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
  • A61K9/14—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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • 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
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/007—Pulmonary tract; Aromatherapy
  • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy

Definitions

• the present invention relates to novel transporters of the active principle (s) -PA-, in particular protein (s) and peptide (s), as well as new modified-release pharmaceutical formulations containing said transporters of PA.
• the therapeutic applications (human and veterinary) of these formulations are numerous.
• the PA reference used throughout this disclosure is directed to at least one active ingredient.
• modified release is meant a prolonged or delayed or pulsatile release.
• novel PA transporters to which the invention relates are microparticles of amphiphilic polymers, for example polyamino acids modified with hydrophobic groups. These microparticles contain at least one PA associated with the polymer and may be in the form of colloidal suspensions or in dry form.
• the plasma concentration of therapeutic protein then has a sawtooth profile, characterized by high concentration peaks and very low concentration minima. Concentration peaks, much higher than the basal concentration in healthy subjects, have very significant adverse effects due to the high toxicity of therapeutic proteins such as interleukin IL-2. In addition, the concentration minima are lower than the concentration required to have a therapeutic effect, which leads to poor therapeutic coverage of the patient and serious consequences in the long term.
• sustained-release forms of therapeutic protein consisting of suspensions, liquid and low viscosity, of nanoparticles. particles loaded with therapeutic proteins. These suspensions allowed the easy administration of native therapeutic proteins.
• the therapeutic protein has been associated with nanoparticles of a copolyamino acid comprising hydrophobic groups and hydrophilic groups.
• US-B-5,904,936 discloses submicron particles (NPV) - average size between 0.01 and 0.5 microns - and micrometric particles (micron) (MPV) - average size between 0.5 and 20 ⁇ m - of amphiphilic copolymer of polyamino acids comprising at least two types of amino acids, one being hydrophilic neutral, the other being ionizable. Proteins such as insulin adsorb spontaneously in aqueous solution to these particles.
• the polyamino acid copolymer is, for example, a block copolymer of sodium poly (L-leucine-bL-glutamate).
• This patent describes the aggregation of NPV in MPV by addition to a colloidal suspension of poly-Leu / Glu of monocation salts (ammonium sulfate), polycationic (Fe 2+ , Fe 3+ , Zn 2+ , Ca 2+ , Al 2+ , Al 3+ or Cu 2+ ), acid (HCl) or cationic polymers (polylysine).
• the patent application WO-A-03/104303 discloses amphiphilic polyamino acids comprising aspartic residues or glutamic residues, at least a portion of these residues carrying grafting agents comprising at least one alpha-tocopherol unit (for example: polyglutamate or polyaspartate grafted with alpha tocopherol of synthetic or natural origin).
• grafting agents comprising at least one alpha-tocopherol unit (for example: polyglutamate or polyaspartate grafted with alpha tocopherol of synthetic or natural origin).
• alpha-tocopherol unit for example: polyglutamate or polyaspartate grafted with alpha tocopherol of synthetic or natural origin.
• the in vivo release time of the active protein (s) (e.g., insulin) vectorized by the suspensions according to US-B-5,904,936 or WO-A-2003/104303 would benefit from being increased.
• the increase in the release time has been partially achieved by the pharmaceutical forms disclosed in PCT application WO-A-05/051416.
• the protein is then slowly released over a period of typically one week.
• the therapeutic protein concentration to be administered is relatively high, as is the case for example for human growth hormone, the release time is limited to a few days only.
• PAs eg proteins, therapeutic peptides and small molecules
• multivalent ions such as Mg 2+ , Ca 2+ , Zn 2+ , Fe 2+ , Cu 2+ or mixtures thereof or Al 3+ , Fe 3+ or mixtures thereof, confer excellent tolerance to such a liquid pharmaceutical formulation for prolonged release of AP.
• n is the valence of said multivalent ions
• [IM] is the molar concentration of multivalent ions
• - [GI] is the molar concentration of ionizable groups GI
• micrometric particles are derived from the aggregation of a large number of amphiphilic copolymer nanoparticles.
• a suspension of microparticles loaded with PA (for example hGH) can thus be manufactured by flocculation with multivalent ions Mg 2+ , Ca 2+ , Zn 2+ , Fe 2+ , Cu 2+ Al 3+ or Fe 3+ , this flocculation being followed by maturation and washing.
• the suspension can then be lyophilized or atomized and then reconstituted with water to make a formulation ready to inject.
• this reconstituted suspension can be easily injected by passage through a hollow needle associated with a syringe or an injection pen (insulin pen type).
• an injection pen insulin pen type
• one of the essential objectives of the invention would be to propose new microparticles obtained by aggregation of amphiphilic polymer nanoparticles, biodegradable and water-soluble, for example of the type according to the formulation described in WO-A-05. / 051416, these microparticles being loaded with PA and having vocation to have improved properties, especially in dry solid form, in particular with regard to their dispersibility (or dispersibility), and, as regards the reconstituted suspension, its stability and its ability to be easily manipulated and injected.
• Another objective of the invention is to propose novel microparticles obtained by aggregation of amphiphilic, biodegradable and water-soluble amphiphilic polymer nanoparticles, said microparticles being loaded with PA and having good properties. dispersion whether in aqueous or organic phase, while maintaining the integrity of said microparticles.
• Another object of the invention is to provide novel microparticles obtained by aggregation of amphiphilic polymer bioparticles, biodegradable and water-soluble, said microparticles being loaded with PA and having excellent stability properties in solid and dry form.
• Another objective of the invention is to propose a new process for the preparation of microparticles in solid and dry form, these microparticles being moreover:
• biodegradable and water-soluble amphiphilic polymer nanoparticles Obtained by aggregation of biodegradable and water-soluble amphiphilic polymer nanoparticles, for example of the type of those according to the formulation described in WO-A-05/051416;
• Another object of the invention is to provide a method for preparing microparticles in solid and dry form, of the type referred to in the previous objective and which is otherwise simple, economic and industrial.
• Another object of the invention is to provide a pharmaceutical formulation comprising novel microparticles obtained by aggregation of amphiphilic polymer nanoparticles, biodegradable and water-soluble, for example of the type according to the formulation described in WO-A-03/104303, these microparticles being otherwise:
• Another object of the invention is to provide a pharmaceutical formulation for the sustained release of PA, remedying deficiencies of the prior art, and in particular allowing after parenteral injection (eg subcutaneous) to obtain a prolonged in vivo release of PA (eg, proteins, therapeutic peptides or small molecules) undenatured.
• Another object of the invention is to provide a pharmaceutical formulation allowing, after parenteral injection (eg subcutaneous), to obtain a prolonged in vivo release time of highly concentrated therapeutic proteins or peptides, for example at several mg / ml.
• Another object of the invention is to provide a sustained-release pharmaceutical formulation of PA in vivo, which is stable to conservation both physico-chemical and biological.
• Another object of the invention is to provide a sustained-release pharmaceutical formulation of PA in vivo, which has at least one of the following properties: biocompatibility, biodegradability, non-toxicity, good local tolerance.
