A suspension of biocompatible particles based on a double-block hydrophilic neutral polyminoacid/hydrophobic neutral polyaminoacid copolymer and suitable for carrying active principles. The carrier particles can combine in colloidal suspension in a non-dissolved state with an active principle and release same, particularly in vivo, in prolonged and/or delayed delivery. The carrier particles may be derived from a powdery solid, and spontaneously form stable aqueous suspensions in the absence of surfactants or organic solvents. The invention also concerns the carrier particles in dry form, the method of preparing them, and pharmaceutical compositions (in dry form or suspension) which include the carrier particles associated with an active principle.

BRIEF DISCLOSURE OF THE INVENTION

With these facts established, one essential objective is to be able to provide novel CP which form stable aqueous suspensions of CP spontaneously, without the aid of surfactants or organic solvents.

Another essential objective of the present invention is to provide novel CP in stable colloidal aqueous suspension or in pulverulent form, based on polyamino acids (PAA), these novel CP preferably meeting specifications 1 to 8 of the specifications sheet mentioned above.

Another essential objective of the invention is to improve the particles disclosed in PCT patent application WO 96/29991.

Another essential objective of the invention is to provide a novel suspension of CP whose characteristics are perfectly controlled, especially in terms of the AP loading factor and in terms of control of the AP release kinetics.

Another essential objective of the invention is to provide injectable medicinal suspensions. The specifications required for such suspensions are a small injection volume and a low viscosity. It is important that the mass of colloidal particles per injection dose be as small as possible, without limiting the amount of active principle, AP, transported by these particles, so as not to detract from the therapeutic efficacy.

Another essential objective of the invention is to provide a colloidal aqueous suspension or a pulverulent solid which comprises active principle carrier particles meeting the above-mentioned specifications, and which constitutes an appropriate galenical form suitable for administration, for example orally, to humans or animals.

Another essential objective of the invention is to provide a colloidal suspension comprising active principle carrier particles that can be filtered on 0.2 .mu.m filters for sterilization purposes.

Another essential objective of the invention is to propose a method of preparing PAA particles (dry or in suspension in a liquid) that are useful especially as active principle carriers, said method being simpler to carry out and non-denaturing towards the active principles and additionally always allowing fine control over the mean size of the particles obtained.

Another essential objective of the invention is to use the above-mentioned particles, in aqueous suspension or in solid form, for the preparation of drugs (e.g. vaccines), especially for oral, nasal, vaginal, ocular, subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, intracerebral or parenteral administration, it being possible in particular for the active principles of these drugs to be proteins, glycoproteins, peptides, polysaccharides, lipopolysaccharides, oligonucleotides and polynucleotides; and/or nutriments; and/or cosmetic or plant health products; and/or medicinal organic molecules.

Another essential objective of the present invention is to provide suspensions of submicron CP based on PAA that are capable of acting as a carrier for an AP, particularly a medicinal AP for administration to a human or animal organism, or alternatively a nutritional, plant health or cosmetic AP.

Another objective of the present invention is to provide a drug, of the type consisting of a system for the prolonged release of active principles, which is easy and economic to produce and which is also biocompatible and capable of assuring a very high level of bioavailability of the AP.

Another essential objective of the invention is to provide a vaccine carrier system which is non-immunogenic intrinsically and in combination with one or more antigens.

The product-related objectives (among others) are achieved by the present invention, which relates first and foremost to a stable colloidal suspension of submicron structured particles capable of being used especially for carrying one or more active principles, AP, these particles being individualized (discrete) supramolecular arrangements that are: based on linear amphiphilic polyamino acids (PAA) having peptide linkages and comprising at least two different types of hydrophilic repeating amino acids, AAI, and hydrophobic repeating amino acids, AAO, the amino acids of each type being identical to or different from one another; capable of associating at least one AP in colloidal suspension, in the undissolved state, and releasing it, especially in vivo, in a prolonged and/or delayed manner; and stable in the aqueous phase at a pH of between 4 and 13, in the absence of surfactant(s), characterized: in that the hydrophilic repeating amino acids, AAI, are hydrophilic neutral amino acids, AANI, with the exclusion of asparagine; in that the hydrophobic repeating amino acids, AAO, are hydrophobic neutral amino acids, AANO; and in that the repeating amino acids of each type, AANI and AANO, are identical to or different from one another.

DETAILED DISCLOSURE OF THE INVENTION

One of the main inventive aspects of these novel carrier particles, CP, in stable colloidal aqueous suspension or in the form of a pulverulent solid, concerns the novel selection of a group of polymers and a novel methodology for obtaining particles of submicron size which form a stable colloidal aqueous suspension in the absence of surfactants or solvents.

