Classifications

• • 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
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
• • 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
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/513—Organic macromolecular compounds; Dendrimers
  • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
• • 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
  • 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
  • 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
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
  • A61K9/1605—Excipients; Inactive ingredients
  • A61K9/1629—Organic macromolecular compounds
  • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers

Definitions

• the present invention relates to novel transporters of active (s) -PA-, in particular protein (s) and peptide (s), as well as new modified-release pharmaceutical formulations containing said PA transporters.
• the application also relates to the applications, particularly therapeutic applications, of these pharmaceutical formulations.
• These active pharmaceutical formulations concern both human and veterinary therapeutics.
• PA used throughout this specification is for at least one active ingredient.
• sustained-release forms of therapeutic protein consisting of suspensions, liquid and low viscosity nanoparticles loaded with therapeutic proteins. These suspensions allowed the easy administration of native therapeutic proteins.
• Another object of the invention is to propose new particles loaded with PA and stable in freeze-dried form.
• Another object of the invention is to provide new particles capable of being preserved in freeze-dried form.
• Another objective of the invention is to propose new easily redispersible particles after lyophilization.
• Another object of the invention is to propose new particles releasing a protein that has preserved its biological activity. Another object of the invention is to propose a new process for the preparation of these microparticles.
• Another object of the invention is to provide a solid pharmaceutical formulation for the sustained release of PA, in particular a dry powder form for inhalation and pulmonary administration.
• the inventors have had the merit of discovering, after long and painstaking researches, that, quite surprisingly and unexpectedly, the mixing under specific conditions of two polymers (for example copolyamino acids) polyelectrolytes of opposite polarity, at least one carrying hydrophobic groups, associated with at least one PA, leads to particles of size between 1 and 100 microns capable of releasing in vitro or in vivo a protein or a peptide over an extended period.
• the invention relates firstly to particles for the sustained release of at least one active ingredient (AP), characterized in that they comprise: a) a first polyelectrolyte polymer (PEI) , preferably a linear alpha-polyamino acid, in a charged state, carrying hydrophobic groups (GH) on the side, said first polyelectrolyte polymer (PE1) spontaneously forming in water a colloidal solution of particles at at least a pHm value of pH inclusive between 3 and 8; b) a second polyelectrolyte polymer (PE2), preferably a linear alpha-polyamino acid, of opposite polarity to that of the first polyelectrolyte polymer (PE1), said second polyelectrolyte polymer (PE2) forming a solution or a colloidal solution in water; minus said pHm value of the pH; at the condition that, if the first electrolyte polymer (PE1) is a polyamino acid, then the second
• the invention also relates to a method for preparing particles for the sustained release of at least one active principle (AP), these particles being in particular those described above, comprising the following steps:
• the invention also relates to particles for the sustained release of at least one active principle (AP), characterized in that they comprise: a) a first polyelectrolyte polymer (PE1), preferably a linear alpha-polyamino acid, in a charged state, bearing hydrophobic groups (GH) on the side, said first polyelectrolyte polymer (PE1) forming spontaneously in water a colloidal solution of particles; at least a pHm value of the pH of between 3 and 8; b) a second polyelectrolyte polymer (PE2), preferably a linear alpha-polyamino acid, of opposite polarity to that of the first polyelectrolyte polymer (PE1), carrying hydrophobic groups (GH) on the side, said second polyelectrolyte polymer (PE2) forming in the water a solution or a colloidal solution at at least said pHm value of the pH; c) at least one active ingredient (PA) non-covalently associated with the particles of the coll
• the invention also relates to a method for preparing particles for the sustained release of at least one active principle (AP), these particles being in particular those described above, comprising the following steps: 1) the preparation, at a value pHm of the pH between 3 and 8, of an aqueous colloidal solution of a first polyelectrolyte polymer (PE1) in the charged state, carrying hydrophobic groups (GH) on the side, said first polyelectrolyte polymer (PEl) being capable of forming spontaneously in water a colloidal solution of particles with at least one pH value, pH of between 3 and 8;
• the invention also relates to a pharmaceutical formulation for the sustained release of at least one active ingredient (AP), said formulation comprising particles as described above.
• AP active ingredient
• the invention also relates to a process for the preparation of medicaments, in particular for parenteral, mucosal, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral or tumoral, or even oral, nasal, pulmonary, vaginal, transdermal or ocular administration. , essentially consisting in implementing at least one formulation as described above.
• a solution is understood to mean a homogeneous solvent and polymer mixture in the form of an individual chain.
• colloidal solution is understood to mean a suspension of particles whose average diameter measured by the T 'test is less than or equal to 0.5 ⁇ m.
• pHm means the pH at which the mixing of the first polyelectrolyte polymer (PEI) with which the active ingredient (PA) is associated with the second polyelectrolyte polymer (PE2).
• the physiological pH is defined as, for example, equal to 7.2 ⁇ 0.4.
• polyelectrolyte means a polymer carrying groups capable of ionizing in water, which creates a charge on the polymer.
• polyampholyte is understood to mean a polyelectrolyte carrying at least two types of groups which dissociate respectively into anionic and cationic groups.
• carrier means that the group carried is pendant, that is to say that said group is a side group with respect to the main chain of the polymer.
