Classifications

• • 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
  • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
  • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
  • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
  • C08G69/10—Alpha-amino-carboxylic acids
• • 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
  • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
• • 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
  • A61K47/62—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 a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• • 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
  • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
  • C08G69/48—Polymers modified by chemical after-treatment
• • 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
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites

Definitions

• the present invention relates to novel biodegradable materials based on copolyamino acids, useful in particular for the vectorization of active principle (s) (PA).
• PA active principle
• the invention also relates to novel pharmaceutical, cosmetic, dietetic or phytosanitary compositions based on these modified polyamino acids. These compositions may be of the type of those allowing the vectorization of AP and are preferably in the form of emulsions, micelles, nanoparticles, microparticles, gels, implants or films.
• the PAs considered are, advantageously, biologically active compounds that can be administered to an animal or human organism orally, parenterally, nasally, vaginally, ocularly, subcutaneously, intravenously, intramuscularly, intradermally, intraperitoneally, intracerebrally, orally, etc.
• PAs more particularly, but not exclusively, concerned by the invention are proteins, glycoproteins, peptides, polysaccharides, lipopolysaccharides, oligo or polynucleotides, and organic molecules. But it can also be cosmetic products or phytosanitary products, such as herbicides, insecticides, fungicides, etc.
• polymers of the polylactic, polylactic-glycolic, polyoxyethylene-oxypropylene, polyamino acid or polysaccharide type are raw materials for manufacturing, for example, mass implants, microparticles, nanoparticles, vesicles, micelles or gels.
• these polymers must be suitable for the manufacture of such systems, they must also be biocompatible, non-toxic, non-immunogenic, economical and they must be easily removed from the body. and / or be biodegradable. On this last aspect, it is moreover essential that the biodegradation in the organism generates non-toxic products.
• US-B-4,652,441 discloses polylactide microcapsules encapsulating the hormone LH-RH. These microcapsules are produced by preparing a water-in-oil-in-water emulsion and comprise an aqueous inner layer containing the hormone, a substance (gelatin) fixing the same, an oily layer of polylactide, and an aqueous outer layer. (polyvinyl alcohol). The release of the AP can be done over a period of more than two weeks after subcutaneous injection.
• compositions based on amphiphilic poly (oxyethylene) -poly (oxypropylene) micelles for the vectorization of anticancer agents such as doxorubicin.
• US-B-4,888,398 discloses polymers based on polyglutamate or polyaspartate, and optionally polyleucine, with pendant groups of alkyloxycarbonyl-methyl type, randomly placed on the polyamino acid chain.
• These polyamino acids, grafted with side groups e.g. methoxycarbonylmethyl, can be used in the form of biodegradable implants containing a sustained release PA.
• US Pat. No. 5,904,936 describes nanoparticles obtained from a polyleucine-polyglutamate block polymer capable of forming stable colloidal suspensions capable of associating spontaneously with biologically active proteins without denaturing them. These can then be released in vivo in a controlled manner over a long period.
• the patent application WO-A-99/61512 describes polylysines and polyornithines functionalized with a hydrophobic group (palmitic acid connected to polylysine or ornithine) and a hydrophilic group (polyoxyethylene).
• These polymers for example polylysine grafted with polyoxyethylene and palmitoyl chains, form, in the presence of cholesterol, vesicles capable of encapsulating doxorubicin or DNA.
• These polymers based on polylysines are cationic in a physiological medium.
• EP-A-963 758 describes polyamino acids functionalized with a cationic group. These polymers are capable of forming complexes with a nucleic acid and can be used in gene therapy.
• the cationic groups are amino derivatives which are not derived from amino acids and the polyamino acids do not contain hydrophobic groups.
• WO-A-03/104303 discloses anionic polyamino acids functionalized with alpha-tocopherol.
• the application WO-A-2004/013206 describes anionic polyamino acids having hydrophobic groups and characterized in that these groups are connected to the polymer via a ball joint containing two amide functions, and more specifically via a lysine-type spacer. or ornithine.
