Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
  • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
  • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
  • C07C323/60—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton with the carbon atom of at least one of the carboxyl groups bound to nitrogen atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
  • A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
  • A61K8/447—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof containing sulfur
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
  • A61K8/81—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds obtained by reactions involving only carbon-to-carbon unsaturated bonds
  • A61K8/8141—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Compositions of derivatives of such polymers
  • A61K8/8158—Homopolymers or copolymers of amides or imides, e.g. (meth) acrylamide; Compositions of derivatives of such polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
  • C11D1/38—Cationic compounds
  • C11D1/52—Carboxylic amides, alkylolamides or imides or their condensation products with alkylene oxides
  • C11D1/528—Carboxylic amides (R1-CO-NR2R3), where at least one of the chains R1, R2 or R3 is interrupted by a functional group, e.g. a -NH-, -NR-, -CO-, or -CON- group
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
  • A61K2800/41—Particular ingredients further characterized by their size
  • A61K2800/413—Nanosized, i.e. having sizes below 100 nm
• • 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
  • A61K9/127—Liposomes
  • A61K9/1274—Non-vesicle bilayer structures, e.g. liquid crystals, tubules, cubic phases, cochleates; Sponge phases

Definitions

• the present invention relates to new telomer type surfactants, their use for the preparation of metastable supramolecular systems.
• These metastable supramolecular systems or nanoparticles can be liposomes, or micellar systems.
• the subject of the invention is also the atypical liposomes and nanoparticles obtained from these surfactants and their use as vectors of active principles, in particular therapeutic active principles.
• Certain amphiphilic molecules, the natural phospholipids have the property of associating in water by forming metastable supramolecular organizations of spherical forms called liposomes which contain an internal aqueous compartment.
• liposomes are capable of containing therapeutic active agents within this internal compartment and can thus be used to transport these active agents to target cells or tissues.
• the study of these particulate vectors has been the subject of an abundant literature in which the problems as well as the potentialities of their use have been widely discussed (Barenholz, Curr. Opin. In Coll. And Int. Sci. 6 (2001 ) 66-77).
• the use of liposomes for the transport of therapeutic active principles has some major drawbacks: These nanostructures generally have a relatively low stability over time, because in the medium in which they are dispersed, they fuse to form larger objects which then rush quickly. This behavior severely limits their capacity for preservation and storage.
• the biological stability of these vectors i.e.
• liposome protection systems In order to reduce their rapid elimination by the reticuloendothelial system, liposome protection systems have been put in place. The most effective consists in using phospholipids substituted by polyethylene glycols of molecular weight between 1000 and 5000 in a proportion of 5 to 10% of the total mixture of phospholipids.
• the “invisible”, so-called stealthy liposomes thus formed (marketed under the brand name Stealth liposomes®) have longer blood retention times than conventional liposomes (45 hours versus a few minutes to a few hours).
• Active targeting of these vectors may be carried out by covering their surface with target molecules such as antibodies, peptides, lectins, sugars, hormones or specific synthetic compounds.
• target molecules such as antibodies, peptides, lectins, sugars, hormones or specific synthetic compounds.
• the literature cites a large number of polymers of amphiphilic nature. These are generally di-block type polymers made up of different hydrophilic and hydrophobic monomers (M. Jones et al, Eur. J. Pharm. Biopharm., 48 (1999) 101-111, VP Torchilin, J. Control. Release, 73 (2001) 137-172).
• Other amphiphiles derived from phospholipids and polymers of polyethylene glycol and polyvinyl pyrrolidone have also been studied (AN Lukyanov et al, Adv. Drug Deliver.
• a first objective of the present invention is the development of nanoparticulate vectors at very low production cost and having the capacity to transport within their internal aqueous cavity a very large family of hydrophilic active agents.
• the nanoparticulate vectors of the invention allow the encapsulation, the retention and the release of dosable substances.
• Targeted applications include the transport of active ingredients, in particular therapeutic active ingredients, epidermal delivery of cosmetic substances, medical diagnosis.
• active ingredients in particular therapeutic active ingredients, epidermal delivery of cosmetic substances, medical diagnosis.