• said pendant group is a side group with respect to the "amino acid” residues and is a substituent of the ⁇ carbonyl function of the "amino acid” residue which door.
• the polarity of a polyelectrolyte is understood to mean the polarity of the overall charge carried by this polyelectrolyte to the pHm value of the pH.
• apparent density is meant the volume occupied by 1 g of particles.
• the apparent density is measured by any method known to those skilled in the art, such as the density gradient method.
• the term "small molecule” is understood to mean a molecule having a molecular weight of less than 1 kDa.
• the T test is used.
• the test T ' is preferably used.
• the result of the test T is a median diameter D50, such that 50% of particles present in the sample have a size less than or equal to this value (D50).
• the result of the test T ' is a mean hydrodynamic diameter.
• Particle solutions are prepared by diluting 400 ⁇ l of the sample to be analyzed in a 5 ml test tube with 600 ⁇ l of deionized water and then vortexing the preparation for 10 s (10 ⁇ 5). These solutions are then introduced drop by drop into the measuring cell until the darkness is between
• the average hydrodynamic diameter of the polymer particles according to the invention is measured according to the procedure Md defined below:
• the polymer solutions are prepared at concentrations of 1 or 2 mg / ml in 0.15 M NaCl medium and left stirring for 24 h. These solutions are then filtered on 0.8-0.2 ⁇ m, before analyzing them in dynamic light scattering using a Malvern Compact Goniometer System, operating with a 632 wavelength He-Ne laser beam. , 8 nm and vertically polarized. The diameter The hydrodynamics of the polymer nanoparticles is calculated from the autocorrelation function of the electric field by the cumulant method, as described in "Surfactant Science Series" volume 22, Surfactant Solutions, Ed R. Zana, ch. 3, M. Dekker, 1984.
• the total mass of released protein can then be plotted by summing the one found in each of the samples and relate it to the total quantity injected.
• the term "protein” refers to a protein as well as a peptide, whether it be an oligo or a polypeptide. This protein or peptide may or may not be modified, for example, by grafting one or more polyoxyethylene groups.
• the first and second polyelectrolyte polymers (PEl) and (PE2) are linear, biocompatible and biodegradable polymers bearing anionic and / or cationic ionizable groups, for example amine functions or carboxylic acids.
• the polymer PE1 or PE2 carries ionizable groups of a given polarity (anionic or cationic).
• Such polymers are, for example, polyamino acids, anionic polysaccharides such as dextran sulfate, carboxymethylcellulose, gum arabic, hyaluronic acid and its derivatives, polygalacturonic compounds, polyglucuronic agents, or cationic polysaccharides such as chitosan, or also collagen and its gelatin derivatives. It is not excluded that a polymer bearing ionizable groups of a given polarity also carries a small fraction of 1 to 30 mol% of ionizable groups of the opposite polarity.
• the first or second polyelectrolyte polymer (PE1) or (PE2) may optionally also carry non-ionizable groups, such as radicals chosen from a hydroxyethylamino radical, an alkylene glycol or a polyalkylene glycol;
• non-ionizable groups such as radicals chosen from a hydroxyethylamino radical, an alkylene glycol or a polyalkylene glycol;
• the net charge of the polymer depends on the pH value relative to the half-neutralization pH of the polymer.
• the net charge of the polymer will be close to zero at a pH of two units below the half-neutralization pH. Virtually all anionic functions will be ionized two pH units above the half-neutralization pH.
• the net charge is canceled when the pH exceeds the half-neutralization pH by about two units.
• the number of charges borne by the first or second polyelectrolyte polymer (PEl, PE2) under the conditions of mixing at the pHm value of the pH is obtained by the conventional method of acid-base titration:
• a solution of polyelectrolyte concentrated at 2 mg / ml and containing 0.15 M of sodium chloride is brought to pH 3 by addition of 1 M acetic acid or 1 M sodium hydroxide.
• This solution is then titrated with a 0.05 M sodium hydroxide solution by recording the evolution of the pH as a function of the volume of sodium hydroxide added.
• the detection of the equivalence point (volume and pH) makes it possible to detect the pH for which all the ionizable groups are ionized, ie where the degree of ionization is equal to 1. From this point, it is then possible to go back to the degree of ionization of the polyelectrolyte for any pH value.
• the pH of half-neutralization that is to say the pH for which the degree of ionization is equal to 0.5. It is also possible to define the degree of ionization of the polyelectrolyte for the pHm value of the pH. In the particular case where the equivalence point is outside a pH range between 3 and 9, it is considered that all the ionizable groups are ionized over this pH range, that is to say that the degree of ionization is equal to 1 for pHs between 3 and 9.
• first and second polyelectrolyte polymers may be linear alpha polyamino acids, recalling that if PE1 is a linear polyamino acid, PE2 is not polylysine or polyethyleneimine.
• polyamino acid covers both natural polyamino acids and synthetic polyamino acids, as well as oligoamino acids comprising from 2 to 20 "amino acid” residues as well as polyamino acids comprising more than 20 residues "amino acid”.
• the polyamino acids used in the present invention are oligomers or homopolymers comprising glutamic or aspartic acid repeating units or copolymers comprising a mixture of these two types of "amino acid” residues.
• the residues considered in these polymers are amino acids having the D or L or D / L configuration and are linked by their alpha or gamma positions for the glutamate or glutamic residue and alpha or beta for the aspartic or aspartate residue.
