantoin, imidazolidinyl urea
  • cationic surfactants preferably benzalkonium chloride.
  • composition according to the present invention is in the form of serum, while the concentration of the ester conjugate according to the general formula I is ranging from 0.001 to 5 % (w/w), preferably from 0.001 to 1 % (w/w); while containing water and at least one water soluble auxiliary active ingredient; at least one thickener, and at least one preservative as stated above.
  • the concentration of the ester conjugate according to the general formula I in the composition according to the present invention is preferably in range from 0.001 to 1 % (w/w).
  • the water-soluble auxiliary active ingredient is chosen from a group comprising vitamin C; B vitamins; amino acids, a-hydroxy and /Lhydroxy acids, peptides; preferably acetyl hexapeptide-8, palmitoyl tripeptide, copper tripeptide- 1, Saccharomyces peptides, hexapeptide- 1 ; and proteins preferably rice protein, soy protein, quinoa protein or wheat protein; and water- soluble polysaccharides, preferably hyaluronic acid or a pharmaceutically and cosmetically acceptable salt thereof, carboxymethyl glucan, schizophyllan, glucomannan, urea; glycerin; pentylene glycol; plant extracts preferably aloe vera extract, cammomile extract, acai extract, green tea extract, algae extract, oat extract, cannabis extract, cranberry extract; extracts, ferments, lysates and filtrates from bacteria preferably from Lactobacillus, Thalassospira
  • composition according to the present invention additionally contains at least one hydrophobic compound selected from a group comprising cannabidiol, tocopherol, curcumin, coenzyme Qio, ethyl ferulate, resveratrol, bakuchiol, retinyl palmitate, that is encapsulated in the self-assembled polymeric particles of the ester conjugate according to the present invention.
  • at least one hydrophobic compound selected from a group comprising cannabidiol, tocopherol, curcumin, coenzyme Qio, ethyl ferulate, resveratrol, bakuchiol, retinyl palmitate, that is encapsulated in the self-assembled polymeric particles of the ester conjugate according to the present invention.
  • composition according to the present invention additionally contains thickener that is selected from a group comprising xanthan gum, sclerotium gum, guar gum, cellulose gum, carbomer, hydroxyethylcellulose, Amorphophallus konjac root extract, carrageenan, pullulan, lecithin, lysolecithin, sodium acrylate, sodium acryloyldimethyl taurate copolymer or mixtures thereof.
  • thickener that is selected from a group comprising xanthan gum, sclerotium gum, guar gum, cellulose gum, carbomer, hydroxyethylcellulose, Amorphophallus konjac root extract, carrageenan, pullulan, lecithin, lysolecithin, sodium acrylate, sodium acryloyldimethyl taurate copolymer or mixtures thereof.
  • composition according to the present invention contains the ester conjugate according to the present invention in the form of a buccal patch.
  • composition according to the present invention preferably contains other auxiliary excipient, preferably hyaluronic acid or pharmaceutically acceptable salt of thereof.
  • auxiliary excipient preferably hyaluronic acid or pharmaceutically acceptable salt of thereof.
  • ester conjugate of sphingolipid and hyaluronan and composition according to the present invention for use in the medical treatment of a skin disease selected from the group comprising atopic dermatitis, psoriasis, ichtyosis and rosacea.
  • ester conjugate of sphingolipid and hyaluronan and composition according to the present invention for use in the medical treatment of a buccal mucosa disease selected from the group comprising aphthous stomatitis and Behcet's disease.
  • composition according to the present invention is topical, buccal, sublingual to the subject, preferably human.
  • Hyaluronic acid is a high-molecular-weight glycosaminoglycan composed of D-glucuronic acid (GlcA) and TV-acetyl-D-glucosamine (GlcNAc) units linked by alternating ?-(l-4)-and /?-(l-3)-glycosidic bonds.
  • GlcA D-glucuronic acid
  • GlcNAc TV-acetyl-D-glucosamine
  • Amphiphilic HA consisting of ceramides or sphingoid base lipids attached to the polysaccharide backbone enhanced the penetration of modified HA through the skin barrier into the epidermal and dermal layers of the skin, thereby allowing the dermal administration of the conjugates without injection or another disruption of the skin.
  • Ceramides are used mainly as moisturizers in various cosmetic products (Choi & Lee, 2015). Thus, it is expected a synergism after covalent binding to HA.
  • the ester conjugates are useful as delivery devices to facilitate the dermal and transdermal delivery of cosmetically and pharmaceutically active substances through the skin barrier, which can be encapsulated in the hydrophobic core.
  • the self-assembling of the ester conjugates, according to the present invention, as stated herein, is forming self-assembled polymeric particles, which can encapsulate hydrophobic compounds.
  • skin permeation is governed by the size, nanostructure and viscosity of the microemulsions. Smaller droplets and less viscous MEs permeated deeper layers of the skin to a higher extent (Sahle et al., 2014).
  • the lower limit of the weight average molecular weight of the hyaluronan useful herein is from 3,000 g/mol, 6,000 g/mol, 10,000 g/mol, 20,000 g/mol, 50,000 g/mol, 60,000 g/mol, 70,000 g/mol, 80,000 g/mol, 90,000 g/mol, or 100,000 g/mol, and the upper limit is 200,000 g/mol, 300,000 g/mol, 400,000 g/mol, 500,000 g/mol, 600,000 g/mol, 700,000 g/mol and 1,600,000 g/mol.
  • HA has a molecular weight of 6,000 g/mol to 20,000 g/mol.
  • R 1 is preferably selected from a group comprising dodecyl, tridecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, more preferably tridecyl.
  • R 2 is preferably selected from a group comprising (Z)-heptadec-8-ene-l-yl, (6Z,9Z)- heptadeca-6,9-diene-l-yl, pentacosyl, tricosyl, henicosyl, nonadecyl, heptadecyl, pentadecyl;
  • 1 -hydroxyalkyl or tu-hydroxyalkyl preferably selected from a group comprising 1- hydroxypentacosyl, 1 -hydroxytricosyl, 1-hydroxyhenicosyl, 1 -hydroxynonadecyl, 1- hydroxyheptadecyl, 1 -hydroxypentadecyl, co-hydroxyheptacosyl, m-hydroxynonacosyl, a>- hydroxyhentriacontyl.
  • the most preferred is (Z)-heptadec-8-ene-l-yl, 1 -hydroxyheptadecyl, 1- hydroxytricosyl, 1 -hydroxypentadecyl.
  • m-hydroxyl of alkyl chains may be esterified with another, typically unsaturated carboxylic acid.
  • This modification creates an internal ester group in the fatty acid chain as might be seen in Ceramides EOS, EOP, EOdS; while preferred R 2 according to general Formula III is 29-[(9Z,12Z)-octadeca-9,12-dienoyloxy]nonacosyl. Examples of other ceramides classes are shown in the following figure.
  • the first step of the method of synthesis of the ester conjugate of sphingolipid and hyaluronan is the reaction of the sphingolipid of the general formula II with the linker, which is a rest of succinic acid or a rest of maleic acid, and which is added to the reaction mixture in the form of succinic anhydride or maleic anhydride.
  • the sphingolipid of the general formula II is dissolved in organic solvents THF or DMSO or dichloromethane, preferably in the presence of an inert atmosphere provided by gasses nitrogen or argon. Afterwards the organic base and succinic anhydride or maleic anhydride is added.
  • the reaction is performed at 0 °C to 50°C for 4 to 20 hours, more preferably for 6 to 8 hours in the range of temperatures from 0 °C to 40°C, followed by stirring at a temperature in the range of 0 °C to 29°C, preferably at room temperature for 5 to 24 hours, more preferably for 16 to 20 hours.
  • the inert atmosphere is not necessary for the successful performance of the reaction.
  • the second step (activation) comprises preparing the activated linked sphingolipid of a general formula VI in an organic solvent.
  • the preferred solvents used in the reaction are isopropanol, /e/7-butanol, THF, 1,4-dioxane, acetonitrile and DMSO.
  • the temperature of the activation is crucial for the formation of the intermediate.
  • the activation reaction is carried out between 5 to 60 °C.
  • the reaction is performed in a time ranging from 0.5 to 24 hours.
  • the activation reaction is performed between 35 to 60°C. More preferably, the activation reaction is performed at 40°C.
  • the activation of sphingolipids can be performed by activating agents described in the art, i.e.
  • CDI 1,1 ’ -carbonyl diimidazole
  • HSTU tetramethyl- ⁇ 9-(iV- succinimidyl)uronium hexafluorophosphate
  • benzoyl chloride Activating agents CDI and HSTU produced a low degree of HA chemical modification (up to 2.8 %). Also, these activators reacted under anhydrous conditions, as shown in the Examples.
  • the activating agent benzoyl chloride provides considerable advantages. The advantage of benzoyl chloride in activation reaction is the short activation time.
  • the third step is the esterification of HA with the activated linked-sphingolipid of a general formula VI, which proceeds at low temperatures (from 0 to 25 °C).
  • This reaction is performed in water mixed with an organic solvent miscible with water or in an organic solvent miscible with water.
  • the advantages of the method are high efficiency in the second step of activation and the third step of esterification and the possibility of performing HA esterification in the mixture of water and water-miscible organic solvent (1 :1 v/v), which allows the dissolution of sodium hyaluronate directly in water.
  • the conversion of sodium hyaluronate to TBA salt or acid form before the modification could be avoided.
  • the esterification reaction is kept under constant stirring for 1-72 hours, more preferably for 2 hours.
  • This reaction is highly selective, and the final esterification products are characterized by at least one of the hydroxyl groups of HA that have been esterified with the activated linked sphingolipid of the general formula VI.
  • the esterification of HA with the activated linked sphingolipid of the general formula VI can reach DS up to 15% (mol/mol) using activating agent benzoyl chloride.
  • activating agent benzoyl chloride activating agent benzoyl chloride.
  • other mentioned activating agents require converting sodium hyaluronate to acid form of hyaluronic acid to be dissolved in anhydrous DMSO.
  • the ester conjugate of sphingolipid and hyaluronan according to the present invention facilitates its penetration in a 1-15% (mol/mol) through the skin barrier into the dermis and epidermis layers.
  • these ester conjugates contain sphingolipids, which are covalently attached to hyaluronan.
  • This invention provides a chemical modification of hyaluronan performed in water- containing solvent industrially applicable.
  • the ester conjugates prepared by the method according to the invention can self-assemble in water or water solutions into self-assembled polymeric particles.
  • the self-assembled ester conjugates according to the present invention can also form self-assembled polymeric particles with encapsulated active substances selected from a group comprising curcumin, tocopherol, cannabidiol, coenzyme Qio, ethyl ferulate and miconazole.
  • the active substance is encapsulated into the conjugates preferably in propan-2-ol water solutions.
  • the average size of the self-assembled polymeric particles in water or water solutions of salts is in the range 10 nm to 10 000 nm by volume.
  • the preferred average size of self-assembled ester conjugate is in the range from 10 nm to 100 nm by volume.
  • ester conjugate is able to suppress the production of proinflammatory cytokine IL-6 up to 95 %.
  • Prepared ester conjugates could be used in various liquid (e.g. serum) or semi-solid (e.g. facial cream) or solid (e.g. buccal patch) forms.
  • Prepared ester conjugates of sphingolipid and hyaluronan according to the present invention are water-soluble and therefore can be included into water phase of the liquid, semisolid or solid compositions in amounts ranging from 0,001 to 7.5 % (w/w), preferably from 0,001 to 5 % (w/w), more preferably from 0,001 to 1 % (w/w); and also can be used with encapsulated hydrophobic active substance.
  • the composition is preferably selected from a group comprising emulsion (oil-in-water, water in oil, a micro- or nano-emulsion, serum (aqueous or hydro-alcoholic solution or gel, sterile or nonsterile), anhydrous gel, paste, dispersion of vesicles, powder, nanofibers, macro-, micro- or nano-capsules, macro-, micro- or nano-spheres, liposomes, oleosomes or chylomicrons, macro-, micro- or nano-sponges which may be adsorbed on organic polymer powders, talcs, bentonites and other inorganic or organic supports.
  • the composition contains at least one cosmetic or pharmaceutical excipient, selected from a group containing oil, wax, butter, emulsifier, auxiliary active ingredient, preservative and thickener.
  • degree of substitution 100 % * molar amount of the bound substituent / molar amount of polysaccharide dimers. The determination of the degree of substitution was performed by NMR.
  • the weight average molecular weight of the native used HA was determined by SEC-MALLS method before chemical modification.
  • room temperature refers to a temperature range of 22°C to 25°C.
  • Equivalent refers to a dimer of hyaluronic acid or a linked sphingolipid of a general formula V. It is a molar equivalent unless otherwise stated.
  • self-assembled polymeric particles refers to the organized macromolecular units of ester conjugate according to the present invention ordered into structures by non-covalent interactions, with particle diameter ranging from 10 to 10 000 nm, by volume.
  • the preferred diameter of self-assembled ester conjugate particles is in the range of 10 nm to 100 nm by volume.
  • Figure 11 Cell viability of NIH-3T3 fibroblasts after treatment with 4-(((2S',3S',4R)-3,4- dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate prepared according to Examples 12, 13, 14, 18 and 17 (from left to right) after 24 h, 48 h, and 72 h.
  • Figure 14 Population sizes of several prepared of sodium salt of the ester conjugates prepared according to Examples 12 to Example 14 Figure 15. Immunomodulatory properties of sodium salt of the ester conjugate prepared according to Examples 14 and 17 compared to corresponding free ceramide NP 18:1 and native sodium hyaluronate (6 kDa).
  • Obr. 16 Cryo-scanning electron microscopy image of self-assembled polymer particles of the conjugate prepared according to Example 12. The image shows white spherical particles of the ester conjugate.
  • reaction mixture was concentrated under reduced pressure, dissolved in CHCI3 (2 mL) and washed with demineralized water (4x 2 mL). The organic fraction was concentrated under reduced pressure to obtain a colourless wax. Yield: 100 %. No regioselectivity was observed.
  • the acylated derivative was isolated by subsequent precipitation using 5-fold absolute isopropanol from the reaction mixture. After decantation, the precipitate underwent 4-fold repeated precipitation and decantation. After that, the precipitate was 4-fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
  • the reaction mixture was diluted with 10 mL of demineralized water containing 3.067 g of NaCl.
  • the acylated derivative was isolated by subsequent precipitation using 5-fold absolute isopropanol from the reaction mixture. After decantation, the precipitate underwent 4-fold repeated precipitation and decantation. After that, the precipitate was 4-fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
  • the reaction mixture was diluted with 10 mL of demineralized water containing 3.067 g of NaCl.
  • the acylated derivative was isolated by subsequent precipitation using 5 -fold absolute isopropanol from the reaction mixture. After decantation, the precipitate underwent 4-fold repeated precipitation and decantation. After that, the precipitate was 4-fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
  • the acylated derivative was isolated by subsequent precipitation using 5-fold absolute isopropanol from the reaction mixture. After decantation, the precipitate underwent 4-fold repeated precipitation and decantation. After that, the precipitate was 4-fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
  • reaction mixture was diluted with 10 mL of demineralized water containing the addition of 3.067 g of NaCl.
  • the acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5 -fold absolute isopropanol. After the decantation, the precipitate underwent 4-fold repeated precipitation and decantation. Finally, the precipitate was 4-fold washed with absolute isopropanol. Afterwards, the precipitate was dried at 40 °C for 48 hours and subsequently spray-dried to remove the residual solvents.
  • the acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5 -fold absolute isopropanol. After the decantation, the precipitate underwent 4-fold repeated precipitation and decantation. Finally, the precipitate was 4-fold washed with absolute isopropanol to remove water from the derivative. Afterwards, the precipitate was dried at 40 °C for 48 hours and subsequently lyophilized to remove the residual solvents.
  • the reaction mixture was diluted with 10 mL of demineralized water containing the addition of 3.067 g of NaCl.
  • the acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5 -fold absolute isopropanol. After the decantation, the precipitate underwent 4-fold repeated precipitation and decantation. Finally, the precipitate was 4-fold washed with absolute isopropanol to remove water from the derivative. Afterwards, the precipitate was dried at 40 °C for 48 hours and subsequently lyophilized to remove the residual solvents.
  • the acylated derivative was isolated by subsequent precipitation using a 5- fold volume of isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (85% by vol.). Afterwards, the precipitate was 3-fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
  • the acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5 -fold isopropanol. After that, the precipitate was decanted and repeatedly washed. First, with absolute isopropanol and subsequently 4-times with an aqueous solution of isopropanol/water (85% by vol.). Afterwards, the precipitate was 3 times washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
  • the reaction mixture was diluted with 6 mL of demineralized water containing the addition of 2.92 g of NaCl.
  • the acylated derivative was isolated from the reaction mixture by subsequent precipitation using the 5-fold of absolute isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with aqueous solution of isopropanol (85% by vol.). Afterwards, the precipitate was 3-fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours and subsequently lyophilized for the purpose of removing the residual solvents.
  • the reaction mixture was diluted with 1 mL of demineralized water containing the addition of 0.7 g of NaCl.
  • the acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5 -fold absolute isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (85% by vol.). Afterwards, the precipitate was 3 -fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
  • the reaction mixture was diluted with 1 mL of demineralized water containing the addition of 0.7 g of NaCl .
  • the acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5 -fold absolute isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (85% by vol.). Afterwards, the precipitate was 3 -fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
  • the reaction mixture was diluted with 0.2 mL of demineralized water containing 29 mg of NaCl.
  • the acylated derivative was isolated by subsequent precipitation using 5 -fold absolute isopropanol from the reaction mixture. After decantation, the precipitate underwent 4-fold repeated precipitation and decantation. After that, the precipitate was 4-fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
  • hyaluronic acid form 5.0 g of sodium hyaluronate (12.5 mmol, 15,000 g mol' 1 ) was dissolved in 250 ml of demineralized water. Subsequently, 30 mL of H + catex was added to the solution. The reaction was kept at 5 °C for 24 h. After that, H + catex was filtered from the hyaluronic acid solution and washed several times with demineralized water. The resulting filtrate was freeze-dried.
  • TEA 0.009 mL; 0.5 eq.
  • CDI 10.72 mg; 0.5 eq.
  • the solution was added to the HA-containing solution and allowed to react for 24 h at room temperature.
  • the reaction mixture was diluted with 0.3 mL of demineralized water containing the addition of 38.65 mg of NaCl.
  • the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-times with an aqueous solution of isopropanol (90 % by vol.). Afterwards, the precipitate was 3-fold washed with absolute isopropanol to remove water from the derivative and dried at 40 °C for 48 hours.
  • TEA 0.009 mL; 0.5 eq.
  • CDI 10.72 mg; 0.5 eq.
  • the solution was added to the solution containing HA and allowed to react for 24 h at room temperature.
  • the reaction mixture was diluted with 0.3 mL of demineralized water containing the addition of 38.65 mg of NaCl.
  • the acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5-fold absolute isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (90% by vol.). Afterwards, the precipitate was 3 -fold washed with absolute isopropanol to remove water from the derivative and was dried at 40 °C for 48 hours.