• anticancer active ingredients, active ingredients for vaccine use genetic material, enzymes, hormones, vitamins, sugars, proteins and peptides, lipids, organic and inorganic molecules.
• a second objective of the present invention is the development of nanoparticulate vectors at very low production cost and having the capacity to transport within their hydrophobic cavity or their lipid sheet a very large family of hydrophobic active agents.
• the nanoparticulate vectors of the invention allow the encapsulation, the retention and the release of dosable substances.
• the targeted applications include the transport of active principles, in particular therapeutic active principles, the epidermal delivery of cosmetic substances, medical diagnosis.
• the subject of the present invention is the compounds corresponding to the formula
• R ' represents H or a hydrophilic group, such as for example a C 4 -C 2 polyhydroxylated hydrocarbon compound; in particular R 'can be chosen from sugars such as for example galactose, glucose, mannose, sialic acid, linked by its anomeric carbon; • R represents a group chosen from: C 4 -C 24 hydrocarbon radicals; C 4 -C 4 fluorinated hydrocarbon radicals; the C - C 24 thioalkyl radicals.
• the group R can in particular be chosen from the following radicals:
• the thiooctyl radical The C -C 24 hydrocarbon radicals such as n-butyl, tert-butyl, isobutyl, n-pentyl, isopentyl, n -hexyl, n-heptyle, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n -hexadecyl, n- heptadecyl, n-octadecyl, the phytyl radical
• C 4 -C 2 fluorinated hydrocarbon radicals such as those corresponding to the formula - (CH 2 ) r (CF 2 ) r F, in which r and t represent two integers with: 24> r + t> 4, as for example: - (CF 2 ) 4 F; - (CF 2 ) 5 F; - (CF 2 ) 6 F; - (CF 2 ) 7 F; - (CF 2 ) 8 F; - (CF 2 ) 9 F; -
• the preferred R chains are those which contribute to giving the surfactant of formula (I) a phase transition temperature greater than 37 ° C.
• these surfactants having a crystal structure at physiological temperature, provide the liposome membrane with greater rigidity and a greater degree of retention of the solutes encapsulated in the internal aqueous compartment.
• R represents a C ⁇ -C 2 hydrocarbon chain or a C 8 -C 24 fluorinated hydrocarbon chain.
• A1 represented below:
• Another object of the invention consists in the use of molecules of formula (I), advantageously molecules of formula (IA) for the manufacture of liposomes.
• the liposomes of the prior art have their walls generally made up of phospholipids. Liposomes are made from surfactants of formula (I), preferably of formula (IA) very easily by the film method (Liposomes, a practical approach, RRC New, Ed., Oxford University Press, New York, 1990) .
• a double sonication and extrusion treatment can also be provided.
• Other conventional methods for preparing liposomes can be used for the preparation of the liposomes of the invention.
• S. Vemuri and CTRhodes Pharmaceutica Acta Helvetiae 70, (1995), 95-111.
• tubular vesicles because of their size, which is of the order of a few tens of nanometers, and of their shape which recalls that of a tube closed at its two ends. .
• the size and mechanical stability over time of the particles obtained in the solution were measured after filtration by dynamic light diffraction. (High Performance Particle Sizer, Malvern).
• a surfactant of formula (IA) given the particle size is substantially homogeneous: it varies in a range of value of + 10%, preferably + 5%, around a central value of length and diameter.
• These tubular vesicles have a relatively high stability since no change in particle size is observed after one year of storage, while liposomes formed from phosphatidyl choline from egg yolk show an evolution after only 5 days of storage.
• the detection of the aqueous internal cavity has been indirectly proven by spectrofluorimetric measurements of the encapsulation and the release kinetics of a hydrophilic fluorescent probe, carboxyfioresoresin.
• the invention further relates to liposomes, or aqueous dispersions of vesicles, characterized in that they comprise one or more compounds of formula (I), advantageously of formula (IA) as constituents of their walls.
• These liposomes have original structural characteristics which give them unexpected properties, in particular improved stability compared to the liposomes of the prior art.
• the liposomes of the invention have also shown an ability to release an active principle over a longer period of time compared to the liposomes of the prior art.
• the subject of the invention is the compounds corresponding to the formula (IB):
• Ri represents a group chosen from the following radicals: in which R ′ represents H or a hydrophilic group, such as for example a C 4 -C 24 polyhydroxy hydrocarbon compound.
