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
  • A61K9/48—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
  • A61K31/00—Medicinal preparations containing organic active ingredients
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
  • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
  • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
  • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
  • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
• • 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
  • A61K9/48—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
  • A61K9/5192—Processes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• • 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

Definitions

• the invention relates to a novel process for preparing nanoparticles from the specific mixture of two polyelectrolytes of opposite polarity, optionally combined with an active ingredient.
• the active formulations must meet a number of tolerance criteria, be sufficiently concentrated in active ingredients, while having a low viscosity to allow easy injection through a small diameter needle, for example a 27 gauge needle. at 31 G.
• microparticles capable of releasing the active ingredient over a prolonged period, are more particularly formed from mixing, under specific conditions, two polyelectrolyte polymers (PE1) and (PE2) of opposite polarity, at least one carrying hydrophobic groups. This mixture leads to microparticles of size between 1 and 100 ⁇ .
• microparticle formulations are unsuitable for intravenous administration and may, in the context of subcutaneous administration, cause problems of intolerance.
• the present invention aims precisely to propose a novel process for obtaining such suspensions of nanoparticles.
• the present invention relates, according to a first aspect, to a method for preparing nanoparticles with an average diameter of less than or equal to 500 nm, comprising at least the steps of:
• anionic and cationic polyelectrolytes having a linear polyamino acid backbone, free from polyalkylene glycol side groups, and having a degree of polymerization of less than or equal to 2,000;
• the molar ratio Z of the number of cationic groups relative to the number of anionic groups of the mixture of the two polyelectrolytes being between 0.1 and 0.75 or between 1.3 and 2;
• the total mass concentration C of polyelectrolytes being strictly less than 2 mg / g of said mixture.
• the method of the invention subsequently comprises, in step (3), one or more concentration steps (4), in particular by tangential or frontal ultrafiltration, centrifugation, evaporation or lyophilization.
• step (1) consists of preparing an aqueous solution of nanoparticles of an anionic polyelectrolyte.
• Step (2) then consists in adding the cationic polyelectrolyte, especially in the form of an aqueous solution, to the solution of the first polyelectrolyte preferably placed under moderate agitation.
• step (1) consists of preparing an aqueous solution of nanoparticles of a cationic polyelectrolyte.
• Step (2) then consists in adding the anionic polyelectrolyte, especially in the form of an aqueous solution, to the solution of the first polyelectrolyte preferably placed under moderate agitation.
• the method of the invention is particularly advantageous, with regard to the specificities of its step (2), to prevent the formation of particles not in accordance with the invention, that is to say of average diameter strictly greater than 500 nm.
• the nanoparticles of the aqueous solution of step (1) are non-covalently associated with an active ingredient.
• Such an aqueous solution of active nanoparticles is obtained by adding the active agent to an aqueous colloidal solution of the first polyelectrolyte, said active associating non-covalently with the nanoparticles of the first polyelectrolyte.
• nanoparticle formulations obtained at the end of the process of the invention prove to be advantageous for several reasons.
• the nanoscale size of the particles obtained by the method of the invention is particularly well suited to administering the active formulation intravenously or subcutaneously.
• the present invention thus proves particularly advantageous with regard to the parenteral administration of active agents used for the treatment of cancers.
• the polyelectrolytes used in the process of the invention are biocompatible. They are perfectly tolerated and degrade rapidly, that is to say on a time scale of a few days to a few weeks.
• nanoparticles obtained by the process of the invention, associated with active agents are particularly advantageous for carrying active agents, in particular proteinaceous, peptide active ingredients, and / or for solubilizing low molecular weight actives.
• these nanoparticles are advantageously capable of releasing the asset over an extended period of time.
• the active-charged nanoparticles obtained according to the process of the invention advantageously have a high density. Such a density makes it possible to slow the release by steric barrier effect (matrix effect), an additional effect on the non-covalent association of the active substance with the nanoparticles of polyelectrolytes. Furthermore, a suspension of nanoparticles according to the invention advantageously has excellent stability.