  • TEA 0.012 mL; 0.5 eq.
  • HSTU 14.26 mg; 0.5 eq.
  • the solution was added to the solution containing HA and allowed to react for 40 h at 25 °C.
  • the reaction mixture was diluted with 0.3 mL of demineralized water containing the addition of 51.41 mg of NaCl.
  • the acylated derivative was isolated from the reaction mixture by subsequent precipitation using the 5-fold of absolute isopropanol.
  • the acylated derivative was isolated from the reaction mixture by subsequent precipitation using the 5-fold of absolute isopropanol.
  • the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (90% by vol.). Afterwards, the precipitate was 3 -fold washed with absolute isopropanol to remove water from the derivative. Finally, the residue was dried at 40 °C for 48 hours.
  • the reaction mixture was diluted with 1 mL of demineralized water containing the addition of 73 mg of NaCl.
  • the acylated derivative was isolated from the reaction mixture by subsequent precipitation using the 5 -fold of absolute isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with aqueous solution of isopropanol (90% by vol.). Afterwards, the precipitate was 3 -fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
  • TEA 0.34 mL, 0,5 eq.
  • CDI 1,1 - carbonyldiimidazole
  • the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (90% by vol.). Afterwards, the precipitate was 3-fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
  • TEA 0.013 mL, 0.2 eq.
  • HSTU 34.24 mg, 0.2 eq.
  • the acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5-fold absolute isopropanol.
  • the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (90% by vol.). Afterwards, the precipitate was 3-fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
  • DS 2.8 % (detennined by NMR).
  • the acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5 -fold absolute isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (90% by vol.). Afterwards, the precipitate was 3-fold washed with absolute isopropanol to remove water from the derivative and was dried at 40 °C for 48 hours.
  • the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (90% by vol.). Afterwards, the precipitate was 3 -fold washed with absolute isopropanol to remove water from the derivative and was dried at 40 °C for 48 hours.
  • TEA 0.004 mL, 0.2 eq.
  • HSTU 11,4 mg, 0.2 eq.
  • the solution was added to the solution containing HA and allowed to react for 72 h at room temperature.
  • the reaction mixture was diluted with 1 mL of demineralized water containing the addition of 46.4 mg of NaCl.
  • the acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5-fold absolute isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (90% by vol.).
  • the amount of bound tocopherol determined by HPLC was 12.3 % (w/w).
  • Tocopherol and cannabidiol were determined by HPLC as 11.4 % and 4.0 % (w/w), respectively.
  • the amount of ethyl ferulate (determined by the UV-Vis method) was 6.1 % (w/w).
  • the amount of bound coenzyme Qio as determined by the UV-Vis method was 11.2 % (w/w).
  • Polymeric micelles containing non-covalently bound Nile red were prepared to determine critical aggregation concentration (cac).
  • 60 mg of ester conjugate prepared as described in examples described in Table 1 was dissolved under continuous stirring in 10 mL of water overnight.
  • the resulting solution was gradually supplemented with the solution of Nile red (0.72 mg in 1.2 mL of CHCI3).
  • CHCI3 was removed from the solution in a continuous evaporation process at 35 °C. After that, the dry residue was rehydrated with water and filtered through a 1 pm glass filter.
  • the filtrate was lyophilized.
  • the lyophilizate was dissolved and diluted to a concentration range from 0.00002 to 1.5 mg.mL -1 .
  • cac was determined by recording emission fluorescence spectra (560-800 nm) on PTI Quanta Master 400 spectrofluorophotometer (PTI) with the set excitation wavelength 543 nm and excitation and emission slit 5 nm.
  • the aggregation onset was determined by the concentration dependence of the maximum position of the NR emission band. Two data sets were collected for each sample and analyzed altogether. The inflexion-point coordinate of a sigmoidal fit of the obtained semilogarithmic (concentration axis) dependence is reported as the cac value (Table 1).
  • a series of pyrene films were prepared in the glass vials containing pyrene film. After that amphiphilic derivative of hyaluronic acid in demineralized water was added to each vial to obtain a concentration from 0.001 to 10 mg.mL’ 1 . The resulting concentration of pyrene in each vial was 0.8 pg.mL’ 1 .
  • the solutions were shaken at room temperature for at least 24 h at room temperature. After that, fluorescence was measured using PTI Quanta Master 400 spectrofluorophotometer. The excitation wavelength was set to 334 nm, and the emission wavelength range to 345 - 550 nm.
  • the interaction of cells with ester conjugates according to the present invention is essential to be investigated before the product application.
  • the derivatives should not be cytotoxic.
  • the cytotoxicity was assessed using the dilution method.
  • the cell toxicity of prepared HA derivatives was tested at NIH-3T3 cells. Cells were seeded into wells of 96-well test plates and cultured for 24 hours. Cell viability was measured 0, 24, 48, and 72 hours after treatment using the 3-(4, 5- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay.
  • MTT 3-(4, 5- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
  • MTT stock solution (20 pL; of concentration 5 mg.mL' 1 ) was added to the cell culture medium (200 pL) in each well. The plates were incubated for 2.5 h at 37 °C. Then, after removing the MTT solution, 220 pL of lysis solution was added, and lysis was carried out for 30 min at room temperature. The optical density was measured by Microplate reader VERSAmax at 570 nm. All assayed derivatives were non-cytotoxic up to the concentration of 1,000 pg.mL -1 .
  • results for conjugates prepared according to examples 9, 11, 10, 40 and 14 are shown in Figure 11, wherein positive effects to cell viability after 24, 48 or 72 h were observed in the whole concentration range tested, while the most positive effects were repeatedly observed at 100 pg.mL -1 indicates an excellent cytocompatibility of the conjugate.
  • Immunomodulatory properties of the sodium salt of the prepared ester conjugates were assessed by measuring the level of the proinflammatory cytokine interleukin 6 (IL-6) produced by LPS-activated macrophages after exposure to these derivatives.
  • THP-1 cells 400 000 cells/well, 6-well plate
  • phorbol myristate acetate 50 ng.mL' 1
  • the macrophages were co-incubated with LPS (1 pg.mL’ 1 ) and ester conjugates (in concentrations of 250 pg.mf/'and 100 pg.mL’ 1 ).
  • Macrophages stimulated with only LPS were used as a positive control. In contrast, non-stimulated macrophages were used as a negative control.
  • Free ceramide NP 18:1 and HA (6 kDa) were used for comparison. They were tested in the same concentrations as ester conjugates (250 pg.mL' 1 and 100 pg.mL' 1 ). The concentrations of free ceramide NP 18:1 were adjusted according to DS of ester conjugates and their concentrations.
  • the ester conjugates prepared according to Examples 14 and 17, and HA (6 kDa) were dissolved in cultivation media; free ceramide NP 18:1 was dissolved in DMSO.
  • the solvent control (DMSO) was also included in the experimental setup. After 24-hour long exposure to LPS and tested substances, the supernatants were collected, and the level of IL-6 was measured by ELISA (Thermo-Fisher).
  • Ester conjugates reduced the level of proinflammatory cytokine IL-6, as shown in Figure 15. The reduction in IL-6 level was found to be concentration-dependent, and the highest inhibition effect in IL-6 production was observed at a concentration of 250 pg. ml/ 1 . At this concentration, ester conjugates statistically significantly (p ⁇ 0.001) inhibited the production of IL-6 by 95.3 % (Example 17) and 91.1 % (Example 14) compared to positive control. Ester conjugate's ability to suppress the inflammation marker IL-6 is unique to these derivatives and was caused by the synergy between HA and ceramide NP 18:1. Both native HA and free ceramide NP 18:1 did not show any significant IL-6 inhibitory effect.
  • Example 44 Example 44
  • composition of emulsion prepared using the ester conjugate of sphingolipid with HA prepared using the ester conjugate of sphingolipid with HA
  • Cream (oil in water emulsion) prepared using the ester conjugate of sphingolipid with HA contains: (a) from 0.001 to 0.1 % by weight of self-assembled polymeric particles made of the ester conjugate of sphingolipid and HA
  • Serum formulation prepared using the ester conjugate of sphingolipid with HA contains: (a) from 0.001 to 0.1 % by weight of self-assembled polymeric particles made of the ester- conjugate of sphingolipid and HA
  • Nanoemulsions were prepared using the homogenization method under high agitation by Ultra- Turrax® equipment (IKA, Germany).
  • the formulation consisted of an oil phase containing an oil emulsifier (0.1 - 10 %), and an aqueous phase containing self-assembled particles of the conjugate of sphingolipid with HA prepared according to Examples 12 to 24 (2 % w/v) and ultrapure water.
  • the phases were homogenized separately with the aid of a magnetic stirrer. Then the oil phase was injected into the aqueous phase under the agitation of 10,000 rpm, which was increased to 17,000 rpm and sustained for 30 min with temperature control.
  • a solution of oxidized HA (HA-OX) prepared according to the patent WO2011069475A2 and self-assembled particles of the conjugate of sphingolipid with HA prepared according to Examples 12 to 24 (1 :1) was prepared in demineralized water in which the final concentrations of the polymers were from 1.5 to 7.5 % (w/v), respectively.
  • To that solution was added (0.1 % w/v) of 0,0 -1,3-propanediylbishydroxylamine dihydrochloride 98% linker was dissolved and homogenised. Then, the solution was transferred to Teflon molds (cylinders, diameter 10 mm, height 5 mm) to form hydrogel.
  • Weight average molecular weight and molecular weight distributions were determined by SECMALLS using an Agilent degasser Model G 1379A, chromatography system composed of an Agilent HPLC pump Model G 1310A, a Rheodyne manual injector Model 7125i, two 7.8 mm ultrahydrogel Linear columns (Waters), chromatographic detectors included a DAWN EOS MALLS, a ViscoStar differential viscometer, and an Optilab T-rEX differential refractive index in series (Wyatt Technology, Santa Barbara, California).
  • the injection volume was 100 pL of 0.015-1% (w/v) of conjugates of sphingolipid and HA.
  • the concentration was previously optimized according to the Mw to be analyzed.
  • the concentration required for low molecular weight (6 - 15 kDa) is 1 % (w/v), while higher molecular weight HA (188 - 442 kDa) needed 0.05% (w/v).
  • the mobile phase was aqueous 50 mM phosphate-buffered saline and 0.02% sodium azide solution at the flow rate of 0.5 mL min -1 .
  • a refractive index increment (dn/dC) of 0.155 mL g 1 was used to calculate the molecular weight and polydispersity (Mw/Mn) of the ester conjugate.
  • a solution of an ester conjugate prepared according to Example 12 at a concentration of 5 g.L" 1 was deposited in an amount of 5 pl on a special cryomicroscopy support.
  • the applied droplet was then covered with a second carrier.
  • the sample thus prepared was transferred to a slush freezer (Leica, Germany) at -210 °C.
  • the sample remained in the slush freezer for a few seconds and was then transferred to the sample preparation station (Leica, Germany). In this station, the sample was attached to a special sample holder for cryomicroscopy.
  • This prepared sample holder was transferred to the EM ACE600 sputter machine (Leica, Germany) using an EM VCT500 (Leica, Germany) shuttier (under vacuum, at -120 °C).
  • ETD secondary electron detector
  • the recorded images are shown in Fig. 16 and Fig. 17.
  • Hyaluronic acid vinyl esters A toolbox toward controlling mechanical properties of hydrogels for 3D microfabrication. Journal of Polymer Science, 58(9), 1288-1298. doi: https://doi.org/10.1002/pol.20200073

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Abstract

The present invention relates to an ester conjugate of sphingolipid and hyaluronan, a composition comprising thereof and its use. Furthermore, it relates to methods of the ester conjugate synthesis.

Description

Ester conjugate of sphingolipid and hyaluronan, method of synthesis thereof, composition containing thereof, use of conjugate or composition.
Field of the invention
The present invention relates to an ester conjugate of sphingolipid and hyaluronan, a composition comprising thereof and its use. Furthermore, it relates to methods of the ester conjugate synthesis.
State of the art
Sphingolipids are a class of lipids containing a backbone of sphingoid bases, a set of aliphatic amino alcohols. Ceramides (CERs) are epidermal sphingolipids necessary for the proper skin barrier function and are composed of fatty acid and a sphingoid base, varying the size and structure of their molecular chains. CERs constituted up to 50% (M'/W) of the lipid matrix of the epidermis in the Stratum corneum (SC). Newer formulations of skin-care products have incorporated CERs into their formulations, intending to exogenously apply CERs to help skin barrier function. CERs support the skin’s natural protective layer renewal and form an effective epidermal barrier.
Several skin diseases, such as psoriasis, ichthyosis, xerosis and atopic dermatitis, are associated with the depletion or disturbance of SC lipids such CERs, free fatty acids or cholesterol (Uchida & Park, 2021 ). Even though CER-containing products have been increasing on the market, there is limited clinical evidence showing the depth and permeation of the exogenous CERs into the skin. Few reports have shown the transport of exogenous CERs in human skin (ex vivo). Furthermore, the penetration of CER using a hydrophilic composition is lower than 10% (Tessema, Gebre-Mariam, Paulos, Wohlrab, & Neubert, 2018).
To facilitate the permeation of ceramides into the SC, surfactants such as lecithin or polyglyceryl-4-laurate are used (Sahle, Wohlrab, & Neubert, 2014). However, polyglycerol esters of fatty acids (PEFA) are toxic and potentially carcinogenic depending on manufacturing processes and starting materials impurities such as epichlorohydrin, glycidol, erucic acid and trans-fatty acids may be present in PEFA. Furthermore, it is challenging to incorporate CER into hydrophilic pharmaceutical formulations due to their poor solubility in pharmaceutically allowed solvents and/or excipients.
In the related art, an oil-in water nanoemulsion-based formulation containing ceramide IIIB using phase-inversion temperature was developed for transdermal delivery. The composition consisted of ethanol, propylene glycol (PG), and glycerol in the cosmetic oil Tegosoft® G20 (octyldodecanol) and Tween® 80 (polysorbate 80). The optimized composition showed an average droplet size from 11.2 to 40.1 nm, and polydispersity was estimated to be 0.12. However, there is an impasse related to skin permeability and penetration of CERs through topical administration. Furthermore, certain classes of CERs do not penetrate through the SC, resulting in no notable clinical responses (Su et al., 2017). These divergences in skin permeation may be related to the presence of penetration enhancers in the formulations, as well as to the skin health condition.
Licciardi et al. synthesized a succinyl derivative of ceramide (Licciardi, Scialabba, Sardo, Cavallaro, & Giammona, 2014). The authors reacted the ceramide AP with succinic anhydride in anhydrous tetrahydrofuran (THF). The resulting mixture was kept at 40 °C for 6 h followed by 16 h at 25 °C. In a second step, the authors reacted bis-(4-nitrophenyl)carbonate (BNPC) with inulin in a reaction under microwaves’ irradiation. However, these conditions cannot be applied to the chemical modification of HA, which is prone to degrade in the presence of external irradiation (Dfimalova, Velebny, Sasinkova, Hromadkova, & Ebringerova, 2005).
A chitosan-ceramide (CS-CE) graft copolymer is suitable for forming polymeric nanoparticles as a carrier for hydrophobic drugs. The conjugate CS-CE formed nanoparticles with a mucoadhesive chitosan corona and hydrophobically stabilized core by CER moieties, wherein hydrophobic drugs were encapsulated, providing an attractive oral drug delivery system. In this work, Battogtokh and Tag Ko carried out the activation of the ceramide by means of N, -disuccinimidyl carbonate (DSC). Battogtokh coupled chitosan with the activated CER NP in a mixture of dimethyl sulfoxide (DMSO)/THF (Battogtokh & Ko, 2014). The activation reaction uses anhydrous solvents, wherein sodium salt of HA is insoluble. Second, DSC reaction is also known to activate secondary alcohols (Z. Li et al., 2003). Thus, it might act as a crosslinking agent of HA.
Cho et al. conjugated CER-Y30 (ceramide NP 18:1, ceramide 3B; mainly N-oleoyl- phytosphingosine) to HA oligomers (4,700 g.mol-1), giving an amphiphilic polymer (Cho et al., 2011). First, the authors prepared tetrabutylammonium salt of HA (HA-TBA). In a second step, the ceramide was reacted with 4-chloromethyl-benzoyl chloride, used as a linker. In a second step, the modified ceramide was conjugated to the carboxyl moiety of HA-TBA via ester bond formation in a reaction performed in a mixture of THF/acetonitrile (4:1, v/v). The degree of substitution (DS) was reported as 2.38%. The authors reported that DS-Y30 ceramide (8.59 mmol) was added to 12.21 mmol of HA, which means the reaction produced a low conversion and needed organic solvents making the process expensive for upscale. Furthermore, the decomposition products of the aromatic spacer are not known, and the polysaccharide was modified on HA's carboxyl moiety, and therefore limiting the ability of the conjugate to interact with the CD44 receptor. Similar conditions were reported by Jin et al. for physicochemical characterization. Critical micellar concentration was reported as 0.042 mg. ml/1 for HA-CE and was determined by fluorescence (Jin et al., 2012).
Turley etal. chemically modified HA with phospholipids or sphingolipids selected from the group consisting of phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, sphingosines, ceramides, and ceramide-based lipids. The most preferred is the use of phosphatidylethanolamines. The modification efficiency would be decreased if the reaction is performed in water (US20150368373-A1).
Generally, as mentioned above, there are several disadvantages and problems concerning the different polarity and solubility of sphingolipid and HA, the necessity of HA conversion into tetrabutylammonium (TBA) salt, the use of costly and anhydrous solvents, insufficient conversion and degree of substitution of HA derivative and the use of linker with unknown biological properties.
Summary of the present invention
The problems mentioned above are solved in the present invention concerning ester conjugate of sphingolipid and hyaluronan, method of synthesis thereof, composition containing thereof, use of conjugate or composition.
The subject-matter of the invention concerns an ester conjugate of sphingolipid and hyaluronan of a general formula I:
Figure imgf000004_0001
(I), wherein n is integer in the range of 7 to 4000 dimers,
R5 is H+ or pharmaceutically acceptable salt,
R4 is -H or linked sphingolipid substituent of a general formula II
Figure imgf000005_0001
(II), wherein
A is CH2-CH2, CH=CH or C(H)OH-CH2, if any of H bonded to C atom of CH2-CH2 or CH=CH or C(H)OH-CH2 is substituted by a hydroxyl group, it can be esterified by R3 that bonds hyaluronic acid or pharmaceutically acceptable salt thereof with a covalent bond,
R1 is a straight chain or branched alkyl group having 10 to 22 carbon atoms which may optionally contain one or more double bonds, any of H bonded to C atom of said alkyl group is optionally substituted with one or more hydroxyl groups, preferably is a straight chain alkyl group having 12 to 18 carbon atoms, more preferably is a straight chain alkyl group having 13 carbon atoms,
R6 is H or COR2, wherein
R2 is a straight chain or branched alkyl group having 13 to 55 carbon atoms, preferably 15 to 50 carbon atoms, more preferably 17 to 23 carbon atoms, that may optionally contain one or more double bonds; the alkyl chain may contain an internal ester group; or H of the alkyl group may optionally be substituted with one or more hydroxyl groups,
R3 is -H or -OC-CH2-CH2-CO- or -OC-CH2=CH2-CO-, and providing that at least one R3 is - OC-CH2-CH2-CO- or -OC-CH2=CH2-CO-, wherein free -OC- part is covalently bound to -O- at R4 position of the conjugate of sphingolipid and hyaluronan of the general Formula I, providing that at least one R4 of the conjugate is the linked sphingolipid substituent of the
Formula II and wherein the degree of substitution of the linked sphingolipid substituent of the general formula II in the conjugate of hyaluronan is in the range from 0.1 to 15 %.