• the preferred "amino acid” residues of the main polyamino acid chain are those having the L-configuration and an alpha-type bond.
• the first and second polyelectrolyte polymers are polyamino acids or a pharmaceutically acceptable salt thereof, the main chain of which is formed by residues chosen from group comprising aspartic residues, glutamic residues and combinations thereof, at least a portion of these residues being modified by grafting at least one hydrophobic group (GH) for at least the first polyelectrolyte polymer (PEI).
• GH hydrophobic group
• polymer PE2 also carries pendant hydrophobic groups.
• these polyamino acids are of the type of those described in patent application PCT WO-A-00/30618, according to which the hydrophobic groups
• (GH) are identical to or different from one another and are selected from the group consisting of: (i) linear or branched, preferably linear, C 1 -C 20 and even more preferably C 2 -C 1 alkyls, acyls or alkenyls; S ;
• hydrocarbon groups containing one or more heteroatoms preferably those containing oxygen and / or sulfur and, more preferably, those of the following formula:
• aryls preferably aryls, aralkyls or alkylaryls, preferably aryls;
• the hydrophobic derivatives preferably the phosphatidylethanolamino radical or the radicals chosen from octyloxy-, dodecyloxy-, tetradecyloxy-, hexadecyloxy-, octadecyloxy-, 9-octadecenyloxy-, tocopheryloxy- or cholesteryloxy-.
• hydrocarbon groups means groups comprising, in particular, hydrogen and carbon atoms.
• the hydrophobic groups are selected from the following group of radicals: methyl, ethyl, propyl, docedyl, hexadecyl, octadecyl.
• hydrophobic groups are chosen from the following group:
• 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 3 o may optionally include at least one unsaturation and / or at least one heteroatom.
• At least one of the hydrophobic groups (GH) is obtained by grafting, starting from a precursor chosen from the group comprising: octanol, dodecanol, tetradecanol, hexadecanol, octadecanol, lec oleyl alcohol, tocopherol or cholesterol.
• one of the polyelectrolyte polymers corresponds to the following formula (I):
• R 1 is H, C 2 -C 10 linear alkyl, C 3 -C 10 branched alkyl, benzyl, -R - [GH], or R together with NH is a terminal amino acid residue;
• R is H, linear C 2 -C 10 acyl, branched C 3 -C 10 acyl, pyroglutamate or -R 4 - [GH];
• R 4 represents a direct bond or a "spacer” based on 1 to 4 amino acid residues
• a 1 and A 2 independently represent -CH 2 - (aspartic residue) or -CH 2 -CH 2 - (glutamic residue);
• N / (n + m) is defined as the molar grafting level and its value is sufficiently low for the polymer dissolved in water at pH 7 and at 25 ° C. to form a colloidal suspension of polymer particles.
• N + m varies from 10 to 1000, preferably from 50 to 300;
• GH represents a hydrophobic group comprising 6 to 30 carbon atoms or is selected from the group comprising the radicals as defined above in paragraphs (i), (ii), (iii) and (iv).
• polyelectrolyte polymer PE 2 corresponds to one of the following formulas (II), (III) and (IV):
• GH represents a hydrophobic group having 6 to 30 carbon atoms
• R 30 is a linear C 2 -C 6 alkyl group
• R 50 is alkyl, dialkoxy or C 2 -C 6 diamino
• R 4 represents a direct bond or a "spacer" based on 1 to 4 amino acid residues
• a 1 and A 2 independently represent a radical -CH 2 - (aspartic residue) or -CH 2 -CH 2 - (glutamic residue); • n '+ m' or n "is defined as the degree of polymerization and varies from 10 to
• one of the polyelectrolyte polymers corresponds to the following formula (V):
• R represents H, a linear C 2 -C 10 alkyl, a branched C 3 -C 10 alkyl or a benzyl, an amino acid residue or a terminal amino acid derivative of formula:
• R 7 is OH, OR 9 or NHR 10 , and R 8 , R 9 and R 1 independently represent H, linear alkyl in
• B is a direct bond or a divalent, trivalent or tetravalent linking group, preferably selected from the following radicals:
• D is H, linear C 2 -C 10 acyl, branched C 3 -C 10 acyl, or pyroglutamate;
• GH represents a hydrophobic group having 6 to 30 carbon atoms
• R 70 represents a radical chosen from the following group:
• X is an oxygen atom or -NH-
• R 1 is H, linear C 2 -C 10 alkyl, branched C 3 -C 10 alkyl or benzyl,
• R 70 is a chloride, a sulfate, a phosphate or an acetate, preferably a chloride;
• R represents a hydroxyethylamino-, an alkylene glycol residue or a polyoxyalkylene; • p, q, r and s are positive integers;
• (P) / (p + q + r + s) is defined as the molar grafting ratio of the hydrophobic groups GH varies from 2 to 99 mol%, and preferably between 5 and 50%, provided that each copolymer chain has on average at least 3 hydrophobic grafts;
• the derivatives of lysine, ornithine, and arginine may be, for example, ethyl and methyl esters, amides, and methylated amides.
• the hydrophobic groups GH and the cationic groups are randomly arranged in pendant groups.