• Another object of the invention is to provide a pharmaceutical formulation for the slow sustained release of PA in vivo, comprising microparticles of amphiphilic PO polymer self-associated with at least one PA (PO / PA microparticles), the PO polymer being a biodegradable, water-soluble polymer carrying hydrophobic groups (GH) and hydrophilic groups (preferably ionizable (GI) at least in part) ionized) forming spontaneously in water a suspension of colloidal nanoparticles, this PO polymer being, for example, a polyamino acid whose main chain is formed by aspartic residues or glutamic residues, at least a portion of these residues being modified by grafting at least one hydrophobic group GH, in the chain or at the end of the chain.
• GH hydrophobic groups
• GI ionizable
• Another objective of the invention is to propose a kit for reconstituting the formulation as defined in the above-mentioned objectives, this kit being for example simple to use, so that it can be easily implemented by the patient or by the medical staff.
• Another object of the invention is to provide a method for reconstituting the formulation as defined in the above-mentioned objectives, this method being for example simple to implement in particular by the patient or the medical staff.
• 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 laborious research, that, quite surprisingly and unexpectedly, the atomization of formulations based on amphiphilic PO (for example of copolyamino acids) and PA leads to very stable, dry PO / PA microparticles, making it possible to reconstitute aqueous suspensions of microparticles in a liquid medium whose size is between 0.5 and 100 ⁇ m.
• the inventors have had the merit of developing means for reconstituting a suspension from these microparticles, said means making it possible to optimize their dispersion both in the aqueous liquid phase and in the organic liquid phase.
• Atomization is a known industrial technique for obtaining dry particles from a solution or suspension of the constituent material of said particles.
• the atomization or spray drying is to evaporate very quickly in a stream of air or hot inert gas nebulized droplets of this solution or suspension.
• the atomization can be delicate because it imposes a thermal stress which may be prohibitive for certain heat-sensitive PAs such as PAs based on proteins or other peptide compounds.
• the use of atomization to produce dry particles based excipients and PA is not therefore obvious, except to select excipients capable of preventing or minimizing the denaturation of PA peptides.
• the application US-A-2005/0158392 discloses the preparation of lipophilic solid microparticles loaded with peptide PA by atomization.
• the latter consists either in atomizing an aqueous solution containing hyaluronic acid, PA and a lipophilic surfactant (eg lecithin) or even another surfactant of Tween 80 type; either, in a first step, atomizing an aqueous solution containing hyaluronic acid, PA and optionally a surfactant like Tween ® 80 to obtain primary particles, and, secondly, atomizing an alcoholic solution of lipophilic surfactant (eg lecithin) in which the primary particles are dispersed.
• a lipophilic surfactant eg lecithin
• hyaluronic acid and lipophilic surfactant eg lecithin
• hyaluronic acid and lipophilic surfactant eg lecithin
• amphiphilic POs and even less amphiphilic copolyamino acids as excipients or carriers of PA together forming the microparticles.
• amphiphilic POs and even less amphiphilic copolyamino acids as excipients or carriers of PA together forming the microparticles.
• Another example of atomization is that described in the article Maa et al, J Pharm
• PA human growth hormone
• microparticles being characterized: a. in that they are obtained by atomization of a solution or a colloidal suspension of PO comprising at least one PA, b. by a size, measured in a T test, between 0.5 and 100 ⁇ m, preferably between 1 and 70 ⁇ m, preferably between 2 and 40 ⁇ m, c. and in that they are dispersible in colloidal suspension in a DPl dispersibility test.
• the invention relates to a process for preparing microparticles of polymer PO associated with at least one active principle (PA), these microparticles PO / PA being in particular those defined above according to the first aspect of the invention.
• PA active principle
• microparticles having a size, measured in a T test, between 0.5 and 100 ⁇ m, preferably between 1 and 70 ⁇ m, preferably between 2 and 40 ⁇ m, characterized in that it consists essentially in atomizing a solution or a colloidal suspension of PO containing PA.
• the microparticles obtained by atomization are redispersed in a substantially aqueous liquid medium (preferably comprising multivalent ions as dispersing means) and then the dispersion obtained is freeze-dried.
• a substantially aqueous liquid medium preferably comprising multivalent ions as dispersing means
• the invention in a third aspect, relates to a liquid pharmaceutical formulation for the sustained release of PA, characterized in that it comprises a low viscosity colloidal suspension based on PO microparticles, containing at least minus one PA, these microparticles being those defined above according to the first aspect of the invention or those obtained by the process defined above according to the second aspect of the invention.
• the invention relates to a reconstitution kit in particular of the formulation as defined above according to a third aspect of the invention, characterized in that it comprises:
• Microparticles of PO containing at least one AP are those defined above according to the first aspect of the invention or those obtained by the process defined above according to the second aspect of the invention;
• the invention relates to a method of reconstitution, in particular of the formulation as defined above according to a third aspect of the invention, characterized in that it consists essentially of:
• the invention relates to a solid pharmaceutical formulation for the sustained release of PA, characterized in that it comprises a dry powder form for inhalation and pulmonary administration:
• microparticles of PO containing at least one PA Based on microparticles of PO containing at least one PA, these microparticles being those defined above according to the first aspect of the invention or those obtained by the process defined above according to the second aspect of the invention;
• hydrophobic groups of the PO polymers which can assemble into hydrophobic domains, play an important role in the PA modified release process, as well as in the stabilization of the latter. .
• the affinity of the protein for the PO polymer makes it possible to limit the protein aggregation phenomena and other degradations that may occur during the atomization process, without the need to resort to other stabilizers (eg sugars, surfactants).
• This protective effect of PO also makes it easy to obtain storage-stable liquid formulations.
• the PO when it has an essentially amorphous character (especially when it is a polyamino acid), gives the microparticles excellent properties of physical stability when they are kept in dry form.
• amphiphilic PO polyamino acids provides additional levers for the modified release of the PA as well as for its stabilization with respect to aggregation or possible chemical degradation.
• amphiphilic nature of the PO polymer makes it possible, during the atomization process, to be able to use either an entirely aqueous phase, or a fully volatile organic phase, or a volatile mixture of aqueous and organic phase, which offers great flexibility in the implementation of the method.
• microparticles formed from a (co) polyamino acid type PO are biodegradable, biocompatible and generally have good local tolerance properties.
• microparticles according to the invention namely the atomization
• the microparticles according to the invention and the formulations containing them are non-toxic and well tolerated locally.
• a linear alkyl which may include at least one heteroatom (O, N or S) or at least one unsaturation may have two heteroatoms, N and S, and unsaturation.
• the term “submicronic particles” or “nanoparticles” designates particles of size (measured in a T test described below), greater than or equal to 1 nm and less than 500 nm, preferably between 5 and 250 nm.
• the term “polyamino acid” covers both natural polyamino acids and synthetic polyamino acids, as well as oligoamino acids comprising from 10 to 20 amino acid residues in the same way as polyamino acids comprising more than 20 amino acid residues.
• the preferred amino acid residues of the main polyamino acid chain are those having the L configuration; the preferred type of binding is the alpha type, i.e., a peptide bond between an alpha-amino group of an amino acid and the carboxyl group at the 1-position of another alpha-amino acid.
