Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
  • A61K8/0291—Micelles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
  • A61K8/14—Liposomes; Vesicles
• • 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
  • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
  • A61K8/90—Block copolymers
• • 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
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • 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
  • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
• • 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
  • A61K9/127—Liposomes
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
  • A61K9/1273—Polymersomes; Liposomes with polymerisable or polymerised bilayer-forming substances
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • 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
• • 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
  • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
  • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
  • C08B37/0021—Dextran, i.e. (alpha-1,4)-D-glucan; Derivatives thereof, e.g. Sephadex, i.e. crosslinked dextran
• • 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
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/10—General cosmetic use

Definitions

• the invention relates to a novel diblock copolymer polysaccharide-b / oc-polypeptide, bioabsorbable or biodegradable and biocompatible, its method of preparation, the nucellar vesicles consisting of this copolymer, and their use for encapsulation, transport, vectorization, and the targeting of molecules of interest, natural or synthetic, with or without a therapeutic activity.
• Naturally occurring organisms exist in vesicular structures resulting from the assembly of amphiphilic biomolecules formed from lipids, proteins and carbohydrates. These natural vesicles are of variable size, the larger ones acting as membranes that protect the intracellular content of the extracellular environment, while small lipid vesicles play a role of transporter of biomolecules inside the cell.
• Stable vesicular structures could be prepared synthetically, starting with liposomes in the 1960s, obtained by assembling natural or synthetic phospholipids, until the recent polymer vesicles formed by the assembly of synthetic block copolymers, called " polymersomes "(BMDischer et al., Science 1999, 284, 1143).
• polymersomes seem very promising for the development of biomedical applications, especially for the transport of therapeutic agents or as microreactors mimicking the behavior of living cells.
• polymersomes have many advantages over liposomes, including a lower critical concentration of aggregation, a unilamellar or multilamellar vesicular structure associated with low size dispersion, and stability. higher membrane decreasing passive leakage of encapsulated molecules.
• the chemical diversity of the block copolymers makes it possible to envisage infinite possibilities of modifying the properties of the vesicle to conform to the desired application.
• polymersomes reported in the literature have been formed by assembling synthetic amphiphilic compounds, such as, for example, polylactide- ⁇ -poly-ethylene oxide, polybutadiene-b / oc poly (ethylene oxide), polycaprolactone-oc-poly-ethylene oxide and poly (2-methyloxazoline) -oc-poly (dimethylsiloxane) -oc-poly (2- methyloxazoline), highly studied because of the biocompatibility or bioresorbability of their respective blocks.
• synthetic amphiphilic compounds such as, for example, polylactide- ⁇ -poly-ethylene oxide, polybutadiene-b / oc poly (ethylene oxide), polycaprolactone-oc-poly-ethylene oxide and poly (2-methyloxazoline) -oc-poly (dimethylsiloxane) -oc-poly (2- methyloxazoline
• polypeptide constructs comprising natural blocks, such as polypeptides or polysaccharides.
• interest for polypeptide constructs lies in the fact that they have unique physicochemical properties: they are optically active by nature, biocompatible and biodegradable in some cases, can be used to mimic natural polypeptide sequences, and are capable of to undergo reversible conformational transitions according to the pH and / or temperature conditions.
• Block copolypeptides represent a particular subclass associating two synthetic polypeptide blocks that combine self-assembly capability with highly ordered three-dimensional structures. They have been described in the literature as promising materials for applications such as biosensors, tissue engineering or selective release of active ingredients. The chemistry of polypeptide-based block copolymers has been extensively studied in the literature using the ring-opening polymerization of the N-carboxyanhydride ("NCA") monomers of the corresponding amino acids initiated by amino derivatives. .
• NCA N-carboxyanhydride
• block copolypeptides could be synthesized, such as poly (L-glutamic acid) -b / oc-poly (L-lysine) (J. Rodriguez-Hernandez and S. Lecommandoux). ,
• dextran- ⁇ b / ⁇ -poly has been reported to self-assemble in water into polydisperse micellar structures having an average diameter of about 100 nm (J. Liu et al. Polymers Carbohydrate, 2007, 69, 196).