• the application WO-A-2004/060968 describes polyamino acids functionalized with at least one oligoamino acid group based on leucine and / or isoleucine and / or valine and / or phenylalanine.
• the invention relates to biodegradable polyamino acids that can be converted into nano- or micro-colloidal vector particles capable of reversibly associating with active principles.
• one of the essential objectives of the present invention is to provide novel amphiphilic copolyglutamates comprising both positive charges at neutral pH or close to neutrality and pendant hydrophobic groups.
• These polymers represent an improvement over those described in the patents or patent applications cited above in terms of vectorization of an active ingredient such as a peptide or a therapeutic protein, a DNA, an RNA or a small molecule.
• Another essential objective of the present invention is that these polymers are capable of being used for the AP active ingredient vectorization and make it possible to optimally satisfy all the specifications of the specification, namely in particular: o capacity:
• polyamino acids or their pharmaceutically acceptable salts, comprising glutamic residues, characterized in that some of the glutamic residues carry a pendant cationic group which, if it is deprotonable, has a pKa greater than or equal to 7, said cationic groups being identical or different from each other, and in that other glutamic residues carry a hydrophobic group (GH) during, said hydrophobic groups ( GH) being identical or different from each other.
• glutamic residues characterized in that some of the glutamic residues carry a pendant cationic group which, if it is deprotonable, has a pKa greater than or equal to 7, said cationic groups being identical or different from each other, and in that other glutamic residues carry a hydrophobic group (GH) during, said hydrophobic groups ( GH) being identical or different from each other.
• GH hydrophobic group
• cationic group will generally refer to cationic groups which are not deprotonable, and cationic groups which are deprotonable and which have a pKa greater than or equal to 7.
• pharmaceutically acceptable salts of a polyamino acid according to the invention means all of the polyamino acids with the counterions associated with the ionized functions of the polymer.
• small molecule refers to a molecule of molecular weight less than 1 kDa.
• the alkyl radicals have 1 to 10 carbon atoms.
• Each polyglutamate according to the invention is therefore functionalized by a multiplicity of cationic groups and hydrophobic groups (GH), pendant and respectively identical or different from each other.
• GH hydrophobic groups
• the term "multiplicity" means that the polyglutamate is functionalized by: at least 1% of cationic groups (mol% relative to the glutamic acid residues) and up to 99%,
• the polyglide glutamic has, in addition to pendant hydrophobic groups (GH), hydrophobic groups (GH) attached to at least one end of the copolymer chains.
• carrier means that the group carried is pendant, that is to say that said group is a lateral group with respect to glutamic residues and is a substituent of the ⁇ carbonyl function of the glutamic residue that carries it.
• Polyglutamate also carries cationic groups. These groups are preferably grafted to glutamic residues through an amide or ester bond.
• other glutamic residues may carry a non-ionizable group during, other than hydrophobic groups (GH), said nonionizable groups being identical or different from each other.
• GH hydrophobic groups
• Such a pendant nonionizable group may be, for example, the hydroxyethylamino group.
• other glutamic residues may carry, also on their carbonyl in ⁇ , a non-ionized group at neutral pH, different from hydrophobic groups (GH), said nonionized groups at pH neutral being the same or different from each other.
• a non-ionized group at neutral pH may for example satisfy the following formula:
• the polyglutamate, carrying both cationic groups and hydrophobic groups, and optionally also nonionic groups (nonionizable or nonionized at neutral pH) may also comprise negative charges (always at neutral pH) resulting from the ionization of pendant groups of polyglutamic acid that have not been functionalized.
• the polymers of the invention can be:
• association or “associate” used to describe the relationship between one or more active ingredients and the modified polyglutamates, mean, in particular, that the active ingredient (s) are related to the (x) polyglutamate (s) in particular by a hydrophobic interaction, and / or are encapsulated by the polyglutamate (s).
• the polyamino acids according to the invention are functionalized homopolymers of L-glutamate or L-glutamic acid; preferably these residues are bonded in the polyamino acid by their carboxyl in the alpha position.