Another main inventive aspect of these novel carrier particles, CP, in stable colloidal aqueous suspension or in the form of a pulverulent solid, concerns the novel selection of two neutral amino acids as hydrophilic repeating monomers, AANI, and hydrophobic repeating monomers, AANO.

Now, contrary to what those skilled in the art might have feared, not having ionizable hydrophilic amino acids, AAII, i.e. negative charges, as in the particles according to WO 96/29991, does not detract from the stability. In fact, contrary to all expectations, the colloidal suspension according to the present invention does not flocculate. The particles based on poly(AANI/AANO) do not self-aggregate. Moreover, it was not at all obvious a priori that these particles of poly(AANI/AANO) would be capable of associating spontaneously with active principles, AP, and releasing these AP at the sites of therapeutic action.

The structure of the PAA polymers and the nature of the neutral amino acids are chosen so that: the polymer chains spontaneously organize themselves into small particles (CP); the particles form a stable colloidal suspension in water and in a physiological medium; the CP associate with proteins or other AP in aqueous media by a spontaneous mechanism that is non-denaturing towards the protein; and the CP release the AP in a physiological medium and, more precisely, in vivo; the release kinetics depend on the nature of the PAA polymer that is the CP precursor.

Thus, by varying the specific structure of the PAA, it is possible to control the AP association and release phenomena from the kinetic and quantitative points of view.

It is to the Applicant's credit to have chosen, as the constituent material of the CP, a specific composition of neutral polyamino acids which are amphiphilic and therefore possess properties of CP made of PAA, namely: the possibility of spontaneously forming colloidal suspensions of CP that are compatible with the pH of the physiological media encountered in the intended therapeutic applications; spontaneous association of the AP with CP in the absence of any agent other than water, which acts as solvent and, in the case of proteins, is non-denaturing; the possibility of releasing the AP from the AP-CP association complex under physiological conditions with pharmacokinetic and pharmacodynamic profiles which lead to the expectation of valuable uses in the therapeutic field (AP transport); filterability with a cut-off threshold less than or equal to 0.2 .mu.m for sterilization purposes; improved biodegradability; and optimized injectability.

These PAA can be of the ordered type with alternate sequences (blocks) or of the disordered type with random sequences.

Thus, in a first embodiment of the CP according to the invention, the constituent PAA are of the "block" type and are characterized by a molar ratio AANO/(AANI+AANO) which is such that: AANO/(AANI+AANO).gtoreq.6%, 10% .ltoreq.AANO/(AANO+AANI).gtoreq.70%, preferably, 20% .ltoreq.AANO/(AANI+AANO).ltoreq.60%, and particularly preferably, 35% .ltoreq.AANO/(AANI+AANO).ltoreq.50%.

Advantageously, the absolute length of each block of AANO, expressed as the number of AANO, is such that: AANO.gtoreq.5, preferably, AANO .gtoreq.10, and particularly preferably, AANO .gtoreq.20.

In a second embodiment of the CP according to the invention, the constituent PAA are of the "random" type, i.e. prepared by the simultaneous copolymerization of AANI and AANO monomers, and the molar ratio AANO/(AANO+AANI) is such that: AANO/(AANO+AANI).gtoreq.10%, preferably, AANO/(AANO+AANI).gtoreq.20%, and particularly preferably, 30% .ltoreq.AANO/(AANI+AANO).ltoreq.70%.

Advantageously, the molecular weight, Mw, of these random PAA is such that: Mw.gtoreq.2000 g/mol, preferably, Mw.gtoreq.5500 g/mol, and particularly preferably, 5500 g/mol .ltoreq.Mw.ltoreq.200,000 g/mol.

According to a preferred characteristic of the invention, the block or random PAA constituting the particles have degrees of polymerization, DP, of between 30 and 600, preferably of between 50 and 200 and particularly preferably of between 60 and 150.

Advantageously, the PAA constituting the particles, CP, are "di-block" PAA.

Preferably, the hydrophilic AANI is (are) selected from the group comprising: natural neutral amino acids, preferably those selected from the group comprising serine, threonine, hydroxyproline and glutamine; the rare or synthetic neutral amino acids, preferably those selected from the group comprising methionine S-oxide and O-glycosidylserine; and derivatives of neutral amino acids, preferably those selected from the group comprising N-hydroxyethylglutamine, N-hydroxypropylasparagine, N-hydroxyethylasparagine and N-hydroxypropylglutamine.