• J. Liu et al. Polymers Carbohydrate, 2007, 69, 196 J. Liu et al. Polymers Carbohydrate, 2007, 69, 196.
• block copolymers combining in a single linear chain a polypeptide segment and a polysaccharide block.
• a glycoprotein is a protein carrying at least one polysaccharide group and a polypeptide chain.
• many glycoproteins are on the outer surface of the plasma membrane, with the glycosylated part oriented towards the extracellular medium.
• Glycoproteins are present, in particular, in microorganisms such as viruses and bacteria.
• many pathogenic microorganisms have evolved to recognize the sugars present on the surface of the cells and use them as anchors and entry points in the cells to be infected.
• the HIV virus enters cells of the immune system by binding to membrane receptors such as the CXCR4 and CXCR5 receptors, which are glycoproteins.
• synthetic biomimetic structures that can mimic glycoproteins and may self-assemble as vesicles. This allows, for example, to form "synthetic viruses” mimicking the structure of viruses and their functional role, and to consider effective intracellular delivery of molecules of interest.
• the object of the invention is therefore, according to a first aspect, a b-polysaccharide / oc-polypeptide diblock copolymer consisting of a block of natural or synthetic saccharide units and a block of natural or synthetic peptide units.
• block copolymer as by “diblock copolymer” is meant a linear structure in which a polysaccharide block and a polypeptide block are joined at their ends.
• the linear structure of the copolymers according to the invention advantageously makes it possible to have a high degree of control and reproducibility of the copolymer structure, thus ensuring a regularity of the properties.
• the diblock polysaccharide-b / oc-polypeptide copolymer according to the invention is bioabsorbable or biodegradable and biocompatible.
• saccharide unit block may be used interchangeably to designate a sequence of saccharide monomeric units forming a polysaccharide or a polysaccharide derivative.
• peptide unit block polypeptide block or polypeptide will be used interchangeably to denote a sequence peptide monomer units forming a polypeptide or polypeptide derivative.
• the diblock copolymer according to the invention may comprise any type of natural or synthetic polysaccharide, or a derivative thereof, and any type of natural or synthetic polypeptide, or a derivative thereof.
• polysaccharide derivatives compounds resulting from a chemical modification of a polysaccharide, capable of conferring, for example, a hydrophobic character on a naturally hydrophilic polysaccharide, or, optionally, of adding new functional groups to the molecule.
• Preferred polysaccharides may be, for example, selected from dextran, hyaluronic acid and their derivatives.
• Polysaccharide derivatives and methods for modifying polysaccharides are described, for example, in the reference books “Polysaccharides I”, Adv. Polym. Sci. (2005), 186, and “Polysaccharides II", Adv. Polym. Sci. (2006), 205.
• Polypeptide derivatives means compounds derived from a chemical modification of a polypeptide, capable of conferring, for example, a hydrophobic character on a naturally hydrophilic polypeptide, or, optionally, of add new functional groups on the molecule.
• Such derivatives may be, for example, ester or ether derivatives, in particular organic esters; aliphatic or aromatic inorganic esters; ether derivatives, such as, for example, alkyl or hydroxyalkyl derivatives, amide or imine derivatives, etc.
• the polysaccharide / oc-polypeptide diblock copolymer may comprise a polysaccharide block which has a hydrophilic character and a polypeptide block which is hydrophobic under physiological conditions, either by nature or by chemical modification giving it hydrophobicity.
• the diblock polysaccharide-ib / oc-polypeptide copolymer may comprise a polysaccharide block which has a hydrophobic character, in particular by chemical modification capable of conferring hydrophobicity on a naturally hydrophilic polysaccharide, and a polypeptide block which has a character hydrophilic under physiological conditions.
• the diblock polysaccharide-fo / oc-polypeptide copolymers according to the invention are amphiphilic compounds able to self-assemble to form a new type of assembled micellar vesicles based on natural or synthetic polymers.