• the cationic groups that can be used to functionalize the glutamate residues are identical or different from each other and correspond to the following general formula:
• Z " chloride, sulphate, phosphate or acetate
• L linear (C 2 -C 6 ) alkylene and optionally substituted by a carboxyl or derivative functional group.
• the cationic groups are obtained from precursors chosen from the group comprising: lysine, subsectionthine, arginine and their derivatives, choline, ethanolamine (bound by oxygen), putrescine and lysine. agmatine.
• the derivatives of lysine, ornithine, and arginine may be, for example, ethyl and methyl esters, amides, and methylated amides.
• cationic groups that can be used in the present invention can be chosen from the following group of radicals:
• Ra represents a hydroxyl, alkoxy or alkylamino group, preferably a group -OMe, -OEt, -NH 2 , -NHCH 3 or
• Z " represents a chloride, a sulfate, a phosphate or an acetate, preferably a chloride, or
• the pendant cationic groups may have the following formulas (where the name of the precursor is indicated under each radical): And ester or ⁇ mld ⁇ Agm ⁇ tine Arghlne ester or ⁇ mlde
• Ra represents a hydroxyl, alkoxy or alkylamino group, preferably a group -OMe, -OEt, -NH 2 , -NH-CH 3 or N (CH 3 ) 2 .
• the polyamino acids of the invention comprise on average at least 3 hydrophobic groups (GH) per polymer chain.
• At least one of the hydrophobic groups GH is included in a hydrophobic graft comprising at least one spacer (or "spacer") balloon (or pattern) for connecting the hydrophobic group GH to a polyglutamate chain (for example a main chain - skeleton-polyglutamate).
• This patella may comprise, e.g., at least one direct covalent bond and / or at least one amide bond and / or at least one ester bond.
• the patella may be of the type belonging to the group comprising, in particular: the amino acid residues different from the constituent monomeric unit of the polyglutamate, the aminoalcohol derivatives, the polyamine derivatives (for example the diamines), the derivatives of the polyols (for example diols) and derivatives of hydroxy acids.
• the grafting of GH on the polyglutamate chain may involve the use of precursors of GH, able to bind to the polyglutamate chain.
• the precursors of GH are, in practice and without being limited to, selected from the group comprising alcohols and amines, these compounds being easily functionalized by those skilled in the art.
• the grafting of GH is explained in more detail below in the description of the process for obtaining modified polyamino acids according to the invention.
• the hydrophobic group GH of the hydrophobic graft comprises from 8 to 30 carbon atoms.
• These hydrophobic groups GH are advantageously and judiciously selected from the group comprising:
• Linear or branched C 8 to C 30 alkyls which may optionally comprise at least one unsaturation and / or at least one heteroatom,
• C 8 -C 30 alkylaryls or arylalkyls which may optionally comprise at least one unsaturation and / or at least one heteroatom,
• the (poly) cyclic C 8 to C 30 may optionally comprise at least one unsaturation and / or at least one heteroatom.
• the GH-forming patellae of the hydrophobic grafts may be di-, tri- or tetravalent (or even pentavalent and more).
• a divalent patella the hydrophobic graft comprises a single GH group, while a trivalent patella gives the hydrophobic graft a bifid character, that is to say that the graft has two "legs" GH.
• a trivalent patella mention may be made, inter alia, of "amino acid” residues, for example "glutamic acid” or polyol residues, for example glycerol.
• two advantageous but non-limiting examples of hydrophobic grafts comprising bifid GHs are dialkylglycerols and dialkylglutamates.
• the hydrophobic groups GH can be, for example, derived from groups selected from the following group: octanol, dodecanol, tetradecanol, hexadecanol, octadecanol, oleyl alcohol, tocopherol or cholesterol.
• the polyglutamates according to the invention may also carry at least one graft of polyalkylene (preferably ethylene) glycol type bonded to a glutamate residue.
• the backbone of the polyglutamate according to the present invention comprises alpha-L-glutamate and / or alpha-L-glutamic acid residues.