At least one linked sphingolipid substituent of the Formula II that it is stated above as possible group R4, binds to hyaluronic acid via carbon of the end group -OC- of R3 substituent, forming an ester bonds between them and to form the ester conjugate of sphingolipid and hyaluronan of the general Formula I according to the present invention.
The weight average molecular weight of the conjugate of the formula I according to the present invention is in the range of 3,000 g/mol to 1,600,000 g/mol, preferably in the range from 3,000 to 20,000 g/mol, more preferably 5,000 to 7,000 g/mol.
The degree of substitution in the conjugate of hyaluronan according to the present invention is in the range from 0.4 to 15 %, preferably the degree of substitution is in the range of 5.5 to 10.5%, more preferably the degree of substitution is in the range of 6.5 to 8.5 %.
The pharmaceutically acceptable salt of the conjugate of hyaluronan according to the present invention, wherein is selected from a group comprising any of ions of alkali metals or ions of alkaline-earth metals, preferably Na+, K+ or Mg2+.
The preferred embodiement of the invention is the ester conjugate wherein A is CH2- CH2, CH=CH or C(H)OH-CH2, if A being C(H)OH-CH2, its hydroxyl group can be esterified with R3 which binds hyaluronic acid or pharmaceutically acceptable salt thereof by a covalent bond.
Another embodiment of the present invention is a method of synthesis of the ester conjugate of sphingolipid and hyaluronan of the present invention wherein a sphingolipid of a general formula III
Figure imgf000006_0001
wherein A is CH2-CH2, CH=CH or C(H)OH-CH2,
R1 is a straight chain or branched alkyl group having 10 to 22 carbon atoms which may optionally contain one or more double bonds, any of H bonded to C atom of said alkyl group is optionally substituted with one or more hydroxyl groups, preferably is a straight chain alkyl group having 12 to 18 carbon atoms, more preferably is a straight chain alkyl group having 13 carbon atoms,
R6 is H or COR2, wherein
R2 is a straight chain or branched alkyl group having 13 to 55 carbon atoms, preferably 15 to 50 carbon atoms, more preferably 17 to 23 carbon atoms; the alkyl chain may contain an internal ester group; may optionally contain one or more double bonds, and may optionally be substituted with one or more hydroxyl groups; is attached to a linker that is a rest of succinic acid or a rest of maleic acid by adding of succinic anhydride or maleic anhydride in a polar solvent in the presence of an organic base or mixtures thereof to form a linked sphingolipid of a general formula IV
Figure imgf000007_0001
wherein
A is CH2-CH2, CH=CH or C(H)OH-CH2,
R1 is a straight chain or branched alkyl group having 10 to 22 carbon atoms which may optionally contain one or more double bonds, any of H bonded to C atom of said alkyl group is optionally substituted with one or more hydroxyl groups, preferably is a straight chain alkyl group having 12 to 18 carbon atoms, more preferably is a straight chain alkyl group having 13 carbon atoms, R6 is H or COR2, wherein
R2 is a straight chain or branched alkyl group having 13 to 55 carbon atoms, preferably 15 to 50 carbon atoms, more preferably 17 to 23 carbon atoms; the alkyl chain may contain an internal ester group; may optionally contain one or more double bonds, and may optionally be substituted with one or more hydroxyl groups;
R7 is -H or -OC-CH2-CH2-COOH or -OC-CH=CH-COOH, and providing that at least one R7 is -OC-CH2-CH2-COOH or -OC-CH=CH-COOH, that is activated with an activation agent selected from a group comprising 1,1'- carbonyldiimidazole or tetramethyl-O-(A-succinimidyl)uronium hexafluorophosphate or benzoyl chloride, in the presence of an organic solvent to form an activated linked sphingolipid of a general formula V
Figure imgf000008_0001
wherein
A is CH2-CH2, CH=CH or C(H)OH-CH2,
R1 is a straight chain or branched alkyl group having 10 to 22 carbon atoms which may optionally contain one or more double bonds, any of H bonded to C atom of said alkyl group is optionally substituted with one or more hydroxyl groups, preferably is a straight chain alkyl group having 12 to 18 carbon atoms, more preferably is a straight chain alkyl group having 13 carbon atoms, R6 is H or COR2, wherein
R2 is a straight chain or branched alkyl group having 13 to 55 carbon atoms, preferably 15 to 50 carbon atoms, more preferably 17 to 23 carbon atoms; the alkyl chain may contain an internal ester group; may optionally contain one or more double bonds, and may optionally be substituted with one or more hydroxyl groups;
R7 is -H or -OC-CH2-CH2-CO- or -OC-CH=CH-CO-, and providing that at least one R7 is -OC- CH2-CH2-CO- or -OC-CH=CH-CO- if R7 is -H, then -O-R9 group is not attached to it;
R9 is substituent selected from a group of substituents comprising a substituent of a general formula VI or a substituent of formula VII or a substituent of formula VIII, wherein R8 is one or more substituents selected from a group comprising H, -NO2, -COOH, halides, Ci-Ce alkyloxy, preferably halides, methoxy or ethoxy, more preferably Cl,
Figure imgf000009_0001
that reacts with hyaluronic acid or a pharmaceutically acceptable salt thereof in the presence of a mixture of water and water-miscible organic solvent or in the presence of an organic solvent, preferably dimethyl sulfoxide.
The concentration of the sphingolipid of the general formula III, is preferably ranging from 0.88 % to 2.5 % (w/v) and the molar equivalent of succinic anhydride or maleic anhydride is ranging from 1.0 to 2.0, preferably 1.5 equivalents with respect to sphingolipid of the general formula III.
The attachment of the linker is preferably earned out in the range of temperatures from 0 °C to 50°C for 4 to 20 hours, more preferably for 6 to 8 hours in the range of temperatures from 0 °C to 40°C, followed by stirring at a temperature in the range of 0 °C to 29 °C, preferably at room temperature for 5 to 24 hours, more preferably for 16 to 20 hours.
The organic base is preferably selected from the group comprising a secondary or tertiary amine having a linear or branched or cyclic or aromatic, saturated or unsaturated C3- C30 alkyl group, and the polar solvent is selected from the group comprising tetrahydrofurane (THF), dioxane, dimethyl sulfoxide, dichloromethane and chloroform or mixtures thereof.
The organic base is preferably selected from the group comprising pyridine, triethylamine, 7V-methylmorpholine, dimethylaminopyridine or mixtures thereof, and the polar solvent is preferably dichloromethane or tetrahydrofurane (THF) or their mixture.
The activation agent is preferably benzoyl chloride that is a substituted or nonsubstituted benzoyl chloride or its derivatives of the general formula IX
Figure imgf000010_0001
wherein R8 is one or more substituents selected from a group comprising H, -NO2, -COOH, halides, Ci-Ce alkyloxy, preferably halides, methoxy or ethoxy, more preferably Cl.
The forming of the activated linked sphingolipid of the general formula V is preferably carried out at the temperature in the range of 5 °C to 60 °C, more preferably 40 °C, for 0.5 to 24 hours.
The molar amount of the activation agent is preferably in the range of 0.03 to 2 molar equivalents, more preferably 1 molar equivalent to a linked sphingolipid of a general formula IV.
The organic solvent is preferably selected from the group comprising isopropanol, tert- butanol, dioxane, dimethyl sulfoxide, acetonitrile and tetrahydrofuran. The concentration of the activated linked sphingolipid of the general formula V that reacts with hyaluronic acid or a pharmaceutically acceptable salt thereof is preferably in the range of 0.25 % to 7.0 % (w/v) in the solution.
The reaction of the activated linked sphingolipid of the formula V and hyaluronic acid or the pharmaceutically acceptable salt thereof is preferably carried out in the range of temperatures 5 °C to 37 °C, for 1 to 72 hours, more preferably at 25°C, for 2 hours.
The water-miscible organic solvent is preferably selected from a group comprising isopropanol, /e/7-butanol, dioxane, dimethylsulfoxide and tetrahydrofuran.
The molar amount of the activated linked sphingolipid of the formula V is 0.01 to 2.0 equivalents, preferably 0.03 to 0.5 equivalents with respect to a dimer of hyaluronic acid.
According to another embodiment of the invention a composition comprises the ester conjugate according to the present invention, preferably the ester conjugate is in a form of a self-assembled polymeric particle.
The concentration of the ester conjugate of the general Formula I in the composition according to the present invention is ranging from 0.001 to 50 % (w/w), preferably from 0.001 to 5 % (w/w), more preferably from 0.001 to 1 % (w/w).
The composition according to the present invention is preferably in the form selected from a group comprising emulsion, serum, hydrogel or buccal patch.
The composition according to the present invention is in the form of emulsion, while the concentration of the ester conjugate according to the general formula I is ranging from 0.001 to 5 % (w/w), preferably from 0.001 to 1 % (w/w).
The composition according to the present invention preferably contains water and at least one cosmetic or pharmaceutical excipient selected from a group containing oil, wax, butter, emulsifier, auxiliary active ingredient, thickener and preservative.
The oil is preferably selected from a group comprising coconut, olive, avocado, sesame, almond, castor, sunflower, hemp, jojoba, argan, apricot, borage, marula, cottonseed, evening primrose, grapeseed, hazelnut, linseed, meadowfoam, moringa, plum, poppy, rice, rosehip, safflower, wheat germ, macadamia oils and squalene; butter is selected from a group comprising cocoa butter, illipe butter, kokum butter, murumuru butter, mango butter, cupuacu butter, avocado butter and shea butter; wax is selected from a group comprising lanolin, beeswax, carnauba wax, candelilla wax and petroleum jelly.
The emulsifier is preferably selected from a group comprising glyceryl stearate, glyceryl caprylate, behenyl alcohol, glyceryl behenate, cetearyl glucoside, methyl glucose sequistearate, glyceryl stearate citrate, polyglyceryl-3 stearate, cetearyl olivate, lecithin, stearyl alcohol, sorbitan oleate, polysorbates, stearic acid, cetyl alcohol and cetearyl alcohol, sodium acrylate, sodium acryloyldimethyltaurate copolymer or mixtures of thererof.
The auxiliary active ingredient is preferably selected from a group comprising vitamins A, D, E, K, C and B group vitamins; curcumin, coenzyme Q10, allantoin, bisabolol, lactic acid, amino acids, a-hydroxy and ^-hydroxy acids, ceramides, peptides, preferably acetyl hexapeptide- 8, palmitoyl tripeptide- 1, palmitoyl tetrapeptide-7, copper tripeptide- 1, palmitoyl pentapeptide-4, Saccharomyces peptides, hexapeptide- 1 and protein, preferably rice protein, soy protein, quinoa protein or wheat protein; polysaccharides, hyaluronic acid or a pharmaceutically and cosmetically acceptable salt thereof, carboxymethyl glucan, schizophyllan, glucomannan; panthenol; urea; glycerin; pentylene glycol; plant extracts, preferably aloe vera extract, chamomile extract, acacia extract, green tea extract, algae extract, oat extract, hemp extract and cranberry extract, bakuchiol, resveratrol; extracts, ferments, lysates or filtrates from bacteria preferably from Lactobacillus, Thalassospira, Bifidobacterium, Halobacterium; or from fungi preferably from Saccharomyces, Agaricus subrufescens, Choiromyces maeandriformis, Cordyceps sinensis, Ganoderma lucidum, Grifola frondosa, Hypsizygus ulmarium, Inonotus obliquus, Lentinula edodes, Polyporus, Trametes versicolor, Tremella fuciformis, Tuber, Schizophyllum commune.
The preservative is preferably selected from a group comprising aromatic acids and their derivatives, preferably benzoic acid, salicylic acid, dehydroacetic acid, potassium sorbate, parabens; alcohols preferably ethanol, isopropanol, benzyl alcohol, phenoxyethanol, phenethyl alcohol; imidazole derivatives preferably hydantoin, imidazolidinyl urea; cationic surfactants preferably benzalkonium chloride.
The composition according to the present invention is in the form of serum, while the concentration of the ester conjugate according to the general formula I is ranging from 0.001 to 5 % (w/w), preferably from 0.001 to 1 % (w/w); while containing water and at least one water soluble auxiliary active ingredient; at least one thickener, and at least one preservative as stated above.
The concentration of the ester conjugate according to the general formula I in the composition according to the present invention is preferably in range from 0.001 to 1 % (w/w).
The water-soluble auxiliary active ingredient is chosen from a group comprising vitamin C; B vitamins; amino acids, a-hydroxy and /Lhydroxy acids, peptides; preferably acetyl hexapeptide-8, palmitoyl tripeptide, copper tripeptide- 1, Saccharomyces peptides, hexapeptide- 1 ; and proteins preferably rice protein, soy protein, quinoa protein or wheat protein; and water- soluble polysaccharides, preferably hyaluronic acid or a pharmaceutically and cosmetically acceptable salt thereof, carboxymethyl glucan, schizophyllan, glucomannan, urea; glycerin; pentylene glycol; plant extracts preferably aloe vera extract, cammomile extract, acai extract, green tea extract, algae extract, oat extract, cannabis extract, cranberry extract; extracts, ferments, lysates and filtrates from bacteria preferably from Lactobacillus, Thalassospira, Bifidobacterium, Halobacterium; or from fungi preferably from Saccharomyces, Agaricus subrufescens, Choiromyces maeandriformis, Cordyceps sinensis, Ganoderma lucidum, Grifola frondosa, Hypsizygus ulmarium, Inonotus obliquus, Lentinula edodes, Polyporus spp., Trametes versicolor, Tremella fuciformis, Tuber, Schizophyllum commune.
The composition according to the present invention additionally contains at least one hydrophobic compound selected from a group comprising cannabidiol, tocopherol, curcumin, coenzyme Qio, ethyl ferulate, resveratrol, bakuchiol, retinyl palmitate, that is encapsulated in the self-assembled polymeric particles of the ester conjugate according to the present invention.
The composition according to the present invention additionally contains thickener that is selected from a group comprising xanthan gum, sclerotium gum, guar gum, cellulose gum, carbomer, hydroxyethylcellulose, Amorphophallus konjac root extract, carrageenan, pullulan, lecithin, lysolecithin, sodium acrylate, sodium acryloyldimethyl taurate copolymer or mixtures thereof.
The composition according to the present invention contains the ester conjugate according to the present invention in the form of a buccal patch.
The composition according to the present invention preferably contains other auxiliary excipient, preferably hyaluronic acid or pharmaceutically acceptable salt of thereof. The use of the ester conjugate of sphingolipid and hyaluronan according to the present invention and composition according to the present invention for treatment of the buccal mucosa. The ester conjugate of sphingolipid and hyaluronan according to the present invention; or the composition according to the present invention for use for treatment of the skin.
The ester conjugate of sphingolipid and hyaluronan and composition according to the present invention for use in the medical treatment of a skin disease selected from the group comprising atopic dermatitis, psoriasis, ichtyosis and rosacea.
The ester conjugate of sphingolipid and hyaluronan and composition according to the present invention for use in the medical treatment of a buccal mucosa disease selected from the group comprising aphthous stomatitis and Behcet's disease.
The application of the composition according to the present invention is topical, buccal, sublingual to the subject, preferably human.
The use of the ester conjugate of sphingolipid and hyaluronan according to the present invention and composition according to the present invention for cosmetic treatment of the skin.
Hyaluronic acid (HA, hyaluronan) is a high-molecular-weight glycosaminoglycan composed of D-glucuronic acid (GlcA) and TV-acetyl-D-glucosamine (GlcNAc) units linked by alternating ?-(l-4)-and /?-(l-3)-glycosidic bonds. HA is found ubiquitously in the human body and is biodegradable and biocompatible. Amphiphilic HA consisting of ceramides or sphingoid base lipids attached to the polysaccharide backbone enhanced the penetration of modified HA through the skin barrier into the epidermal and dermal layers of the skin, thereby allowing the dermal administration of the conjugates without injection or another disruption of the skin. Through their ability to deliver HA to the epidermal and dermal layers in the present formulations is suitable for use in dermal rejuvenation, hyaluronan and ceramides replenishment and protection therapy. Ceramides are used mainly as moisturizers in various cosmetic products (Choi & Lee, 2015). Thus, it is expected a synergism after covalent binding to HA.
Furthermore, according to the present invention, the ester conjugates are useful as delivery devices to facilitate the dermal and transdermal delivery of cosmetically and pharmaceutically active substances through the skin barrier, which can be encapsulated in the hydrophobic core. The self-assembling of the ester conjugates, according to the present invention, as stated herein, is forming self-assembled polymeric particles, which can encapsulate hydrophobic compounds. In previously reported art, it was reported that skin permeation is governed by the size, nanostructure and viscosity of the microemulsions. Smaller droplets and less viscous MEs permeated deeper layers of the skin to a higher extent (Sahle et al., 2014). The ester conjugates, according to the present invention, can self-assemble in the water or in the water solutions while forming self-assembled polymeric particles (size average 65.1 nm and polydispersity pdi= 0.384 (see Figure 13)). Moreover, it suppresses the production of proinflammatory cytokine IL-6 up to 95.3 %. (see Figure 15). Also, the ester conjugates, according to the present invention, could be freeze-dried and form buccal patches for internal application on the buccal mucosa. In one aspect, the lower limit of the weight average molecular weight of the hyaluronan useful herein is from 3,000 g/mol, 6,000 g/mol, 10,000 g/mol, 20,000 g/mol, 50,000 g/mol, 60,000 g/mol, 70,000 g/mol, 80,000 g/mol, 90,000 g/mol, or 100,000 g/mol, and the upper limit is 200,000 g/mol, 300,000 g/mol, 400,000 g/mol, 500,000 g/mol, 600,000 g/mol, 700,000 g/mol and 1,600,000 g/mol. In one aspect, HA has a molecular weight of 6,000 g/mol to 20,000 g/mol.
Preferred are those sphingolipids that can be found in the human skin, such as IV-oleoyl- phytosphingosine - Ceramide NP 18:1
Figure imgf000015_0001
or /V-(2-hydroxyoctadecanoyl)phytosphingosine - Ceramide AP 18
Figure imgf000015_0002
Furthermore, other sphingolipids, according to general Formula III can be used. R1 is preferably selected from a group comprising dodecyl, tridecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, more preferably tridecyl.