• protein designates, for example, a protein as well as a peptide, whether it be an oligo or a polypeptide.
• This protein (or this peptide) can be modified or not, for example, by grafting one or more polyoxyethylene groups.
• association For the purposes of the invention and throughout this presentation, the terms "association" or
• "associate" employees to qualify the relationship between one or more PA and PO polymers mean in particular that the active ingredient (s) are bound to the polymer (s) PO by a non-covalent bond, for example by electrostatic or hydrophobic interaction or hydrogen bond or steric hindrance.
• the bactericidal microparticles (a) the bactericidal microparticles (a);
• the PA for example a protein or another peptide compound
• an amphiphilic PO polymer such as a (co) polyamino acid amphiphilic
• the physical atomization treatment confers on the solid dry microparticles obtained a particular structure at the origin of many of their advantageous properties. This structure is correctly characterized by this method of obtaining by atomization, as well as by the functions attached to these advantageous properties.
• Characteristic (b) This characteristic is defined objectively by the T test described below. T test for measuring the size of the microparticles by laser diffraction:
• the circulating fluid stored in the dispersion system liquid channel at rest is drained and replaced with heptane.
• the agitation of the Hydro 2000SM module is positioned at 2400 rpm.
• Data on the D50 is obtained, which is the diameter below which 50% of the objects analyzed are found.
• the sample to be analyzed is introduced into the cell as it is or may optionally be rediluted by water in the case of highly diffusing samples. 3 Analysis of the sample
• the circulating fluid stored in the liquid dispersion system at rest is drained and replaced with fresh demineralized water.
• the agitation of the Hydro 2000SM module is positioned at 2400 rpm.
• Data on the D50 is obtained, which is the diameter below which 50% of the objects analyzed are found.
• test sample 400 ⁇ L of the test sample should be diluted in a 5 mL test tube with 600 ⁇ L of heptane and vortexed for 10 ⁇ 5 s.
• the circulating fluid stored in the dispersion system liquid channel at rest is drained and replaced with heptane.
• the agitation of the Hydro 2000SM module is positioned at 2400 rpm.
• the entire suspension is aspirated through a 1 ml syringe (Braun Injeckt-F Luer type 1 ml - ref 9166017V) equipped with a 25G needle and transferred to a new vial beforehand. calibrated and the mass m 3 of solution recovered is determined.
• the powder has good dispersion properties in the liquid if: the suspension obtained contains microparticles with a mean diameter of between 2 and 40 ⁇ m, the appearance of the suspension is homogeneous, it can be injected through a needle 25G and if - at least 80% of the suspension can be recovered.
• microparticles according to the invention can be defined objectively by the fact that they are stable in an ST1 test or in an ST2 test.
• the size of the microparticles present in the atomized dry powder is measured in the week following the atomization of the powder on a laser granulometer according to the TO test.
• the powder is then placed in an oven at 30 ° C for one week.
• a control sample is left at 5 ° C.
• a measurement of size is carried out again after dispersion under the same conditions as time tO. The particle distributions before and after aging are compared.
• the microparticle powder is dispersed in the dispersion liquid with magnetic stirring at the concentration at which it is desired to observe its stability and is allowed to stir with moderate magnetic stirring for at least 2 hours (measurement t0).
• the size of the particles is measured on a laser granulometer according to the Tl or T2 test depending on the medium (aqueous or organic) dispersion.
• the suspension is then allowed to stand for 7 days at 5 ° C.
• the sedimentation pellet is dispersed by stirring for a few minutes (until a homogeneous suspension is obtained after visual observation).
• a measurement of size is carried out again after dispersion under the same conditions as time tO.
• the PO polymers according to the invention are biodegradable, water-soluble polymers carrying hydrophobic groups GH and groups hydrophilic (preferably ionizable GI groups, at least partially ionized).
• the hydrophobic groups GH may be in reduced number vis-à-vis the rest of the chain and may be located laterally to the chain or interspersed in the chain, and be randomly distributed (random copolymer) or distributed in the form of sequences or grafts (block copolymers or block copolymers).
• the PO polymer is an amphiphilic (co) polyamino acid.
• Such a choice of PO provides excellent compatibility with PAs of the protein (s) / peptide (s) type. It is thus possible to greatly simplify the composition of the starting solution or suspension of the protein (s) / peptide (PA) -type PA with the PO polymer.
• This starting solution or suspension may comprise only PA and PO in an aqueous or organic phase. The absence of other ingredients allows such a solution or starting suspension can be filtered (pore size: 0.2 microns) before atomization, which allows the latter to be carried out under aseptic conditions.
• the PO is chosen from the
• the polyamino acids according to the present invention are oligomers or homopolymers comprising recurring glutamic or aspartic acid residues or copolymers comprising a mixture of these two types of residues.
• the residues considered in these polymers are amino acids having the D, 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 polymer PO is a polyamino acid formed by aspartic residues or glutamic residues, at least a portion of these residues carrying scions comprising at least one hydrophobic group GH.
• These polyamino acids are in particular of the type described in PCT patent application WO-A-00/30618.
• the (or) PO is (are) set (s) by the formula ggéénnéérraallee ((II)) ssuuiivvaannttee ((tthhee rraaddiiccaall - CCOOOORR 33 i includes forms wherein the bond between the carboxyl and R 3 is an ionic bond - COO "+ R 3 ):
• R 1 is selected from the group consisting of H, linear C2 to C 10 alkyl, branched C 3 -C 10, benzyl, and -R 4 - [GH], or "NHR 1 is a terminal amino acid residue;
• R 2 is selected from the group consisting of H, C 2 -C 10 linear acyl, C 3 -C 10 branched acyl, and -R 4 - [GH] or "R 2 is a terminal pyroglutamate residue;
• R + is preferably selected from the group comprising: metal cations advantageously chosen from the subgroup comprising: sodium, potassium, calcium, magnesium, organic cations advantageously chosen from the subgroup comprising:
• the cations based on amino acid (s) advantageously chosen from the class comprising cations based on lysine or arginine, or cationic polyamino acids advantageously chosen from the subgroup comprising polylysine or oligolysine ;
• R 4 represents a direct bond or a spacer based on 1 to 4 amino acid residues
• A is independently -CH 2 - (aspartic residue) or - CH 2 -CH 2 - (glutamic residue);
• GH is selected from the group consisting of alkoxy radicals of the type: OCH 2 (CH 2 -CH 2 ) 3 g -CH 3 , oleyl, tocopheryl or cholesteryl, and R 4 represents a single valence bond.
• GH represents a hydrophobic group having 6 to 30 carbon atoms
• R 30 is a C2-CO bivalent linear alkylene chain;
• R 3 ' is H, or "1 H 3' is preferably selected from the group consisting of:
• the metal cations advantageously chosen from the subgroup comprising: sodium, potassium, calcium, magnesium, the organic cations advantageously chosen from the subgroup comprising:
• cations based on amino acid (s) advantageously chosen from the class comprising cations based on lysine or arginine,
• R 50 is a C1 to C8 divalent linear alkylene chain in which one or two methylenes, preferably at each end of R 50 , may be independently replaced by -O- or -NH-;
• R 4 represents a direct bond or a spacer based on 1 to 4 amino acid residues
• A independently represents 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 1000, preferably between 50 and 300.