• polyglutamates according to the invention have the following formula (I):
• A represents independently:
• -R 7 is -OH, -OR 9 or -NHR 10 , and
• R 8 , R 9 and R 10 independently represent H, linear C 2 -C 10 alkyl, branched C 3 -C 10 alkyl or benzyl;
• B is a direct bond, a divalent linking group, trivalent or tetravalent, preferably chosen from the following radicals: -O-, -NH-, -N (C ⁇ 5 alkyl) -, an amino acid residue (of natural preference), diol, triol, diamine, triamine, aminoalcohol or hydroxyacid having 1 to 6 carbon atoms;
• D is H, linear C 2 -C 10 acyl, branched C 3 -C 10 acyl, or pyroglutamate;
• the hydrophobic groups GH each independently represent a radical chosen from: • linear or branched C 8 to C 3 o alkyls which may optionally comprise at least one unsaturation and / or at least one heteroatom (preferably O and / or N and / or S), or C 8 -C
• the C 8 to C 30 (poly) cyclic compounds possibly comprising at least one unsaturation and / or at least one heteroatom (preferably
• this radical is selected from the following group: octyloxy-, dodecyloxy-, tetradecyloxy-, hexadecyloxy-, octadecyloxy-, 9-octadecenyloxy-, tocopheryloxy- or cholesteryloxy-, B being then a direct bond;
• R 1 represents a radical chosen from the group having the following formulas:
• X is an oxygen atom or a -NH-
• R is H, linear C 2 to Ci 0 alkyl, branched C 3 to Ci 0 or benzyl,
• R 3 represents a hydroxyethylamino-, an alkylene glycol, a polyalkylene glycol or a radical of formula:
• hydrophobic groups GH and the cationic groups are randomly arranged in pendant groups.
• the molar grafting rate of the polyglutamates according to the invention in the hydrophobic groups is between 2 and 99%, and preferably between 5 and 50%, provided that each polymer chain has an average of at least 3 hydrophobic grafts.
• the ratio (q) / (p + q + r + s) of the polyglutamates according to the invention means that they can contain from 1 to about 97 mol% of groups containing a cationic charge.
• the ratio (s) / (p + q + r + s) of the polyglutamates according to the invention means that they can be anionic, neutral or cationic at neutral pH.
• the polymers according to the invention have a molar mass which is between 2000 and 200000 g / mol, and preferably between 5000 and 100000 g / mol.
• the invention also covers mixtures of modified polyamino acids as defined above.
• the polyglutamates of the invention are likely to be used in several ways depending on the nature of the hydrophobic groups and cationic groups, the charge and the degree of polymerization of the polyglutamate.
• Methods for shaping a polymer for the encapsulation of an active ingredient in the various forms contemplated by the invention are known to those skilled in the art. For more details, we can refer, for example to these few particularly relevant references:
• polyglutamates in the form of particles or not
• active ingredients such as proteins, peptides, DNA, RNA or small molecules.
• the preferred shaping is that described in US Pat. No. 6,630,171, which consists of dispersing the copolymer in water and incubating the solution in the presence of an active ingredient (PA).
• PA active ingredient
• polymer is cationic and soluble at acid pH due to an excess of cationic charge and that this charge is partially or completely neutralized at neutral pH, such a polymer is said to be pH dependent.
• This type of polymer can therefore be used to form a deposit after administration, for example in the subcutaneous tissue.
• the carboxylic residual functions of the modified polyglutamate are either neutral (COOH form) or ionized (COO anion), depending on pH and composition.
• the counter-cation may be a metal cation such as sodium, calcium or magnesium, or an organic cation such as triethanolamine, tris (hydroxymethyl) aminomethane or a polyamine such as polyethyleneimine. If it is divalent, a counter cation can salt two nearby monovalent cationic groups.
• the counter anion of the cationic groups is preferably chosen from the group comprising a chloride, a sulfate, a phosphate or an acetate. If he is divalent, a counteranion can salify two nearby monovalent cationic groups.
