Colloidal suspension of submicronic particles as vectors for active principles and method for preparing same

Abstract

A suspension of vector particles (PV) based on polyamino acids and have a mean hydrodynamic diameter between 30 and 120 nm, and an insulin load factor of from 5 to 25% of associated insulin volume relative to the polyamino acid volume forming the vector particles. The polyamino acids are double-block polymers containing hydrophilic and hydrophobic monomers. The suspension may be prepared by copolymerizing N-carboxy anhydrides of hydrophobic monomers and precursors of hydrophilic monomers, in the presence of N-methyl pyrrolidone and methanol. The copolymer is optionally neutralized, subjected to dialysis, concentrated and water is eliminated to produce a solid powder, which can be suspended in a liquid to produce the colloidal suspension. Active principles such as insulin or vaccines are associated with the carrier particles to prepare special pharmaceutical products.

1 - 19 . (canceled)  
     
     
         20 . A pulverulent solid, obtained from a colloidal suspension of submicronic particles which can be used, in particular for carrying active principle(s) (PA(s)), these particles being individualized supramolecular arrangements: 
 based on linear, amphiphilic polyamino acids (PAA), with a-peptide linkages and comprising at least two different types of recurring amino acids: hydrophilic AAI and hydrophobic neutral AAO, the amino acids of each type being mutually identical or different,    and capable of combining in colloidal suspension, in the nondissolved state, at least PA and of releasing it, in particular in vivo, in a prolonged and/or delayed manner characterized: 
 in that the AAI(s) is (are) chosen from amino acids with an ionizable side chain, the natural amino acids Glu an Asp in carboxylic form and/or in the form of salts being particularly preferred,  
 in that the AAO(s) is(are) chosen from the group comprising natural neutral amino acids, preferably those belonging to the subgroup comprising: Leu, Ile, Val, Ala, Gly, Phe;  
 in that it is stable at pH between 4 and 13 in the absence of surfactant(s),  
 by a load factor Ta with insulin, expressed as % of combined insulin mass relative to the mass and measured according to a procedure Ma, Ta being such that: 
 7≦Ta,  
 preferably, 8≦Ta≦50,  
 and, still more preferably, 10≦Ta≦30,  
 
 and by a mean hydrodynamic diameter Dh expressed in nm and measured according to a procedure Md, Dh being such that: 
 10 nm≦Dh≦150 nm,  
 preferably, 20 nm≦Dh≦1100 nm.  
 
   
     
     
         21 . A method for preparing the pulverulent solid of  claim 20 , comprising: 
 1) copolymerization of monomers consisting of anhydrides of N-CarboxyAmino acids (NCA) of at least two different types, on the one hand, NCAs-pAAI (“pAAI” designated precursors of AAI) and, on the other hand NCAs-AAO, is carried out in the presence: 
 of at least one nonaromatic polar solvent, preferably chosen from the group comprising: N-MethylPyrrolidone (NMP), DiMethylFormamide (DMF), DiMethyl SulfOxide (DMSO), DiMethylAcetamide (DMAc), pyrrolidone; NMP being most particularly preferred;  
 and optionally of at least one cosolvent selected from aprotic solvents (preferably 1,4-dioxane) and/or protic solvents (preferably pyrrolidone) and/or water and/or alcohols, methanol being particularly preferred;  
   2) the recurring pAAI motifs of the copolymer obtained in step 1 are converted to recurring AAI motifs, using hydrolysis, preferably acid hydrolysis, for which the copolymer obtained in step 1 is brought into contact with an aqueous phase for acid hydrolysis+water;    3) the reaction medium is neutralized;    4) optionally, the reaction medium is dialyzed in order to purify the aqueous suspension of structured particles;    5) optionally, this suspension of step 4 is concentrated;    6) the liquid medium is removed in order to collect the pulverulent solid comprising the particles.    
     