• the hydrophobic mole ratio of the polyglutamate is between 2 and 99%, and preferably between 5 and 50% provided that each polymer chain has on average at least 3 hydrophobic grafts.
• the ratio (q) / (p + q + r + s) of the polyglutamates means that they may contain from 1 to about 97 mole percent of cationic charge containing groups.
• the ratio (s) / (p + q + r + s) of polyglutamates means that they can be anionic, neutral or cationic at neutral pH.
• the group R 4 or B represented in the preceding formulas represents a direct bond.
• one of the polyelectrolyte polymers comprises hydroxyalkyl (preferably ethyl) glutamine residues and a multiplicity of hydrophobic groups (GH) pendant and identical or different from each other.
• the hydroxyalkylglutamine residues also carry hydroxyalkylamine groups. These hydroxyalkylamine groups are preferably bonded to the copolymer via an amide bond.
• hydroxyalkylamine groups that can be used to functionalize the glutamate residues of these hydroxyalkylglutamine residues are identical or different from each other and are for example chosen from the following groups: 2-hydroxyethylamino, 3-hydroxypropylamino, 2,3-dihydroxypropylamino, tris (hydroxymethyl) methylamino and 6-hydroxyhexylamino.
• at least one of the hydrophobic groups GH used in the present invention is included in a hydrophobic graft comprising at least one spacer (or "spacer") to connect the hydrophobic group GH to a chain of copolyglutamates (eg a main chain - skeleton-copolyglutamates).
• This patella may comprise, eg 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 copolyglutamate, the aminoalcohol derivatives, the polyamine derivatives (for example the diamines), the derivatives of polyols (for example diols) and derivatives of hydroxy acids.
• the grafting of the GH on the copolyglutamate or polyalkylglutamine chain may involve the use of GH precursors that are capable of binding to the copolyglutamate chain or the hydroxyalkylglutamine residues.
• 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.
• hydroxyalkyl (preferably hydroxyethyl) glutamine residues reference is made to FR 2,881,140.
• hydrophobic groups (GH) used in the present invention are independently selected from the group of radicals comprising:
• a linear or branched alkoxy having from 6 to 30 carbon atoms and which may comprise at least one heteroatom (preferably O and / or N and / or S) and / or at least one unsaturation,
• the hydrophobic group (GH) is derived from an alcoholic precursor chosen from the group comprising: octanol, dodecanol, tetradecanol, hexadecanol, octadecanol, oleyl alcohol, tocopherol or cholesterol, and R represents a direct linkage.
• hydrophobic groups (GH) in particular according to at least one of the various abovementioned possibilities, each independently represent a monovalent radical of the following formula:
• R represents a methyl (alanine), isopropyl (valine), isobutyl (leucine), secbutyl (isoleucine), benzyl (phenylalanine);
• R 6 represents a hydrophobic radical containing from 6 to 30 carbon atoms
• - 1 varies from 0 to 6.
• hydrophobic radicals R 6 of the hydrophobic groups (GH) are independently selected from the group of radicals comprising:
• said hydrophobic radical R 6 is derived from an alcoholic precursor chosen from the group comprising: octanol, dodecanol, tetradecanol, hexadecanol, octadecanol, oleyl alcohol, tocopherol or cholesterol.
• the main chain of the polyelectrolyte polyamino acids (PE1, PE2) used in the invention is chosen from the group comprising alpha-L-glutamate homopolymers, alpha-L-glutamic homopolymers, alpha-L-glutamic homopolymers and L-aspartate, alpha-L-aspartic homopolymers, alpha-L-aspartate / alpha-L-glutamate copolymers and alpha-L-aspartic / alpha-L-glutamic copolymers.
• the distribution of the aspartic and / or glutamic residues of the main polyamino acid chain of the polyelectrolyte polymer PE1 or PE2 is such that the polymer thus formed is either random, either of the block type or of the multiblock type.
• the polyelectrolyte polymer PE1 or PE2 has a molar mass which is between 2,000 and 100,000 g / mol, and preferably between 5,000 and 40,000 g / mol.
• the degree of polymerization of the first and second polyelectrolyte polymers is between 50 and 500, preferably between 70 and 300.
• the mole fraction of the chain members of the main chain substituted with hydrophobic groups is between 1 and 40 mol%, preferably between 3 and 30 mol%.
• the polymers used in the present invention are chosen from the various families described above so that they are globally cationic or anionic at the pH value equal to pHm.
• An essential characteristic of the first polyelectrolyte polymer (PEl) bearing lateral hydrophobic groups is to be able to spontaneously form in water a colloidal solution.
• each nanoparticle consists of one or more polymer chains PEl more or less condensed around its hydrophobic domains.
• the polymers used in the invention contain ionizable functions which are, depending on the pH and the composition, either neutral
• aqueous solution in the case of carboxylic functions, the counter-ion 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
• the counteranion of the cationic groups is preferably selected from the group comprising a chloride, a sulfate, a phosphate or an acetate.
• the polyelectrolytes of the polyamino acid type which can be used in the present invention are obtained, for example, by methods known to those skilled in the art.
• the random polyamino acids can be obtained by grafting the hydrophobic graft, previously functionalized by the "spacer", directly onto the polymer. by a conventional coupling reaction.
• the block or multiblock polyamino acid polyelectrolytes can be obtained by sequential polymerization of the corresponding N-carboxy-amino acid anhydrides (NCA).