• pharmaceutically acceptable salts of the polymer according to the invention all of the polymers with the counter-ions associated with the ionized functions of the polymer. It is also conceivable, for certain structures where there is co-existence of positive and negative charges that there is a total or partial neutralization of charges. A polymer having an equivalent number of positive charges and negative charges (isoelectric point) can exist without the presence of counter-anion or counter-cation.
• the copolymers of the invention are for example obtained by methods known to those skilled in the art.
• N-carboxy-amino acid anhydrides NCA
• the polymers are then hydrolyzed under appropriate conditions to obtain the polymer in its acid form.
• the copolymers of the invention are synthesized according to 2 routes.
• the cationic group for example pargininamide
• the group B-GH for example dodecylamine
• This reaction can be carried out in a solvent such as DMF, DMSO or NMP according to the following scheme.
• the precursor of the R 1 group such as nitrogen-bound ethanolamine, is introduced during the synthesis together with the cationic group.
• the cationic group contains two chemically undifferentiated amino functions (e.g. linear diamine), it can be introduced in a form in which one of the two functions is protected. A last step of cleavage of the protecting group is then added to the above scheme.
• two chemically undifferentiated amino functions e.g. linear diamine
• the poly (L-glutamic acid) can be synthesized according to the route described in the patent application FR 2801 226.
• the HB-GH group is linked via an ester function, it is easier to graft first.
• the B-GH group by a conventional coupling reaction using a carbodiimide before grafting the cationic group.
• the precursor of the group R 1 such as ethanolamine bonded with nitrogen, is introduced during the synthesis together with the cationic group.
• the cationic group contains two chemically undifferentiated amino functions (e.g. linear diamine), it can be introduced in a form in which one of the two functions is protected. A last step of cleavage of the protecting group is then added to the above scheme.
• two chemically undifferentiated amino functions e.g. linear diamine
• the degree of polymerization is defined by the molar ratio of the initiator to that of the monomer.
• the coupling of the hydrophobic graft GH with an acidic function of the polymer is easily achieved by reacting the polyamino acid in the presence of a carbodiimide as a coupling agent and optionally a catalyst such as 4-dimethylaminopyridine and in a suitable solvent such as dimethylformamide ( DMF), N-methyl pyrrolidone (NMP) or dimethylsulfoxide (DMSO).
• a carbodiimide is, for example, dicyclohexylcarbodiimide or diisopropylcarbodiimide.
• Coupling reagents such as chloro Formates can also be used for the formation of amide bonds (see, for example, Bodanszky: “Principles of Peptide Synthesis” Springer Verlag, 1984).
• the degree of grafting is controlled by the stoichiometry of the constituents and reactants or the reaction time.
• Hydrophobic grafts functionalized with an amino acid other than that of the polymer are obtained by conventional peptide coupling or by direct condensation by acid catalysis. These techniques are well known to those skilled in the art.
• the invention relates to a pharmaceutical, cosmetic, dietetic or phytosanitary composition
• a pharmaceutical, cosmetic, dietetic or phytosanitary composition comprising at least one polyglutamate as defined above and optionally at least one active ingredient, which may be therapeutic, cosmetic, dietetic or phytosanitary.
• the active principle is associated with (x) polyamino acid (s) modified with a cationic group by one or more bonds other than (or that) chemical bond (s) (s) ( s) covalent (s).
• the active principle is chosen from the group comprising: proteins, glycoproteins, proteins linked to one or more polyalkylene glycol chains [preferably polyethylene glycol (PEG): "PEGylated proteins”], peptides, polysaccharides, liposaccharides, oligonucleotides, polynucleotides and mixtures thereof, and more preferably still in the erythropoietin subgroup, such as epoetin alpha, epoetin beta, darbepoetin, hemoglobin, their analogues or their derivatives; oxytocin, vasopressin, adrenocorticotropic hormone, epidermal growth factor, platelet derived growth factor (PDGF), stimulating factors of hematopo
• active ingredients are polysaccharides (eg, heparin) and oligo- or polynucleotides, DNA, RNA, iRNA, antibiotics, and living cells.