Advantageously, the hydrophobic AANO is (are) selected from the group comprising: natural neutral amino acids, preferably those selected from the group comprising Leu, Ile, Val, Ala, Pro and Phe; rare or synthetic neutral amino acids, preferably those selected from the group comprising norleucine and norvaline; and derivatives of polar amino acids, preferably those selected from the group comprising methyl glutamate, ethyl glutamate, benzyl aspartate and N-acetyllysine.

According to an advantageous characteristic, the carrier particles (CP) of the suspension have a mean size of between 0.01 and 0.5 .mu.m, preferably of between 0.01 and 0.2 .mu.m.

Another preferred characteristic of the suspension is that it is aqueous and stable.

The present invention relates not only to suspensions of bare particles as defined above, but also to suspensions of particles comprising at least one active principle, AP. Preferably, the suspension according to the invention is aqueous and stable. These particles, whether loaded with AP or not, are advantageously in a form dispersed in a liquid (suspension), preferably an aqueous liquid, but can also be in the form of a pulverulent solid obtained from the suspension of CP as defined above.

It follows from this that the invention relates not only to a colloidal (preferably aqueous) suspension of CP, but also to a pulverulent solid comprising CP which is obtained from the suspension according to the invention.

Another essential object of the invention concerns the preparation of: selected particles as described above; and other selected particles which are structured, submicron and capable of being used especially for carrying one or more active principles, AP, these particles being individualized (discrete) supramolecular arrangements that are: based on linear amphiphilic polyamino acids (PAA) having peptide linkages and comprising at least two different types of hydrophilic repeating amino acids, AAI, and hydrophobic repeating amino acids, AAO, the amino acids of each type being identical to or different from one another; capable of associating at least one AP in colloidal suspension, in the undissolved state, and releasing it, especially in vivo, in a prolonged and/or delayed manner; and stable in the aqueous phase at a pH of between 4 and 13, in the absence of surfactant(s), where: the hydrophilic repeating amino acids, AAI, consist at least partly of asparagine; and the hydrophobic repeating amino acids, AAO, are hydrophobic neutral amino acids, AANO, which are identical to or different from one another, it being possible for these particles to be either in the form of a colloidal suspension or in the form of a pulverulent solid obtained from a stable colloidal suspension of particles.

The method of preparation in question consists essentially in synthesizing precursor PAA and converting them to structured particles.

More precisely, the method of preparation is first and foremost a method of preparing submicron structured particles capable of being used especially for carrying one or more active principles, these particles being discrete supramolecular arrangements that are: based on linear amphiphilic polyamino acids (PAA) having peptide linkages and comprising at least two different types of hydrophilic repeating amino acids, AAI, and hydrophobic repeating amino acids, AAO, the amino acids of each type being identical to or different from one another; capable of associating at least one AP in colloidal suspension, in the undissolved state, and releasing it, especially in vivo, in a prolonged and/or delayed manner; and stable in the aqueous phase at a pH of between 4 and 13, in the absence of surfactant(s).

This method is characterized in that: 1) a copolymerization is carried out between monomers formed of amino acid N-carboxy anhydrides (NCA) of at least two different types: on the one hand starting NCA monomers comprising NCA-Glu-OR and/or NCA-Asp-OR and/or NCA-AANI, and on the other hand NCA-AANO, in the presence of: at least one non-aromatic polar solvent preferably selected from the group comprising N-methylpyrrolidone (NMP), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dimethylacetamide (DMAc) and pyrrolidone, NMP being more particularly preferred; and optionally 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) in the case where the starting NCA monomers are NCA-Glu-OR and/or NCA-Asp-OR (R=alkyl), an aminolysis is carried out which consists in bringing the copolymer obtained in step 1 into contact with an aqueous phase comprising at least one amine, and which makes it possible to convert Glu-OR to Gin and Asp-OR to Asn; 3) the reaction medium is optionally dialyzed to purify the aqueous suspension of structured particles; 4) this suspension of step 3 is optionally concentrated; and 5) the liquid medium is removed so that the pulverulent solid comprising the particles can be collected.

The first step of the method is based on the known techniques of polymerizing .alpha.-amino acid N-carboxy anhydrides (NCA), which are described for example in the article "Biopolymers, 15, 1869 (1976)" and in the work by H. R. KRICHELDORF entitled ".alpha.-Amino acid N-carboxy anhydride and related heterocycles", Springer Verlag (1987).