• R ' can be chosen from sugars such as for example galactose, glucose, mannose, sialic acid, linked by its anomeric carbon.
• R represents a group chosen from: the hydrocarbon radicals in
• R chains are those which contribute to giving the surfactants of formula (IB) a Critical Micellar Concentration (CMC) of less than 10 "5 M.
• CMC Critical Micellar Concentration
• a low CMC in fact provides the nanoparticle with greater thermodynamic stability as well as a capacity greater retention of the solutes encapsulated in the internal hydrophobic compartment.
• R represents a C 1 -C 2 hydrocarbon chain or a C 8 -C 4 fluorinated hydrocarbon chain.
• the molecules of formula (IB) can be synthesized in a simple way using the conventional methods of organic synthesis. Several examples of synthesis are illustrated in the experimental part.
• the preferred compounds of formula (IB) are those for which Y represents S.
• Another preferred variant is that in which p represents an integer ranging from 5 to 15.
• Another subject of the invention consists in the use of the compounds of formula (I), advantageously of the compounds of formula (IB) for the preparation of nanoparticles with hydrophobic cavity and the nanoparticles thus obtained.
• the particles are made from surfactants of formula (I) or (IB) very easily by the film method which is well known to those skilled in the art and which is described in the work Liposomes, a practical approach, RRC New , Ed., Oxford University Press, New York, 1990. The procedure is described above for the compounds of formula (IA).
• the size and mechanical stability over time of the particles obtained in the solution were measured after filtration by dynamic light diffraction (High Performance Particle Sizer, Malvern).
• the nature of the particles obtained was studied by transmission electron microscopy after negative staining of the sample or after cryofracture.
• the Critical Aggregation Concentration of these surfactants was determined by tensiometry and spectrofluorimetry by the fluorescent marker method.
• the tensiometry technique of Wilhelmy made it possible to determine the limit surface tensions and the area of the pole head at the water-air interface (FIG. 8).
• These compounds have relatively low CMCs of the order of 10 ⁇ 5 M.
• the CMC of these surfactants hardly changes as a function of p (FIG. 8).
• These ellipsoidal particles have original structural characteristics and their average hydrodynamic diameter can be easily modulated by the variation of p, that is to say of the number of monomeric units constituting the polymeric hydrophilic part.
• the particles of the invention have also shown an ability to encapsulate hydrophobic active agents. This incorporation can be carried out using techniques well known to those skilled in the art. For example, the encapsulation can be carried out by dissolving the active ingredient in a preformed solution of ellipsoids or micelles, by the oil-in-water procedure or by dialysis.
• the therapeutic compounds which can be encapsulated are all the compounds, preferably hydrophobic, which can be stably incorporated into these micellar or ellipsoid structures.
• Different families of active ingredients that are not very hydrophilic or hydrophobic can be encapsulated or dissolved via these objects, including anticancer drugs, antibiotics, immunomodulators, steroids, anti-inflammatories or nucleotides.
• Hydrophilic compounds liable to complex with the polar part of the nanoparticles can also be encapsulated or vectorized.
• the dose of active ingredient effectively encapsulated in these nanoparticles is determined after filtration of the active ingredient not encapsulated by HPLC, by UN or fluorescence spectrometry as well as by RM ⁇ 1H.
• the nanoparticles, micellar, ellipsoidal or liposomes, of the invention may preferably also comprise at least one compound corresponding to formula (II) below:
• - Y represents a sulfur atom or the group -NH-CO- (CH 2 ) nX- in which X represents a sulfur atom S or a group -CH 2 -, n is an integer ranging from 0 to 10; - represents a group -NH- or -CH 2 - - x represents 0 or an integer ranging from 1 to 30; - y represents 0 or an integer ranging from 1 to 10; - R !
• R ' represents a hydrophilic group chosen from the following radicals in which R 'represents H or a hydrophilic group, such as for example a C 4 -C 2 polyhydroxylated hydrocarbon compound; in particular R 'can be chosen from sugars such as for example galactose, glucose, mannose, sialic acid, linked by its anomeric carbon; - R represents a recognition group which is chosen as a function of the cell target, preferably it is chosen from groups having a pronounced affinity for the biological target of the active principle carried in the nanoparticle.