• Another class of active ingredients includes pharmaceutical substances acting on the central nervous system, for example, risperidone, zuclopenthixol, fluphenazine, perphenazine, flupentixol, haloperidol, fluspirilene, quetiapine, clozapine, amisulprid, sulpiride, ziprasidone, etc.
• the active ingredient is a small hydrophobic, hydrophilic or amphiphilic organic molecule.
• a "small" molecule is especially a small nonprotein molecule.
• PA that may be associated with the polyamino acids according to the invention, whether or not in the form of (nano or micro) particles, mention may be made of: o proteins such as insulin, interferons, hormones growth, interleukins, erythropoietin or cytokines; peptides such as leuprolide or cyclosporine; o small molecules such as those belonging to the family of anthracyclines, taxoids or camptothecins; o and their mixtures.
• proteins such as insulin, interferons, hormones growth, interleukins, erythropoietin or cytokines
• peptides such as leuprolide or cyclosporine
• small molecules such as those belonging to the family of anthracyclines, taxoids or camptothecins
• o and their mixtures o proteins such as insulin, interferons, hormones growth, interleukins, erythropoietin or cytokines
• the active principle is chosen from at least one of the following families of active substances: alcohol abuse treatment agents, agents for treating Alzheimer's disease, anesthetics, acromegaly treatment agents, analgesics, anti-asthmatics, allergy-treating agents, anti-cancer agents, anti-inflammatories, anticoagulants and antithrombotics, anticonvulsants, antiepileptics, antidiabetics, antiemetics, anti-glaucoma, antihistamines, antiparasitics, antibiotics, antifungals, antivirals, antiparkinsonians, anticholinergics, antitussives, carbonic anhydrase inhibitors, cardiovascular agents, lipid-lowering agents, anti-arrhythmics, vasodilators, anti-hypertensives, vasoprotective agents, cholinesterase inhibitors, anti-inflammatory agents, disorders of central nervous system, central nervous system stimulants, contraceptives, fertility promoters, uterine labor inducers and inhibitors,
• the composition of the invention is in the form of a gel, a solution, a suspension, an emulsion, micelles, nanoparticles, microparticles, an implant, a d a powder, a suspension or a film.
• the composition whether loaded or not with active ingredient (s), is a stable colloidal suspension of nanoparticles and / or microparticles and / or polyamino acid micelles, in an aqueous or oily phase.
• Microparticles can be obtained by various methods such as coacervation in the presence of an aggregating agent (divalent or trivalent ions or polyelectrolytes), precipitation by change of pH or ionic strength, extraction / evaporation or by atomization.
• an aggregating agent divalent or trivalent ions or polyelectrolytes
• precipitation by change of pH or ionic strength
• extraction / evaporation or by atomization.
• composition according to the invention may be a colloidal solution of nanoparticles in an aqueous phase at acidic pH and which precipitates at physiological pH.
• a polyamino acid of the invention having an excess of cationic charge can condense an anionic active principle such as DNA, a DNA fragment, an RNA or an oligo RNA in the form of nano- or microparticles and these particles can be internalized in a cell.
• the composition of the invention is in the form of a solution in a biocompatible solvent and can be injected subcutaneously, intramuscularly or into a tumor.
• composition according to the invention since it is pharmaceutical, can be administered orally, pulmonary, parenterally, nasally, vaginally, ocularly, subcutaneously, intravenously, intramuscularly, intradermally, intraperitoneally, intracerebrally or buccally.
• composition is in the form of a solution in a solvent or a mixture of biocompatible solvents that can be injected subcutaneously, intramuscularly or into a tumor.
• the composition may contain an excipient for pH adjustment and / or osmolarity and / or to improve stability (antioxidants) and / or as an antimicrobial agent.
• excipients are well known to those skilled in the art (see Injectable Drug Development, P.G. Gupta et al., Interpharm Press, Denver, 1999).