R2 is preferably selected from a group comprising (Z)-heptadec-8-ene-l-yl, (6Z,9Z)- heptadeca-6,9-diene-l-yl, pentacosyl, tricosyl, henicosyl, nonadecyl, heptadecyl, pentadecyl;
1 -hydroxyalkyl or tu-hydroxyalkyl, preferably selected from a group comprising 1- hydroxypentacosyl, 1 -hydroxytricosyl, 1-hydroxyhenicosyl, 1 -hydroxynonadecyl, 1- hydroxyheptadecyl, 1 -hydroxypentadecyl, co-hydroxyheptacosyl, m-hydroxynonacosyl, a>- hydroxyhentriacontyl. The most preferred is (Z)-heptadec-8-ene-l-yl, 1 -hydroxyheptadecyl, 1- hydroxytricosyl, 1 -hydroxypentadecyl.
Furthermore, m-hydroxyl of alkyl chains may be esterified with another, typically unsaturated carboxylic acid. This modification creates an internal ester group in the fatty acid chain as might be seen in Ceramides EOS, EOP, EOdS; while preferred R2 according to general Formula III is 29-[(9Z,12Z)-octadeca-9,12-dienoyloxy]nonacosyl. Examples of other ceramides classes are shown in the following figure.
Figure imgf000017_0001
The first step of the method of synthesis of the ester conjugate of sphingolipid and hyaluronan, according to the present invention, is the reaction of the sphingolipid of the general formula II with the linker, which is a rest of succinic acid or a rest of maleic acid, and which is added to the reaction mixture in the form of succinic anhydride or maleic anhydride. In this step, the sphingolipid of the general formula II is dissolved in organic solvents THF or DMSO or dichloromethane, preferably in the presence of an inert atmosphere provided by gasses nitrogen or argon. Afterwards the organic base and succinic anhydride or maleic anhydride is added. The reaction is performed at 0 °C to 50°C for 4 to 20 hours, more preferably for 6 to 8 hours in the range of temperatures from 0 °C to 40°C, followed by stirring at a temperature in the range of 0 °C to 29°C, preferably at room temperature for 5 to 24 hours, more preferably for 16 to 20 hours. However, the inert atmosphere is not necessary for the successful performance of the reaction.
The second step (activation) comprises preparing the activated linked sphingolipid of a general formula VI in an organic solvent. The preferred solvents used in the reaction are isopropanol, /e/7-butanol, THF, 1,4-dioxane, acetonitrile and DMSO. The temperature of the activation is crucial for the formation of the intermediate. The activation reaction is carried out between 5 to 60 °C. The reaction is performed in a time ranging from 0.5 to 24 hours. Preferably, the activation reaction is performed between 35 to 60°C. More preferably, the activation reaction is performed at 40°C. The activation of sphingolipids can be performed by activating agents described in the art, i.e. 1,1 ’ -carbonyl diimidazole (CDI), tetramethyl-<9-(iV- succinimidyl)uronium hexafluorophosphate (HSTU) or benzoyl chloride. Activating agents CDI and HSTU produced a low degree of HA chemical modification (up to 2.8 %). Also, these activators reacted under anhydrous conditions, as shown in the Examples. The activating agent benzoyl chloride provides considerable advantages. The advantage of benzoyl chloride in activation reaction is the short activation time.
The third step is the esterification of HA with the activated linked-sphingolipid of a general formula VI, which proceeds at low temperatures (from 0 to 25 °C). This reaction is performed in water mixed with an organic solvent miscible with water or in an organic solvent miscible with water.
Among the advantages of the method, according to the present invention, are high efficiency in the second step of activation and the third step of esterification and the possibility of performing HA esterification in the mixture of water and water-miscible organic solvent (1 :1 v/v), which allows the dissolution of sodium hyaluronate directly in water. Thus, the conversion of sodium hyaluronate to TBA salt or acid form before the modification could be avoided.
According to the method of the present invention, the esterification reaction is kept under constant stirring for 1-72 hours, more preferably for 2 hours. This reaction is highly selective, and the final esterification products are characterized by at least one of the hydroxyl groups of HA that have been esterified with the activated linked sphingolipid of the general formula VI. The esterification of HA with the activated linked sphingolipid of the general formula VI can reach DS up to 15% (mol/mol) using activating agent benzoyl chloride. In contrast, other mentioned activating agents require converting sodium hyaluronate to acid form of hyaluronic acid to be dissolved in anhydrous DMSO.
The ester conjugate of sphingolipid and hyaluronan according to the present invention facilitates its penetration in a 1-15% (mol/mol) through the skin barrier into the dermis and epidermis layers. Particularly, these ester conjugates contain sphingolipids, which are covalently attached to hyaluronan. This invention provides a chemical modification of hyaluronan performed in water- containing solvent industrially applicable. The ester conjugates prepared by the method according to the invention can self-assemble in water or water solutions into self-assembled polymeric particles. The self-assembled ester conjugates according to the present invention can also form self-assembled polymeric particles with encapsulated active substances selected from a group comprising curcumin, tocopherol, cannabidiol, coenzyme Qio, ethyl ferulate and miconazole. The active substance is encapsulated into the conjugates preferably in propan-2-ol water solutions. The average size of the self-assembled polymeric particles in water or water solutions of salts is in the range 10 nm to 10 000 nm by volume. The preferred average size of self-assembled ester conjugate is in the range from 10 nm to 100 nm by volume.
Furthermore, the ester conjugate, according to the present invention, is able to suppress the production of proinflammatory cytokine IL-6 up to 95 %. Prepared ester conjugates could be used in various liquid (e.g. serum) or semi-solid (e.g. facial cream) or solid (e.g. buccal patch) forms.
Prepared ester conjugates of sphingolipid and hyaluronan according to the present invention are water-soluble and therefore can be included into water phase of the liquid, semisolid or solid compositions in amounts ranging from 0,001 to 7.5 % (w/w), preferably from 0,001 to 5 % (w/w), more preferably from 0,001 to 1 % (w/w); and also can be used with encapsulated hydrophobic active substance. The composition is preferably selected from a group comprising emulsion (oil-in-water, water in oil, a micro- or nano-emulsion, serum (aqueous or hydro-alcoholic solution or gel, sterile or nonsterile), anhydrous gel, paste, dispersion of vesicles, powder, nanofibers, macro-, micro- or nano-capsules, macro-, micro- or nano-spheres, liposomes, oleosomes or chylomicrons, macro-, micro- or nano-sponges which may be adsorbed on organic polymer powders, talcs, bentonites and other inorganic or organic supports. The composition contains at least one cosmetic or pharmaceutical excipient, selected from a group containing oil, wax, butter, emulsifier, auxiliary active ingredient, preservative and thickener.
Definitions
DS = degree of substitution = 100 % * molar amount of the bound substituent / molar amount of polysaccharide dimers. The determination of the degree of substitution was performed by NMR.
The weight average molecular weight of the native used HA was determined by SEC-MALLS method before chemical modification.
The term “room temperature” refers to a temperature range of 22°C to 25°C.
Equivalent (eq.) refers to a dimer of hyaluronic acid or a linked sphingolipid of a general formula V. It is a molar equivalent unless otherwise stated.
The term “self-assembled polymeric particles” refers to the organized macromolecular units of ester conjugate according to the present invention ordered into structures by non-covalent interactions, with particle diameter ranging from 10 to 10 000 nm, by volume. The preferred diameter of self-assembled ester conjugate particles is in the range of 10 nm to 100 nm by volume.
Detailed description of drawings
Figure 1. Edited 'H NMR of 4-(((2S,3S,4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4- oxobutanoic acid in CDCI3.
Figure 2. FT-IR spectrum of 4-(((2S,3S,4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4- oxobutanoic acid.
Figure 3. (A) 4-(((2S,3)S',4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative dissolved in D2O and (B) ]H NMR spectra of native 6,000 g/mol sodium hyaluronate dissolved in D2O. Figure 4. Edited heteronuclear single quantum coherence (HSQC) NMR spectrum of 4- (((2>S',3S,4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate (15,000 g/mol) with signals assigned to the structure.
Figure 5. FT-IR spectrum of (A) native sodium hyaluronate (6,000 g mol'1), (B) 4-(((2S',3S,4R)-
3.4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate and (C) 4-(((2S',3>S',4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid
Figure 6. Edited 'H NMR spectrum of 4-oxo-4-(((2S,3S,4R)-l,3,4-trihydroxyoctadecan-2- yl)amino)butanoic acid in DMSO-fi with signals assigned to the structure.
Figure 7. FT-IR spectrum of 4-oxo-4-(((2S,3S,4R)-l,3,4-trihydroxyoctadecan-2- yl)amino)butanoic acid.
Figure 8. Edited ’H NMR spectrum of 4-oxo-4-(((2S’,3S,4R)-l,3,4-trihydroxyoctadecan-2- yl)amino)butanoic acid derivative of sodium hyaluronate.
Figure 9. DOSY NMR spectrum of 4-oxo-4-(((2S,3S,4R)-l,3,4-trihydroxyoctadecan-2- yl)amino)butanoic acid derivative of sodium hyaluronate (15,000 g mol-1) in deuterated water: acetonitrile (4:1). Signals ordered approximately in one line (especially methylene and methyl groups) suggests modification of hyaluronan.
Figure 10. (A) FT-IR spectrum of native sodium hyaluronate and (B) 4-oxo-4-(((2<S,,3S,4R)-
1.3.4-trihydroxyoctadecan-2-yl)amino)butanoic acid derivative of sodium hyaluronate.
Figure 11. Cell viability of NIH-3T3 fibroblasts after treatment with 4-(((2S',3S',4R)-3,4- dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate prepared according to Examples 12, 13, 14, 18 and 17 (from left to right) after 24 h, 48 h, and 72 h.
Figure 12. Penetration of 4-(((2S,3S,4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4- oxobutanoic acid derivatives of sodium hyaluronate with non-covalently bound Nile red in swine skin.
Figure 13. Size determination of self-assembled polymeric particles of the ester conjugate in water, prepared according to Example 10, using dynamic light scattering.
Figure 14. Population sizes of several prepared of sodium salt of the ester conjugates prepared according to Examples 12 to Example 14 Figure 15. Immunomodulatory properties of sodium salt of the ester conjugate prepared according to Examples 14 and 17 compared to corresponding free ceramide NP 18:1 and native sodium hyaluronate (6 kDa).
Obr. 16. Cryo-scanning electron microscopy image of self-assembled polymer particles of the conjugate prepared according to Example 12. The image shows white spherical particles of the ester conjugate.
Obr. 17. Cryo-scanning electron microscopy image of self-assembled polymer particles of the conjugate prepared according to Example 12. The image shows white spherical particles of the ester conjugate with diameters of 29.5 nm and 37.1 nm.
Notes to Figure 15:
Concentration of free ceramide NP 18:1 corresponding to ceramide NP 18:1 bound to the conjugate prepared according to Example 14.
“Concentration of free ceramide NP 18:1 corresponding to ceramide NP 18:1 bound to the conjugate prepared according to Example 11.
Examples
Example 1
Preparation of 4-(((28',3>S',47?)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid
To a solution of (Z)-M[(2S,3S,4 ?)-l,3,4-trihydroxyoctadecan-2-yl]octadec-9-enamide (200 mg, 0.344 mmol) in 2.2 mL of THF and triethylamine (TEA, 20 pL, 0.148 mmol), was dropwise added a solution of succinic anhydride (51.6 mg, 0.516 mmol) in 0.5 mL THF under nitrogen. The resulting mixture was heated at 40 °C for 6 h followed by stirring for 16 h at 25 °C. The reaction mixture was concentrated under reduced pressure, dissolved in CHCI3 (10 mL) and washed with demineralized water (4x 10 mL). The organic fraction was concentrated under reduced pressure to obtain a colorless wax. Yield: 96 %. Regioselectivity of the primary hydroxyl modification: 75 % 'H NMR (CDC13, 700 MHz) signals: 8 11.51 ppm (br. s, 1H), 6.48-6.40 ppm (d, 1H), 5.32-5.23 (m, 2H), 4.88-4.80 (m, 0.25H), 4.57-4.50 (m, 0.25H), 4.47-4.42 (dd, J=11.9 Hz, 0.75H), 4.24- 4.20 (dd, J=11.6 Hz, 0.75H), 4.18-4.13 (m, 1H), 3.60-3.50 (m, 2H), 2.65-2.54 (m, 4H) 2.19- 2.09 (m, 2H), 2.0-1.88 (m, 4H) 1.60-1.40 (m, 4H), 1.32-1.05 (m, 44H), 0.81 (m, 6H). Example 2 Preparation of 4-(((25,35,4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid
To a solution of (Z)-/V-[(2>S',3.S',47?)-l ,3,4-trihydroxyoctadecan-2-yl]octadcc-9-enamide (20 g, 34.367 mmol) in 690 mL of THF and triethylamine (TEA, 3.832 mL, 27.494 mmol), was added a solution of succinic anhydride (5.155 g, 51.551 mmol) in 110 mL of THF. The resulting mixture was heated at 40 °C for 6 h followed by stirring for 16 h at 25 °C. The reaction mixture was concentrated under reduced pressure, dissolved in CHCh (600 mL) and washed with demineralized water (4x 200 mL). The organic fraction was concentrated under reduced pressure. The residue was dissolved in 50 ml of propan-2-ol and concentrated under reduced pressure to obtain a colorless wax. Yield: 99 %. Regioselectivity of the primary hydroxyl modification: 65 % ^ NMR ^DCh, 700 MHz) signals: 8 11.51 ppm (br. s, 1H), 6.48-6.40 ppm (d, 1H), 5.32-5.23 (m, 2H), 4.88-4.80 (m, 0.35H), 4.57-4.50 (m, 0.35H), 4.47-4.42 (dd, J=11.9 Hz, 0.65H), 4.24- 4.20 (dd, J=11.6 Hz, 0.65H), 4.18-4.13 (m, 1H), 3.60-3.50 (m, 2H), 2.65-2.54 (m, 4H) 2.19- 2.09 (m, 2H), 2.0-1.88 (m, 4H) 1.60-1.40 (m, 4H), 1.32-1.05 (m, 44H), 0.81 (m, 6H). Example 3
Preparation of 4-(((2S',3S',4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid
To a solution of (Z)-A-[(2S,3S,47?)-l,3,4-trihydroxyoctadecan-2-yl]octadec-9-enamide (20 g, 34.367 mmol) in 690 mL of 1,4-dioxane and triethylamine (TEA, 3.832 mL, 27.494 mmol), was added a solution of succinic anhydride (5.155 g, 51.551 mmol) in 110 mL of 1,4- dioxane. The resulting mixture was heated at 40 °C for 6 h followed by stirring for 16 h at 25 °C. The reaction mixture was concentrated under reduced pressure, dissolved in CHCh (600 mL) and washed with demineralized water (4x 200 mL). The organic fraction was concentrated under reduced pressure. The residue was dissolved in 50 ml of propan-2-ol and concentrated under reduced pressure to obtain a colorless wax. Yield: 99 %. Regioselectivity of the primary hydroxyl modification: 63 % ’HNMR (CDCI3, 700 MHz) signals: 8 11.51 ppm (br. s, 1H), 6.48-6.40 ppm (d, 1H), 5.32-5.23 (m, 2H), 4.88-4.80 (m, 0.37H), 4.57-4.50 (m, 0.37H), 4.47-4.42 (dd, J=1 L9 Hz, 0.63H), 4.24- 4.20 (dd, J=11.6 Hz, 0.63H), 4.18-4.13 (m, 1H), 3.60-3.50 (m, 2H), 2.65-2.54 (m, 4H) 2.19- 2.09 (m, 2H), 2.0-1.88 (m, 4H) 1.60-1.40 (m, 4H), 1.32-1.05 (m, 44H), 0.81 (m, 6H). Example 4
Preparation of 4-(((21S',3.S',4/ )-3,4-dihydroxy-2-olcamidooctadecyl)oxy)-4-oxobutanoic acid
To a solution of (Z)-A-[(2S,3S,4R)-l,3,4-trihydroxyoctadecan-2-yl]octadec-9-enamide (25 mg, 0.043 mmol) in 1.5 mL of dichloromethane (DCM), pyridine (6.9 pL, 0.148 mmol) and DMAP (1 mg, 0.009 mmol), was dropwise added a solution of succinic anhydride (8.6 mg, 0.086 mmol) in 1 mL DCM under nitrogen. The resulting mixture was heated and refluxed for 24 h. The reaction mixture was concentrated under reduced pressure, dissolved in CHCI3 (2 mL) and washed with demineralized water (4x 2 mL). The organic fraction was concentrated under reduced pressure to obtain a colourless wax. Yield: 100 %. No regioselectivity was observed.
*H NMR (CDCI3, 700 MHz) signals: 8 11.51 (br. s, 1H), 6.48-6.40 (d, 1H), 5.32-5.23 (m, 2H), 5.16-5.10 (m, 0.13H), 5.05-4.96 (m, 0.73H), 4.96-4.87 (m, 0.59H), 4.66-4.56 (m, 0.50H), 4.34-4.21 (m, 0.48H), 4.13-4.08 (m, 0.65H), 3.80-3.64 (m, 0.49H), 3.64-3.57 (m, 0.52H) 2.65-2.54 (m, 7H) 2.19-2.09 (m, 2H), 2.0-1.88 (m, 4H) 1.60-1.40 (m, 4H), 1.32-1.05 (m, 44H), 0.81 (m, 6H).
Example 5
Preparation of 4-(((2S,3S,4 ?)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid
A solution of (Z)->-[(2S,3S,47?)-l,3,4-trihydroxyoctadecan-2-yl]octadec-9-enamide (50 mg, 0.086 mmol) was prepared in THF (2.5 mL), pyridine (13.8 pL, 0.172 mmol ) and DMAP (2.1 mg, 0.017 mmol), were dropwise added a solution of succinic anhydride (19.9 mg, 0.129 mmol) in 0.5 mL THF. The resulting mixture was heated at 40 °C for 6 h followed by stirring for 16 h at 25 °C. The reaction mixture was concentrated under reduced pressure, dissolved in CHCI3 (2 mL), and washed with demineralized water (4x 2 mL). The organic fraction was concentrated under reduced pressure to obtain a colourless wax. Yield: 98 %. Regioselectivity of the primary hydroxyl group modification was 61%.
'H NMR (CDCI3, 700 MHz) signals: 8 11.51 (br. s, 1H), 6.69-6.64 (d, 0.229H), 6.59-6.47 (d, 0.58H), 6.22-6.15 (m, 0.13H) 5.32-5.23 (m, 2H), 4.93-4.81 (dd, 0.38H), 4.57-4.50 (m 0.35H), 4.47-4.42 (dd, J=11.9 Hz, 0.61H), 4.24-4.20 (dd, J=11.6 Hz, 0.61H), 4.18-4.13 (m, 0.61H), 3.60-3.50 (m, 2H), 2.65-2.54 (m, 4H) 2.19-2.09 (m, 2H), 2.0-1.88 (m, 4H) 1.60-1.40 (m, 4H), 1.32-1.05 (m, 44H), 0.81 (m, 6H).