Using carefully chosen polar, non-aromatic aprotic copolymerization solvents that avoid any precipitation, and using acid hydrolysis in the presence of water and a non-aromatic, polar organic solvent, affords submicron, discrete structured particles with a high AP loading capacity which form a stable colloidal suspension in aqueous media. These particles are in no way comparable to a macroscopic agglomerated precipitate of the kind referred to above in respect of earlier proposal (d).

In one variant, after step 1, the poly(AANO/AANI) copolymer obtained is precipitated--preferably in water--and this precipitate is collected. This variant corresponds to a batch mode of preparing particles in which the poly-(AANO/AANI) copolymer is isolated in the form of a precipitate constituting a stable intermediate. This precipitate can be filtered off, washed and dried, for example.

Particularly preferably, the NCA-pAAI are NCA of O-alkylated glutamic or aspartic acid, for example NCA-Glu-O-Me, NCA-Glu-O-Et or NCA-Glu-O-Bz (Me=methyl--Et=ethyl--Bz=benzyl).

In known manner, the copolymerization takes place at a temperature of between 20 and 120.degree. C., at atmospheric pressure and in the presence of an amine initiator, e.g. NH.sub.3.

Other experimental parameters, such as the concentration of NCA and/or polymer in the non-aromatic polar solvent (preferably NMP) and/or the concentration or nature of the protic cosolvent, during the synthesis, will be adjusted according to the desired effects known to those skilled in the art.

The acid hydrolysis (step 2) is carried out with water and at least one mineral acid such as phosphoric or hydrochloric acid--the latter being preferred--and/or at least one organic acid such as trifluoroacetic acid (TFA), acetic acid, dichloroacetic acid or organosulfonic acids.

The proportions of water/acid--expressed in parts by weight--in an acidic aqueous hydrolysis phase are advantageously: from 60/1 to 2/1, preferably 40/1 to 2/1, and particularly preferably 20/1 to 2/1.

The proportions of acidic aqueous hydrolysis phase/NMP--expressed in parts by weight--are advantageously: from 5/100 to 200/100, preferably 10/100 to 100/100, and particularly preferably from 20/100 to 80/100.

Other parameters, such as the polymer concentration, the temperature of the reaction mixture, the mode of addition of the acidic aqueous hydrolysis phase, the use of reduced pressure, the reaction time, etc., are adjusted according to the desired effects well known to those skilled in the art.

In practice, the neutralization (step 3) is carried out e.g. with sodium hydroxide.

The salt formed at the end of the neutralization, and the solvent, are then removed by any appropriate physical separation treatment, for example by diafiltration (dialysis) (step 4), filtration, pH modification, chromatography, etc.

This yields an aqueous suspension of structured particles which can be concentrated, for example by distillation or any other suitable physical means such as ultrafiltration or centrifugation.

In step 6, to separate the particles from their liquid suspension medium, the aqueous phase is optionally removed, for example by drying (e.g. in an oven), lyophilization or any other suitable physical means such as ultrafiltration or centrifugation. A white pulverulent solid is recovered at the end of this step 6.

In one variant, the concentration step can be carried out by means of a chemical treatment, such as a lowering of the pH, which converts the hydrophilic part of the glutamate monomers to acid, making them insoluble in water. These acidic PAA intermediates can be filtered off, washed and dried. Said acidic intermediates can be neutralized with a chemical base in a subsequent step to give a suspension of particles.

It is pointed out that the implementation of steps 1, 2, 3, 4 and optionally 5 of the above method corresponds to a preparation of a colloidal suspension of submicron particles with a high AP loading factor.

In this preparation of a colloidal suspension, the amphiphilic PAA, poly(AANO/AANI), of step 2 are placed in an aqueous medium in which at least part of the AANI is soluble and at least part of the AANO is insoluble. The PAA exist in the form of nanoparticles in this aqueous medium.

An alternative preparation of the suspension of CP according to the invention consists in bringing the pulverulent solid, as described above as a product and by its method of preparation, into contact with an aqueous medium that is a non-solvent for the AANO.

One or more AP can be associated with the particles by using several methods according to the invention. Non-limiting examples of these methods are listed below.

According to a first method, an AP is associated with the particles by bringing a liquid phase (aqueous or non-aqueous) containing the AP into contact with the colloidal suspension of particles which may or may not comprise Asp as AANI.

According to a second method, the AP is associated with the particles by bringing an AP in the solid state into contact with the colloidal suspension of particles which may or may not comprise Asp as AANI. The solid AP can be e.g. in the form of a lyophilizate, precipitate or powder or in another form.