• a polyamino acid, homopolyglutamate, homopolyaspartate or a glutamate / aspartate, block, multiblock or random copolymer is prepared according to conventional methods.
• NCA N-carboxy-amino acid anhydrides
• 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-methylpyrrolidone (NMP) or dimethylsulfoxide (DMSO).
• a carbodiimide is, for example, dicyclohexylcarbodiimide or diisopropylcarbodiimide.
• the degree of grafting is chemically controlled by the stoichiometry of the constituents and reactants or the reaction time.
• Hydrophobic grafts functionalized by a "spacer” are obtained by conventional peptide coupling or by direct condensation by acid catalysis.
• the coupling of the cationic and optionally neutral groups with an acid function of the polymer is carried out simultaneously in a second step in the presence of a chloroformate as coupling agent and in a suitable solvent such as dimethylformamide, N-methylpyrrolidone (NMP) or dimethylsulfoxide (DMSO).
• the cationic group contains two chemically undifferentiated amino functions (eg 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.
• the polymerization chemistry and coupling reactions of the groups are conventional and well known to those skilled in the art (see for example the patents or patent applications of the applicant mentioned above).
• NCA derivatives previously synthesized with the hydrophobic graft are used.
• the NCA-hydrophobic derivative is copolymerized with the NCA-O-Benzyl and then the benzyl groups are selectively removed by hydrolysis.
• PE1 and PE2 examples of particularly preferred combinations of polyelectrolyte polymers (PE1 and PE2) according to the invention are described in the examples hereinafter.
• the first polyelectrolyte polymer (PE1) is in the form of a colloidal solution
• the second polyelectrolyte polymer (PE2) is in the form of a solution or colloidal solution for at least one value, pHm, the pH between 3 and 8.
• the half-neutralization pH of the cationic polymer will be sufficiently high, for example greater than 5.5, preferably greater than 6, or even greater than 8
• the half-neutralization pH of the anionic polymer will be sufficiently low, for example less than 6.5, preferably less than 6.0 or even less than 5.5.
• the first polyelectrolyte polymer (PEI) is anionic
• the latter is chosen so that it has a half-neutralization pH of between 3 and 6.5, and preferably between 4.5 and 6.5.
• the first polyelectrolyte polymer (PE1) forms a colloidal solution for a pHm value of the pH of between 6 and 8.
• Such a PEl polymer is described in particular in Example 1a).
• the second electrolyte polymer (PE2) is cationic and forms a colloidal solution with a pH of less than 8.
• the second polyelectrolyte polymer (PE2) is chosen so that its pH of half neutralization is greater than 8.
• Such a polymer PE2 is in particular described in Example Id).
• the first polyelectrolyte polymer (PE1) forms a colloidal solution
• the second polyelectrolyte polymer (PE2) forms a solution or a colloidal solution for the pHm value of the pH between 6 and 8.
• the ratio of the mass of the first polyelectrolyte polymer (PEl) to the mass of the second polyelectrolyte polymer (PE2) is chosen so that the load ratio Z, ratio of the number of moles cationic ionized groups to the number of moles of anionic ionized groups, measured at pHm, between 0.25 and 3, and preferably between 0.25 and 1.5.
• the second polyelectrolyte polymer (PE2) is cationic and forms a colloidal solution with a pH below 6 and a precipitate with a pH greater than 6.5.
• the second polyelectrolyte polymer (PE2) is chosen so that its pH of half-neutralization is between 5.5 and 7.
• a polymer PE2 is described in particular in Example Ic).
• the first polyelectrolyte polymer (PE1) forms a colloidal solution
• the second polyelectrolyte polymer (PE2) forms a solution or a colloidal solution for a pH value, pHm, of between 3 and 6.
• the ratio of the mass of the first polyelectrolyte polymer (PEl) to the mass of the second polyelectrolyte polymer (P E2) is chosen so that the charge ratio Z, measured at pHm, is between 3.5 and 30, of preferably between 5 and 15, and even more preferably between 8 and 12.
• the first polyelectrolyte polymer (PEI) is cationic
• the latter is chosen so that its pH of half-neutralization is greater than 5.
• the second electrolyte polymer (PE2) is anionic and is chosen so that its pH of half-neutralization is between 3 and 6.5, and preferably between
• the particles according to the invention have, at physiological pH, a size, measured in a T test, of between 1 and 100 ⁇ m.
• the particles according to the invention are not chemically crosslinked.
• the particles have, at physiological pH, an apparent density in high polymer, of between 0.15 and 1.1, preferably of between 0.3 and 1.0, and even more preferentially between 0.5 and 1.0.
• a high polymer density reflects the existence within the particles of a dense network of polymer chains. Without wishing to be bound by the theory, it can be supposed that this dense network slows the diffusion of the active ingredient (PA) contained in the particles according to the invention to the external environment and thus contributes to slowing down its release.
• PA active ingredient
• a surprising aspect of the dense particles according to the invention is that the network of polymer chains which constitutes them makes it possible to slow the release of the AP without trapping this same PA in the heart of the particles.
• the carrier according to the invention makes it possible to obtain both prolonged release of the AP and good bioavailability.