Example 6
Preparation of (Z)-4-(((2S,3>S',4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobut-2-enoic acid using A-methylmorpholine
To a solution of (Z)-A/-[(25',31S',4/?)-l ,3,4-trihydroxyoctadccan-2-yl]octadec-9-enamide (200 mg, 0,344 mmol) in 11.5 mL of THF and AMnethyl-morpholine (42 pL, 0.378 mmol), a solution of maleic anhydride (50 mg, 0.516 mmol) in 0.5 mL THF was dropwise added under nitrogen. The reaction was kept at room temperature for 22 h. The reaction mixture was concentrated under reduced pressure, dissolved in CHCI3 (15 mL) and washed with demineralized water (4x 15 mL). The chloroform fraction was concentrated under reduced pressure to obtain a colourless wax. Yield: 96 %. Regioselectivity of the primary hydroxyl group modification was 70 %.
*H NMR (DMSO-d6, 700 MHz) signals: 5 12.75 (br. s, 1H), 7.87-7.81 (d, 0.25H), 7.68-7.61 (d, 0.61H), 6.48-6.08 (m, 2H), 5.41-5.31 (m, 2H), 4.28-4.04 (m, 1H), 3.65-3.60 (m, 1H) 3.42-3.37 (m, 1H), 3.36-3.30 (m, 1H), 2.16-2.06 (m, 2H), 2.05-1.79 (m, 4H) 1.65-1.42 (m, 4H), 1.32-1.05 (m, 44H), 0.90-0.82 (t, 6H)
Example 7
Preparation of (Z)-4-(((2S,3S,4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobut-2-enoic acid using TEA
To a solution of (Z)-Y-[(2S,3S,4R)-l,3,4-trihydroxyoctadecan-2-yl]octadec-9-enamide (40 mg, 0.069 mmol) in 1.6 mL of THF and TEA (4 pL, 0.03 mmol), a solution of maleic anhydride (8.8 mg, 0.089 mmol) in 0.2 mL THF was dropwise added under nitrogen. The reaction was kept at 0 °C for 7 h, and subsequently 17 h at 25 °C. The reaction mixture was concentrated under reduced pressure, dissolved in CHCh (2 mL) and washed with demineralized water (4x 2 mL). The chloroform fraction was concentrated under reduced pressure to obtain a colorless wax. Yield: 94 %. Regioselectivity of the primary hydroxyl group modification was 60 %.
*H NMR (CDCI3, 700 MHz) signals: 8 12.75 (br. s, 1H), 7.48-7.36 (1H), 6.48-6.08 (m, 2H), 5.41-5.31 (m, 2H), 4.44-4.40 (dd, 0.64H), 4.40-0.35 (dd, 0.60H), 4.27-4.16 (m, 0.75H), 4.09- 4.05 (m, 0.25H), 3.62-3.53 (2H), 2.16-2.06 (m, 2H), 2.05-1.79 (m, 4H), 1.65-1.42 (m, 4H), 1.32-1.05 (m, 44H), 0.90-0.82 (t, 6H) Example 8
Preparation of 4-oxo-4-[(2S,,3iS',47?)-l,3,4- trihydroxyoctadecan-2-yl]aminobutanoic acid using pyridine
(2S,3S,4R)-2-aminooctadecane-l,3,4-triol (30 mg, 0.094 mmol) was dissolved in 3.1 mL of THF/CH2CI2 (3:2; v/v). Subsequently, pyridine (0.002 ml, 0.019 mmol) and succinic anhydride (9.5 mg, 0.094 mmol) in 0.2 mL of THF was added dropwise under argon. The reaction was kept at 0 °C for 7 h. Afterwards, the mixture was allowed to warm spontaneously to room temperature, followed by stirring for 17 h. The reaction mixture was concentrated under reduced pressure, and the residue was crystallized from ethyl acetate. The white precipitate was centrifuged, decanted and dried to give a white solid. Yield 79 %.
'H NMR (DMSO-d6, 700 MHz) signals: 8 12.05 (br. s, 1H), 7.57 (d, J=8.9 Hz, 1H), 4.59 (s, 1H), 4.46 (s, 1H), 4.30 (s, 1H), 3.93-3.85 (m„ 1H), 3.57-3.52 (dd, J=10.7 HZ, 1H), 3.49-3.43 (dd, J=8.4 HZ, 1H), 3.40-3.20 (m, 2H), 2.42-2.38 (m, 2H), 2.36-2.32 (m, 2H), 1.56-1.47 (m, 1H), 1.46-1.37 (m, 1H), 1.32-1.14 (m, 24H), 0.87-0.82 (m, 3H).
Example 9
Preparation of 4-oxo-4-(((2S,3S',47?)-l,3,4- trihydroxyoctadecan-2-yl)amino)butanoic acid using triethylamine
(2S,3S,4R)-2-aminooctadecane-l,3,4-triol (40 mg, 0.126 mmol) was dissolved in 4.3 mL of THF/CH2Q2 (3:2; v/v). Subsequently, TEA (0.002 mL, 0.025 mmol) and succinic anhydride (0.0139 mg, 0.139 mmol) in 0.2 mL of THF was added dropwise under argon. The reaction was kept at 0 °C for 7 h. Afterwards, the mixture was allowed to warm spontaneously to room temperature, followed by stirring for 17 h. The reaction mixture was concentrated under reduced pressure, and the residue was crystallized from ethyl acetate. The white precipitate was centrifuged, decanted, and dried to give a white solid. Yield 85 %. The product contained 36 % of the ester side product.
'l l NMR (DMSO-<76, 700 MHz) signals: 8 12.05 (br. s, 1H), 7.66 (d, J=8.18 Hz, 0.36H), 7.57 (d, J-8.8 Hz, 0.64H), 4.18-4.13 (m, 0.66H) 4.03 (dd, J 6.7 Hz, 0.37H) 3.93-3.85 (m., 0.64H), 3.57-3.52 (dd, JM0.7 HZ, 0.64H), 3.49-3.43 (dd, J=8.4 HZ, 0.64H), 3.40-3.20 (m, 2H), 2.42- 2.38 (m, 2H), 2.36-2.32 (m, 2H), 1.56-1.47 (m, 1H), 1.46-1.37 (m, 1H), 1.32-1.14 (m, 24H), and 0.87-0.82 (m, 3H).
Example 10
Preparation of (Z)-4-oxo-4-(((2S,3S,47?)-l ,3,4-trihydroxyoctadecan-2-yl)amino)but-2-enoic acid using pyridine
(2S',31S,,47?)-2-aminooctadecane-l,3,4-triol (150 mg, 0.472 mmol) was dissolved in 16.6 mL solution of THF/CH2CI2 (3:2; v/v). Subsequently, pyridine (0.008 mL, 0.094 mmol) and a solution of maleic anhydride (46.3 mg, 0.472 mmol) in 0.2 mL of THF were added dropwise under an argon atmosphere at 0 °C. The resulting mixture was kept at 0 °C for 7 h. Afterwards, the mixture was allowed to warm spontaneously to room temperature, followed by stirring for 17 h. The reaction mixture was concentrated under reduced pressure, and the residue was crystallized from ethyl acetate. The white precipitate was centrifuged, decanted, and dried to give a white solid. Yield 85 %.
’H NMR (DMSO-t/6, 700 MHz) signals: 8 15.74 (br. s, 1H), 9.35 (br. s, 1H), 6.50 (d, J=12.58 Hz, 1H), 6.23 (d, J=12.8 Hz, 1H), 4.82 (d, J=6.0 Hz, 1H), 4.63 (s, 1H), 4.47 (d, J=6.3 HZ, 1H), 4.12-4.05 (m, 1H), 3.69-3.64 (dd, J=11.2 Hz, 1H), 3.50-3.55 (dd, J=11.2 Hz, 1H), 3.41-3.20 (m, 2H), 1.56-1.36 (m, 2H), 1.32-1.12 (m, 24H), and 0.89-0.82 (m, 3H)
11 Preparation of 4-(((2S',3S,4R)-3,4-dihydroxy-2-(2-hydroxyoctadecanamido)octadecyl)oxy)-4- oxobutanoic acid
To a solution of 2-hydroxy-N-((2S,3S,4R)-l,3,4-trihydroxyoctadecan-2- yl)octadecanamide (60 mg, 0.100 mmol) in 2.5 mL of THF and triethylamine (TEA, 6 pL, 0.043 mmol), was dropwise added a solution of succinic anhydride (15 mg, 0.15 mmol) in 1.5 mL THF under nitrogen. The resulting mixture was heated at 40 °C for 6 h followed by stirring for 16 h at 25 °C. The reaction mixture was concentrated under reduced pressure, dissolved in CHCI3 (10 mL) and washed with demineralized water (4x 10 mL). The organic fraction was concentrated under reduced pressure to obtain a colourless solid. Yield: 69 %.
*H NMR (CDCI3, 700 MHz) signals: 8 11.51 ppm (br. s, 1H), 7.4-7.000 ppm (d, 1H), 5.14- 5.02-4.42 (dd, 1H), 4.45-4.31 (dd, 1H), 4.05-.91 (m, 1H), 3.560-3.40 (m, 2H), 2.65-2.54 (m, 4H), 1.80-1.40 (m, 4H), 1.32-1.05 (m, 58H), 0.81 (m, 6H).
Example 12
Preparation of 4-(((2S,,3S',47?)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate using benzoyl chloride.
3.0 g of sodium hyaluronate (7.5 mmol; 6,000 g.moL1) was dissolved in 37.5 mL of demineralized water. Afterwards, 18.75 mL of THF were gradually added. Then, TEA (3.136 mL, 3 eq.) and DMAP (46 mg; 0.05 eq.). Simultaneously, 4-(((2<S)35,4R)-3,4-dihydroxy-2- oleamidooctadecyl)oxy)-4-oxobutanoic acid (5.115 g, 1 eq.) was dissolved in 18.75 mL of THF and TEA (1.045 mL; 1 eq.), and then benzoyl chloride (0.871 mL; 1 eq.) was added to the solution. After 30 min at 60 °C, the solution was added to the solution containing HA and allowed to react for 2 h at 25 °C. Afterwards, the reaction mixture was diluted with 10 mL of demineralized water containing 3.067 g of NaCl. The acylated derivative was isolated by subsequent precipitation using 5-fold absolute isopropanol from the reaction mixture. After decantation, the precipitate underwent 4-fold repeated precipitation and decantation. After that, the precipitate was 4-fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
DS = 14.2 % (determined by NMR)
Mw of the ester conjugate: 8 800 g/mol
'l l NMR (D2O, 700 MHz) signals of acyl: 8 5.5-5.22 (m, 2H, -CH=CH-), 2.9-2.36 (m, 4H, - OOC-CH2-CH2-COO), 1.8-1.2 (m. 48H. -CH2-), 0.9-0.8 (m, 6H, -CH3)
Figure imgf000027_0001
Preparation of 4-(((2S',3<S',4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate using benzoyl chloride. 3.0 g of sodium hyaluronate (7.5 mmol; 6,000 g mol-1) was dissolved in 37.5 mL of demineralized water. Afterwards, 18.75 mL of isopropyl alcohol were gradually added. Then, TEA (3.136 mL, 3 eq.) and DMAP (46 mg; 0.05 eq.). Simultaneously, 4-(((2S’,3S,4R)-3 ,4- dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid (5.115 g, 0.6 eq.) was dissolved in 18.75 mL of isopropylalcohol and TEA (1.045 mL; 0.6 eq.), and then benzoyl chloride (0.871 mL; 0.6 eq.) was added to the solution. After 30 min at 60 °C, the solution was added to the solution containing HA and allowed to react for 2 h at 25 °C. Afterwards, the reaction mixture was diluted with 10 mL of demineralized water containing 3.067 g of NaCl. The acylated derivative was isolated by subsequent precipitation using 5-fold absolute isopropanol from the reaction mixture. After decantation, the precipitate underwent 4-fold repeated precipitation and decantation. After that, the precipitate was 4-fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
DS = 12.0 % (determined by NMR)
Mw of the ester conjugate: 6 600 g/mol
*H NMR (D2O, 700 MHz) signals of acyl: 8 5.5-5.22 (m, 2H, -CH=CH-), 2.9-2.36 (m, 4H, - OOC-CH2-CH2-COO), 1.8-1.2 (m. 48H. -CH2-), 0.9-0.8 (m, 6H, -CH3) Example 14
Preparation of 4-(((2S',31S',4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate using benzoyl chloride.
3.0 g of sodium hyaluronate (7.5 mmol; 6,000 g mol'1) was dissolved in 37.5 mL of demineralized water. Afterwards, 18.75 mL of isopropyl alcohol were gradually added. Then, TEA (3.136 mL, 3 eq.) and DMAP (46 mg; 0.05 eq.). Simultaneously, 4-(((2S,3S',47?)-3,4- dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid (5.115 g, 0.4 eq.) was dissolved in 18.75 mL of isopropylalcohol and TEA (1.045 mL; 1 eq.), and then benzoyl chloride (0.871 mL; 1 eq.) was added to the solution. After 30 min at 60 °C, the solution was added to the solution containing HA and allowed to react for 2 h at 25 °C. Afterwards, the reaction mixture was diluted with 10 mL of demineralized water containing 3.067 g of NaCl. The acylated derivative was isolated by subsequent precipitation using 5 -fold absolute isopropanol from the reaction mixture. After decantation, the precipitate underwent 4-fold repeated precipitation and decantation. After that, the precipitate was 4-fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
DS = 8.8 % (determined by NMR)
Mw of the ester conjugate: 5 600 g/mol *H NMR (D2O, 700 MHz) signals of acyl: 5 5.5-5.22 (m, 2H, -CH=CH-), 2.9-2.36 (m, 4H, - OOC-CH2-CH2-COO), 1.8-1.2 (m. 48H. -CH2-)> 0.9-0.8 (m, 6H, -CH3) Example 15
Preparation of 4-(((2S',31S',4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate using benzoyl chloride.
50 mg of sodium hyaluronate (0.125 mmol; 3,000 g mol-1) was dissolved in 0.625 mL of demineralized water. Afterwards, 0.313 mL of isopropyl alcohol were gradually added. Then, TEA (0.052 mL, 3 eq.) and DMAP (1 mg; 0.05 eq.). Simultaneously, 4-(((2S,35,4 ?)-3,4- dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid (34 mg, 0.4 eq.) was dissolved in 0.313 mL of isopropylalcohol and TEA (0.007 mL; 1 eq.), and then benzoyl chloride (0.006 mL; 1 eq.) was added to the solution. After 30 min at 60 °C, the solution was added to the solution containing HA and allowed to react for 2 h at 25 °C. Afterwards, the reaction mixture was diluted with 0.2 mL of demineralized water containing 51 mg of NaCl. The acylated derivative was isolated by subsequent precipitation using 5-fold absolute isopropanol from the reaction mixture. After decantation, the precipitate underwent 4-fold repeated precipitation and decantation. After that, the precipitate was 4-fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
DS = 3 % (determined by NMR)
’H NMR (D2O, 700 MHz) signals of acyl: 5 5.5-5.22 (m, 2H, -CH=CH-), 2.9-2.36 (m, 4H, - OOC-CH2-CH2-COO), 1.8-1.2 (m. 48H. -CH2-), 0.9-0.8 (m, 6H, -CH3) Example 16
Preparation of 4-(((2S’,3>S',4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate using benzoyl chloride.
3.0 g of sodium hyaluronate (7.5 mmol; 9,000 g.mol-1) was dissolved in 33.3 mL of demineralized water. Afterwards, 16.67 mL of THF were gradually added. Then, TEA (3.136 mL, 3 eq.) and DMAP (46 mg; 0.05 eq.). Simultaneously, 4-(((2S,35,4R)-3,4-dihydroxy-2- oleamidooctadecyl)oxy)-4-oxobutanoic acid (5.115 g, 1 eq.) was dissolved in 16.67 mL of THF and TEA (1.045 mL; 1 eq.) and then benzoyl chloride (0.871 mL; 1 eq.) was added to the solution. After 30 min at 60 °C, the solution was added to the solution containing HA and allowed to react for 2 h at 25 °C. Afterwards, the reaction mixture was diluted with 10 mL of demineralized water containing the addition of 3.067 g of NaCl. The acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5 -fold absolute isopropanol. After the decantation, the precipitate underwent 4-fold repeated precipitation and decantation. Finally, the precipitate was 4-fold washed with absolute isopropanol. Afterwards, the precipitate was dried at 40 °C for 48 hours and subsequently spray-dried to remove the residual solvents.
DS = 8.3 % (determined by NMR)
Mw of the ester conjugate: 10 000 g/mol
’H NMR (D2O, 700 MHz) signals of acyl: 5 5.5-5.22 (m, 2H, -CH=CH-)> 2.9-2.36 (m, 4H, - OOC-CH2-CH2-COO-), 1.8-1.2 (m. 48H. -CH2-), 0.9-0.8 (m, 6H, -CH3)
Example 17
Preparation of 4-(((2.S',35',47?)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate using benzoyl chloride.
3.0 g of sodium hyaluronate (7.5 mmol; 15,000 g.mol'1) was dissolved in 60 mL of demineralized water. Afterwards, 30 mL of isopropanol were gradually added. Then, TEA (3.136 mL, 3 eq.) and DMAP (46 mg; 0.05 eq.). Simultaneously, 4-(((2S,3S,4 ?)-3,4-dihydroxy- 2-oleamidooctadecyl)oxy)-4-oxobutanoic acid (5.115 g, 1 eq.) was dissolved in 30 ml of THF and TEA (1.045 mL; 1 eq.), and then benzoyl chloride (0.871 mL; 1 eq.) was added to the solution. After 30 min at 60 °C, the solution was added to the solution containing HA and allowed to react for 2 h at 25 °C. Afterwards, the reaction mixture was diluted with 10 mL of demineralized water containing the addition of 3.067 g of NaCl. The acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5 -fold absolute isopropanol. After the decantation, the precipitate underwent 4-fold repeated precipitation and decantation. Finally, the precipitate was 4-fold washed with absolute isopropanol to remove water from the derivative. Afterwards, the precipitate was dried at 40 °C for 48 hours and subsequently lyophilized to remove the residual solvents.
DS = 7.7 % (determined by NMR)
Mw of the ester conjugate: 21 000 g/mol
'H NMR (D2O, 700 MHz) signals of acyl: 8 5.5-5.22 (m, 2H, -CH=CH-), 2.9-2.36 (m, 4H, - OOC-CH2-CH2-COO-), 1.8-1.2 (m. 48H. -CH2-), 0.9-0.8 (m, 6H, -CH3) Example 18
Preparation of 4-(((25',35',4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate using benzoyl chloride.