• R 2 can be saccharide in nature (targeting specific membrane lectins which are found in particular tissues and which selectively recognize either galactose - cases of the liver, bones, certain cancerous tumors - or mannose - in the case of macrophages, heart-, ie sialic acid- case of erythrocytes -%), of a hormonal nature (such as steroids), of a synthetic nature such as gleevek to target kinases, specific antibodies, biotin which binds to certain specific proteins, and more generally any substrate whose previous research has demonstrated the specificity of recognition.
• saccharide in nature targeting specific membrane lectins which are found in particular tissues and which selectively recognize either galactose - cases of the liver, bones, certain cancerous tumors - or mannose - in the case of macrophages, heart-, ie sialic acid- case of erythrocytes -
• a hormonal nature such as steroids
• a synthetic nature such as gleevek to
• the RGD sequence known for its affinity for the ⁇ N ⁇ 3 integrins. It is possible to provide that the same molecule of formula (II) comprises one or more identical recognition groups R 2 or several different recognition groups R 2 , which makes it possible to direct the particles towards several distinct biological targets.
• the group R obeys the same rules as those previously defined for the structure of the compound of formula (I).
• - Z is a spacer arm which connects the recognition group R to the polymer chain.
• the spacer arm Z may consist of a peptide chain.
• This spacer arm comprises 1 to 5 amino acids, preferably 1 to 3 amino acids.
• the amino acids constituting the spacer arm Z are chosen from natural amino acids such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or unnatural amino acids such as hydroxyproline, norleucine , ornithine, citrulline, cyclohexylalanine.
• This spacer arm Z can consist of a tyrosine residue allowing in vivo monitoring of the vector after labeling with 125 I OR 131 I. It is also possible to use as group Z ⁇ -amino acids such as 3- aminopropionic and 4-amino-butyric acid, but also ethanolamine, 3-propanolamine or diamines of formula -NH- (CH 2 ) r -
• -ZR 2 is constituted by an NTA group of formula:
• the preferred compounds of formula (II) are those of formula
• the nanoparticles (liposomes, tubular vesicles, micelles or ellipsoidal particles) of the invention comprise from 1 to 5% of one or more compounds of formula (II) which makes it possible to promote the targeting of these nanoparticles towards their target. biological without altering their organization.
• lipid telomeres of formula (II) have the advantage thanks to their hydrophilic oligomeric part of being able to distance the grafted recognition agents from the surface of the tubular vesicles, thus promoting their recognition by the target cells or tissues.
• the other advantage linked to the use of these targeting lipids (II) is the possibility of multiplying the recognition patterns on a single compound thanks to the telomerization technique.
• Factors x and y are indeed easy to control and will depend quite closely on the proportion of monomers and telogen agent reacted.
• the ligation of the recognition agents can be carried out before the telomerization of the hydrophilic head if there is compatibility with the reaction conditions.
• the recognition agents can also be attached to the oligomeric polar head after formation of the tubular vesicles.
• the telomerized hydrophilic part is then functionalized by groups capable of ensuring coupling with these recognition agents.
• the different coupling techniques that can be used are well known to those skilled in the art and they are described in particular in: Allen et al, Biochim. Biophys. Acta, 1237 (1995) 99-108; Sapra, Prog. Lipids Res., 42 (2003) 439-462, Hansen et al, Biochim. Biophys. Acta, 1239 (1995) 133-144.
• the compounds of formula (II) constitute another object of the invention.
• the compounds of formula (I) and of formula (II) described above can be grouped under the same formula (III):
• R 3 represents a group chosen from:
• the liposomes or tubular vesicles of the invention formed from the compounds of formula (IA), and optionally (IIA) are stabilized by telomerization or polymerization of a monomer of acrylic type contained in their internal watery cavity.
• oligomeric or polymeric matrix is produced after encapsulation of the constituent monomer (s) in the tubular vesicles and elimination of the nonencapsulated monomers by separation techniques on size exclusion gel.
• Telomerization which consists of forming the polymer in the presence of a chain transfer agent, provides access to small polymers of controlled size. The reduced molecular weight of this polymer promotes its elimination via the kidneys. By avoiding the accumulation of polymer in the lysosome, problems of toxicity are also avoided.