• R 4 represents a simple valence bond.
• PO comprises at least one essentially neutral copolyhydroxyalkylglutamine (preferably alkyl is ethyl) comprising a multiplicity of pendant hydrophobic groups (GH), which are identical or different from each other.
• the copolyhydroxyalkylglutamine carries hydroxyalkylamino groups. These hydroxyalkylamino groups are preferably bonded to the copolymer via an amide bond.
• hydroxyalkylamines that can be used to amidify the carboxyl of glutamate residues and to give this copolyhydroxyalkylglutamine are identical or different from each other and are, for example, chosen from 2-hydroxyethylamine, 3-hydroxypropylamine, 2,3-dihydroxypropylamine, tris (hydroxymethyl) aminomethane and 6-hydroxyhexylamine.
• At least one of the hydrophobic groups GH is included in a hydrophobic graft comprising at least one patella (or pattern) spacing (spacer) for connecting the hydrophobic group GH to a main chain of copolyglutamates.
• This patella may comprise, e.g. at least one direct covalent bond or at least one amide bond or at least one ester bond.
• the patella may be of the type belonging to the group comprising in particular: residues amino acids, aminoalcohol derivatives, polyamine derivatives (eg diamines), polyol derivatives (eg diols) and hydroxy acid derivatives.
• the grafting of the GH on the copolyglutamate or polyhydroxyalkylglutamine chain can be carried out by the use of precursors of GH, able to bind to the copolyglutamate or copolyhydroxyalkylglutamine 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.
• this copolhydroxyalkylglutamine preferably alkyl is ethyl
• all or part of the hydrophobic radicals GH of the POs are independently selected from the group of radicals comprising: a linear or branched alkoxy having from 6 to 30 atoms of carbon and may comprise at least one heteroatom (preferably O, N or S) or at least one unsaturation,
• alkoxy having 6 to 30 carbon atoms and having one or more annealed cycloalkyls and optionally containing at least one unsaturation or at least one heteroatom (preferably O, N or S),
• the hydrophobic group GH is chosen from the group comprising the alkoxy radicals of the type: OCH 2 (CH 2 -CH 2 ) 3 g -CH 3 , oleyl, tocopheryl or cholesteryl, and R 4 represents a single valence bond.
• n GH groups of the PO in particular according to at least one of the three aforementioned contingencies, each independently represent a monovalent radical of the following formula:
• R 5 is methyl (alanine residue), 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.
• all or part of the hydrophobic radicals R 6 of the PO 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 minus one heteroatom (preferably O, N or S) or at least one unsaturation,
• alkoxy having 6 to 30 carbon atoms and having one or more annealed cycloalkyls and optionally containing at least one unsaturation or at least one heteroatom (preferably O, N or S),
• the hydrophobic radical R 6 of the PO graft is chosen from the group comprising the alkoxy radicals of the type: OCH 2 (CH 2 -CH 2 ) 3 g -CH 3 , oleyl, tocopheryl or cholesteryl.
• the main chain of the polyamino acid is: a homopolymer of alpha-L-glutamate or alpha-L-glutamic acid; a homopolymer of alpha-L-aspartate or alpha-L-aspartic acid
• the distribution of the aspartic or glutamic residues of the main polyamino acid chain of the PO is such that the polymer thus constituted is either random, or of the block type, or of the multiblock type.
• PO has a molar mass which is between 2,000 and 100,000 g / mol, and preferably between 5,000 and 40,000 g / mol.
• PO carries at least one graft of polyalkylene glycol type bound to a glutamate or aspartate residue.
• this graft is of polyalkylene glycol type is of formula (V) below.
• X is a heteroatom selected from the group consisting of oxygen, nitrogen or sulfur;
• R 7 and R 8 independently represent H, linear C 1 -C 4 alkyl
• the polyalkylene glycol is for example a polyethylene glycol.
• the molar percentage of grafting of the polyalkylene glycol ranges from 1 to 30%.
• PA active principles such as proteins, peptides or small molecules can spontaneously associate with PO polyamino acid nanoparticles.
• POs contain ionizable groups which are, depending on the pH and the composition, either neutral (for example -COOH) or ionized (for example -COO " ).
• the solubility in one phase Aqueous solution is directly a function of the fraction of ionized functions and therefore of the pH
• the counterion may be a metal cation such as sodium, calcium or magnesium, or an organic cation such as the protonated forms of triethanolamine, tris (hydroxymethyl) aminomethane or a polyamine such as polyethyleneimine.
• PO polyamino acid type 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 POs 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
• polymers usable according to the invention for example of the poly (alpha-L-aspartic), poly (alpha-L-glutamic), poly (alpha-D-glutamic) and poly (gamma-L-glutamic) type variable masses are commercially available.
• Alpha-beta polyaspartic acid is obtained by condensation of aspartic acid (to obtain polysuccinimide) followed by basic hydrolysis (see Tomida et al., Polymer, 1997, 18:
• Coupling of the graft with an acidic function of the polymer is easily achieved by reaction of the polyamino acid in the presence of a carbodiimide as 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. These techniques are well known to those skilled in the art.
• NCA derivatives previously synthesized with the hydrophobic graft are used.
• the NCA-hydrophobic derivative is copolymerized with the NCA-O-Bz and then the benzyl groups are selectively removed by hydrolysis.
• PA 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 subgroup of erythropoietin, oxytocin, vasopressin, adrenocorticotropic hormone, epidermal growth factor, platelet growth factor (PDGF), hematopoietic growth factors and their mixtures, factors VIII and IX, hemoglobins, cytochromes , albumin, prolactin, luteinizing hormone releasing hormone (LHRH), LHRH antagonists, LHRH agonists, human growth hormone (GH), porcine or bovine, growth hormone releasing hormone, insulin , somatostatin, glucagon, interleukins or their
• PA 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 cyclosporine. and their mixtures
• a small molecule is especially a small non-protein molecule, for example, free of amino acids.
• the PA 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, acromegaly treatment agents, analgesics, anti-asthmatics, allergy treatment agents, anti-cancer agents, anti-inflammatories, anticoagulants and antithrombotics, anticonvulsants, antiepileptics, antidiabetics, antiemetics, antiglaucoma, antihistamines, antiinfectives, antibiotics, antifungals, antivirals, antiparkinsonians, anti-cholinergics, antitussives, carbonic anhydrase inhibitors, cardiovascular agents, lipid-lowering agents, anti-arrhythmics, vasodilators, anti-anginal medications, antihypertensives, vasoprotectants, cholinesterase inhibitors, dice central nervous system
• the mass fraction of PA not associated with micrometric particles [non-associated PA] in% by weight is such that: o [PA not associated] ⁇ 1 o preferably [PA not associated] ⁇ 0.5.