• the applications of these vesicles based on diblock copolymer polysaccharide-b / oc-polypeptide offer new possibilities thanks to the intrinsic properties of the polymers used.
• the vesicles based on diblock polysaccharide-b / oc-polypeptide copolymer have the following advantages in particular: a biocompatible and bioresorbable or biodegradable character, a high stability in aqueous solution, an ability to encapsulate hydrophilic and hydrophobic species, and a diversity of chemical functions that can be easily used to modify the surface of the vesicles and thus give them a particular property such as the affinity for a biological receptor.
• polysaccharides or polypeptides having the property of being ligands of cellular receptors also makes it possible to prepare micellar vesicles allowing the targeted release of molecules of interest, said polysaccharide being able, by its nature, to be involved in receptor-mediated endocytosis (RME) mechanisms.
• RME receptor-mediated endocytosis
• the copolymers according to the invention may comprise, in particular, from 5 to 100 saccharide units and from 5 to 100 peptide units, in particular from 10 to 100 saccharide units and from 10 to 100 peptide units and, preferably, from about 10 to about 50 saccharide units and about 10 to 50 peptide units.
• the hydrophilic polymer / hydrophobic polymer mass ratio can be, for example, about 1/1.
• Advantageous copolymers according to the invention have a molar mass of 1000 to 50000 g / mol.
• the polysaccharide may be chosen, for example, from dextran, chitin, chitosan, hyaluronic acid, pullulan, fucan, sulphated fucan, succinoglycan, galactan, arabinogalactan, sulphated galactan or alginates.
• glucan polysialic acid, heparin, heparan sulfate, chondroitin sulfate, acharan sulfate, keratan sulfate, dermatan sulfate, xanthan, pectin, xylan, amylose, amylopectin, cellulose, agarose, mannan, galactomannans, curdlane, arabinan, carrageenans, schizophyllan and their derivatives.
• polypeptide in which the amino acid which constitutes it can be in optically active form L or D or in DL racemic form, can be chosen, for example, from poly (alanine), poly (arginine), poly ( aspartic acid), poly (asparagine), poly (aspartate), poly (cysteine), poly (glutamic acid), poly (glutamate), poly (glutamine), poly (glycine), poly ( histidine), poly (isoleucine), poly (leucine), poly (lysine), poly (methionine), poly (phenylalanine), poly (proline), poly (pyrrolysine), poly (selenocysteine) , poly (serine), poly (threonine), poly (tryptophan), poly (tyrosine), poly (valine), poly (benzyl glutamate), poly (alkyl glutamate), poly (trifluoroacetyl-Lysine), poly (sarcosine) poly (hydroxyethylasparagine
• Preferred hydrophobic polypeptides are, for example, poly ( ⁇ -benzyl L-glutamate) and poly ( ⁇ -trifluoroacetyl-L-lysine). According to another advantageous aspect, said polypeptide is hydrophilic under physiological conditions.
• Preferred hydrophilic polypeptides are, for example, poly (L-glutamic acid), poly (L-glutamate) and poly (L-lysine).
• the polypeptide can be obtained by a ring-opening polymerization process of the N-carboxyanhydride (NCA) monomer of the corresponding amino acid, cited above.
• NCA N-carboxyanhydride
• the invention also relates, in a further aspect, to a process for the preparation of the bioabsorbable or biodegradable biocompatible diblock polysaccharide- ⁇ / polypeptide copolymers described above.
• the synthesis of the copolymers according to the invention implements well-controlled chemical reactions, in particular "chemistry-click” reactions allowing an efficient and quantitative coupling under mild conditions.
• cycloadditions of unsaturated species for example, 1,3-dipolar azide-alkyne cycloadditions, Diels Aider reactions between a diene and a dienophile.