• the mixture obtained at the end of the process of the invention may subsequently undergo one or more concentration steps, in particular by tangential or frontal ultrafiltration, centrifugation, evaporation or lyophilization, without impairing the physicochemical properties of the suspension, in particular in terms of viscosity, particle size, colloidal or chemical stability. It is thus possible according to the invention to access a stable suspension of nanoparticles, fluid and sufficiently concentrated.
• suspension of nanoparticles according to the invention can be formed extemporaneously at the time of administration by simple mixing of two liquid suspensions prepared as described above.
• these suspensions of nanoparticles can easily be stored, allowing to consider a production cost limited to the industrial scale.
• the active ingredient is used in an aqueous process that does not require excessive temperature, high shear, surfactant or organic solvent, which advantageously makes it possible to avoid any potential degradation of the active ingredient.
• active agents such as peptides and proteins, which can potentially be degraded when they are subjected to the abovementioned conditions.
• the process of the invention involves the mixing of at least two polyelectrolytes of opposite polarity, that is to say, at least one anionic polyelectrolyte and at least one cationic polyelectrolyte.
• polyelectrolyte in the sense of the present invention, a polymer bearing groups capable of ionizing in water, in particular at pH ranging from 5 to 8, which creates a charge on the polymer. So, in solution in a polar solvent like water, a polyelectrolyte dissociates, showing charges on its skeleton and counter-ions in solution.
• the carboxylic acid and amine functions of the polyelectrolyte are respectively in the forms -COOH or -COO " and NH 2 or NH 3 + as a function of the pH of the solution, the neutrality being ensured by the counter-cations. and counter-anions present in solution.
• the compound is likely to be present in a salified form.
• the countercations may in particular be monovalent metal cations, preferably sodium or potassium ions.
• the counteranions may in particular be chloride, acetate or ammonium ions.
• the polyelectrolytes according to the invention may comprise a set of identical or different electrolyte groups.
• polyelectrolytes are described throughout the remainder of the description as they occur at the mixing pH value of the anionic and cationic polyelectrolytes in step (2) of the process of the invention.
• the qualification of a "cationic” or “anionic” group is for example considered with regard to the charge borne by this group to this value of the mixing pH of the anionic and cationic polyelectrolytes.
• the polarity of a polyelectrolyte is defined with respect to the overall load carried by this polyelectrolyte to this pH value.
• anionic polyelectrolyte means a polyelectrolyte having a negative overall charge at the pH value of the mixture of the two polyelectrolytes.
• cationic polyelectrolyte means a polyelectrolyte having a positive overall charge at the pH value of the mixture of the two polyelectrolytes.
• the value of the mixing pH of the anionic and cationic polyelectrolytes leading to the formation of the nanoparticles ranges from 5 to 8, preferably from 6 to 7.5.
• the aqueous solution (1) has a pH value ranging from 5 to 8, in particular from 6 to 7.5 and more particularly of approximately 7.
• step (2) of the method of the invention comprises at least:
• the first polyelectrolyte carries hydrophobic side groups.
• This polyelectrolyte is particularly capable of spontaneously forming, when it is dispersed in an aqueous medium of pH ranging from 5 to 8, in particular water, nanoparticles.
• Each nanoparticle is thus constituted by one or more chains of polyelectrolytes more or less condensed around these hydrophobic domains.
• the nanoparticles formed by the first polyelectrolyte, bearing hydrophobic side groups have a mean diameter ranging from 10 to 100 nm, in particular from 10 to 70 nm, and more particularly ranging from 10 to 50 nm.
• the second polyelectrolyte of step (2) of the process of the invention also carries hydrophobic groups. It may also be capable of forming, when it is dispersed in an aqueous medium of pH ranging from 5 to 8, in particular water, nanoparticles. Linear polyamino acid skeleton
• the polyelectrolytes considered according to the invention have a linear polyamino acid backbone, that is to say comprising amino acid residues.
• the polyelectrolytes according to the invention are biodegradable.
• polyamino acid covers both natural polyamino acids and synthetic polyamino acids.
• the polyamino acids are linear polymers, advantageously composed of alpha-amino acids bound by peptide bonds.
• the polyamino acid chain consists of a homopolymer of alpha-L-glutamate or alpha-L-glutamic acid.
• the polyamino acid chain consists of a homopolymer of alpha-L-aspartate or of alpha-L-aspartic acid.