• 2 nd aspect of the invention the process for obtaining the microparticles by atomization of a solution or a colloidal suspension of PO containing PA.
• the PO contained in the solution or the colloidal suspension intended to be atomized is at least partly in the form of PO nanoparticles having a size, measured in the Tl test, of less than 500 nm. preferably between 10 and 300 nm, and more preferably still between 10 and 100 nm.
• the concentration of PO in the solution or the colloidal suspension to be atomized is, for example, between 5 mg / ml and 100 mg / ml, preferably between 10 mg / ml and 40 mg / ml.
• the PA / PO combination is carried out before or during the atomization step.
• the PO or PA may be in solid form (preferably a powder) or in the form of a liquid suspension.
• the PO polymer microparticles associated with at least one active principle (PA) are dispersed in a substantially aqueous liquid medium, said medium preferably containing a dispersion means M1, and the dispersion obtained is freeze-dried.
• PA active principle
• the lyophilisate thus obtained has the advantage of facilitating the preparation of liquid formulations based on microparticles (3 rd aspect of the invention described below) because it lyophilisate disperses rapidly in the useful reconstituting liquid for preparing the above liquid formulations .
• the dispersion means M1 is chosen from the group consisting of: multivalent ions whose polarity is opposite to the polarity of the ionizable groups of the PO polymer and which are contained in the aqueous continuous phase; ii- at least one hydrophilic compound (preferably usable for an injectable preparation) added to the suspension / PO solution to be atomized and thus contained in the atomized PO / PA microparticles; iii- at least one coating of the microparticles with at least one film of at least one hydrophilic compound (preferably used for an injectable preparation); iv- change of pH; v- and combinations of at least two of the means (i) to (iv); the means (i) being particularly preferred.
• liquid dispersion medium it may contain the same additives as those described for the reconstitution liquid defined below.
• Liquid Formulations based microparticles obtained by spray-drying a solution or a colloidal suspension of PO containing PA.
• the formulation according to the invention may be a liquid pharmaceutical form for the prolonged release of PA comprising a low viscosity colloidal suspension, based on PO microparticles associated with at least one PA, these microparticles being those as defined above or those obtained by the process also as defined above and in the examples infra.
• the qualifiers "low” or “very low viscosity” correspond, advantageously, to a dynamic viscosity at 20 ° C. of less than or equal to 1000 mPa.s.
• the dynamic viscosity of the formulation measured at 20 ° C, for a shear rate of 1000 s -1 , is preferably less than or equal to 500 mPa ⁇ s, preferably between 2 and 200 mPa ⁇ s. for example, between 1, 0 and 100 mPa.s, or between 1, 0 and 50 mPa.s.
• the reference measurement for the dynamic viscosity can be carried out, for example, at 20 ° C. using an AR1000 rheometer (TA Instruments) equipped with a cone-plane geometry (4 cm, 2 °). Viscosity v is measured for a shear rate of 10 s * 1 .
• This low viscosity makes the formulations of the invention easily injectable parenterally, particularly mucosally, subcutaneously, intramuscularly, intradermally, intraperitoneally, intracerebrally or into a tumor.
• the formulation according to the invention can also be administered orally, nasally, pulmonary, vaginal or ocular, among others.
• the viscosity of the liquid formulations of the invention is low both at injection temperatures corresponding to ambient temperatures, for example between 4 and 30 ° C, than at physiological temperature.
• dry microparticles formed by atomization of amphiphilic PO may further be readily redispersed to form low viscosity suspensions or microparticulate solutions.
• said formulation comprises a means M2 for dispersing the microparticles of PO / PA.
• M2 of dispersion may differ depending on the nature of the continuous phase (that is to say the reconstitution liquid) of the suspension forming part of the formulation.
• the continuous phase of the suspension is essentially aqueous
• the continuous phase of the suspension is an essentially organic phase miscible with water.
• the continuous phase of the suspension is an essentially organic phase immiscible with water.
• the continuous phase of the suspension is an essentially organic phase miscible or not with water.
• continuous essentially aqueous phase is meant, for example, a liquid containing at least 60% by weight of water.
• essentially organic continuous phase is meant, for example, a liquid containing at least 60% by weight of organic phase.
• dispersion means M2 is for example chosen from the group consisting of: multivalent ions whose polarity is opposite to the polarity of the ionizable groups PO polymer and which are contained in the aqueous continuous phase; ii- at least one hydrophilic compound (preferably usable for an injectable preparation) added to the suspension / PO solution to be atomized and thus contained in the atomized PO / PA microparticles; iii- at least one coating of the microparticles with at least one film of at least one hydrophilic compound (preferably used for an injectable preparation); iv- change of pH; v- and combinations of at least two of the means (i) to (iv); the means (i) being particularly preferred.
• the dispersion means M2 is for example chosen from the group consisting of: multivalent ions whose polarity is opposite to the polarity of the ionizable groups PO polymer and which are contained in the aqueous continuous phase; ii- at least one hydrophilic compound (
• the dispersion means M2 may comprise multivalent ions whose polarity is opposite to the polarity of the ionizable groups GI (at least partially ionized) of the polymer PO and which are introduced into the phase aqueous continuous, when reconstituting the suspension / solution that forms the suspension.
• multivalent ions denotes, for example, divalent ions, trivalent ions, tetravalent ions and mixtures of these ions.
• the multivalent ions are multivalent cations, preferably divalent cations, more preferably still selected from the group consisting of: Mg + , Ca + , Zn + , Fe + , Cu 2+ or mixtures thereof, or trivalent cations, more preferably still selected from the group consisting of: Al 3+ , Fe 3+ or mixtures thereof.
• the formulation according to the invention contains monovalent ions (eg cations), possibly active in the aggregation of nanoparticles into microparticles.
• multivalent ions are brought into the formulation of the invention, preferably in the form of aqueous saline solution (organic or inorganic), for example a solution of sulfate, chloride, acetate, gluconate or glutamate (or other anionic amino acid) of cations. multivalent.
• This multivalent ion-dispersing means M2 is more preferable in the case where the amphiphilic PO (for example a (co) polyamino acid) is relatively hydrophilic.
• the amphiphilic PO for example a (co) polyamino acid
• the dispersion means M2 can essentially comprise:
• At least one hydrophilic compound (preferably used for an injectable preparation) added to the suspension / PO solution to be atomized and thus contained in the atomized PO / PA microparticles;
• M2 of dispersion can be incorporated in the formulation according to several modes.
• At least one hydrophilic compound chosen from those used in an injectable preparation preferably selected from the group comprising:
• ⁇ amino acids eg lysine, arginine, glycine
• Polyalkylene glycols preferably polyethylene glycols
• Copolyalkylene glycols preferably ethylene glycol-propylene glycol copolymers (of the Poloxamer, Pluronic or Lutrol type);
• Cellulosic polymers and their derivatives preferably carboxyalkylcelluloses (eg carboxymethylcelluloses) or alkylcelluloses (eg methylcelluloses);
• Polyols such as propylene glycol or glycerol
• Nitrogen-containing (co) polymers preferably those contained in the group comprising polyacrylamides, poly-N-vinylamides, polyvinylpyrrolidones (PVP) and polyvinylvinyl lactams;
• the microparticles are coated with at least one layer of at least one hydrophilic compound as defined above.