     
         22 . The method of  claim 21 , wherein, at the end of step 1, the copolymer poly(AAO) (PAAI) obtained is precipitated—preferably in water—and the precipitate is recovered.  
     
     
         23 . A method for preparing the pulverulent solid of  claim 20 , comprising 
 1) copolymerization of monomers consisting of anhydrides of N-CarboxyAmino acids (NCA) of at least two different types, on the one hand, NCAs-pAAI (“pAAI” designating precursors of AAI) and, on the other hand, NCAs-AAO, is carried out in the presence: 
 of at least one nonaromatic polar solvent, preferably chosen from the  
   group comprising: N-MethylPyrrolidone (NMP), DiMethylFormamide (DMF), DiMethyl SulfOxide (DMSO), DiMethylAcetamide (DMAc), pyrrolidone; NMP being most particularly preferred; 
 and optionally of at least one cosolvent selected from aprotic solvents (preferably 1,4-dioxane) and/or protic solvents (preferably pyrrolidone) and/or water and/or alcohols, methanol being particularly preferred;  
   2) the recurring pAAI motifs of the copolymer obtained in step 1 are converted to recurring AAI motifs, using hydrolysis, preferably acid hydrolysis, for which the copolymer obtained in step 1 is brought into contact with an aqueous phase for acid hydrolysis+water;    3) the reaction medium is neutralized;    4) optionally, the reaction medium is dialyzed in order to purify the aqueous suspension of structured particles.    
     
     
         24 . A method for preparing the pulverulent solid of  claim 20 , wherein the combination of PA with the particles is carried out by bringing a liquid phase containing the PA into contact with the colloidal suspension of particles.  
     
     
         25 . A method for preparing the pulverulent solid of  claim 20 , wherein the combination of the PA with the particles is carried out by bringing a PA in the solid state into contact with the colloidal suspension of particles.  
     
     
         26 . A method for preparing a colloidal suspension of submicronic particles, comprising contacting the pulverulent solid of  claim 20  with a liquid phase containing the PA.  
     
     
         27 . A method for preparing a colloidal suspension of submicronic particles, comprising contacting the pulverulent solid of  claim 20  with the PA in solid form to form a mixture, and dispersing this mixture of solids in a liquid phase, preferably an aqueous solution.  
     
     
         28 . An intermediate product of the method of  claim 21 , comprising PAA copolymers which are precursors of particles.  
     
     
         29 . A pharmaceutical, nutritional, plant-protection or cosmetic proprietary product, comprising the pulverulent solid of  claim 20 , comprising at least one active principle selected from the group consisting of: 
 Vaccines,    Proteins and/or peptides, among which those most preferably selected are:    hemoglobins, cytochromes, albumins, interferons, antigens, antibodies, erythropoietin, insulin, growth hormones, factors VIII and IX, interleukins or mixtures thereof, hematopoiesis-stimulating factors,    polysaccharides, heparin being more particularly selected,    nucleic acids and, preferably, RNA and/or DNA oligonucleotides,    non-petido-protein molecules belonging to various anticancer chemotherapy classes and, in particular, anthracyclines and taxoids,    and mixtures thereof.    
     
     
         30 . A method for preparing a copolymer comprising precursors of hydrophilic amino acids with ionizable side chains, comprising 
 copolymerizing N-carboxy anhydride (NCA) forms of i) monomers of precursors of hydrophilic amino acids with ionizable side chains and ii) monomers of hydrophobic neutral amino acids in the presence of at least one non-aromatic polar solvent selected from the group consisting of N-methylpyrrolidone (NMP)), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dimethylacetamide (DMAc) and pyrrolidone, and optionally in the presence of at least one cosolvent selected from the group consisting of aprotic solvents, protic solvents, water, and alcohols,    precipitating the copolymer to thereby recover a copolymer comprising precursors of hydrophilic amino acids with ionizable side chains.    
     
     
         31 . A copolymer comprising precursors of hydrophilic amino acids with ionizable side chains produced by the method of  claim 30.