• the polyamino acid chain consists of a copolymer of alpha-L-aspartate / alpha-L-glutamate, alpha-L-aspartic acid / alpha-L-glutamic acid, alpha / beta -L-aspartate or alpha / beta-L-aspartic acid.
• the polyamino acid chain consists of a homopolymer of poly-L-lysine.
• polyamino acids are described in particular in WO 03/104303, WO 2006/079614 and WO 2008/135563, the contents of which are incorporated by reference. These polyamino acids may also be of the type described in patent application WO 00/30618. These polymers can be obtained by methods known to those skilled in the art.
• polymers that can be used according to the invention, for example, of poly (alpha-L-glutamic acid), poly (alpha-D-glutamic acid), poly (alpha-D, L-glutamate), poly (acid) gamma-L-glutamic) and poly (L-lysine) of variable masses are commercially available.
• the poly (L-glutamic acid) can be further synthesized according to the route described in the patent application FR 2801 226.
• the anionic polyelectrolyte considered according to the invention has the following formula (I) or a pharmaceutically acceptable salt thereof,
• R a represents a hydrogen atom, a linear C 2 to C 10 acyl group, a branched C 3 to C 10 acyl group, a pyroglutamate group or a hydrophobic group G as defined below;
• R b represents a group -NHR 5 or a terminal amino acid residue bonded by nitrogen and whose carboxyl is optionally substituted by an alkylamino-NHR 5 or an alkoxy-OR 6 radical, in which:
• R 5 represents a hydrogen atom, a linear C 1 -C 10 alkyl group, a branched C 3 -C 10 alkyl group, or a benzyl group;
• R 6 represents a hydrogen atom, a linear C 1 -C 10 alkyl group, a branched C 3 -C 10 alkyl group, a benzyl group or a G group;
• G represents a hydrophobic group chosen from: octyloxy-, dodecyloxy-, tetradecyloxy-, hexadecyloxy-, octadecyloxy-, 9-octadecenyloxy-, tocopheryl- and cholesteryl-, preferably alpha-tocopheryl-;
• Si corresponds to the average number of ungrafted, anionic glutamate monomers at neutral pH
• Pi is the average number of glutamate monomers carrying a hydrophobic group G
• the linking of the monomers of said general formula (I) can be random, of monoblock or multiblock type.
• the anionic polyelectrolyte considered according to the invention has the following formula ( ⁇ ) or a pharmaceutically acceptable salt thereof,
• G ' represents a hydrophobic group chosen from: octyl-, dodecyl-, tetradecyl-, hexadecyl-, octadecyl- and 9-octadecenyl-;
• Pi ( ⁇ '+ Pi ") corresponds to the average number of aspartate monomers carrying a hydrophobic group G' and can be optionally zero,
• the sequence of the monomers of said general formula ( ⁇ ) can be random, monoblock or multiblock type.
• the cationic polyelectrolyte according to the invention has the following formula (II) or a pharmaceutically acceptable salt thereof,
• R a represents a hydrogen atom, a C 2 to C 10 linear acyl group, a C 3 to C 10 branched acyl group, a pyroglutamate group or a hydrophobic group G as defined below;
• R b represents a group -NHR 5 or a terminal amino acid residue linked by nitrogen and whose carboxyl is optionally substituted with an alkylamino-NHR 5 radical or an alkoxy-OR 6 , in which:
• R 5 represents a hydrogen atom, a linear C 1 -C 10 alkyl group, a branched C 3 -C 10 alkyl group, or a benzyl group;
• R 6 represents a hydrogen atom or a linear C 1 to C 6 alkyl group
• G represents a hydrophobic group chosen from: octyloxy-, dodecyloxy-, tetradecyloxy-, hexadecyloxy-, octadecyloxy-, 9-octadecenyloxy-, tocopheryl- and cholesteryl-, preferably alpha-tocopheryl;
• R 2 represents a cationic group, in particular argininamide linked by the amine function
• R 3 represents a neutral group chosen from: hydroxyethylamino-, dihydroxypropylamino-linked by the amine function;
• P 2 corresponds to the average number of glutamate monomers carrying a hydrophobic group G
• R 2 corresponds to the average number of monomers of glutamate carrying a cationic group R 2 .