• the hydrophilic compound preferably comprises at least one hydrophilic polymer chosen from hydrophilic compounds as defined above.
• the M2 (ii) dispersion means based on at least one hydrophilic compound has proved particularly suitable for amphiphilic POs having a sufficiently high hydrophobicity, for example a level of GH groups greater than or equal to 10 mol% for a PO constituted by at least one polyamino acid.
• the coating (average M2 (iii)) of the microparticles with such a layer of hydrophilic and biocompatible compound can be, for example, carried out by two successive atomizations; the second atomization, carried out on a suspension of particles in a solvent immiscible with said microparticles, can contribute to facilitating the dispersion of these particles.
• M2 (iii) of dispersion by hydrophilic coating of the microparticles, allows the suspension of microparticles to remain intact and stable for at least a few days, which makes its handling easier.
• the combination of a means M2 (ii) or (iii) for dispersing the microparticles based on hydrophilic compound (s) and a divalent ion-based dispersion medium M2 (i), contained in the aqueous phase during the reconstitution of the formulation allows accelerated dispersion.
• the aqueous continuous phase or the hydrophilic coating may also contain at least one injectable surfactant such as
• Tween® 80 a lecithin, phosphatidylethanolamine, phosphatidylserine or a polyoxypropylene-polyoxyethylene copolymer (trade name: Poloxamer, Pluronic, Lutrol).
• Another dispersible means that can be envisaged according to the invention consists of a means of dispersion by pH change, preferably before atomization.
• This type of means has proved suitable especially in the case where the amphiphilic PO (for example polyamino acid) is a compound bearing ionizable functions and is otherwise very hydrophilic, that is to say comprising, for example, less than 10 mol% of hydrophobic groups GH.
• this pH changing dispersion means (iv) before atomization is applied to the suspension / PO solution just before the atomization.
• M2 (ii) or (iii) of dispersion based on hydrophilic compound preferably by coating M2 (iii) microparticles with at least one hydrophilic polymer.
• the dispersing means consists of this organic phase which is miscible with water.
• This phase can thus contain, for example, ethanol, N-methylpyrrolidone, dimethylsulfoxide, isopropanol, glycerol or glycofurol (tetrahydrofurfuryl alcohol polyethylene glycol ether).
• the dispersing means comprises a lipophilic liquid, whose melting temperature is preferably less than or equal to 15 C, contained in the continuous organic phase immiscible with water.
• the lipophilic liquid comprises at least one mixture of triglycerides of saturated fatty acids or at least one vegetable oil or at least one lipid or at least one lipid derivative or at least one fatty acid or at least one acid derivative.
• the lipophilic liquid comprises:
• At least one vegetable oil preferably soybean oil, palm oil, linseed oil, cottonseed oil, sesame oil, sunflower oil, peanut, • at least one lipid, preferably liquid lecithin, synthetic or natural vitamin E;
• At least one lipid derivative preferably arachidoylphosphatidylcholine and stearoylphosphatidylcholine, At least one fatty acid, preferably oleic acid, myristic acid, palmitic acid, stearic acid and their salts;
• At least one fatty acid derivative preferably a mono-, di- or triglyceride derivative, ethyl oleate, lauryl lactate, glyceryl stearate, sorbitan palmitate, sorbitan stearate, sorbitan monoleate, polysorbate;
• the triglyceride derivatives of fatty acids are particularly preferred, in particular a mixture of triglycerides of saturated C 8 -C 0 0 fatty acids derived from coconut oil, for example that marketed under the name Miglyol 812 by Sasol) .
• Other long-chain fatty acid triglycerides that can be used include vegetable oils such as soybean oil, palm oil, linseed oil, cottonseed oil, sesame oil, and the like. , sunflower oil or peanut oil.
• Atomized PO / PA powders disperse readily into this water-immiscible organic phase to give stable suspensions of microparticles which retain their integrity for several days and are, therefore, again easily manipulable. This dispersion is particularly rapid without the need to add other means (s) of dispersion, even if this possibility is possible.
• the dispersing means comprises a coating of the microparticles with at least one film-forming coating compound (preferably used for an injectable preparation).
• the film-forming compound comprises at least one hydrophobic polymer chosen from the group comprising polylactides, polyglycolides, poly (lactide-co-glycolide), polyorthoesters, polyanhydrides, polyhydroxybutyric acids and polycaprolactones. polyalkylcarbonates, water-insoluble PO polymers, their derivatives and mixtures thereof.
• the film-forming coating compound is of lipidic nature and has a melting point of preferably greater than or equal to 15 ° C. and comprises at least one mixture of saturated fatty acid triglycerides or at least one vegetable oil. or at least one lipid or at least one lipid derivative or at least one fatty acid or at least one fatty acid derivative or a mixture of these products.
• the organic continuous phase for example hydrophobic, or hydrophobic coating bit (s) may also contain at least one injectable surfactant such as Tween ® 80, lecithin, phosphatidylethanolamine, phosphatidylserine or a polyoxypropylene-polyoxyethylene copolymer (trade name: Poloxamer, Pluronic, Lutrol).
• injectable surfactant such as Tween ® 80, lecithin, phosphatidylethanolamine, phosphatidylserine or a polyoxypropylene-polyoxyethylene copolymer (trade name: Poloxamer, Pluronic, Lutrol).
• the injectable formulation comprises other additives, on the one hand those hereafter referred to as "excipients / stabilizers", and on the other hand conventional excipients.
• excipients / stabilizers may be selected from the group consisting of:
• Polyalkylene glycols preferably polyethylene glycols; Copolyalkylene glycols, preferably ethylene glycol-propylene glycol copolymers (of the Poloxamer, Pluronic or Lutrol type);
• Cellulosic polymers and their derivatives preferably carboxyalkylcelluloses (eg carboxymethylcelluloses) or alkylcelluloses (eg methylcelluloses);
• sorbitan esters of fatty acid preferably polyoxyalkylene (eg ethylene) glycol esters and at least one acid (eg oleic acid), of the Tween type, (or polysorbate);
• Polyols such as propylene glycol or glycerol; -> gelatin preferably hydrolysed; nitrogen-containing (co) polymers, preferably in the group comprising polyacrylamides, poly-vinyl-vinylamides, polyvinylpyrrolidones (PVP) and poly-N-vinyl-lactams;
• PVA polyvinyl alcohols
• One of the excipients (stabilizers) preferred according to the invention is formed by nanoparticles of at least one PO polymer, identical or different (preferably identical) from that constituting the microparticles.
• the amount of excipient / stabilizer used in the formulation is preferably between 0.01 and 10% by weight, and more preferably between 0.1 and 5% by weight.
• the stabilizers comprising nanoparticles
• they are advantageously used in the formulation in a proportion of 1.5 to 3.5% by weight, for example 2.0 to 3.0 ( ⁇ 2.5)% by weight.