• Nanoparticles, liposomes or tubular vesicles, comprising, in addition to the surfactants of formula (IA), at least one oligomer or telomer as described below constitute another object of the invention.
• the oligomer or telomer consists of monomeric, hydrophilic, ionic or nonionic building blocks, chosen from acrylic acid, methacrylic acid, methacrylamide derivatives, as well as the acrylate, methacrylate, acrylamide and methacrylamide derivatives of C ⁇ alcohols.
• sugars can be simple sugars such as: glucose, ribose, arabinose, xylose, lyxose, allose, altrose, mannose, galactose, fructose, talose.
• Disaccharides such as maltose, sucrose, or lactose
• Amino acids can be chosen from natural amino acids such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, acid glutamic, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or unnatural amino acids such than hydroxyproline, norleucine, ornithine, citrulline, cyclohexylalanine.
• Tris (hydroxy) methyl acrylamidomethane sodium acrylate, hydroxyethyl acrylate, glucose monoacrylate, glucose-1 - (N -methyl) acrylamide, 2-acrylamide glucose, 1-acrylamide maltose, sorbitol monoacrylate.
• telomer In order to modify the retention and stabilization capacities of the telomer, it is possible to use, in the manufacture of the telomer, water-soluble crosslinking agents such as: glucose-1,2-diacrylami, sorbitol diacrylate, sucrose diacrylate, sucrose di (ethylenediamine acrylamide), kanamycin tetracrylamide, kanamycin diacrylamide, or other sugars di or polyfunctionalized with acrylates or acrylamides.
• water-soluble crosslinking agents such as: glucose-1,2-diacrylami, sorbitol diacrylate, sucrose diacrylate, sucrose di (ethylenediamine acrylamide), kanamycin tetracrylamide, kanamycin diacrylamide, or other sugars di or polyfunctionalized with acrylates or acrylamides.
• all hydrophilic compounds capable of receiving at least two acrylate or acrylamide groups can be used. This is the case for example of the tris (hydroxymethyl) acrylamidomethane
• crosslinking agents are used in proportions ranging from 1 to 5% by weight relative to the weight of the monomer (s).
• the size of the telomer is controlled using one or more hydrophilic or hydrophobic transfer agents which are inserted into the membrane or the internal aqueous cavity of nanoparticles and particularly tubular vesicles.
• the chain transfer agent can be hydrophilic or hydrophobic, of thiol or phosphite type.
• the chain transfer agents used are chosen from hydrophilic thiols such as thiol acetic acid, mercaptopropionic acid, thioethylene glycol, cystamine, cysteine or hydrophobic thiols such as alkane thiols from C 2 to C 30 , such as compound D (derived from cholesterol) which is known for its ability to integrate in phospholipid membranes.
• hydrophilic thiols such as thiol acetic acid, mercaptopropionic acid, thioethylene glycol, cystamine, cysteine or hydrophobic thiols such as alkane thiols from C 2 to C 30 , such as compound D (derived from cholesterol) which is known for its ability to integrate in phospholipid membranes.
• compound D derived from cholesterol
• the chain transfer agent can also be chosen from the double-stranded thiols previously described.
• the phosphites can, for their part, be hydrophilic such as diethyl phosphite or hydrophobic such as dioctyl, didodecyl, dihexadecyl phosphites.
• hydrophobic structure of these hydrophobic thiols approximate that of the surfactants constituting it.
• the common motif of these telogen agents advantageously consists of an aminoglycerol motif onto which fatty chains are grafted by carbamate bonds according to formula (VI):
• the internal aqueous cavity of the tubular vesicles comprises a hydrophilic telomer, optionally provided with a hydrogenic phogenous part integrated into the internal membrane sheet.
• the physico-chemical description of these stabilized tubular vesicles is dominated in the experimental part and their superior retention capacity of an encapsulated solute and their increased mechanical stability are demonstrated.
• the targeted applications include the transport of active principles, in particular therapeutic active principles, the epidermal delivery of cosmetic substances, medical diagnostic agents. In particular the transport of anticancer active ingredients, vaccines, genetic material, enzymes, hormones, vitamins, sugars, proteins and peptides, lipids, organic and inorganic molecules.