• T 2 corresponds to the average number of monomers of glutamate carrying a neutral group R 3 ,
• the degree of polymerization DP 2 (s 2 + p 2 + r 2 + t 2 ) is less than or equal to
• the linking of the monomers of said general formula (II) may be random, of monoblock or multiblock type.
• the cationic polyelectrolyte corresponding to formula (II) is such that the overall charge of the polyelectrolyte (r 2 -s 2 ) is positive.
• anionic and cationic polyelectrolytes used in the process of the invention is such that at least one of the two polyelectrolytes carries hydrophobic side groups G.
• the average molar mass of the polymers is measured by means of a static light diffusion detector coupled to a size exclusion chromatography equipment.
• the average molar mass retained is the molar mass at the peak (Mp).
• the grafted poly (glutamic acid) sample in aqueous solution, is precipitated by addition of 0.1 N hydrochloric acid, lyophilized and then dissolved in N-methylpyrrolidone (NMP) and analyzed.
• NMP N-methylpyrrolidone
• the average peak molar mass is measured by means of an 18-angle static light scattering detector (MALLS) coupled to steric exclusion chromatography equipment in N-methylpyrrolidone having 3 sequential polystyrene-co-chromatographic columns.
• MALLS 18-angle static light scattering detector
• divinylbenzene 5 ⁇ / 100 00 ⁇ ⁇ , 5 ⁇ / 10,000 ⁇ and 5 ⁇ / 1,000 ⁇ .
• the mole fractions x; corresponding to each of the monomer units (grafted or not) AAi constituting the polyelectrolyte are measured by proton NMR in a suitable solvent.
• solvent suitable for the polyelectrolyte to be analyzed and to define the analysis conditions.
• the polymer sample is freeze-dried, dissolved in deuterated trifluoroacetic acid and then analyzed by means of a 300 MHz NMR spectrometer equipped with a probe proton 1H.
• x p is the molar fraction of grafted monomer units with hydrophobic groups which is the average molar grafting rate of the hydrophobic group
• x is the mole fraction of anionic monomer units
• x c is the molar fraction of cationic monomer units.
• This average molar mass of a pattern is the average of the molar masses of the units comprising the polyelectrolyte, each being weighted by the molar fraction of this unit.
• the average mass ⁇ ⁇ will be given by the following formula:
• the amount and nature of the anionic and cationic polyelectrolyte implemented in the method of the invention are such that the molar ratio, denoted Z, of the number of cationic groups relative to the number of anionic groups in the mixture of the two polyelectrolytes is between 0.1 and 0.75 or between 1.3 and 2.
• the molar ratio Z is between 0.3 and 0.75, more particularly between 0.5 and 0.75, or between 1.3 and 1.5.
• the molar ratio Z may be defined with regard to the quantities and the nature of the polyelectrolytes introduced during the preparation of the nanoparticles according to the method of the invention, by the following formula:
• nii and m 2 respectively represent the mass quantities of the solutions before mixing the anionic polyelectrolyte and the cationic polyelectrolyte with respective mass concentrations of polymer (before mixing) Ci and C 2 ;
• DPi and DP 2 respectively represent the degrees of polymerization of the anionic polyelectrolyte and of the cationic polyelectrolyte;
• M 2 respectively represent the molar masses of the anionic polyelectrolyte and the cationic polyelectrolyte
• - x c2 represents the molar fraction of monomers carrying cationic groups in the cationic polyelectrolyte
• x a i and x a 2 represent respectively the molar fractions in monomers bearing anionic groups of the anionic polyelectrolyte and the cationic polyelectrolyte.
• the quantities and the nature of the anionic and cationic polyelectrolytes used in the process of the invention are such that the total mass concentration C of polyelectrolytes is strictly less than 2 mg / g of the mixture.
• the total mass concentration C of polyelectrolytes is between 0.5 and 1.8 mg / g, in particular between 1 and 1.5 mg / g of the mixture.
• the total mass concentration C of polyelectrolytes according to the invention is strictly less than 2 mg / g of the aqueous solution obtained at the end of step (2) of method of the invention.