3.0 g of sodium hyaluronate (7.5 mmol; 15,000 g.mol'1) was dissolved in 60 mL of demineralized water. Afterwards, 30 mL of isopropanol were gradually added. Then, TEA (3.136 mL, 3 eq.) and DMAP (46 mg; 0.05 eq.). Simultaneously, 4-(((2S,3S,47?)-3,4-dihydroxy- 2-oleamidooctadecyl)oxy)-4-oxobutanoic acid (1.279 g, 0.25 eq.) was dissolved in 30 ml of THF and TEA (0.261 mL; 0.25 eq.), and then benzoyl chloride (0.218 mL; 0.25 eq.) was added to the solution. After 30 min at 60 °C, the solution was added to the solution containing HA and allowed to react for 2 h at 25 °C. Afterwards, the reaction mixture was diluted with 10 mL of demineralized water containing the addition of 3.067 g of NaCl. The acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5 -fold absolute isopropanol. After the decantation, the precipitate underwent 4-fold repeated precipitation and decantation. Finally, the precipitate was 4-fold washed with absolute isopropanol to remove water from the derivative. Afterwards, the precipitate was dried at 40 °C for 48 hours and subsequently lyophilized to remove the residual solvents.
DS = 3.2 % (determined by NMR)
Mw of the ester conjugate: 12 200 g/mol
*H NMR (D2O, 700 MHz) signals of acyl: 5 5.5-5.22 (m, 2H, -CH=CH-), 2.9-2.36 (m, 4H, - OOC-CH2-CH2-COO-), 1.8-1.2 (m. 48H. -CH2-), 0.9-0.8 (m, 6H, -CH3) Example 19
Preparation of 4-(((21S’,3S,4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate using benzoyl chloride.
2.0 g of sodium hyaluronate (5 mmol, 188,000 g.mol’1) was dissolved in 100 mL of demineralized water. Afterwards, 50 mL of THF were gradually added. Then, TEA (2.091 mL, 3 eq.) and DMAP (30.6 mg; 0.05 eq.). Simultaneously, 4-(((25,35,47?)-3,4-dihydroxy-2- oleamidooctadecyl)oxy)-4-oxobutanoic acid (3.41 g, 1 eq.) was dissolved in 50 mL of THF and TEA (0.697 mL; 1 eq.), and then benzoyl chloride (0.581 mL; 1 eq.) was added to the solution. After 30 min at 60 °C, the solution was added to the solution containing HA and allowed to react for 2 h at 25 °C. Afterwards, the reaction mixture was diluted with 6 mL of supersaturated solution of NaCl. The acylated derivative was isolated by subsequent precipitation using a 5- fold volume of isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (85% by vol.). Afterwards, the precipitate was 3-fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
DS = 4.6 % (determined by NMR)
Mw of the ester conjugate: 184 400 g/mol
'HNMR (D2O, 700 MHz) signals of acyl: 8 1.8-1.2 (m. 48H. -CH2-), 0.9-0.8 (m, 6H, -CH3) Example 20
Preparation of 4-(((25,3S,4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate using benzoyl chloride. 0.2 g of sodium hyaluronate (0.5 mmol, 300,000 g.mof1) was dissolved in 10 mL of demineralized water. Afterwards, 5 mL of THF were gradually added. Then, TEA (0.209 mL, 3 eq.) and DMAP (3 mg; 0.05 eq.). Simultaneously, 4-(((2S,3S, 47^-3, 4-dihydroxy-2- oleamidooctadecyl)oxy)-4-oxobutanoic acid (0.256 g, 0.75 eq.) was dissolved in 5 mL of THF and TEA (0.052 mL; 0.75 eq.), and then benzoyl chloride (0.044 mL; 0.75 eq.) was added to the solution. After 30 min at 60 °C, the solution was added to the solution containing HA and allowed to react for 2 h at 25 °C. Afterwards, the reaction mixture was diluted with 5 mL of demineralized water containing supersaturated solution of NaCl. The acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5 -fold isopropanol. After that, the precipitate was decanted and repeatedly washed. First, with absolute isopropanol and subsequently 4-times with an aqueous solution of isopropanol/water (85% by vol.). Afterwards, the precipitate was 3 times washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
DS = 6.4 % (determined by NMR)
Mw of the ester conjugate: 298 500 g/mol
’H NMR (D2O, 700 MHz) signals of acyl: 8 1.8-1.2 (m. 48H. -CH2-), 8 0.9-0.8 (m, 6H, -CH3) Example 21
Preparation of 4-(((2S,3S',47?)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate using benzoyl chloride.
2.0 g of sodium hyaluronate (5 mmol; 416,000 g.moF1) was dissolved in 100 mL of demineralized water. Afterwards, 50 mL of THF were gradually added. Then, TEA (2.091 mL, 3 eq.) and DMAP (30.6 mg; 0.05 eq.). Simultaneously, 4-(((2S,3S,47?)-3,4-dihydroxy-2- oleamidooctadecyl)oxy)-4-oxobutanoic acid (1.705 g, 0.5 eq.) was dissolved in 50 mL of THF and TEA (0.348 mL; 0.5 eq.) and then benzoyl chloride (0.290 mL; 0.5 eq.) was added to the solution. After 30 min at 60 °C, the solution was added to the solution containing HA and allowed to react for 2 h at 25 °C. Afterwards, the reaction mixture was diluted with 6 mL of demineralized water containing the addition of 2.92 g of NaCl. The acylated derivative was isolated from the reaction mixture by subsequent precipitation using the 5-fold of absolute isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with aqueous solution of isopropanol (85% by vol.). Afterwards, the precipitate was 3-fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours and subsequently lyophilized for the purpose of removing the residual solvents.
DS = 1.9 % (determined by NMR) ’H NMR (D2O, 700 MHz) signals of acyl: 5 1.8-1.2 (m. 48H. -CH2-), 8 0.9-0.8 (m, 6H, -CH3) Example 22
Preparation of 4-(((2S’,3>S',4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate using benzoyl chloride
0.5 g of sodium hyaluronate (1.3 mmol; 1,000,000 g.rnoF1) was dissolved in 100 mL of demineralized water. Afterwards, 50 mL of isopropanol were gradually added. Then, TEA (0.523 mL, 3 eq.) and DMAP (7.6 mg; 0.05 eq.). Simultaneously, 4-(((2iS,,3S',4 ?)-3,4- dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid (426 mg, 0.5 eq.) was dissolved in 50 ml of THF and TEA (0.087 mL; 0.5 eq.) and then benzoyl chloride (0.073 mL; 0.5 eq.) was added to the solution. After 30 min at 60 °C, the solution was added to the solution containing HA and allowed to react for 2 h at 25 °C. Afterwards, the reaction mixture was diluted with 1 mL of demineralized water containing the addition of 0.7 g of NaCl. The acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5 -fold absolute isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (85% by vol.). Afterwards, the precipitate was 3 -fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
DS = 2.5 % (determined by NMR) ’H NMR (D2O, 700 MHz) signals of acyl: 6 1.8-1.2 (m. 48H. -CH2-), 0.9-0.8 (m, 6H, -CH3) Example 23
Preparation of 4-(((2S',3>S',47?)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxo butanoic acid derivative of sodium hyaluronate using benzoyl chloride
0.5 g of sodium hyaluronate (1.3 mmol; 1,600,000 g.mol"’) was dissolved in 100 mL of demineralized water. Afterwards, 50 mL of isopropanol were gradually added. Then, TEA (0.523 mL, 3 eq.) and DMAP (7.6 mg; 0.05 eq.). Simultaneously, 4-(((2S',3S,4R)-3,4- dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid (426 mg, 0.5 eq.) was dissolved in 50 ml of THF and TEA (0.087 mL; 0.5 eq.) and then benzoyl chloride (0.073 mL; 0.5 eq.) was added to the solution. After 30 min at 60 °C, the solution was added to the solution containing HA and allowed to react for 2 h at 25 °C. Afterwards, the reaction mixture was diluted with 1 mL of demineralized water containing the addition of 0.7 g of NaCl . The acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5 -fold absolute isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (85% by vol.). Afterwards, the precipitate was 3 -fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
DS = 0.7 % (determined by NMR)
]H NMR (D2O, 700 MHz) signals of acyl: 5 1.8-1.2 (m. 48H. -CH2-), 0.9-0.8 (m, 6H, -CH3) Example 24
Preparation of 4-(((2S,3S',4 ?)-3,4-dihydroxy-2-(2-hydroxyoctadecanamido)octadecyl)oxy)-4- oxobutanoic acid derivative of sodium hyaluronate using benzoyl chloride.
40 mg of sodium hyaluronate (0.1 mmol; 6,000 g.mol'1) was dissolved in 0.5 mL of demineralized water. Afterwards, 0.25 mL of THF were gradually added. Then, TEA (0.042 mL, 3 eq.) and DMAP (1 mg; 0.05 eq.). Simultaneously, 4-(((2S',3»S',42?)-3,4-dihydroxy-2-(2- hydroxyoctadecanamido)octadecyl)oxy)-4-oxobutanoic acid (35 mg, 0.5 eq.) was dissolved in 0.25 mL of THF and TEA (0.007 mL; 0.5 eq.), and then benzoyl chloride (0.006 mL; 0.5 eq.) was added to the solution. After 30 min at 40 °C, the solution was added to the solution containing HA and allowed to react for 2 h at 25 °C. Afterwards, the reaction mixture was diluted with 0.2 mL of demineralized water containing 29 mg of NaCl. The acylated derivative was isolated by subsequent precipitation using 5 -fold absolute isopropanol from the reaction mixture. After decantation, the precipitate underwent 4-fold repeated precipitation and decantation. After that, the precipitate was 4-fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
DS = 0.6 % (determined by NMR)
*H NMR (D2O, 700 MHz) signals of acyl: 2.9-2.36 (m, 4H, -OOC-CH2-CH2-COO-), 1.8-1.2 (m. 62H. -CH2-), 0.9-0.8 (m, 6H, -CH3) Example 25
Preparation of 4-(((2S,3S',4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate using CDI
Preparation of hyaluronic acid form In brief, 5.0 g of sodium hyaluronate (12.5 mmol, 15,000 g mol'1) was dissolved in 250 ml of demineralized water. Subsequently, 30 mL of H+ catex was added to the solution. The reaction was kept at 5 °C for 24 h. After that, H+ catex was filtered from the hyaluronic acid solution and washed several times with demineralized water. The resulting filtrate was freeze-dried.
50 mg of acid form of hyaluronan (0.132 mmol, 15,000 g mol'1) was dissolved in 0.8 mL of DMSO. Subsequently, TEA (0.009 mL; 0.5 eq.) and DMAP (1 mg; 0,05 eq.) were added. Simultaneously, 4-(((2S',3S',47?)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid (45 mg; 0.066 eq.) was dissolved in 0,8 mL of DMSO. After that, TEA (0.009 mL; 0.5 eq.) and CDI (10.72 mg; 0.5 eq.) were added to the solution. After 30 min at 60 °C, the solution was added to the HA-containing solution and allowed to react for 24 h at room temperature. Afterwards, the reaction mixture was diluted with 0.3 mL of demineralized water containing the addition of 38.65 mg of NaCl. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-times with an aqueous solution of isopropanol (90 % by vol.). Afterwards, the precipitate was 3-fold washed with absolute isopropanol to remove water from the derivative and dried at 40 °C for 48 hours.
DS = 0.8 % (determined by NMR)
*H NMR (D2O, 700 MHz) signals of acyl: 8 1.8-1.2 (m. 48H. -CH2-), 0.9-0.8 (m, 6H, -CH3) Example 26
Preparation of (Z)-4-(((2S',3S',4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobut-2-enoic acid derivative of sodium hyaluronate using CDI
50 mg of acid form of hyaluronan (0.132 mmol, 15,000 g.mol’1) was dissolved in 0.8 mL of DMSO. Subsequently, TEA (0.009 mL; 0.5 eq.) and DMAP (1 mg; 0.05 eq.) were added. Simultaneously, (Z)-4-(((2S',3>S',4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobut-2- enoic acid (45 mg; 0.5 eq.) was dissolved in 0.8 mL of DMSO. After that, TEA (0.009 mL; 0.5 eq.) and CDI (10.72 mg; 0.5 eq.) were added to the solution. After 30 min at a temperature of 60 °C, the solution was added to the solution containing HA and allowed to react for 24 h at room temperature. Afterwards, the reaction mixture was diluted with 0.3 mL of demineralized water containing the addition of 38.65 mg of NaCl. The acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5-fold absolute isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (90% by vol.). Afterwards, the precipitate was 3 -fold washed with absolute isopropanol to remove water from the derivative and was dried at 40 °C for 48 hours.
DS = 1.6 % (determined by NMR) 'l I NMR (D2O, 700 MHz) signals of acyl: 8 7.75-7.40 (m, 2H, -OOC-CH=CH-COO-) 5.5-5.22 (m, 2H, -CH=CH-), 1.8- 1.2 (m. 48H. -CH2-), 0.9-0.8 (m, 6H, -CH3) Example 27 (Z)-4-(((2S,3lS',4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobut-2-enoic acid derivative of sodium hyaluronate using HSTU
66.50 mg of acid form of hyaluronan (0.176 mmol, 15,000 g.moL1) was dissolved in 0.85 mL of DMSO. Subsequently, TEA (0.012 mL; 0.5 eq.) was added. Simultaneously, (Z)-4- ( (2S,3S, 4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobut-2-enoic acid (59.8 mg; 0.5 eq.) was dissolved in 0.8 mL of DMSO. After that, TEA (0.012 mL; 0.5 eq.) and HSTU (14.26 mg; 0.5 eq.) were added to the solution. After 6 h at 60 °C, the solution was added to the solution containing HA and allowed to react for 40 h at 25 °C. Afterwards, the reaction mixture was diluted with 0.3 mL of demineralized water containing the addition of 51.41 mg of NaCl. The acylated derivative was isolated from the reaction mixture by subsequent precipitation using the 5-fold of absolute isopropanol. The acylated derivative was isolated from the reaction mixture by subsequent precipitation using the 5-fold of absolute isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (90% by vol.). Afterwards, the precipitate was 3 -fold washed with absolute isopropanol to remove water from the derivative. Finally, the residue was dried at 40 °C for 48 hours.
DS = 2.6 % (determined by NMR):
’H NMR (D2O, 700 MHz) signals of acyl: 6 5.5-5.22 (m, 2H, -CH=CH-), 1.8-1.2 (m. 48H. - CH2-), 0.9-0.8 (m, 6H, -CH3) Example 28
Preparation of (Z)-4-(((2S,3S',4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobut-2-enoic acid derivative of sodium hyaluronate using benzoyl chloride
100 mg of sodium hyaluronate (0.30 mmol, 6,000 g.mol"1) was dissolved in 1.25 mL of demineralized water. Afterwards, 0.625 mL of THF were gradually added. Then, TEA (0.105 mL, 3 eq.) and DMAP (15.3 mg, 0.05 eq.). Simultaneously, (Z)-4-(((2S,3S,4R)-3,4-dihydroxy- 2-oleamidooctadecyl)oxy)-4-oxobut-2-enoic acid (85 mg, 0.5 eq.) was dissolved in 0.625 mL of THF and TEA (0.017 mL, 0.5 eq.) and then benzoyl chloride (0.015 mL, 0.5 eq.) was added to the solution. After 1 h at 60 °C, the solution was added to the HA-containing solution and allowed to react for 3 h at 25 °C. Afterwards, the reaction mixture was diluted with 1 mL of demineralized water containing the addition of 73 mg of NaCl. The acylated derivative was isolated from the reaction mixture by subsequent precipitation using the 5 -fold of absolute isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with aqueous solution of isopropanol (90% by vol.). Afterwards, the precipitate was 3 -fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
DS = 1.3 % (determined by NMR)
'H NMR (D2O, 700 MHz) signals of acyl: 8 6.8-6.4 (m, 2H, -OOC-CH=CH-COO-), 5.5-5.22 (m, 2H, -CH=CH-), 1.8-1.2 (m. 48H. -CH2-), 0.9-0.8 (m, 6H, -CH3) Example 29 Preparation of 4-oxo-4-(((25,35,47?)-l,3,4- trihydroxyoctadecan-2-yl)amino)butanoic acid derivative of sodium hyaluronate using CDI
279 mg of acid form of hyaluronan (0.738 mmol, 15,000 g.mol'1) was dissolved in 2.7 mL of DMSO. Subsequently, TEA (0.051 mL, 0.5 eq.) was added. Simultaneously, 4-oxo-4- (((25, 35, 47?)- 1,3, 4- trihydroxyoctadecan-2-yl)amino)butanoic acid (154.11 mg, 0,5 eq.) was dissolved in 2.8 mL of DMSO. After that, TEA (0,034 mL, 0,5 eq.) and 1,1 - carbonyldiimidazole (CDI, 59.84 mg, 0.5 eq.) were added to the solution. After 3 h at room temperature, the solution was added to the solution containing HA and allowed to react for 24 h at 25°C. Afterwards, the reaction mixture was diluted with 1 mL of demineralized watercontaining the addition of 215.67 mg of NaCl. The acylated derivative was isolated from the reaction mixture by subsequent precipitation using the 5-fold of absolute isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (90% by vol.). Afterwards, the precipitate was 3-fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours.
DS = 1.9% (determined by NMR)
‘H NMR (D2O, 700 MHz) signals of acyl: 8 2.8-2.3 ppm (4H, OOC-CH2-CH2-COO), 1.8-1.2 ppm (m. 26H, -CH2-), 0.9-0.8 (m, 3H, -CH3)
Example 30
Preparation of 4-oxo-4-(((25,35,47?)-l,3,4- trihydroxyoctadecan-2-yl)amino)butanoic acid derivative of sodium hyaluronate using HSTU
180 mg of acid form of hyaluronan (0.476 mmol, 15,000 g.mol'1) was dissolved in 1.8 mL of DMSO. Subsequently, TEA (0.066 mL, 1 eq.) and DMAP (3 mg, 0.05) were added. Simultaneously, 4-oxo-4-(((25,35,47?)-l,3,4-trihydroxyoctadecan-2-yl)amino)butanoic acid (39.77 mg, 0.2 eq.) was dissolved in 1.5 mL of DMSO. After that, TEA (0.013 mL, 0.2 eq.) and HSTU (34.24 mg, 0.2 eq.) were added to the solution. After 7 h at room temperature, the solution was added to the solution containing HA and allowed to react for 72 h at room temperature. Afterwards, the reaction mixture was diluted with 1 mL of demineralized watercontaining the addition of 139.14 mg of NaCl. The acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5-fold absolute isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (90% by vol.). Afterwards, the precipitate was 3-fold washed with absolute isopropanol to remove water from the derivative. Finally, the precipitate was dried at 40 °C for 48 hours. DS = 2.8 % (detennined by NMR).
NMR (D2O, 700 MHz) signals of acyl: 8 2.8-2.S ppm (4H, OOC-CH2-CH2-COO), 1.8-1.2 ppm (m. 26H, -CH2-), 0.9-0.8 (m, 3H, -CH3) Example 31
Preparation of 4-oxo-4-(((2S,3S',47?)-l,3,4- trihydroxyoctadecan-2-yl)amino)butanoic acid derivative of sodium hyaluronate using benzoyl chloride.