• reactions involving an electrophilic non-aldol carbonyl group for example, formation of oxime ethers from an oxyamine, hydrazones from a hydrazine, or thiosemicarbazones from a thiosemicarbazine
• reactions involving a thiol group formation of thioethers from an alkene and mixed disulfides
• reactions involving thiocarboxylic acid functions or thioesters formation of thioester bonds and amides
• Staudinger ligations involving phosphine and azide functions for example, formation of oxime ethers from an oxyamine, hydrazones from a hydrazine, or thiosemicarbazones from a thiosemicarbazine
• reactions involving a thiol group formation of thioethers from an alkene and mixed disulfides
• reactions involving thiocarboxylic acid functions or thioesters
• the process according to the invention for the preparation of the diblock polysaccharide-b / oc-polypeptide copolymers comprises the steps of:
• said method comprises the steps of:
• Reductive amination can be carried out, for example, using an amine such as propargylamine in the presence of a reducing agent such as sodium borocyanohydride.
• the azotur ⁇ function can be introduced at the end of the polypeptide chain using a bifunctional agent, such as 3-azidoaminopropane, the amine function serving to initiate the ring opening polymerization of the monomer NCA of the acid. corresponding amine.
• the length of the polypeptide block can thus be controlled by the monomeric NCA / bifunctional agent molar ratio.
• a solvent for the coupling step of the polysaccharide chain and the polypeptide chain it is possible to use, for example, an organic solvent such as dimethylsulfoxide (DMSO).
• DMSO dimethylsulfoxide
• This reaction is preferably catalyzed by copper derivatives, such as, for example, CuBr, in the presence of a ligand, for example pentamethyldiethylenetriamine.
• the hydrophilic chain, polypeptide or polysaccharide can be added in slight excess, of the order of 2 molar equivalents, then removed by dialysis, in order to ensure a quantitative coupling reaction and the formation of pure block copolymers.
• the invention also relates, in a further aspect, to new micellar vesicles formed from diblock polysaccharide-fe / oc-polypeptide copolymers consisting of a block of natural or synthetic saccharide units and a block of natural peptide units. or synthetic, connected by their end in a linear chain structure, as described above.
• said copolymers comprise from 5 to 100 saccharide units and from 5 to 100 peptide units, especially from 10 to 100 saccharide units and from 10 to 100 peptide units, and preferably from about 10 to about 50 saccharide units and about 10 about 50 peptide units.
• said copolymers are bioresorbable or biodegradable, and biocompatible.
• micellar vesicles may be prepared by various conventional methods such as, for example, direct dissolution, film hydration, emulsification / diffusion process or nanoprecation. Nanoprecipitation, which consists of mixing a polymer solution in a water-miscible organic solvent, will preferably be used.
• an organic solution of the diblock copolymer according to the invention in water with moderate stirring simultaneously induces a phase separation and a self-assembly process which progresses as the organic solvent diffuses into the water.
• aqueous phase the latter being in excess relative to the organic phase.
• a suitable organic solvent is, for example, dimethylsulfoxide (DMSO) or formamide.
• DMSO dimethylsulfoxide
• the vesicles are recovered directly in aqueous solution. They can then be lyophilized in order to obtain them in the form of a redispersible solids. Their size can be modulated by adjusting the emulsification / diffusion or nanoprecation process (addition direction, nature of the organic solvent, phase volume, copolymer concentration, etc.).
• micellar vesicles according to the invention can be characterized by dynamic and static scattering of light, scattering of neutrons at small angles and transmission electron microscopy. Such techniques for the characterization of micellar systems are described, for example, in G.Riess, Progress in Polymer Science, 2003, 28, 1107.
• said micellar vesicle formed from diblock polysaccharide-6 / oc copolymers -polypeptide contains at least one molecule of interest, natural or synthetic, with or without activity therapeutic.
• This molecule can be introduced, depending on its polarity, in the aqueous phase or in the organic phase before the nanoprecipitation process. It is thus encapsulated during the manufacturing process of the vesicle. It can also be introduced after vesicle formation by pH gradient or emulsification / diffusion methods.