• the total mass concentration C in polyelectrolytes can be defined by:
• the anionic and cationic polyelectrolytes are such that:
• ⁇ the degree of polymerization of the anionic and cationic polyelectrolytes is between 50 and 220; ⁇ the anionic polyelectrolyte door 4 to 12% molar of hydrophobic side groups distributed randomly.
• the anionic and cationic polyelectrolytes are such that:
• the degree of polymerization of the anionic and cationic polyelectrolytes is between 50 and 220;
• ⁇ t 2 is zero, that is to say that the cationic polyelectrolyte is devoid of neutral groups
• the cationic polyelectrolyte and the anionic polyelectrolyte both carry from 4 to 12 mol% of hydrophobic side groups, statistically distributed.
• the anionic and cationic polyelectrolytes are such that:
• the degree of polymerization of the anionic and cationic polyelectrolytes is between 50 and 220;
• the cationic polyelectrolyte carries from 30 to 60 mol% of cationic side groups, in particular arginine.
• the anionic and cationic polyelectrolytes are such that:
• the degree of polymerization of the anionic and cationic polyelectrolytes is between 50 and 220;
• the nanoparticles formed according to the invention have an average diameter of less than or equal to 500 nm.
• the size of the nanoparticles can vary from 20 to 300 nm, in particular from 50 to 200 nm.
• the size of the nanoparticles can be measured by quasi-elastic light scattering.
• the size of the particles is characterized by the volume average hydrodynamic diameter, obtained according to measurement methods well known to those skilled in the art, for example using a device of the ALV CGS-3 type.
• the measurements are carried out with polymer solutions prepared at concentrations of 1 mg / g in 0.15 M NaCl medium and left stirring for 24 h. These solutions are then filtered on 0.8-0.2 ⁇ , before analyzing them in dynamic light scattering.
• the diffusion angle is 140.degree.
• signal acquisition time is 10 minutes. The measurement is repeated 3 times on two solution samples. The result is the average of the 6 measurements.
• anionic nanoparticles means nanoparticles whose overall charge at neutral pH is negative; and "cationic nanoparticles", nanoparticles whose overall charge at neutral pH is positive.
• the method of the invention may further implement at least one active.
• nanoparticle formulations obtained by the process of the invention can thus be used for the purpose of conveying active substances.
• the active agent is used in the aqueous solution of step (1).
• the active ingredient is non-covalently associated with the nanoparticles of the aqueous solution of step (1).
• association or “associate” used to describe the relationships between one or more active ingredients and the polyelectrolyte (s), mean that the active (s) are associated with the (x) polyelectrolyte (s) through physical interactions non-covalent, in particular hydrophobic interactions, and / or electrostatic interactions and / or hydrogen bonds and / or via steric encapsulation by polyelectrolytes.
• This active agent may be a molecule of therapeutic, cosmetic, prophylactic or imaging interest.
• polyalkylene glycol chains preferably polyethylene glycol (PEG)
• PEG polyethylene glycol
• the active agent is chosen from the erythropoietin subgroup, the hemoglobin refinery, their analogues or their derivatives; oxytocin, vasopressin, adrenocorticotropic hormone, growth factor, blood factors, hemoglobin, cytochromes, prolactin albumins, luliberin (luteinizing hormone releasing hormone or LHRH) or its analogues, such as as leuprolide, goserelin, triptorelin, buserelin, nafarelin; LHRH antagonists, LHRH competitors, human growth hormones (GH), porcine or bovine hormones, growth hormone releasing hormone, insulin, somatostatin, glucagon, interleukins or their mixtures, interferons, such as interferon alpha, alpha-2b, beta, beta, or gamma; gastrin, tetragastrin, pentagastrin, urogastrone, secretin, calcitonin
• active ingredients are polysaccharides (for example, heparin) and oligo- or polynucleotides, DNA, RNA, iRNA, antibiotics and living cells, risperidone, zuclopenthixol, fiuphenazine, perphenazine, flupentixol, haloperidol, fluspirilene, quetiapine, clozapine, amisulpride, sulpiride, ziprasidone, etc.