• the release of the protein or peptide can be facilitated when the polymer PE1 or PE2, of a given polarity, is also a carrier of ionizable groups of the opposite polarity and / or nonionizable groups such as groups substituted by a hydroxyethylamino radical.
• one of the two PEl or PE2 polymers simultaneously comprises: from 15 to 50 mol% of glutamate monomers;
• nonionizable monomers such as groups substituted with a hydroxyethylamino radical
• from 3 to 15 mol% of nonionizable monomers substituted with a hydrophobic group from 20 to 55 mol% of nonionizable monomers such as groups substituted with a hydroxyethylamino radical
• the polymer PE1 or PE2 is cationic and simultaneously comprises: from 0 to 5 mol% of glutamate monomers; from 50 to 85 mol% of nonionizable monomers such as groups substituted with a hydroxyethylamino radical; from 10 to 40 mol% of monomers carrying cationic groups of neutralization pH greater than 8; from 3 to 15 mol% of nonionizable monomers substituted with a hydrophobic group.
• the total concentration of polymer (PE1 + PE2) contained in the formulation is between 4 and 15 mg / ml, especially when the active ingredient is a therapeutic protein.
• the formulation is easily injectable by a small diameter needle, for example a Gauge 27, even 29, and even 31 needle. Examples 3 and 4 describe in detail such formulations.
• the active principle it is preferably 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 erythropro-retin subgroup, such as epoetin alpha, epoetin beta, darbepoetin, hemoglobin, and the like.
• PEG polyethylene glycol
• oxytocin vasopressin, adrenocorticotropic hormone, epidermal growth factor, platelet growth factor (PDGF), stimulatory factors of hematopoiesis and mixtures thereof, blood factors, such as alteplase, tenecteplase, factor V ⁇ I (a), factor VII; hemoglobin, cytochromes, prolactin albumins, luliberin (luteinizing hormone releasing hormone or LHRH) or the like, such as leuprolide, goserelin, triptorelin, buserelin, nafarelin; LHRH antagonists, competitors of LHRH, human growth hormones (GH), porcine or bovine hormones, growth hormone releasing hormone, insulin, somatostatin, glucagon, interleukins or their mixtures (IL-2, IL-1, IL-12), interferons, such as interferon alpha, alpha-2b, beta, beta, or ⁇ ; gastrin, t
• 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, sulpirid, ziprasidone, etc.
• the active ingredient is a small hydrophobic, hydrophilic or amphiphilic organic molecule of the type belonging to the family of anthracyclines, taxoids or camptothecins or of the type belonging to the family of peptides such as leuprolide or cyclosporin and mixtures thereof.
• a small molecule is especially a small non-protein molecule, for example, free of amino acids.
• the active ingredient is advantageously chosen from at least one of the following families of active substances: alcohol abuse treatment agents, agents for treating Alzheimer's disease, anesthetics, Pacromegaly treatment agents, analgesics, antiasthmatics, allergy treatment agents, anticancer agents, anti-inflammatories, anticoagulants and antithrombotics, anticonvulsants, anti-epileptics, antidiabetics, antiemetics, antiglaucoma , antihistamines, antihistamines infections, antibiotics, antifungals, antivirals, antiparkinsonians, anti-cholinergics, antitussives, carbonic anhydrase inhibitors, cardiovascular agents, lipid-lowering agents, anti-arrhythmics, vasodilators, anti-angines, antihypertensives , vasoprotectants, cholinesterase inhibitors, central nervous system treatment agents, central nervous system stimulants, contraceptives, fertility promoters, uterine labor inducers and inhibitors,
• step 2 adding at least one active ingredient (AP) to the first polyelectrolyte polymer (PEI) obtained in step 1, said active ingredient non-covalently associating with the particles of the colloidal solution of said first polyelectrolyte polymer ( PEI);
• the subject of the invention is also a method for preparing particles for the sustained release of at least one active principle (AP), these particles corresponding in particular to certain ones described above, comprising the following steps:
• step 2 adding at least one active ingredient (AP) to the first polyelectrolyte polymer (PEI) obtained in step 1, said active ingredient non-covalently associating with the particles of the colloidal solution of said first polyelectrolyte polymer ( PEI);
• An essential characteristic of the process according to the invention is to form particles, spontaneously, by simple mixing at pH m of a colloidal solution of particles of the first polyelectrolyte polymer (PEI) loaded with active ingredient (PA) and a solution or d a colloidal solution of the second polyelectrolyte polymer (PE2) of opposite polarity.
• the active ingredients such as proteins, peptides or small molecules can spontaneously associate with the first polymer (PE1) of polyamino acid type.
• the loading the nanoparticles of the first polyelectrolyte polymer (PEI) with the active ingredient (PA) is carried out by simple mixing of a solution of active principle (PA) with a colloidal solution of the first polyelectrolyte polymer (PEI).
• PA active principle
• PEI colloidal solution of the first polyelectrolyte polymer
• This association is purely physical and does not imply the creation of a co-valent bond between the active ingredient (PA) and the polymer (PEI). Without being bound by theory, it can be assumed that this non-specific association is effected by hydrophobic and / or electrostatic interaction between the polymer (PEl) and the active ingredient (PA).
• the process according to the invention comprises a step of dehydration of the suspension of particles obtained (for example by lyophilization or atomization), in order to obtain them in the form of a dry powder.