According to a third method, the pulverulent solid (PAA which may or may not comprise Asp as AANI), as described above as a product and by its preparative characteristics, is brought into contact with a liquid phase (aqueous or non-aqueous) containing the AP.

According to a fourth method, the pulverulent solid, as described above as a product and by its preparative characteristics (PAA which may or may not comprise Asp as AANI), is brought into contact with the AP in solid form. This mixture of solids is then dispersed in a liquid phase, preferably an aqueous solution.

In all these methods, the AP used can be in the pure form or a preformulated form.

Given the nanometric size of the particles, the suspension can be filtered on sterilization filters, enabling sterile injectable medicinal liquids to be obtained easily and at lower cost. The ability, afforded by the invention, to control the particle size and reach Dh values of between 25 and 100 nm is an important asset.

The present invention further relates to novel intermediates of the method described above, characterized in that they consist of PAA copolymers (which may or may not comprise Asp as AANI) that are particle precursors.

INDUSTRIAL APPLICATION

According to another of its features, the invention relates to a suspension and/or a pulverulent solid (PAA which may or may not comprise Asp as AANI) as defined above and/or as obtained by the method described above, this suspension and this solid comprising at least one active principle preferably selected from: vaccines; proteins and/or peptides, among which the following are more preferably selected: hemoglobins, cytochromes, albumins, interferons, antigens, antibodies, erythropoietin, insulin, growth hormones, factors VIII and IX, interleukins or mixtures thereof, and hemopoiesis-stimulating factors; polysaccharides, heparin being more particularly selected; nucleic acids and preferably RNA and/or DNA oligonucleotides; non-peptido-protein molecules belonging to various anticancer chemotherapy categories, particularly anthracyclines and taxoids; and mixtures thereof.

The invention further relates to a suspension and/or a pulverulent solid (PAA which may or may not comprise Asp as AANI) loaded with a nutritional, plant health or cosmetic AP.

Finally, the invention relates to a pharmaceutical, nutritional, plant health or cosmetic proprietary product, characterized in that it contains a suspension and/or pulverulent solid as defined above, loaded with AP.

According to another of its objects, the invention further relates to the use of these CP (in suspension or in solid form: PAA which may or may not comprise Asp as AANI), loaded with AP, for the manufacture of drugs of the type consisting of systems for the controlled release of AP.

In particular, the invention relates to the use of a stable colloidal suspension of submicron structured particles loaded with one or more active principles, AP, these particles being individualized (discrete) supramolecular arrangements that are: based on linear amphiphilic polyamino acids (PAA) having peptide linkages and comprising at least two different types of hydrophilic repeating amino acids, AAI, and hydrophobic repeating amino acids, AAO, the amino acids of each type being identical to or different from one another; capable of associating at least one AIP in colloidal suspension, in the undissolved state, and releasing it, especially in vivo, in a prolonged and/or delayed manner; and stable in the aqueous phase at a pH of between 4 and 13, in the absence of surfactant(s), where: the hydrophilic repeating amino acids, AAI, consist at least partly of asparagine; and the hydrophobic repeating amino acids, AAO, are hydrophobic neutral amino acids, AANO, which are identical to or different from one another, for the preparation of an aqueous suspension or a pulverulent solid as above defined, loaded with at least one AP.

Examples of drugs are those that can preferably be administered by the oral, nasal, vaginal, ocular, subcutaneous, intravenous, intramuscular, intradermal, intra-peritoneal, intracerebral or parenteral route.

Examples of cosmetic applications that can be considered are compositions which comprise an AP associated with the CP according to the invention and which can be administered transdermally.

Examples of possible plant health products are herbicides, pesticides, insecticides, fungicides, etc.
 

Claim 1 of 24 Claims

1. A stable colloidal suspension comprising submicron particles, wherein the particles: are individualized supramolecular arrangements comprising linear, peptide-linked, amphiphilic polyamino acids (PAA) comprising at least hydrophilic repeating neutral amino acids (AANI), other titan aspangine, and hydrophobic repeating neutral amino acid (ANNO); wherein the AANI are identical to or different from one another and the AANO are identical to or different from one another; are stable in the aqueous phase at a pH of between 4 and 13 in the absence of a surfactant; have a degree of polymerization (DP) between 50 and 200; spontaneously associate with at least one active principle when at least one active principle in the undissolved state is added to the colloidal suspension; and when associated with at least one active principle, release it in a prolonged manner, in a delayed manner; or in a prolonged and delayed manner.

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