• polysaccharides for example, heparin
• oligo- or polynucleotides DNA, RNA, iRNA, antibiotics and living cells
• risperidone zuclopenthixol
• fiuphenazine perphenazine
• flupentixol haloperidol
• fluspirilene quetiapine
• clozapine clozapine
• amisulpride sulpiride
• ziprasidone etc.
• the active ingredient is selected from growth hormone, interferon alpha, calcitonin and fulvestrant.
• the nanoparticle suspension obtained according to the process of the invention is suitable for parenteral administration, in particular intravenously.
• it has a viscosity, measured at 20 ° C. and at a shear rate of 10 sec -1 , ranging from 1 to 500, preferably from 2 to 200 mPa.s.
• the viscosity can be measured at 20 ° C., using conventional equipment, such as, for example, an imposed stress-type rheometer (Gemini, Bohlin) on which a cone-plane geometry (4 cm) has been installed. and 2 ° angle), following the manufacturer's instructions.
• an imposed stress-type rheometer Gemini, Bohlin
• the suspension of nanoparticles obtained at the end of step (2) of the process according to the invention described above is subjected to one or more concentration steps, in particular by tangential or frontal ultrafiltration. centrifugation, evaporation or lyophilization.
• the process according to the invention may comprise a subsequent dehydration step of the suspension of the particles obtained (for example by lyophilization or atomization), in order to obtain them in the form of a dry powder.
• the nanoparticles according to the invention are stable in freeze-dried form. Moreover, they are easily redispersible after lyophilization. Thus, the suspension of nanoparticles obtained according to the invention can be lyophilized and then reconstituted in aqueous solution, without affecting the properties of the nanoparticles obtained.
• the method of the invention may allow the preparation of new pharmaceutical, phytosanitary, food, cosmetic or dietetic preparations prepared from the compositions according to the invention.
• the suspension of nanoparticles obtained at the end of step (2) of the invention can thus undergo one or more subsequent processing steps, to prepare a composition in the form of a powder, a solution, a suspension, a tablet or capsule.
• composition obtained at the end of the process of the invention may in particular be intended for the preparation of medicament.
• Step 1 A polysuccinimide is synthesized according to a protocol analogous to that described in Polymer 1997, 38 (18), 4733-4736 using L-aspartic acid.
• Step 2 aminolysis with stearylamine and then hydrolysis of the residual polysuccinimide groups, according to a protocol similar to that described in Langmuir 2001, 17, 7501.
• Table 1 describes the characteristics of the anionic polyelectrolytes PA (the notation pi and Si refer to the formulas (I) and ( ⁇ ) of the description, the notations x pl , x a i, DPi are those defined in the description).
• Polyglutamates grafted with vitamin E and arginine (PCK PC TM and PCV) The synthesis of these polymers is described in particular in the international application WO 2008/135563 of the applicant. Polyglutamates grafted with vitamin E, arginine and ethanolamine
• PC (p 2 notations, r 2, s 2 and t 2 refer to formula (II) of the description; DP 2 notations, M 2, x p2, are x a2 and x c2 those defined previously in the description ).
• ⁇ T 2 refers in this case to neutral grafts hydroxyethylamino
• the anionic polyelectrolyte PA is diluted in a solution of 10 mM NaCl to obtain a solution at the concentration Ci.
• the cationic polyelectrolyte PC is diluted in a solution of 10 mM NaCl to obtain a solution at the concentration C 2 .
• the process then differs in the order of addition according to whether the target final mixture is in excess of anionic charge or in excess of cationic charge:
• an average mass of anionic polyelectrolyte PA at the concentration Ci is placed in a beaker with moderate stirring and a mass m 2 cationic polyelectrolyte PC at the concentration C 2 is then added.
• a mass m 2 of cationic polyelectrolyte PC at the concentration C 2 is placed in a beaker with moderate stirring and a mass of the anionic polyelectrolyte PA at the concentration Ci is then added.
• the diameter of the nanoparticles obtained is measured by quasi-elastic light scattering, as described above.
• the overall Zeta load is measured by measuring the Zeta potential at neutral pH.
• anionic and cationic polyelectrolytes used are chosen from the polyelectrolytes described above.