• the subject of the invention is a pharmaceutical formulation for the sustained release of at least one active ingredient (AP), comprising an aqueous suspension of particles as described above or those obtained by the process described above.
• AP active ingredient
• the present invention also relates to a solid pharmaceutical formulation for the sustained release of at least one active ingredient (AP), comprising a dry powder form:
• such a solid pharmaceutical formulation is used for inhalation and pulmonary administration.
• the subject of the invention is a process for preparing medicaments, in particular for parenteral, mucosal, subcutaneous, intramuscular, intradermal, transdermal, intraperitoneal, intracerebral or tumor administration, or even orally. , nasal, pulmonary, vaginal or ocular, said method essentially consisting in implementing at least one of the formulations described above.
• FIG. 1 in vitro release of IFN- ⁇ from the particle formulations of Example 2 (white circles), Example 3.1 (black triangles), Example 3.2 (black diamonds), the Example 3.3 (black squares), Example 4 (black circles) and Example 5 (lines).
• 15 g of an alpha-L-polyglutamic acid are solubilized with respect to a polyoxyethylene standard and obtained by polymerization of NCA-GIuOMe followed by hydrolysis as described in the FR-patent application.
• the solution is cooled to 15 ° C. and 2.5 g of D, L-alpha-tocopherol (> 98% obtained from Fluka®) preliminarily solubilized in 8 ml of DMF, 280 mg of 4-dimethylaminopyridine, which has been solubilized beforehand, is added successively.
• the solution of activated polymer is then added to the suspension of histidinamide.
• the reaction medium is stirred for 2 h at 0 ° C. and then overnight at 20 ° C. 0.62 ml of 35% HCl are then added, followed by 83 ml of water.
• the solution obtained is then poured into 500 ml of water at a pH of between 3 and 4.
• the solution is then diafiltered against 8 volumes of salt water (0.9% NaCl) and 4 volumes of water.
• the polymer solution is then concentrated to a volume of 300 mL (the polymer concentration is 18 mg / g).
• the percentage of grafted histidinamide determined by 1 H NMR in D 2 O is 95%.
• Example 1 preparation of particles with polyelectrolytes having no hydrophobic group (1) Preparation of a colloidal solution of PEl-B polymer:
• the PEl-B polymer obtained according to the synthesis b) above is used.
• This polymer has a half-neutralization pH equal to 5.985.
• a colloidal solution of polymer PE1-B is obtained by solubilizing it in water by adjusting the pH to 7.63 by adding a solution of NaOH.
• the osmolarity of the solution is adjusted to 100 mOsm by introducing the necessary amount of an aqueous solution of NaCl.
• the polymer concentration PE1-B is adjusted to 8.38 mg / g.
• the combination is carried out overnight at 25 0 C with stirring.
• This polymer has a pH of half-neutralization greater than 9.
• a colloidal solution of poly-L-arginine is obtained by solubilizing it in water by first adjusting the pH to 0.92 with a solution of HCl, then back at pH equal to 6.91 with a solution of NaOH and heating the solution at 45 0 C for 15 min.
• the poly-L-arginine polymer concentration is adjusted to 5.13 mg / g.
• the charge ratio Z is the ratio of the number of moles of cationic ionized groups to the number of moles of anionic ionized groups, measured at pHm equal to 6.95.
• the particle size is measured according to the T test. Table I
• the suspension is centrifuged for 15 min at 8000 rpm and the IFN- ⁇ is assayed in the supernatant by the method described in the European Pharmacopoeia (colorimetric assay by UV absorbance).
• a colloidal solution of PEI-A polymer is obtained by solubilizing it in water by adjusting the pH to 7.53 by adding a solution of NaOH.
• the osmolarity of the solution is adjusted to 101 mOsm by introducing the necessary amount of an aqueous solution of NaCl.
• the polymer concentration PEI-B is adjusted to 8.41 mg / g.
• the combination is carried out overnight at 25 ° C with stirring.
• the charge ratio Z is measured at pHm equal to 6.88.
• the particle size is measured according to the T test.
• the suspension is centrifuged for 15 min at 8000 rpm and PIFN- ⁇ is assayed in the supernatant by the method described in the European Pharmacopoeia (colorimetric assay by UV absorbance).
• All of the introduced protein is encapsulated in the formed microparticles.
• Example 3 Preparation of particles based on PEI-A and PE2-A, containing IFN-a 4.1)
• the PEI-A polymer obtained according to the synthesis a) above is used. This polymer has a half-neutralization pH equal to 5.445.
• a colloidal solution of PEI-A polymer is obtained by solubilizing it in water by adjusting the pH to 7.45 by adding a solution of NaOH.
• the osmolarity of the solution is adjusted to 108 mOsm by introducing the necessary amount of an aqueous solution of NaCl.
• the PEl polymer concentration is adjusted to 23.88 mg / g.
• the polymer PE2-A obtained according to the synthesis c) above is used. This polymer has a half-neutralization pH of 6.05.
• a colloidal solution of PE2-A polymer is obtained by solubilizing it in water by adjusting the pH to 5.17.
• the osmolarity of the solution is adjusted to 289 mOsm and the polymer concentration PE2-A is adjusted to 5.70 mg / g.
• the charge ratio Z is measured at pHm equal to 5.17.