0.176 g of sodium hyaluronate (0.4 mmol, 15,000 g.mol'1) was dissolved in 2.93 mL of demineralized water. Afterwards, 1.47 mL of THF were gradually added. Then, TEA (0.184 mL, 3 eq.) and DMAP (5.4 mg; 0,1 eq.). Simultaneously, 4-oxo-4-(((2S', 3S, 4R)~ 1,3,4- trihydroxyoctadecan-2-yl)amino)butanoic acid (92 mg, 0.5 eq.) was dissolved in 1.47 mL of THF and TEA (0.031 mL; 0.5 eq.) and then benzoyl chloride (0.026 mL; 0.5 eq.) was added to the solution. After 30 min at 60 °C, the solution was added to the solution containing HA and allowed to react for 3 h at room temperature. Afterwards, the reaction mixture was diluted with 0.5 mL of demineralized water containing the addition of 129 mg of NaCl. The acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5 -fold absolute isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (90% by vol.). Afterwards, the precipitate was 3-fold washed with absolute isopropanol to remove water from the derivative and was dried at 40 °C for 48 hours.
DS = 0.5 % (determined by NMR)
]H NMR (D2O, 700 MHz) signals of acyl: 3 2.8-2.3 ppm (4H, CH2), 1.8-1.2ppm (m. 26H, - CH2-), 0.9-0.8 (m, 3H, -CH3) Example 32
Preparation of (Z)-4-oxo-4-(((2S,3S,47?)-l ,3,4-trihydroxyoctadecan-2-yl)amino)but-2-enoic acid derivative of sodium hyaluronate using benzoyl chloride
360 mg of sodium hyaluronate (0.9 mmol, 6,000 g.mol'1) was dissolved in 3.6 mL of demineralized water. Afterwards, 1.8 mL of THF were gradually added. Then, TEA (0.251 mL, 2 eq.) and DMAP (5.5 mg; 0.05 eq.). Simultaneously, (Z)-4-oxo-4-(((2S',3S',47?)-l,3,4- trihydroxyoctadecan-2-yl)amino)but-2-enoic acid (187 mg, 0.5 eq.) was dissolved in 18.75 mL of THF and TEA (0.063 mL; 0.5 eq.) and then benzoyl chloride (0.052 mL; 0.5 eq.) was added to the solution. After 30 min at 60 °C, the solution was added to the HA-containing solution and allowed to react for 2 h at room temperature. The acylated derivative was isolated from the reaction mixture by subsequent precipitation using the 5-fold of absolute isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (90% by vol.). Afterwards, the precipitate was 3 -fold washed with absolute isopropanol to remove water from the derivative and was dried at 40 °C for 48 hours.
DS = 0.4 % (determined by NMR)
!H NMR (D2O, 700 MHz) signals of acyl: 8 1.8-1.2 ppm (m, 26H, -CH2-), 0.9-0.8 (m, 3H, - CH3)
Example 33 Preparation of (Z)-4-oxo-4-(((2S',3S',4R)-l,3,4- trihydroxyoctadecan-2-yl)amino)but-2-enoic acid derivative of sodium hyaluronate using HSTU
60 mg of the acid form of hyaluronan (0.159 mmol, 15,000 g.mol’1) was dissolved in 0.6 mL of DMSO. Subsequently, TEA (0.022 mL, 1 eq.) and DMAP (1 mg, 0.05 eq.) were added. Simultaneously, (Z)-4-oxo-4-(((2S,3S,47?)-l,3,4-trihydroxyoctadecan-2-yl)amino)but- 2-enoic acid (13,19 mg, 0.2 eq.) was dissolved in 0.5 mL of DMSO. After that, TEA (0.004 mL, 0.2 eq.) and HSTU (11,4 mg, 0.2 eq.) were added to the solution. After 7 h at room temperature, the solution was added to the solution containing HA and allowed to react for 72 h at room temperature. Afterwards, the reaction mixture was diluted with 1 mL of demineralized water containing the addition of 46.4 mg of NaCl. The acylated derivative was isolated from the reaction mixture by subsequent precipitation using 5-fold absolute isopropanol. After that, the precipitate was decanted and repeatedly washed initially with absolute isopropanol and subsequently 4-fold with an aqueous solution of isopropanol (90% by vol.). Afterwards, the precipitate was 3-fold washed with absolute isopropanol was dried at 40 °C for 48 hours. DS=1.4 % (determined by NMR) ‘HNMR (CDCI3) signals of acyl: 8 1.8-1.2 ppm (m, 26H, -CH2-), 0.9-0.8 (m, 3H, -CH3) Example 34
Encapsulation of tocopherol (vitamin E) into 4-(((2S,35,4R)-3,4-dihydroxy-2- oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate
100 mg of the 4-(((2S,3S,4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate, which has been prepared according to Example 16, was dissolved in 10 mL of water under continuous stirring overnight. The resulting solution was gradually supplemented with the solution of tocopherol (15 mg in 3 mL of isopropanol) under continuous stirring and under the temperature ranging between 25 and 40°C. Subsequently, isopropanol was removed from the solution in a continuous evaporation process at 35 °C. After that, the dry residue was rehydrated with demineralized water and filtered through a 1 pm glass filter. The filtrate was lyophilized.
The amount of bound tocopherol determined by HPLC was 12.3 % (w/w).
Example 35
Co-loading of tocopherol (vitamin E) and cannabidiol into 4-(((25,35,47?)-3,4-dihydroxy-2- oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate.
100 mg of the 4-(((2lS,3S,47?)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate, which has been prepared according to Example 12, was dissolved in 10 mL of water under continuous stirring overnight. The resulting solution was gradually supplemented with the solution of tocopherol (15 mg) and cannabidiol (5 mg) in 4 mL of isopropanol under continuous stirring and a temperature ranging between 25 and 40°C. Subsequently, isopropanol was removed from the solution in a continuous evaporation process at 35 °C. After that, the dry residue was rehydrated with water and filtered through a 1 pm glass filter. The filtrate was lyophilized.
Tocopherol and cannabidiol were determined by HPLC as 11.4 % and 4.0 % (w/w), respectively.
Example 36
Encapsulation of curcumin into 4-(((25,35,,47?)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4- oxobutanoic acid derivative of sodium hyaluronate
100 mg of the 4-(((2S,3>S,47(')-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate, which has been prepared according to Example 12, was dissolved in 10 mL of water under continuous stirring overnight. The resulting solution was gradually supplemented with the curcumin solution (5 mg in 5 mL of isopropanol) under continuous stirring and a temperature ranging between 25 and 40°C. Subsequently, isopropanol was removed from the solution in a continuous evaporation process at 35 °C. After that, the dry residue was rehydrated with demineralized water and filtered through a 1 pm glass filter. The filtrate was lyophilized.
The amount of curcumin determined by UV-Vis was 1.9 % (w/w).
Figure imgf000040_0001
Encapsulation of ethyl ferulate into 4-(((2S,3S,4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy) 4-oxobutanoic acid derivative of sodium hyaluronate
100 mg of the 4-(((2S,35,47?)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate, which has been prepared according to Example 16, was dissolved in 10 mL of water under continuous stirring overnight. The resulting solution was gradually supplemented with the solution of ethyl ferulate (15 mg in 15 mL of isopropanol) under continuous stirring and under the temperature ranging between 25 and 40°C. Subsequently, isopropanol was removed from the solution in a continuous evaporation process at 35 °C. After that, the dry residue was rehydrated with demineralized water and filtered through a 1 pm glass filter. The filtrate was lyophilized.
The amount of ethyl ferulate (determined by the UV-Vis method) was 6.1 % (w/w).
Example 38
Encapsulation of coenzyme Qio into 4-(((2S,3S,4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)- 4-oxobutanoic acid derivatives of sodium hyaluronate
100 mg of the 4-(((2S’,3S',4R)-3,4-dihydroxy-2-oleamidooctadecyl)oxy)-4-oxobutanoic acid derivative of sodium hyaluronate, which has been prepared according to Example 12, was dissolved in 10 mL of water under continuous stirring overnight. The resulting solution was gradually supplemented with the solution of coenzyme Qio (20 mg in 5 mL of CHCh) under continuous stirring and under the temperature ranging between 25 and 40°C. Subsequently, CHCI3 was removed from the solution in a continuous evaporation process at 35 °C. After that, the dry residue was rehydrated with demineralized water and filtered through a 1 pm glass filter. The filtrate was lyophilized.
The amount of bound coenzyme Qio as determined by the UV-Vis method was 11.2 % (w/w).
Example 39
Determination of critical aggregation concentration (cac) of ester conjugates and Nile red.
Polymeric micelles containing non-covalently bound Nile red were prepared to determine critical aggregation concentration (cac). In brief, 60 mg of ester conjugate prepared as described in examples described in Table 1 was dissolved under continuous stirring in 10 mL of water overnight. The resulting solution was gradually supplemented with the solution of Nile red (0.72 mg in 1.2 mL of CHCI3). Subsequently, CHCI3 was removed from the solution in a continuous evaporation process at 35 °C. After that, the dry residue was rehydrated with water and filtered through a 1 pm glass filter. The filtrate was lyophilized. The lyophilizate was dissolved and diluted to a concentration range from 0.00002 to 1.5 mg.mL-1. After 24 hours of equilibration, cac was determined by recording emission fluorescence spectra (560-800 nm) on PTI Quanta Master 400 spectrofluorophotometer (PTI) with the set excitation wavelength 543 nm and excitation and emission slit 5 nm. The aggregation onset was determined by the concentration dependence of the maximum position of the NR emission band. Two data sets were collected for each sample and analyzed altogether. The inflexion-point coordinate of a sigmoidal fit of the obtained semilogarithmic (concentration axis) dependence is reported as the cac value (Table 1).
Table 1. Determination of critical aggregation concentration for ester conjugates of this application.
Figure imgf000042_0001
Example 40
Determination of the pyrene binding constant of ester conjugates.
A series of pyrene films were prepared in the glass vials containing pyrene film. After that amphiphilic derivative of hyaluronic acid in demineralized water was added to each vial to obtain a concentration from 0.001 to 10 mg.mL’1. The resulting concentration of pyrene in each vial was 0.8 pg.mL’1. The solutions were shaken at room temperature for at least 24 h at room temperature. After that, fluorescence was measured using PTI Quanta Master 400 spectrofluorophotometer. The excitation wavelength was set to 334 nm, and the emission wavelength range to 345 - 550 nm. The ratios of the first and third fluorescence peaks of pyrene (I1/I3) were plotted against ester conjugate concentration prepared as the examples described on table 2, to obtain sigmoidal dependence as reported before (Kwon et al., 2003). The reciprocal value of inflex point on the x-axis was reported as pyrene binding constant Kv.
Table 2. Determination of pyrene binding constant Kv.
Figure imgf000042_0002
Figure imgf000043_0001
The results indicated that the hydrophobicity of the inner core of self-assembled polymeric particles increase with increasing the degree of substitution and decreased Mw.
Example 41
Cytotoxicity evaluation of selected ester conjugates
The interaction of cells with ester conjugates according to the present invention, is essential to be investigated before the product application. After the chemical modification of hyaluronic acid, the derivatives should not be cytotoxic. In this work, the cytotoxicity was assessed using the dilution method. The cell toxicity of prepared HA derivatives was tested at NIH-3T3 cells. Cells were seeded into wells of 96-well test plates and cultured for 24 hours. Cell viability was measured 0, 24, 48, and 72 hours after treatment using the 3-(4, 5- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. MTT stock solution (20 pL; of concentration 5 mg.mL'1) was added to the cell culture medium (200 pL) in each well. The plates were incubated for 2.5 h at 37 °C. Then, after removing the MTT solution, 220 pL of lysis solution was added, and lysis was carried out for 30 min at room temperature. The optical density was measured by Microplate reader VERSAmax at 570 nm. All assayed derivatives were non-cytotoxic up to the concentration of 1,000 pg.mL-1. For example, results for conjugates prepared according to examples 9, 11, 10, 40 and 14 are shown in Figure 11, wherein positive effects to cell viability after 24, 48 or 72 h were observed in the whole concentration range tested, while the most positive effects were repeatedly observed at 100 pg.mL-1 indicates an excellent cytocompatibility of the conjugate.
Example 42
Skin penetration of the conjugates
According to OECD guidelines in FDA-certified vertical Franz diffusion cells, skin penetration experiments were performed using full-thickness skin (approx. 1 mm) from porcine auricles donated by a local slaughterhouse. The receptor was filled with PBS (pH 7.4) held at 32 °C, and the excised tissue was clamped between donor and receptor with SC facing upwards and exposing a diffusion area of 1 cm2. After 30 min of equilibration, the donor compartment was slowly filled with 0.2 mL of control (PBS) or Nile red-loaded dispersion of self-assembled polymeric particles prepared according to examples 12 and 14 with a concentration 1 mg.mL'1 in PBS (pH 7.4) and closed by a lid. After an application lasting 24 h, the cells were dismantled, and the skin was washed with PBS, frozen and cryo-sectioned for further microscopic examination. Nile red fluorescence in the dermis was observed after topical application of the Nile red-loaded self-assembled nanoparticles. Figure 12 shows skin penetration of conjugates prepared according to examples 11 (DS = 8.2 %) and 9 (DS = 14.2 %).
Example 43
Immunomodulatory properties of the ester conjugates
Immunomodulatory properties of the sodium salt of the prepared ester conjugates were assessed by measuring the level of the proinflammatory cytokine interleukin 6 (IL-6) produced by LPS-activated macrophages after exposure to these derivatives. THP-1 cells (400 000 cells/well, 6-well plate) were differentiated into macrophages by cultivation in phorbol myristate acetate (50 ng.mL'1) for 24 hours, followed by 24 hour-long resting phase. Afterwards, the macrophages were co-incubated with LPS (1 pg.mL’1) and ester conjugates (in concentrations of 250 pg.mf/'and 100 pg.mL’1). Macrophages stimulated with only LPS were used as a positive control. In contrast, non-stimulated macrophages were used as a negative control. Free ceramide NP 18:1 and HA (6 kDa) were used for comparison. They were tested in the same concentrations as ester conjugates (250 pg.mL'1 and 100 pg.mL'1). The concentrations of free ceramide NP 18:1 were adjusted according to DS of ester conjugates and their concentrations. The ester conjugates prepared according to Examples 14 and 17, and HA (6 kDa) were dissolved in cultivation media; free ceramide NP 18:1 was dissolved in DMSO. The solvent control (DMSO) was also included in the experimental setup. After 24-hour long exposure to LPS and tested substances, the supernatants were collected, and the level of IL-6 was measured by ELISA (Thermo-Fisher).
Ester conjugates reduced the level of proinflammatory cytokine IL-6, as shown in Figure 15. The reduction in IL-6 level was found to be concentration-dependent, and the highest inhibition effect in IL-6 production was observed at a concentration of 250 pg. ml/1. At this concentration, ester conjugates statistically significantly (p < 0.001) inhibited the production of IL-6 by 95.3 % (Example 17) and 91.1 % (Example 14) compared to positive control. Ester conjugate's ability to suppress the inflammation marker IL-6 is unique to these derivatives and was caused by the synergy between HA and ceramide NP 18:1. Both native HA and free ceramide NP 18:1 did not show any significant IL-6 inhibitory effect. Example 44
Composition of emulsion prepared using the ester conjugate of sphingolipid with HA
Cream (oil in water emulsion) prepared using the ester conjugate of sphingolipid with HA contains: (a) from 0.001 to 0.1 % by weight of self-assembled polymeric particles made of the ester conjugate of sphingolipid and HA
(b) at least one fat selected from the group comprising oil, wax or butter,
(c) at least one emulsifier,
(d) at least one auxiliary active ingredient, (e) at least one preservative
(f) q.s.p. 100% by weight of gel base or water.
Examples of cosmetic formulations are resumed in Tables 3 - 5.
Table 3
Figure imgf000045_0001
Figure imgf000046_0001
Table 4
Figure imgf000046_0002
Table 5
Figure imgf000046_0003
Figure imgf000047_0001
Example 45.
Serum formulation prepared using the ester conjugate of sphingolipid with HA
Serum formulation prepared using the ester conjugate of sphingolipid with HA contains: (a) from 0.001 to 0.1 % by weight of self-assembled polymeric particles made of the ester- conjugate of sphingolipid and HA
(b) at least one auxiliary active ingredient,
(c) at least one preservative
(d) at least one thickener- (e) q.s.p. 100% by weight of water.
Examples of cosmetic formulations are resumed in Tables 6 - 7.
Table 6
Figure imgf000047_0002
Table 7
Figure imgf000048_0001
Example 46
Development of nanoemulsion made of the ester conjugate of sphingolipid with HA Nanoemulsions were prepared using the homogenization method under high agitation by Ultra- Turrax® equipment (IKA, Germany). The formulation consisted of an oil phase containing an oil emulsifier (0.1 - 10 %), and an aqueous phase containing self-assembled particles of the conjugate of sphingolipid with HA prepared according to Examples 12 to 24 (2 % w/v) and ultrapure water. The phases were homogenized separately with the aid of a magnetic stirrer. Then the oil phase was injected into the aqueous phase under the agitation of 10,000 rpm, which was increased to 17,000 rpm and sustained for 30 min with temperature control.
Example 47
Formulation of hydrogel containing the ester conjugate of sphingolipid with HA
A solution of oxidized HA (HA-OX) prepared according to the patent WO2011069475A2 and self-assembled particles of the conjugate of sphingolipid with HA prepared according to Examples 12 to 24 (1 :1) was prepared in demineralized water in which the final concentrations of the polymers were from 1.5 to 7.5 % (w/v), respectively. To that solution was added (0.1 % w/v) of 0,0 -1,3-propanediylbishydroxylamine dihydrochloride 98% linker was dissolved and homogenised. Then, the solution was transferred to Teflon molds (cylinders, diameter 10 mm, height 5 mm) to form hydrogel.
Example 48
Preparation of buccal patch
300 mg of ester conjugate prepared according to Examples 21 and 22 was dissolved in 60 ml of demineralized water over 12 h. After that, the solution was freeze-dried to form a buccal patch for internal application on the buccal mucosa.
Example 49
Determination of weight average molecular weight by size-exclusion chromatography (SEC)- multi-angle laser scattering (MALLS)
Weight average molecular weight and molecular weight distributions were determined by SECMALLS using an Agilent degasser Model G 1379A, chromatography system composed of an Agilent HPLC pump Model G 1310A, a Rheodyne manual injector Model 7125i, two 7.8 mm ultrahydrogel Linear columns (Waters), chromatographic detectors included a DAWN EOS MALLS, a ViscoStar differential viscometer, and an Optilab T-rEX differential refractive index in series (Wyatt Technology, Santa Barbara, California). The injection volume was 100 pL of 0.015-1% (w/v) of conjugates of sphingolipid and HA. The concentration was previously optimized according to the Mw to be analyzed. So far, the concentration required for low molecular weight (6 - 15 kDa) is 1 % (w/v), while higher molecular weight HA (188 - 442 kDa) needed 0.05% (w/v). The mobile phase was aqueous 50 mM phosphate-buffered saline and 0.02% sodium azide solution at the flow rate of 0.5 mL min-1. A refractive index increment (dn/dC) of 0.155 mL g 1 was used to calculate the molecular weight and polydispersity (Mw/Mn) of the ester conjugate.