• the invention also relates to a preparation process
• drugs especially for oral, nasal, vaginal, ocular, subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal or intracerebral
• the active principles of these drugs may be, in particular, proteins, glycoproteins, proteins linked to one or more polyalkylene glycol chains (for example PolyEthyleneGlycol (PEG), it is called “PEGylated” proteins), peptides hydrophobic, hydrophilic or amphiphilic polysaccharides, liposaccharides, oligonucleotides, polynucleotides and small organic molecules;
• PEG PolyEthyleneGlycol
• the invention also relates to a method of therapeutic treatment consisting essentially of administering the composition as described herein, orally, pulmonally, parenterally, nasally, vaginally, ocularly, subcutaneously, intravenously, intramuscularly, intradermally, intraperitoneally, intracerebral or oral.
• this method of therapeutic treatment essentially consists in using the composition as described above in the form of a solution in a biocompatible solvent and then injecting it subcutaneously, intramuscularly or into a tumor, preferably so that it forms a deposit on the injection site.
• Comparative compound C1 is the precursor (in its anionic form) of the polyglutamate modified with a cationic group, ie DP20 polyglutamate grafted at 5% statistically with racemic alpha-tocopherol. This compound is obtained by the method described in application WO-A-03/104303.
• aqueous solution containing 10 mg of polymer per milliliter at pH 7.4 and 200 IU insulin (7.4 mg) is prepared.
• the solutions are incubated for 2 hours at room temperature and the free insulin is separated from the associated insulin by ultrafiltration (threshold at 100 kDa, 15 min at 10,000 ° C. at 18 ° C.).
• the free insulin recovered in the filtrate is then assayed by HPLC (High Performance Liquid Chromatography) and the amount of insulin associated is deduced.
• HPLC High Performance Liquid Chromatography
• Example 13 Measurement of the viscosity (mPa / s) under shear of an aqueous solution at 29 mg / g with a gradient of speed of 10 s -1
• a therapeutic RNA of 1433 nucleotides are incubated for 2 h at 37 ° C. and then analyzed by electrophoresis on 1% agarose gel under denaturing conditions (revelation of the RNAs with ethidium bromide).
• RNA alone serves as a positive control of integrity.
• RNA incubated with commercial RNAses serves as a control of degraded RNA.
• RNA In a 2nd time is added to the RNA the amount of polymer (1) or (3) to fully involve (conditions where the RNA is no longer visible to the expected size on gel) and allowed to these mixtures incubate for 2 h at 37 ° C. After 2 h at 37 ° C., increasing amounts of compound C1 are added to these mixtures and a new incubation is carried out for 16 h at 37 ° C. The mixtures obtained are analyzed by electrophoresis. on 1% agarose gel under denaturing conditions (revelation of RNA ethidium bromide). The results show that the expected size is observed increasing amounts of RNA correlated with the amount of Cl polymer added to the RNA / polymer mixture (1) or (3).
• Example 16 Study of the crossing of the cell membrane of a model oligonucleotide
• Opti-MEM ® medium without fetal calf serum is mixed with a Cy3-labeled 30-base RNA oligonucleotide at an amount of polymer (3) or (7) close to the minimum amount to associate the entire oligonucleotide. .
• This mixture is contacted on Huh-7 human hepatocarcinoma cells grown in 24-well plate at 25,000 cells / well. After 4 hours of incubation at 37 ° C., 5% CO 2 , D-MEM medium is added to 20% fetal calf serum (FCS) so that the final concentration of FCS is 10%.
• FCS fetal calf serum
• the cells After 24 hours of incubation of the cells in the presence of oligonucleotide / polymer mixtures, the cells are washed, membrane-labeled with biotinylated concanavalin and then fixed for 3 min with 3.7% paraformaldehyde. After 2 washes with PBS, cells were incubated with DAPI (nuclear DNA) for 10 min, washed 3 times with PBS and then incubated with streptavidin labeled with Alexa Fluor ® 488 which reveals the biotinylated concanavalin. The cells are observed by confocal microscopy.
• DAPI nuclear DNA
• the localization of the cells is possible by observation of their membrane labeled with Alexa Fluor ® 488 and their marked nucleus at the DAPI.
• the entry of the Cy3-labeled oligonucleotide into the cell is visualized by observation of Cy3 fluorescence (excitation at 550 nm, emission at 570 nm).

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