• the particle size is measured according to the T test.
• Example 3.2 final polymer concentration equal to 5 mg / g, Z being equal to about 10
• the PEI-A polymer obtained according to the synthesis a) above is used.
• a colloidal solution of PEl polymer is obtained by solubilizing it in water by adjusting the pH to 7.52 by adding a solution of NaOH.
• the osmolarity of the solution is adjusted to 108 mOsm by introducing the necessary amount of an aqueous solution of NaCl.
• the PEl polymer concentration is adjusted to 20.21 mg / g.
• the combination is carried out overnight at 25 0 C with stirring.
• the association is then adjusted to a pH of 4.88.
• the charge ratio Z is measured at pHm equal to 4.81.
• the particle size is measured according to the T test.
• Example 3.3 final polymer concentration equal to 10 mg / g, Z being equal to about 10
• a colloidal solution of PEI-A polymer is obtained by solubilizing it in water by adjusting the pH to 7.52 by adding a solution of NaOH.
• the osmolarity of the solution is adjusted to 108 mOsm by introducing the necessary amount of an aqueous solution of NaCl.
• the polymer concentration PEI-A is adjusted to 20.21 mg / g.
• the charge ratio Z is measured at pHm equal to 4.95.
• the particle size is measured according to the T test.
• a colloidal solution of PEI-A polymer is obtained by solubilizing it in water by adjusting the pH to 7.52 by adding a solution of NaOH.
• the osmolarity of the solution is adjusted to 108 mOsm by introducing the necessary amount of an aqueous solution of NaCl.
• the polymer concentration PEI-A is adjusted to 20.21 mg / g.
• the PE2-B polymer obtained according to the synthesis d) above is used. This polymer has a neutralization pH greater than 9.
• a colloidal solution of PE2-B polymer is obtained by solubilizing it in water by adjusting the pH to 6.98.
• the osmolarity of the solution is adjusted to 288 mOsm and the PE2-B polymer concentration is adjusted to 6.33 mg / g.
• the charge ratio Z is measured at pHm equal to 6.85.
• the particle size is measured according to the T test.
• Example 5 preparation of particles based on PEI-A alone, containing VIFN a
• the PEI-A polymer obtained according to the synthesis a) above is used.
• a colloidal solution of PEI-A polymer is obtained by solubilizing it in water by adjusting the pH to 7.52 by adding a solution of NaOH.
• the osmolarity of the solution is adjusted to 108 mOsm by introducing the necessary amount of an aqueous solution of NaCl.
• the polymer concentration PEI-A is adjusted to 29.05 mg / g.
• the release of the active ingredient from the particles according to the invention is measured using the L-test.
• Figure 1 shows the release in the L-test as the percentage of protein released over time.
• Comparative Example 2 The formulation of Comparative Example 2 in which only one of the polymers carrying hydrophobic groups has a very low release profile, with 1.6% of the protein released after 23 hours.
• C max represents the maximum average plasma concentration of protein on all animals.
• Median T max represents the median time for which the plasma concentration passes through its maximum.
• AUC represents the mean area under the curve of plasma concentration as a function of time.
• T50% AUC represents the average time at which the area under the curve reaches 50% of its total value.
• RBA is the ratio of the area under the curve of the formulation considered to the area under the curve of the IFN IR formulation.
• PEI-A polymer obtained according to the synthesis a) above is used.
• a colloidal solution of polymer PEI-A is obtained by solubilizing it in water by adjusting the pH to 7.15 by adding a solution of NaOH.
• the osmolarity of the solution is adjusted to 145 mOsm by introducing the necessary amount of an aqueous solution of NaCl.
• the polymer concentration PEI-A is adjusted to 3.10 mg / g.
• the combination is carried out overnight at 25 ° C with stirring.
• the association is then adjusted to a pH of 7.0.
• the PE2-C polymer obtained according to the synthesis e) above is used.
• a colloidal solution of PE2-C polymer is obtained by diluting and adjusting the PE2-C polymer to pH 7.04, 288 mOsm and 7.96 mg / g in 140 mOsm PBS.
• the charge ratio Z is measured at pHm equal to 7.
• the size of the particles is measured according to the T test. The table below groups together the characteristics of the particles obtained:
• PEl-A polymer obtained according to the synthesis a) above is used.
• a colloidal solution of polymer PEI-A is obtained by solubilizing it in water by adjusting the pH to 7.02 by addition of a solution of NaOH.
• the osmolarity of the solution is adjusted to 101 mOsm by introducing the necessary amount of an aqueous solution of NaCl.
• the polymer concentration PEI-A is adjusted to 2.0 mg / g.
• the combination is carried out overnight at 25 0 C with stirring.
• the association is then adjusted to a pH of 7.0.
• the charge ratio Z is measured at pHm equal to 7.
• the size of the particles is measured according to the T test. The table below groups together the characteristics of the particles obtained:
• Example 8 (Comparison) release rate of the PE1-A / PE2-D-based and PE1-A / PE2-C-containing particles containing IFN- ⁇ .
• this example shows that it is possible, in particular by selecting a cationic polymer containing more or fewer neutral and / or anionic groups, to modulate the release rate of the AP.
• the release is 9.5% for the microparticles obtained from the PE2-C polymer and 31% for the microparticles obtained from PE2-D.

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