Example 50
Observation of self-assembled polymer particles by cryo-scanning electron microscopy
A solution of an ester conjugate prepared according to Example 12 at a concentration of 5 g.L" 1 was deposited in an amount of 5 pl on a special cryomicroscopy support. The applied droplet was then covered with a second carrier. The sample thus prepared was transferred to a slush freezer (Leica, Germany) at -210 °C. The sample remained in the slush freezer for a few seconds and was then transferred to the sample preparation station (Leica, Germany). In this station, the sample was attached to a special sample holder for cryomicroscopy. This prepared sample holder was transferred to the EM ACE600 sputter machine (Leica, Germany) using an EM VCT500 (Leica, Germany) shuttier (under vacuum, at -120 °C). There, the prepared sample was broken with a knife. Subsequently, the knife temperature was raised by 3 °C and the knife was passed just above the sample for 30 seconds. The next step was to coat the sample with 11 nm Cu. Then, the sample was transferred to a Quattro S electron microscope (Thermo Fisher, Czech Republic) using a VCT500 shuttle (at -120 °C). In the electron microscope, the sample was placed on a cryostick (Leica, Germany) which was tempered to -135 °C. The sample was scanned with an ETD (secondary electron detector) under the following parameters: distance from the sample surface (WD) = 5 mm, electron accelerating voltage (HV) = 10 kV, spot size = 2.5, pressure 10'3 Pa. The current was set to 23 pA and the aperture to 30 pm.
The recorded images are shown in Fig. 16 and Fig. 17.
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Claims

Claims
1. An ester conjugate of sphingolipid and hyaluronan of a general formula I:
Figure imgf000052_0001
(I), wherein n is integer in the range of 7 to 4000 dimers,
R5 is H+ or pharmaceutically acceptable salt,
R4 is -H or linked sphingolipid substituent of a general formula II
Figure imgf000052_0002
(II), wherein
A is CH2-CH2, CH=CH or C(H)OH-CH2, if any of H bonded to C atom of CH2-CH2 or CH=CH or C(H)0H-CH2 is substituted by a hydroxyl group, it can be esterified by R3 that bonds hyaluronic acid or pharmaceutically acceptable salt thereof with a covalent bond,
R1 is a straight chain or branched alkyl group having 10 to 22 carbon atoms which may optionally contain one or more double bonds, any of H bonded to C atom of said alkyl group is optionally substituted with one or more hydroxyl groups, preferably is a straight chain alkyl group having 12 to 18 carbon atoms, more preferably is a straight chain alkyl group having 13 carbon atoms,
R6 is H or COR2, wherein
R2 is a straight chain or branched alkyl group having 13 to 55 carbon atoms, preferably 15 to 50 carbon atoms, more preferably 17 to 23 carbon atoms, that may optionally contain one or more double bonds; the alkyl chain may contain an internal ester group; or H of the alkyl group may optionally be substituted with one or more hydroxyl groups,
R3 is -H or -OC-CH2-CH2-CO- or -OC-CH2=CH2-CO-, and providing that at least one R3 is - OC-CH2-CH2-CO- or -OC-CH2=CH2-CO-, wherein free -OC- part is covalently bound to -O- at R4 position of the conjugate of sphingolipid and hyaluronan of the general Formula I, providing that at least one R4 of the conjugate is the linked sphingolipid substituent of the formula II and wherein the degree of substitution of the linked sphingolipid substituent of the general formula II in the conjugate of hyaluronan is in the range from 0.1 to 15 %.
2. The conjugate of Claim 1 , wherein the weight average molecular weight of the conjugate of the formula I is in the range of 3,000 g/mol to 1,600,000 g/mol, preferably in the range from 3,000 to 20,000 g/mol, more preferably 5,000 to 7,000 g/mol.
3. The conjugate of any one of Claims 1 to 2, wherein the degree of substitution in the conjugate of hyaluronan is in the range from 0.4 to 15 % preferably the degree of substitution is in the range of 5.5 to 10.5%, more preferably the degree of substitution is in the range of 6.5 to 8.5 %.
4. The conjugate of any one of Claims 1 to 3, wherein the pharmaceutically acceptable salt is selected from a group comprising any of ions of alkali metals or ions of alkaline-earth metals, preferably Na+, K+ or Mg2+.
5. A method of synthesis of the ester conjugate of sphingolipid and hyaluronan according to any of Claims 1 to 4 characterized in that a sphingolipid of a general formula III
Figure imgf000054_0002
wherein
A is CH2-CH2, CI I-CH or C(H)OH-CH2,
R1 is a straight chain or branched alkyl group having 10 to 22 carbon atoms which may optionally contain one or more double bonds, any of H bonded to C atom of said alkyl group is optionally substituted with one or more hydroxyl groups, preferably is a straight chain alkyl group having 12 to 18 carbon atoms, more preferably is a straight chain alkyl group having 13 carbon atoms,
R6 is H or COR2, wherein
R2 is a straight chain or branched alkyl group having 13 to 55 carbon atoms, preferably 15 to 50 carbon atoms, more preferably 17 to 23 carbon atoms; the alkyl chain may contain an internal ester group; may optionally contain one or more double bonds, and may optionally be substituted with one or more hydroxyl groups; is attached to a linker that is a residue of succinic acid or a rest of maleic acid by adding of succinic anhydride or maleic anhydride in a polar solvent in the presence of an organic base or mixtures thereof to form a linked sphingolipid of a general formula IV
Figure imgf000054_0001
wherein
A is CH2-CH2, CH=CH or C(H)OH-CH2,
R1 is a straight chain or branched alkyl group having 10 to 22 carbon atoms which may optionally contain one or more double bonds, any of H bonded to C atom of said alkyl group is optionally substituted with one or more hydroxyl groups, preferably is a straight chain alkyl group having 12 to 18 carbon atoms, more preferably is a straight chain alkyl group having 13 carbon atoms,
R6 is H or COR2, wherein
R2 is a straight chain or branched alkyl group having 13 to 55 carbon atoms, preferably 15 to 50 carbon atoms, more preferably 17 to 23 carbon atoms; the alkyl chain may contain an internal ester group; may optionally contain one or more double bonds, and may optionally be substituted with one or more hydroxyl groups;
R7 is -H or -OC-CH2-CH2-COOH or -OC-CH=CH-COOH, and providing that at least one R7 is -OC-CH2-CH2-COOH or -OC-CH=CH-COOH, that is activated with an activation agent selected from a group comprising 1,1'- carbonyldiimidazole or tetramethyl-O-(A-succinimidyl)uronium hexafluorophosphate or benzoyl chloride, in the presence of an organic solvent to form an activated linked sphingolipid of a general formula V
Figure imgf000055_0001
wherein
A is CH2-CH2, CH=CH or C(H)0H-CH2,
R1 is a straight chain or branched alkyl group having 10 to 22 carbon atoms which may optionally contain one or more double bonds, any of H bonded to C atom of said alkyl group is optionally substituted with one or more hydroxyl groups, preferably is a straight chain alkyl group having 12 to 18 carbon atoms, more preferably is a straight chain alkyl group having 13 carbon atoms,
R6 is H or COR2, wherein
R2 is a straight chain or branched alkyl group having 13 to 55 carbon atoms, preferably 15 to 50 carbon atoms, more preferably 17 to 23 carbon atoms; the alkyl chain may contain an internal ester group; may optionally contain one or more double bonds, and may optionally be substituted with one or more hydroxyl groups;
R7 is -H or -OC-CH2-CH2-CO- or -OC-CH=CH-CO-, and providing that at least one R7 is -OC- CH2-CH2-CO- or -OC-CH=CH-CO- if R7 is -H, then -O-R9 group is not attached to it;
R9 is substituent selected from a group of substituents comprising a substituent of a general formula VI or a substituent of formula VII or a substituent of formula VIII, wherein R8 is one or more substituents selected from a group comprising H, -NO2, -COOH, halides, Ci-Ce alkyloxy, preferably halides, methoxy or ethoxy, more preferably Cl,
Figure imgf000056_0001
ii),
Figure imgf000057_0001
(VIII), that reacts with hyaluronic acid or a pharmaceutically acceptable salt thereof in the present of a mixture of water and water-miscible organic solvent or in the presence of an organic solvent, preferably dimethyl sulfoxide.
6. Method according to Claim 5 characterized in that, the concentration of the sphingolipid of the general formula III, is ranging from 0.88 % to 2.5 % (w/v) and the molar equivalent of succinic anhydride or maleic anhydride is ranging from 1.0 to 2.0, preferably 1.5 equivalents with respect to sphingolipid of the general formula III.
7. The method according to Claim 5 or Claim 6 characterized in that, attachment of the linker is carried out in the range of temperatures from 0 °C to 50°C for 4 to 20 hours, more preferably for 6 to 8 hours in the range of temperatures from 0 °C to 40°C, followed by stirring at a temperature in the range of 0 °C to 29 °C, preferably at room temperature for 5 to 24 hours, more preferably for 16 to 20 hours.
8. The method according to any one of Claims 5 to 7 characterized in that, the organic base is selected from the group comprising a secondary or tertiary amine having a linear or branched or cyclic or aromatic, saturated or unsaturated C3-C30 alkyl group, and the polar solvent is selected from the group comprising tetrahydrofuran, dioxane, dimethyl sulfoxide, dichloromethane and chloroform or mixtures thereof.
9. The method according to Claim 8, characterized in that the organic base is selected from the group comprising pyridine, triethylamine, 7V-methylmorpholine, dimethylaminopyridine or mixtures thereof, and the polar solvent is preferably dichloromethane or tetrahydrofuran or their mixture.
10. The method according to any one of Claims 5 to 9, characterized in that the activation agent is benzoyl chloride that is a substituted or non-substituted benzoyl chloride or its derivatives of the general formula IX
Figure imgf000058_0001
wherein R8 is one or more substituents selected from a group comprising H, -NO2, -COOH, halides, Ci-Ce alkyloxy, preferably halides, methoxy or ethoxy, more preferably Cl.
11. The method according to any one of Claims 5 to 10, characterized in that, forming of the activated linked sphingolipid of the general formula V is earned out at the temperature in the range of 5 °C to 60 °C, preferably 40 °C, for 0.5 to 24 hours.
12. The method according to any one of Claims 5 to 11, characterized in that, the molar amount of the activation agent is in the range of 0.03 to 2 molar equivalents, preferably 1 molarequivalent to a linked sphingolipid of a general formula IV.
13. The method according to any one of Claims 5 to 12, characterized in that, the organic solvent is selected from the group comprising isopropanol, tert-butanol, dioxane, dimethyl sulfoxide, acetonitrile and tetrahydrofuran.
14 . The method according to any one of Claims 5 to 13, characterized in that, the concentration of the activated linked sphingolipid of the general formula V that reacts with hyaluronic acid or a pharmaceutically acceptable salt thereof is in the range of 0.25 % to 7.0 % (w/v) in the solution.
15. The method according to any one of Claims 5 to 14, characterized in that, the reaction of the activated linked sphingolipid of the formula V and hyaluronic acid or the pharmaceutically acceptable salt thereof is carried out in the range of temperatures 5 °C to 37 °C for 1 to 72 hours, preferably at 25 °C, for 2 hours.
16. The method according to any one of Claims 5 to 15, characterized in that, the water- miscible organic solvent is selected from a group comprising isopropanol, /e/7-butanol, dioxane, dimethylsulfoxide and tetrahydrofuran.
17. The method according to any one of Claims 5 to 16, characterized in that the molar amount of the activated linked sphingolipid of the foimula V is 0.01 to 2.0 equivalents, preferably 0.03 to 0.5 equivalents with respect to a dimer of hyaluronic acid.
18. A composition characterized by comprising the ester conjugate of any one of Claims 1 to 4, preferably the ester conjugate is in a form of a self-assembled polymeric particle.
19. The composition according to Claim 18, characterized in that the concentration of the ester conjugate of the general Formula I is ranging from 0.001 to 7.5 % (w/w), preferably from 0.001 to 5 % (w/w), more preferably from 0.001 to 1 % (w/w).
20. The composition according to any one of the Claims 18 to 19, characterized in that it is in the form selected from a group comprising emulsion, serum, hydrogel or buccal patch.
21. The composition according to the Claim 20, characterized in that it is in the form of emulsion, while the concentration of the ester conjugate according to the general formula I is ranging from 0.001 to 5 % (w/w), preferably from 0.001 to 1 % (w/w).
22. The composition according to the Claim 21 , characterized in that it contains water and at least one cosmetic or pharmaceutical excipient selected from a group containing oil, wax, butter, emulsifier, auxiliary active ingredient, and preservative.
23. The composition according to the Claim 22, characterized in that, oil is selected from a group comprising coconut, olive, avocado, sesame, almond, castor, sunflower, hemp jojoba, argan, apricot, borage, marula, cottonseed, evening primrose, grapeseed, hazelnut, linseed, meadow foam, moringa, plum, poppy, rice, rosehip, safflower, wheat germ, macadamia oils and squalene; butter is selected from a group comprising cocoa butter, illipe butter, kokum butter, murumuru butter, mango butter, cupuacu butter, avocado butter and shea butter; waxes are selected from a group of lanolin, beeswax, carnauba wax, candelilla wax and petroleum jelly.
24. The composition according to any one of the Claim 22, characterized in that, the emulsifier is selected from a group comprising glyceryl stearate, glyceryl caprylate, behenyl alcohol, glyceryl behenate, cetearyl glucoside, methyl glucose sequistearate, glyceryl stearate citrate, polyglyceryl-3 stearate, cetearyl olivate, lecithin, stearyl alcohol, sorbitan oleate, polysorbates, stearic acid, cetyl alcohol and cetearyl alcohol, sodium acrylate, sodium acryloyldimethyltaurate copolymer or mixtures of thererof.
25. The composition according to any one of the Claim 22, characterized in that auxiliary active ingredient is selected from a group comprising vitamins A, D, E, K, C and B group vitamins; curcumin, coenzyme Q10, allantoin, bisabolol, lactic acid, amino acids, « -hydroxy and /-hydroxy acids, ceramides, peptides, preferably acetyl hexapeptide- 8, palmitoyl tripeptide- 1, palmitoyl tetrapeptide-7, copper tripeptide- 1, palmitoyl pentapeptide-4, Saccharomyces peptides, hexapeptide- 1 and protein, preferably rice protein, soy protein, quinoa protein or wheat protein; polysaccharides, hyaluronic acid or a pharmaceutically and cosmetically acceptable salt thereof, carboxymethyl glucan, schizophyllan, glucomannan; panthenol; urea; glycerin; pentylene glycol; plant extracts, preferably aloe vera extract, chamomile extract, acacia extract, green tea extract, algae extract, oat extract, hemp extract and cranberry extract, bakuchiol, resveratrol; extracts, ferments, lysates or filtrates from bacteria preferably from Lactobacillus, Thalassospira, Bifidobacterium, Halobacterium; or from fungi preferably from Saccharomyces, Agaricus subrufescens, Choiromyces maeandriformis, Cordyceps sinensis, Ganoderma lucidum, Grifola frondosa, Hypsizygus ulmarium, Inonotus obliquus, Lentinula edodes, Polyporus, Trametes versicolor, Tremella fuciformis, Tuber, Schizophyllum commune
26. The composition according to any one of the Claim 22, characterized in that, the preservative is selected from a group comprising aromatic acids and their derivatives, preferably benzoic acid, salicylic acid, dehydroacetic acid, potassium sorbate, parabens; alcohols preferably ethanol, isopropanol, benzyl alcohol, phenoxyethanol, phenethyl alcohol; imidazole derivatives preferably hydantoin, imidazolidinyl urea; cationic surfactants preferably benzalkonium chloride.
27. The composition according to the Claim 20, characterized in that it is in the form of serum, while the concentration of the ester conjugate according to the general formula I is ranging from 0.001 to 5 % (w/w), preferably from 0.001 to 1 % (w/w); while containing water and at least one water soluble auxiliary active ingredient; at least one thickener, and at least one preservative according to Claim 26.
28. The composition according to the Claim 27, characterized in that, the concentration of the ester conjugate according to the general formula I is preferably in range from 0.001 to 1 (w/w).
29. The composition according to the Claim 27, characterized in that water-soluble auxiliary active ingredient is selected from a group comprising vitamin C; B vitamins; amino acids, a-hydroxy and /-hydroxy acids, peptides; preferably acetyl hexapeptide-8, palmitoyl tripeptide, copper tripeptide- 1, Saccharomyces peptides, hexapeptide- 1; and proteins preferably rice protein, soy protein, quinoa protein or wheat protein; and water-soluble polysaccharides, preferably hyaluronic acid or a pharmaceutically and cosmetically acceptable salt thereof, carboxymethyl glucan, schizophyllan, glucomannan, urea; glycerin; pentylene glycol; plant extracts preferably aloe vera extract, cammomile extract, acai extract, green tea extract, algae extract, oat extract, cannabis extract, cranberry extract; extracts, ferments, lysates and filtrates from bacteria preferably from Lactobacillus, Thalassospira, Bifidobacterium, Halobacterium or from fungi preferably from Saccharomyces, Agaricus subrufescens, Choiromyces maeandriformis, Cordyceps sinensis, Ganoderma lucidum, Grifola frondosa, Hypsizygus ulmarium, Inonotus obliquus, Lentinula edodes, Polyporus spp., Trametes versicolor, Tremella fuciformis, Tuber, Schizophyllum commune.
30. The composition according to any one of the Claims 18 to 29 characterized by that it additionally contains at least one hydrophobic compound selected from a group comprising cannabidiol, tocopherol, curcumin, coenzyme Qio, ethyl ferulate, resveratrol, bakuchiol, retinyl palmitate, that is encapsulated in the self-assembled polymeric particles of the ester conjugate according to claims 1 to 4.
31. The composition according to claim 27, characterized in that the thickener is selected from a group comprising xanthan gum, sclerotium gum, guar gum, cellulose gum, carbomer, hydroxy ethylcellulose, Amorphophallus konjac root extract, carrageenan, pullulan, lecithin, lysolecithin, sodium acrylate, sodium acryloyldimethyl taurate copolymer or mixtures thereof.
32. The ester conjugate of sphingolipid and hyaluronan according to any one of Claims 1 to 4; or the composition according to any one of the Claims 18 to 31 for use for treatment of the skin or buccal mucosa.
33. The ester conjugate of sphingolipid and hyaluronan and composition according to Claim 32 for use in the medical treatment of a skin disease selected from the group comprising atopic dermatitis, psoriasis, ichtyosis and rosacea or for use in the medical treatment of a buccal mucosa diseases selected from the group comprising aphthous stomatitis and Behcet's disease.
34. The use of the ester conjugate of sphingolipid and hyaluronan according to any one of Claims 1 to 4 and composition according to any one of the Claims 18 to 31 for cosmetic treatment of the skin.
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