• molecule having a therapeutic activity is meant, for example, a natural or synthetic molecule used for the prevention or treatment of a pathology or the restoration of a biological function, in vitro or in vivo, in particular in humans. animal, including humans, or isolated cells.
• Such molecules may be, for example, a drug active ingredient, such as an antibiotic, an analgesic, an anti-inflammatory, an antitumoral, etc. or a peptide, a protein, a hormone, an enzyme, a nucleic acid, an oligonucleotide, an antigen, an antibody, an interferon, a growth factor, an enzymatic activity modulator, an activator or a cell receptor inhibitor , a vitamin etc.
• a drug active ingredient such as an antibiotic, an analgesic, an anti-inflammatory, an antitumoral, etc. or a peptide, a protein, a hormone, an enzyme, a nucleic acid, an oligonucleotide, an antigen, an antibody, an
• Such molecules having a therapeutic activity can be used, for example, in the pharmaceutical or medical field.
• the encapsulation of said molecule having a therapeutic activity in a micellar vesicle according to the invention makes it possible to significantly reduce the toxicity of said molecule.
• antitumor agents such as, in particular, their cardiotoxicity, for example in the case of the use of doxorubicin or mitoxantrone or their neurotoxicity, for example in the case of the use of vincristine
• molecule of interest having no therapeutic activity is meant, for example, a natural or synthetic molecule which is not used for the prevention or treatment of a pathology or the restoration of a function. biological, in vitro or in vivo, especially in animals, including humans, or isolated cells.
• diagnostic agents such as, for example, contrast agents, isotopes, fluorescent probes
• olfactory molecules pigments, dyes, antioxidants, antibacterial agents, antiseptic agents, emollients, exfoliating agents, moisturizers, etc .;
• cleaning and detergency surfactants, dyes, antioxidants, antibacterials, antiseptics, whiteners, etc .;
• phytopharmacy pesticides, fungicides, herbicides, insecticides, growth accelerators, etc.
• - agrifood coloring agents, flavor enhancers etc
• organic chemistry organic, metallic or inorganic catalytic derivatives, etc., or in any other field of activity in which it is desired to encapsulate a molecule in a micellar vesicle, the latter having the advantage of being bioabsorbable or biodegradable and biocompatible .
• micellar vesicles formed from diblock polysaccharide-fc / oc-polypeptide copolymers consisting of a block of natural or synthetic saccharide units and a block of natural or synthetic polypeptide units, as described above, as vehicles for encapsulation, transport, vectorization and / or targeting of at least one molecule of interest, natural or synthetic, with or without a therapeutic activity, represents another aspect of the invention.
• the invention also relates to pharmaceutical compositions comprising micellar vesicles formed from diblock polysaccharide - /> / oc-polypeptide copolymers consisting of a block of natural or synthetic saccharide units and a block of natural or synthetic polypeptide units. and containing at least one molecule of interest, natural or synthetic, with or without a therapeutic activity, as described above, and a pharmaceutically acceptable excipient.
• the pharmaceutically acceptable excipients may be chosen according to the method of administration envisaged from the usual excipients in the pharmaceutical field.
• the excipients may be selected from binders, fillers, disintegrants, adjuvants or retarders and for oral administration as suspensions, solutions or emulsions.
• binders for oral administration in tablet form
• the excipients may be selected from binders, fillers, disintegrants, adjuvants or retarders and for oral administration as suspensions, solutions or emulsions.
• the compositions may also contain suspending agents; emulsifiers, non-aqueous vehicles or preservatives
• the pharmaceutical compositions may be in the form of aqueous and non-aqueous sterile injectable solutions which may contain antioxidants, buffers, bacteriostatic agents and solutes which render the formulation isotonic with the blood of the selected recipient; or in the form of sterile aqueous or non-aqueous suspensions which may include suspending agents and thickening agents.
• compositions according to the invention may also be administered by other routes, such as the oral or sublingual route using a suitable excipient, the topical route on the epidermis, in the form of cream or gel, ointment, lotion or transdermal patch, the intranasal route, in the form of a spraying liquid, a powder or drops or by inhalation, using a suitable propellant.