• the epitopes and peptides may be incorporated into the internal aqueous matrix or expressed on the surface of the tubular vesicles by a covalent binding system in order to improve the immune response of the epitopes.
• the present invention therefore further relates to any composition, in particular any therapeutic, vaccine or cosmetic diagnostic composition comprising at least one active principle in association with a nanoparticle, liposome, tubular vesicle, ellipsoid or micelle, as described above, and in particular any composition comprising at least one active principle encapsulated in a liposome or tubular vesicle, ellipsoid or micelle according to the present invention.
• FIG. 1 illustrates the release kinetics of the carboxyfluorescein encapsulated in phosphatidyl choline (+) liposomes and tubular vesicles consisting of the compound Al (•) measured by spectrofluorimetry.
• FIG. 2 represents the infrared spectrum in the liquid phase of a 9 -A solution of compound Al in CC1 4 at different concentrations (1.10 " to 2.5.10 M).
• FIG. 3 represents the distribution curve in size by volume of tubular vesicles, measured by electron microscopy
• Figure 4 is a photograph obtained by electron microscopy by phase transmission after negative staining with uranyl acetate 2% of tubular vesicles formed by dispersion of the compound Al (2.5 mg.ml "1 ) in the water.
• FIG. 5 is a photograph obtained by electron microscopy by phase transmission after cryofracture of a sample of tubular vesicles formed by dispersion of the compound Al (2.5 mg.ml "1 ) in water.
• FIG. 1 represents the distribution curve in size by volume of tubular vesicles, measured by electron microscopy
• Figure 4 is a photograph obtained by electron microscopy by phase transmission after negative staining with uranyl acetate 2% of tubular vesicles formed by dispersion of the compound Al (2.5 mg.ml "1 ) in the water.
• FIG. 5 is a photograph obtained by electron microscopy by phase
• FIG. 6 is a photograph obtained by electron transmission phase microscopy after negative staining with 2% uranyl acetate of a sample of tubular vesicles formed by dispersion of the compound of structure B (2.5 mg.ml "1 ) in water.
• Figure 7 shows pictures of electron microscopy by
• FIG. 9 represents the phase transition temperatures measured by light scattering and recall of the values measured in spectrofluorimetry
• Example 1 synthesis of the Al derivative The synthesis of the Al derivative is summarized in scheme 1 • Trityl mercaptopropionic acid (1): available molecule commercially.
• the crude reaction product is then washed with a saturated sodium bicarbonate solution and then with a normal hydrochloric acid solution, saturated with sodium chloride before being dried over sodium sulfate. After filtration on sintered glass, the product is purified by chromatography on silica gel with elution with a gradient of pure ethyl acetate to ethyl acetate / methanol 9: 1 (v: v)). The pure product is obtained in the form of a white powder (2.12 g, Yield: 87%). The product can also be obtained pure, with an identical yield, by crystallization at room temperature of the reaction crude in an ethyl acetate / methanol mixture 8: 2 (v: v) in 8 days.
• the reaction medium is brought to reflux and a tip of a spatula of 1,4-diaza bicyclo- [2,2,2] -octane (DABCO) (cat.) Is added to the mixture. After 6 hours, the crude is evaporated to dryness and taken up in a minimum of ether where the product crystallizes at room temperature. After filtration, the product is obtained pure in the form of a white powder (4.04 g, Yield: 86%).
• DABCO 1,4-diaza bicyclo- [2,2,2] -octane
• the medium is then neutralized by adding formic acid, evaporated to dryness and purified by chromatography on silica gel using an elution gradient of ethyl acetate / cyclohexane 7: 3 to 5: 5.
• the pure product is obtained in the form of a yellow oil (1.1 g, Yield: 87%).
• Synthesis diagram of the targeting G lipid telomer • Synthesis of the telomerized lipid G 0.767 g of tris- (acetoxymethyl) acrylamidomethane 6 monomer (2.55 mmol, 12 eq) and 0.2 g of monomer 5 are dissolved in a 100 ml two-necked flask surmounted by a condenser. 0.63 mmol, 3 eq) in 20 mL of freshly distilled acetonitrile. Tris- (acetoxymethyl) acrylamidomethane 6 was prepared in accordance with the teaching of the document A.Polidori et al, New J. Chem., 1994, 18, 839-848.