• the reconstitution liquid used in the preparation of the formulation according to the invention may comprise, for example: at least one buffer or at least one injectable salt (phosphate buffer, citrate buffer, chloride of sodium) in concentration for example between 0.001 M and 0.1 M, preferably between 0.005 M and 0.02 M, this buffer or injectable salt for adjusting the pH of the solution; at least one injectable surfactant, preferably of the polysorbate type, such as
• the reconstitution liquid may further contain densifying agents such as saccharides, ie e.g. sucrose, D-mannitol or trehalose in concentrations of between 0.1% and 10%, preferably between 0.5 and 5%.
• densifying agents such as saccharides, ie e.g. sucrose, D-mannitol or trehalose in concentrations of between 0.1% and 10%, preferably between 0.5 and 5%.
• the reconstitution solution may also contain an injectable viscosifying polymer selected from the group consisting of polysaccharides, synthetic polymers (eg, sodium carboxymethylcellulose), polyvinyl alcohol, polyvinylpyrrolidone, polyalkylene glycols (e.g. polyethylene glycols) and mixtures thereof.
• amphiphilic PO eg polyamino acid
• ad hoc means of reconstitution thus makes it possible to meet the double objective which is to be able to obtain pharmaceutical formulations both stable and easily dispersible to allow their parenteral injection.
• the other conventional excipients which can be added to the suspension / solution to be atomized or to the formulation according to the invention are, for example, antimicrobials, buffers, antioxidants, or isotonicity adjusting agents known to those skilled in the art.
• antimicrobials for example, antimicrobials, buffers, antioxidants, or isotonicity adjusting agents known to those skilled in the art.
• buffers for example, buffers, antioxidants, or isotonicity adjusting agents known to those skilled in the art.
• isotonicity adjusting agents known to those skilled in the art.
• formulation according to the invention is preferably pharmaceutical, this does not exclude cosmetic, dietary or phytosanitary formulations comprising at least one PO as defined above and at least one PA.
• the PO / PA microparticles and the essentially aqueous reconstitution liquids, the essentially organic and water miscible reconstitution liquids and the essentially water-immiscible organic liquids are defined above.
• the PO / PA microparticles and the essentially aqueous reconstitution liquids, the essentially organic and water miscible reconstitution liquids and the essentially water-immiscible organic liquids are defined above.
• This formulation for the sustained release of PA comprises a dry powder form for inhalation and pulmonary administration based on PO / PA microparticles defined above.
• the present invention also relates to solid products derived from the microparticles of PO according to the invention.
• these derived products may in particular be constituted by powders, gels, implants or films, among others.
• the invention relates to products derived from the formulation according to the invention, taken as such, regardless of their method of production.
• the invention also relates to a method of therapeutic treatment consisting essentially of administering a therapeutically effective amount of the formulation as described herein, parenterally, in particular mucosally, subcutaneously, intramuscularly, intradermally, intraperitoneally. , intracerebral or in a tumor.
• the formulation according to the invention can also be administered orally, nasally, pulmonary, vaginal or ocular, among others.
• this method of therapeutic treatment consists in administering the formulation as described above by parenteral injection: subcutaneously, intramuscularly, intradermally, intraperitoneally, intracerebrally or in a tumor, preferably so that it forms a deposit at the injection site.
• the solution was cooled to 15 ° C and successively 2.5 g of D, L-alpha-tocopherol (> 98%, obtained from Fluka ®), previously solubilized in 8 ml of DMF, 280 mg of 4-dimethylaminopyridine dissolved beforehand in 1 ml of DMF and 1.6 g of diisopropylcarbodiimide previously solubilized in 6 ml of DMF.
• the precipitated polymer is then recovered by filtration, washed with 0.1 N hydrochloric acid, water and with diisopropyl ether.
• the polymer is then dried in an oven under vacuum at 40 ° C. A yield of the order of 90% is obtained.
• the molar mass measured by size exclusion chromatography is 15500 relative to a polyoxyethylene standard.
• the grafted tocopherol level, estimated by proton NMR spectroscopy, is 5.1 mol%.
• a suspension of nanoparticles of the polymer in water is obtained by solubilizing it in water and adjusting the pH to 7 ⁇ 1 (neutralization of the carboxylates).
• EXAMPLE 2 SYNTHESIS OF AN AMPHIPHILE PO-B POLYMER
• Example 2 is adapted from Example 1, aiming for a grafting rate of 20%
• the mixture is left for 14 hours at room temperature.
• the solution is filtered through a 0.2 ⁇ m sterilizing filter.
• the solution is atomized on a spray-drying apparatus of B ⁇ chi B290 type.
• the liquid solution is aspirated at a rate of 5 ml / min and nebulized through a spray nozzle fed with nitrogen (700 kPa - 900 1 / h).
• the suction flow rate (drying air) is 40 m 3 / h.
• the inlet temperature is maintained at 90 ° C., which induces under these conditions a temperature at the outlet of 45 ° C.
• the HFN assay by HPLC is identical to that of the solution before atomization. No degraded form is detected.
• the PO polymer and interferon solution is prepared in the same manner as in Example 3. 0.9 g of polysorbate 80 are added to the solution before atomization.
• the IFN assay by HPLC is identical to that of the solution before atomization. No degraded form is detected.
• the PIFN assay by HPLC is identical to that of the solution before atomization. No degraded form is detected.
• a powder of dry microparticles of PO-A and IFN polymer is obtained from a PO-A / IFN mixture according to the protocol used in Example 3.
• the IFN assay by HPLC is identical to that of the solution before atomization. No degraded form is detected.
• the PIFN assay by HPLC is identical to that of the solution before atomization. No degraded form is detected.
• EXAMPLE 8 PREPARATION OF DRY MICROMETRIC PARTICLES OF POLYMER PO-A CONTAINING HGH
• a recombinant human growth hormone solution (concentration 3.9 mg / g) are thawed for 90 min at 25 ° C. and concentrated approximately 6 times by frontal ultrafiltration on an exclusion limit membrane of 10 kD until to obtain a concentration of 24 mg / g (controlled by HPLC).
• the HPLC assay of hGH is identical to that of the solution before atomization. No degraded form is detected.
• the mixture containing 20 mg / g of PO polymer and 1.25 mg / g of IL-2 is left overnight at room temperature and then filtered through a 0.2 ⁇ m sterilizing filter.
• the IL-2 assay by HPLC is identical to that of the solution before atomization. No degraded form is detected.
• the solution is then atomized on a spray-drying apparatus of B ⁇ chi B290 type.
• the liquid solution is aspirated at a rate of 5 ml / min and nebulized through a spray nozzle fed with nitrogen (700 kPa - 900 1 / h).
• the suction flow rate (drying air) is 40 m 3 Vh.
• the inlet temperature is maintained at 120 ° C., which induces under these conditions a temperature at the outlet of 70 ° C.
• the insulin assay by HPLC is identical to that of the solution before atomization. No degraded form is detected.
• the size of the microparticles is measured according to the TO test and the stability according to the S 1 test.
• the evaluation of the degradation of the protein is carried out by HPLC by comparing a chromatogram of the formulation before and after atomization.