• routes such as the oral or sublingual route using a suitable excipient, the topical route on the epidermis, in the form of cream or gel, ointment, lotion or transdermal patch, the intranasal route, in the form of a spraying liquid, a powder or drops or by inhalation, using a suitable propellant.
• micellar vesicles according to the invention can be used both for the encapsulation of hydrophobic molecules and hydrophilic molecules, because they have two compartments of different polarity, namely the membrane which is hydrophobic and the internal cavity which is hydrophilic.
• said micellar vesicle is formed from a diblock polysaccharide-6 / oc-polypeptide copolymer in which the polysaccharide has affinity for at least one cellular receptor.
• said micellar vesicle is formed from a b-polysaccharide / oc-polypeptide diblock copolymer in which the polypeptide has affinity for at least one cellular receptor.
• micellar vesicles may advantageously be used for targeting a molecule of interest.
• hyaluronic acid which has an affinity for CD44 receptors.
• CD44 receptors are present at elevated levels in tumor cells of various carcinomas, melanomas, lymphomas, etc. ("Chemistry and Biology of Hyaluronan", H.G. Garg and CA.Haies Ed. (2004), Chapter 5, Elsevier Ltd.).
• Polypeptides of interest in this regard are, for example, polyglutamic acid or polyglutamate.
• polyglutamic acid or polyglutamate are, for example, glutamate receptor ligands (Mornet C, Briley M, Trends Pharmacol Sci., 1988; 9: 278-279) or TLR4 receptors ( Poo et al 22 (2) 517 - The FASEB Journal).
• micellar vesicle is formed from a diblock polysaccharide-b / oc-polypeptide copolymer wherein said polysaccharide is capable of being involved in a receptor-controlled endocytosis mechanism ("Receptor”). mediated endocytosis "or RME).
• Receptor receptor-controlled endocytosis mechanism
• RME mediated endocytosis
• micellar vesicle is formed from a b-polysaccharide / oc-polypeptide diblock copolymer wherein said polypeptide is capable of being involved in a receptor controlled endocytosis mechanism.
• Receptor-controlled endocytosis is a biological mechanism by which molecules in the extracellular space bind to cellular receptors and are internalized, also allowing the targeting of a molecule of interest.
• a polysaccharide of interest for the purposes of the invention is arabinogalactan, which interacts with hepatocyte receptors involved in controlled endocytosis mechanisms.
• dextran-block-poly ( ⁇ -benzyl L-glutamate) copolymer Two equivalents of alkynyl dextran (0.4 g, 6.06.10 5 mol) and one equivalent of PBLG-azide (0.147 g, 3.03 ⁇ 10 5 mol) ) are solubilized in 25 mL of anhydrous DMSO. The mixture is stirred for 20 minutes then 12.6 ⁇ l of pentamethyldiethylenetriamine (6.06 ⁇ 10 5 mol) are added under a nitrogen atmosphere. The mixture is then degassed 3 times by freeze / thaw cycles and transferred under nitrogen into a Schlenk-type flask containing CuBr (9 mg, 6.06 ⁇ 10 5 mol).
• the reaction mixture is left stirring at ambient temperature for 3 days and then dialyzed against milliQ water using a Spectra / Por® dialysis membrane 6 characterized by a cutoff threshold of 50 kDa.
• the copolymer is then recovered by lyophilization.
• the mass of product recovered is 0.302 g (yield: 87%).
• a solution of vesicles is thus obtained and characterized by dynamic and static scattering of light, diffusion of small angle neutrons and transmission electron microscopy.
• Polymeric vesicles of 200 nm in radius with a small size distribution and having a membrane thickness of about 9 nm are thus obtained.
• FIG. 2 shows the size distribution of the vesicles (hydrodynamic radius) obtained by light scattering.
• FIG. 3 represents an image of transmission electron microscopy obtained on these same vesicles.