• the reaction medium is degassed under argon and brought to reflux. 7 mg of AIBN (4.29.10 " mmol, 0.2 eq) and 0.157 g of thiol 4 (0.21 mmol, 1 eq) dissolved in 5 ml of freshly distilled and degassed acetonitrile are added. The reaction is maintained at reflux for 4 hours until total consumption of the monomers (detected by TLC). The reaction medium is concentrated under reduced pressure and the crude reaction product is filtered through a sephadex column (MeOH / CH 2 Cl2 1: 1).
• the product is then dissolved in 100 ml of methanol in the presence of a catalytic amount of sodium methylate After 5 hours of stirring, the reaction medium is neutralized by addition of acid resin IRC 50. The resin is removed by filtration and the solvent removed under reduced pressure. product is then reacted cold in an acid mixture TFA CH 2 C1 (20%) for 3 hours The reaction medium is concentrated under reduced pressure The oil obtained is taken up several times in ether until precipitation of the telomeres under the has the form of a white powder The product is dissolved in water and lyophilized until compound G is obtained in the form of a white powder.
• the average degree of polymerization (DPn) and the concentration ratio of each monomer in the macromolecule (x and y) was determined by 1 H NMR by comparing the integrations of the signals of the methyls of the two alkyl chains at 1.1 ppm with that of Tris ( ⁇ H? OH) signals at 4.3 ppm and lysine (CH? NH) at 3.37 ppm.
• x and y x: 30 and y: 10
• 6-Acryloylamino-2- [bis- (2-carboxy-ethyl) -amino] -hexanoic acid 8 In a 50 mL flask, 1.9 g of compound 7 (6.33 mmol) are dissolved in 20 mL of '' a 1: 1 trifluoroacetic acid - dichloromethane mixture. After 2 hours of stirring the solvent is evaporated under reduced pressure and the oil obtained is taken up several times in chloroform and evaporated until a powder is obtained. In a 25 mL flask, 1.94 g of bromoacetic acid (12.66 mmol) are dissolved in 7 mL of 2N sodium hydroxide. The solution is cooled to 0 ° C.
• Example 5 Preparation of tubular vesicles from the derivative Al Material used. • Measurement of the size of tubular vesicles. The particle size distribution was measured by photon correlation spectroscopy of the sample light diluted in water using a device
• the film obtained is then dried under reduced pressure using a vane pump.
• the lipid film is rehydrated with distilled water at 65 ° C (10 ° C above the phase transition temperature of the lipid) at a concentration of 2.5 mg.mL "1.
• the mixture is homogenized using a vortex for 5 minutes then subjected to ultrasound for 30 minutes at 70 ° C.
• Tm 54 ° C.
• tubular vesicles have a high stability since no change in particle size is observed after one year of storage, while under the same conditions, liposomes formed from phosphatidyl choline from egg yolk evolve after only 5 days. .
• the detection of the aqueous internal cavity has been indirectly proven by spectrofluorimetric measurements of the encapsulation and the kinetics of release of a hydrophilic fluorescent probe, carboxyfluorescein (FIG. 1). The measurements clearly show a slower release kinetics of the fluorescent probe compared to traditional encapsulation in a mixture of phosphatidyl choline from egg yolk.
• Example 6 Encapsulation of Carboxyfluorescein in Tubular Vesicles Manufactured from the Al Derivative
• the encapsulation power of the various compounds is determined by spectrofluorimetry using a fluorescent probe: 5 (6) -carboxyfluorescein.
• the release of this fluorescent marker is measured from the vesicles prepared according to standardized methods. This study requires the preparation of a Tris buffer (15 mM and 150 mM
• the non-encapsulated fluorescent probe is eliminated by passage over a Sephadex G25 column previously equilibrated with Tris buffer.
• the fraction of vesicle collected is immediately studied by spectrofluorimetry.
• the fluorescence measurements were carried out using a Jobin-Yvon spectrofluorimeter (spectrofluoromax 2), equipped with a 150 W Xenon lamp. All the measurements were carried out in a quartz tank, thermostatically controlled at 25 ° C.