• a quantity of a solution of the anionic polyelectrolyte PA described in Example 1 is added to the concentration Ci in a solution of 10 mM NaCl to a quantity m 2 of a solution of the cationic polyelectrolyte PC described in the example 2 previously diluted to a C 2 concentration in 10 mM NaCl solution.
• Formulation e 1.9 of Example 3 having a total polymer concentration of 1.65 mg / g is concentrated by a factor of about 8 by frontal ultrafiltration on a membrane having a cutoff of 10 kDa.
• the final polymer concentration obtained (measured by dry extract) is 13.4 mg / g.
• the particle size (volume mean diameter) after concentration is 332 nm and the Zeta potential is -37 mV. This example shows that it is possible to concentrate by ultrafiltration the formulation obtained without significantly altering the size and Zeta potential of the particles constituting this formulation.
• Formulations according to the invention incorporating salmon calcitonin (sCT) as active agent,
• the sCT is mixed at first with the anionic polyelectrolyte PA and the PA / sCT complex thus obtained is mixed in a second step with the cationic polyelectrolyte PC.
• the anionic polyelectrolyte PA is diluted in a 10 mM phosphate buffer solution and mixed with a solution containing 10 mg / g of sCT (Polypeptide Laboratories AB) so as to obtain a PA / sCT mixture having a Ci concentration in polyelectrolyte.
• anionic PA and a concentration C p1 of protein sCT The mixture is stirred for 1 h at room temperature with gentle stirring.
• the final mixture has a total polymer concentration C and a protein concentration C p .
• the concentration of non-polyelectrolyte-associated active material is determined after separation by ultracentrifugation on ultrafilters with a cut-off of 30 kDa and by HPLC filtrate determination. It is in all cases strictly less than 5%.
• Formulations according to the invention incorporating salmon calcitonin (sCT) as active agent,
• the sCT is mixed at first with the anionic polyelectrolyte PA and the PA / sCT complex thus obtained is mixed in a second step with the cationic polyelectrolyte PC.
• the anionic polyelectrolyte PA is diluted in a solution of 10 mM NaCl and mixed with a solution containing 10 mg / g of sCT (Polypeptide Laboratories AB) so as to obtain a PA / sCT mixture having a concentration C1 in anionic polyelectrolyte PA and a C p concentration of sCT protein.
• the mixture is stirred for 1 h at room temperature with gentle stirring.
• a mass m 2 of PC cationic polyelectrolyte previously diluted at the concentration C 2 in a solution of 10 mM NaCl is added.
• the final mixture has a total polymer concentration C and a protein concentration C p .
• the non-polyelectrolyte concentration of the active substance is determined after separation by ultracentrifugation on ultrafilters with a cut-off of 30 kDa and by HPLC filtrate determination. It is in all cases strictly less than 5%.
• the characteristics of the anionic and cationic polyelectrolytes used for this example are described in Examples 1 and 2.
• the IFNa is mixed initially with the anionic polyelectrolyte PA and the PA / IFNa complex thus obtained is mixed in a second step with the cationic polyelectrolyte PC. More precisely :
• the anionic polyelectrolyte PA is diluted in 10 mM NaCl solution. Then a solution containing 2.3 mg / g of IFNa (Biosidus) is added so as to have a PA / IFNa mixture having a concentration C1 of anionic polyelectrolyte PA and a concentration C p1 of IFNa protein. The mixture is kept under moderate stirring for 14 hours at room temperature.
• the final mixture has a total polymer concentration C and a protein concentration C p .
• the characteristics of the anionic and cationic polyelectrolytes used for this example are described in Examples 1 and 2.
• Formulations according to the invention incorporating as active fulvestrant.
• Fulvestrant is mixed firstly with the anionic polyelectrolyte AP 5 and the complex PAs / fulvestrant thus obtained is mixed in a second step with the cationic polyelectrolyte PCi.
• the anionic polyelectrolyte PA 5 is diluted in 10 mM NaCl solution and mixed with powdered fulvestrant (ScimoPharm Taiwan) so as to obtain a PAs / fulvestrant mixture having a concentration Ci anionic polyelectrolyte PA 5 and a concentration C p active ingredient.
• the mixture is stirred for 24 h at 30 ° C. with gentle stirring.
• the final mixture has a total polymer concentration C and a concentration C p active ingredient.

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