• the ⁇ -benzyl dextran-b / ⁇ -poly ( ⁇ -benzyl L-glutamate) copolymer of Example 2 is dissolved in 1 ml of DMSO at a concentration of 0.5 mg / ml, then 9 ml of milliQ water are gradually added to the solution. using a syringe pump at 18 mL / h with shaking.
• the DMSO is then removed by dialysis against milliQ water using a Spectra / Por® dialysis membrane 6 characterized by a 2kDa cut-off.
• the vesicles then formed are analyzed by dynamic light scattering and transmission electron microscopy.
• Polymeric vesicles of 40 nm in radius are thus obtained with a small size distribution.
• FIG. 4 shows the size distribution of the vesicles (hydrodynamic radius) obtained by light scattering.
• FIG. 5 represents an image of transmission electron microscopy obtained on these same vesicles.
• Example 6 Encapsulation of an Active Principle in a Micellar Vesicle
• Doxorubicin and docetaxel were encapsulated in micellar vesicles based on the hyaluronic acid-b / oc-poly ( ⁇ -benzyl L-glutamate) copolymer of Example 1 by nanoprecipitation.
• the active ingredient was dissolved in DMSO or Tris buffer in bulk ratios of active ingredient: copolymer of 0.1: 1, 0.2: 1 and 0.3: 1.
• the amount of encapsulated doxorubicin (in the hydrophobic or hydrophilic portion of the vesicles) and the encapsulation efficiency were determined by breaking the charged vesicles into a DMSO: Tris (80:20) mixture. After centrifugation for 1 h, the sample was filtered and measured by UV spectroscopy at 485 nm. Quantification was performed from the calibration curve of doxorubicin in the DMSO: Tris (80:20) mixture. The amount of encapsulated docetaxel and the encapsulation efficiency were determined from the reconstitution of a dry vesicle extract containing docetaxel in ethanol. Then, the filtered sample was measured by UV spectroscopy at 230 nm. Quantification was performed from the calibration curve of doxorubicin in ethanol.
• Encapsulated quantity (mass of active principle in the charged vesicle / mass of vesicle) x100
• the desired amount for example, a volume of 4mL at a concentration of 1mg / mL
• doxorubicin and docetaxel loaded vesicles is poured into a dialysis tube (Spectra / Por® Float-A-Lyzer® Dialysis
• the dialysis tube is introduced vertically into a measuring cylinder of 50 ml.
• the system is maintained at 37 ° C + 2 and covered with parafilm to prevent evaporation.
• a measuring cylinder of 50 ml.
• sampling is done in the dialysis tube and then reinserted as is after analysis.
• sampling is done outside the dialysis tube.
• the dialysis medium contained 50 mL of Tris buffer (pH 7.4) with 2% v / v ethanol to increase the solubility of the released docetaxel and to avoid aggregation of the free docetaxel.
• Quantification is performed by the calibration curve of free active principles in their respective solvents.
• the release profiles expressed as a percentage of cumulative release as a function of time (in second 1/2 ) are represented in FIGS. 6 (doxorubicin) and 7 (docetaxel).
• the free active ingredient is represented by the symbol -o- (empty circle) and the release of the encapsulated active ingredient is represented by the symbol - • - (full circle).
• toxicity and in vitro release experiments were carried out from vesicles based on hyaluronic acid- ⁇ / ⁇ -poly ( ⁇ -benzyl L-glutamate) diblock copolymer prepared in Example 6, containing 11% of doxorubicin.
• rat C6 glioma cells were placed in 10 cm diameter falcons and grow in 10 ml of the culture medium containing penicillin (10000 ⁇ g / ml), streptomycin (10000 ⁇ g / ml) and amphotericin.
• B 25 ⁇ g / ml [Invitrogen Corporation] in an incubator at 37 ° C. in a controlled atmosphere at 5% CO 2 .
• C6 cells were incubated in a 24-well plate (15 ⁇ 10 4 cells per well) for 24 hours.
• Viability was determined by a standard tetrazolium salt test MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide).
• MTT 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide.