• the samples were analyzed at an excitation wavelength of 480 nm, and an emission wavelength of 530 nm for 4 hours.
• the bandwidths were set at 0.5 nm for both the excitation and the emission.
• the initial fluorescence intensity (F 0 ) is determined 30 seconds after column filtration.
• the release is studied over a period of 4 hours and the fluorescence intensity (F t ) is measured at regular intervals.
• the maximum fluorescence intensity (F max ) which corresponds to 100%> of salting out, is obtained after the lysis of the tubular vesicles obtained by adding Triton XI 00 (10% v / v).
• Example 7 Polymerization of a monomer encapsulated in tubular vesicles obtained from the lipid Al The composition of the solutions used is summarized in Table 1.
• Table 1 Preparation of the film 20 mg of lipids Al are dissolved in 2 mL of a solution of Cumene hydroperoxide in dichloromethane freshly distilled and degassed by bubbling with argon (2.5.10 " M), in a heart-shaped flask The solution is evaporated to dryness on a rotary evaporator (the bath temperature does not exceed 40 ° C) then the film is dried with a vane pump (1 hour) and placed under an inert atmosphere until use.
• the film in the case of priming with sodium dithionite, the film is prepared in freshly distilled dichloromethane and degassed by bubbling with Argon • Dispersion 2 mL of monomer solution in distilled water (0.1 M), previously deoxygenated by bubbling Argon are added The mixture is stirred for 1 minute then placed in an ultrasonic bath for 60 minutes at 70 ° C.
• the preparation is deposited on a Sephadex G50 column (2 cm in diameter for a height of 10 cm of gel) previously equilibrated with a 0.1 M NaCl buffer deoxygenated for 30 minutes by bubbling Argon. 2 ml of bluish fraction corresponding to an elution of 25 to 27 ml are recovered in a flask. 2 mL of sodium meta bisulfite solution in NaCl buffer (2.5.10 "4 M) are then added to initiate the polymerization which takes place under an inert atmosphere, at 37 ° C.
• Table 2 • Preparation of the film 18 mg of lipid Al and 2 mg of telogen compound D are dissolved in 2 ml of a solution of Cumene hydroperoxide in dichloromethane freshly distilled and degassed by bubbling of Argon (2.5 ⁇ 10 ⁇ 4 M), in a heart flask. The solution is evaporated to dryness on a rotary evaporator (the bath temperature does not exceed 40 ° C) then the film is dried with a vane pump (1 hour) and placed under an inert atmosphere until use. In the case of priming with sodium dithionite, the film is prepared in freshly distilled dichloromethane and degassed by bubbling with Argon.
• NaCl (2.5.1G “4 M) are then added to initiate the telomerization which takes place under an inert atmosphere, at 37 ° C. and for 16 to 20 hours.
• Triethylamine TEA
• 27.07 g 97.23 mmol, 1.05 equ.
• triphenylmethyl chloride dissolved in 10 ml of THF are added dropwise to the mixture at a temperature below 30 ° C.
• the reaction medium is left under stirring, cold, maintaining the pH at 8-9 by adding TEA.
• the excess triphenylmethyl chloride is eliminated by adding a saturated NaHCO 3 solution before evaporating the THF under reduced pressure.
• the crude product is taken up in CH 2 C1 2 before being washed with a normal solution of HCl then NaHCO 3 and being dried over Na 2 SO 4 .
• the product is finally purified by chromatography on silica gel column eluted by gradient (cyclohexane / AcOEt 7: 3 to "1: 1) 28.8 g of pure product are obtained in the form of white powder Rfpr o d u i.. t : 0.4 (TLC - AcOEt / cyclohexane 7: 3). Yield: 89%.
• TE 17-20 compound 1.09 g (6.25 mmol, 5 equ.) Of Tris (hydroxymethyl) acrylamidomethane is dissolved in 15 ml of freshly distilled MeOH. The mixture is stirred and bubbled with argon and then heated. As soon as it boils, a solution containing 0.04 g (0.25 mmol, 0.2 equ.) Of AIBN and 0.8 g (1.25 mmol) of Compound 12 in a minimum of freshly distilled THF (approximately 1 mL ) and previously degassed with a stream of argon is injected.

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