• Atomization is a method that can degrade the protein. HPLC analysis shows that no form of degradation is observed by comparing the chromatograms before and after atomization.
• the atomization of a protein in the presence of an amphiphilic polyamino acid leads to microparticles that are stable over time and does not induce degradation of the protein.
• powders can be used in solid form (inhalation, needleless injection by a pressure gun for example) or in the form of an injectable suspension after reconstitution in a suitable medium.
• EXAMPLE 12 SUSPENSION RECONSTITUTION FROM THE POWDERS OF EXAMPLES 3 TO 10
• the samples are then evaluated by carrying out the following steps: dispersion in one of the abovementioned media transfer into a syringe and injection through a 25G syringe
• the suspension is considered to have good properties if at least 80% is recovered by suction / injection through a 25G needle.
• EXAMPLE 13 VISCOSITY MEASUREMENT OF THE RECONSTITUTED FORMULATIONS
• a dispersion of the powders at 30 mg / ml in the media B and C of the preceding examples (except for 5 in the medium B) give stable, low-viscosity suspensions; in medium B, the viscosities measured are all less than 10 mPa.s and in medium C, the viscosities are all of the order of 30 to 40 mPa.s.
• a composition of the same nanoparticulate polymers according to the prior art can not be obtained at this concentration (30 mg / mL) in the acceptable viscosity values ( ⁇ 100 mPa.s).
• the particles are first prepared as described in Example 3.
• the atomization step is performed under aseptic conditions and the particles are recovered in a sterile container.
• the particles are recovered and redispersed under aseptic conditions in an aqueous solution of MgCl 2 0.10 M.
• the amount of MgCl 2 solution added is adjusted so that the polymer concentration PO-A in the suspension is approximately 40 mg / ml.
• the pH is adjusted to 6.5 by adding 1N sodium hydroxide. Lyophilization
• the suspension is divided into Lyoguard ® containers to keep the suspension sterile during lyophilization: the containers are then sterilized lyophilized during a 72 h cycle on a laboratory freeze-dryer (Christ benchtop module, Osterode, Germany). The powder is sterilely distributed in vials before use.
• EXAMPLE 15 COMPARISON OF THE RECONSTITUTION OF THE POWDERS OF MICROPARTICLES OBTAINED FROM EXAMPLE 3 AND THOSE OF EXAMPLE 14
• the volumes of reconstituted suspension are identical in both cases (about 1 ml) and the reconstitution vials are identical (3 ml glass vials).
• the amounts of powder are adjusted so that the two suspensions finally contain 40 mg / ml of polymer and 0.5 mg / ml of IFN- ⁇ -2b.
• the powder of Example 3 is reconstituted in an aqueous solution of MgCl 2 0.1 M while the powder of Example 14 (which already contains Mg 2+ ions) is reconstituted in pure water.
• the bottles are stirred manually. While the powder of Example 3 requires at least 15 min to redisperse, the dispersion is faster for the powder of Example 14 and in less than 5 min, a homogeneous suspension is obtained. A magnetic bar is then inserted into the flasks and the two flasks are shaken in an identical manner for 1 h.
• the characteristics of the two suspensions are compared: the particle size and the viscosity of the two suspensions are similar.
• EXAMPLE 16 PHARMACOKINETICS OF IFN IN THE DOG AFTER SUB-CUTANEOUS INJECTION OF A FORMULATION BASED ON AMPHIPHILIC POLYAMINOACIDES IN THE MICROPARTICULAR FORM Eight naive Beagle dogs (weight of 9 ⁇ 0.6 kg) were treated with the following formulations:
• IR-IFN IR for immediate release, ie immediate release
• PCGen a recombinant human interferon solution
• the particles of the formulation 1 are prepared according to Example 14 from the same batch of interferon PCGen: the lyophilized powder is resuspended in water sterilely (in a laminar flow hood) according to a process similar to that described in example 15.
• C max is the maximum serum concentration in IFN
• T> 50 ⁇ g / ml is the time when the serum concentration of IFN is greater than
• AUC represents the area under the serum IFN concentration versus time curve
• RBA represents bioavailability compared to an Immediate Release formulation
• T 50 o / oauc represents the time required to release 50% of released PIFN in total.
• the IFN IR has a rapid release profile with a peak serum concentration of 25.2 ⁇ 0.4 ng / mL achieved after a median time of 5 h (range: 3-5 h).
• the circulating IFN is no longer quantifiable beyond 24 hours.
• Formulation 1 provides a major change in the pharmacokinetic profile of IFN, with very slow release, and a peak serum concentration of 0.5 ⁇ 0.2 ng / mL (50 times lower than that of the IR form) reached after a median time of 60 h (range: 48 - 96 h).
• the general pattern of pharmacokinetics is a flat profile pseudoplatter.
• the circulating IFN level returns to an unquantifiable concentration between 192 h and 240 h (8 and 10 days).
• the T 5 o % aUc is about 20 times higher than that of the IFN IR.
• EXAMPLE 17 PHARMACOD YNAMIE OF INSULIN IN DOGS AFTER SUBCUTANEOUS INJECTION OF A FORMULATION BASED ON AMPHIPHILIC POLYAMINO ACIDS IN MICROP ARTICULAR FORM
• Lantus ® is a modified insulin analog (insulin glargine - Sanofi-Aventis).
• the modification of two amino acids on the primary structure of human insulin gives Lantus ® extended release properties over a 24 hour period via in situ precipitation.
• a group of 6 healthy Beagle dogs (weight of 11.4 ⁇ 1 kg) were treated with the Lantus formulation in a 2-period crossover trial and a group of 8 healthy Beagle dogs (weight 1 1, 8 ⁇ 1 kg) was treated with Formulation 2, two by two, in a 4-period crossover trial.
• the summary table of treatments is summarized below:
• the particles of the formulation 2 are prepared according to Example 10 by aiming at a ratio of 30 mg of PO polymer for 3.5 mg (100 IU) of insulin.
• the formulation is prepared by dispersing the atomized powder in 0.1 M MgCl 2 and magnetic stirring of the suspension for 1 h.
• the pH of the formulation is 6.2 and the osmolality is 348 mOsm.
• the blood glucose is measured by enzymatic method (hexokinase) on an automaton of biochemical blood tests (Advia 1650, Bayer Diagnostics).
• the analysis of pharmacodynamic results is performed on the percentage of basal glucose as a function of time.
• the pharmacodynamic data are grouped in the following table:
• Area Percent Glycemia Curve represents the area between the basal blood glucose (100%) and the curve representing the evolution of blood glucose
• - T 50 % APGC represents the time required to obtain 50% of the APGC 0-36I1 .
• the administration of the Lantus ® reference leads to a rapid decrease in blood glucose levels in the first hour.
• the hypoglycemic action of insulin glargine is then maintained over a period of between 18 and 36 hours (return of blood glucose to basal level after 30 hours on average).
• the duration of action of the formulation 2 is clearly greater than that of the long-acting reference Lantus ® . This is illustrated by an average value of T50% APGC higher for formulation 2.

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