• the tetrazolium it contains is reduced in fomnazan by the mitochondrial succinate dehydrogenase of living living living cells.
• the color of the medium then changes from yellow to purplish blue.
• the intensity of this staining is proportional to the number of living cells present during the test.
• analysis is performed spectrophotometrically (U-2800A, HITACHI) by measuring the absorbance at 570 nm.
• the measurements are standardized by a control experiment, for which the MTT test is performed without cells.
• the value of IC50 is thus determined for a time of 48 hours, corresponding to the concentration for which half of
• the follow-up of cellular intemalisation was performed in vitro by fluorescence microscopy.
• the C6 cells were incubated (15 ⁇ 10 4 cells) in a 4-well plate of 16 mm.
• the vesicles containing doxorubicin at a concentration of 4.54 ⁇ M were incubated at times of 3h, 6h and 24h at 37 ° C. in a controlled atmosphere at 5% CO 2 .
• the cells are washed twice with PBS buffer, fixed on a microscope slide cover with 4% paraformaldehyde (PF4) in PBS buffer, left in the dark at room temperature for 30 min, and then washed. once with PBS buffer and once with pure water.
• PF4 paraformaldehyde
• This dispersion was applied to a dose of O 1 SmL 1 under semi-occlusive dressing, for 4 hours on a healthy skin area in 3 rabbits.
• the experimental protocol is that of OECD Directive No. 404 of 24 April 2002 and Test Method B.4 of EC Directive 440/2008.
• Example 1 A 1% (w / v) dispersion of hyaluronic acid-6 / oc-poly ( ⁇ -benzyl L-glutamate) diblock copolymer of Example 1 was used.
• the copolymer dispersion was applied to the dorsal surface of the ear of a female mouse line.
• the copolymer dispersion was administered intravenously in a male and a female Swiss mouse at an administration volume of 10 ml / kg. Considering the density of the formulation, this administration corresponds to a dose of 10 g / kg.
• MTD Maximum tolerated dose of the copolymer dispersion according to the invention intravenously is greater than 10 g / kg in the mouse.
• the human cell line MCF-7 which significantly expresses CD44 receptors, was used.
• a competitive inhibition assay was performed by previously adding free hyaluronic acid to the incubation medium in order to block to some extent the receptors for hyaluronic acid on the surface of the cells.
• the cells were then washed twice with PBS buffer and collected by centrifugation (1000 rpm for 10 min). The cells were then resuspended in 300 ⁇ l of PBS buffer prior to measurement.
• Samples were collected on a Calibur FACS flow cytometer (Beckton Dickinson) and analyzed using CeII Quest software provided by the manufacturer. For each analysis, at least 10,000 cells were counted.
• FIG. 9 represents the relative fluorescence intensity measured in arbitrary units (AU) in the MCF-7 cells measured after incubation in the presence of encapsulated doxorubicin (black column), free doxorubicin
• DMBA 7,12-dimethylbenz [ ⁇ ] anthracene
• the tumor-bearing animals were separated and randomly divided into different treatment groups, and then treated with a single dose of 250 ⁇ l intravenous doxorubicin at 5mg / kg, either in the form of doxorubicin free, either in the form of doxorubicin encapsulated in the micellar vesicles of Example 6.
• the control group received a single dose of 250 ⁇ l of buffer
• PBS (pH 7.4) intravenously.
• Figure 10 reports the change in tumor volume as a function of time, following treatment with free doxorubicin (curve represented by the symbol -V-), encapsulated doxorubicin (curve represented by the symbol -D-) or PBS (curve represented by the symbol - • -).
• free doxorubicin and encapsulated doxorubicin significantly inhibit tumor growth compared to the PBS treated control group.
• encapsulated doxorubicin causes tumor suppression significantly higher than free doxorubicin.
• doxorubicin One of the main well-known side effects of doxorubicin is its cardiotoxicity. This can be evaluated by measuring the production of specific enzymes, mainly lactate dehydrogenase and creatine kinase.

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