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
  • A61K9/10—Dispersions; Emulsions
  • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
  • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/513—Organic macromolecular compounds; Dendrimers
  • A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates

Definitions

• the subject of the invention is polymerized micelles, processes for their preparation and their applications.
• a large number of molecules with therapeutic activity and in particular anti-cancer molecules have a low solubility in water, derived from the hydrophobic intrinsic properties of the molecule. Indeed, these molecules must have a certain degree of hydrophobicity in order to be internalized in the cells.
• the purpose of vectorization of the therapeutic molecules is to circumvent the problems associated in particular with the solubility, stability, pharmacokinetics, biodistribution of these molecules and their specific targeting.
• One of these vectorization techniques consists in encapsulating an active ingredient in a microsphere formed by a matrix of polymers such as poly (alkylcyanoacrylates), poly (anhydrides), poly (lactic acid). These microspheres have sizes of 20 .mu.m to 100 .mu.m excluding their intravenous use and allow relatively low inclusion rates between 0.2% and 3.5% of hydrophobic compounds.
• Polymer micelles designate colloidal dispersions composed of amphiphilic polymers with distinct hydrophilic and hydrophobic domains. These polymer micelles are supramolecular structures in the heart / shell. Polymer micelles are composed of polyethers used in combination with poly (ethylene glycol) to form amphiphilic polymers in the form of diblock copolymers (pluronic: PPO-co-PEG) or triblock copolymers (poloxamers: PEG-co-PPO-co-PEG). These micelles of polymers have a size of between 50 nm and 100 nm.
• the inclusion rates of compounds in the polymer micelles are between 0.1% and 40% depending on the inclusion method used: inclusion by evaporation, inclusion by dialysis or inclusion by nanoprecipitation.
• the micelles of polymers are therefore vectors allowing the incorporation of significant amounts of hydrophobic compounds but requiring implementation (synthesis and inclusion) technical and industrial difficult.
• Liposome and polymer vesicle delivery systems are intensively studied and several drug formulations by these systems are currently in the clinical phase, some of which have even been approved for clinical use.
• Liposomes and polymer vesicles may include hydrophilic active ingredients in the aqueous heart of the vesicle or hydrophobic molecules in the polymer bilayer.
• Liposomes and polymer vesicles have structures (MLV multilamellar vesicles, SUV small unilamellar vesicles, LUV large unilamellar vesicles, GUV giant vesicles) and very different sizes between 100nm and 1000nm.
• Liposomes and polymer vesicles are vehicles of important active principles in the vectorization of hydrophilic molecules.
• Nanobagues ie a structuration of amphiphiles in rings along the entire length of the nanotubes.
• Nanobagues are rings of polymerized surfactants formed on the surface of nanotubes then separated from their carbon support for use as solubilizing agents of hydrophobic active ingredients (WO 2004/092231). Nanobagues, however, are nanovectors that are difficult to industrialize.
• the delivery vectors described supra more or less respond to the challenge of vectorization which is to transport enough active ingredient effectively and low toxicity to treat the pathology. These vectors respond to the problem of the solubility of the hydrophobic active ingredients without solving the problems of vector size, implementation or industrialization.
• the present invention therefore aims to propose a new strategy to obtain nanovectors having a capacity for inclusion of hydrophobic active ingredients greater than that of conventional vectors of the literature and having a simple formulation facilitating future industrialization.
• the present invention relates to polymerized micelles characterized in that they comprise polymerized amphiphilic molecules obtained from amphiphilic molecules comprising one or two lipid chains each comprising one or two polymerizable units, of diacetylene, vinyl, acrylate or styrene type, linked to a polar head.
• the term “micelles” means self-assembled spherical objects with a hydrophilic surface and a lipophilic core and whose size is less than 100 nm.
• amphiphiles or "surfactants” means organic molecules having the particularity of being both hydrophilic and hydrophobic. Amphiphiles, characterized by their antagonistic properties, have particular properties in solution and spontaneously organize in aqueous media under different microstructures.
• the invention relates preferably to polymerized micelles comprising polymerized amphiphilic molecules obtained from amphiphilic molecules of general formula A - X - B - L - Z
• CH 2 CH-C 6 H 4 -, n and m, identical or different, being integers from 1 to
• X is CO-NH or NH-CO or a bond, X is a bond if B is a bond and L is a bond; where B is OR bond, n and m, identical or different, being integers from 1 to 16; where L is - (CH 2 ) ( -CH [NH-CO-A '] - or a bond, where r is an integer of 1 to 16 and A' is A, where Z is
• R 2 representing COOH or SO 3 H or OSO 3 H or OPO 3 H 2 or OPO 2 H 2
• R 1 representing H or a COOH or SO 3 H or OSO 3 H radical or OPO 3 H 2 or OPO 2 H 2 or a group ⁇ CO-NH- (CH 2 ) t -CH 3
• t is an integer of 1 to 16
• Z may also be neutral hydrophilic polar heads, sugar or polysaccharide, as well as their addition salts with a pharmaceutically acceptable acid or base.
• pharmaceutically acceptable acids mention may be made, without limitation, of hydrochloric, hydrobromic, sulfuric, phosphiral, acetic, trifluoroacetic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, tartaric, maleic, citric, ascorbic, oxalic and methanesulphonic acids. , benzenesulfonic, camphoric.
• pharmaceutically acceptable bases mention may be made, without limitation, of sodium hydroxide, potassium hydroxide, triethylamine and tertbutylamine.
• s being an integer of 1 to 16 with R 1 representing H or a COOH radical or a -CO-NH- (CH 2 ) t -CH 3 group , t being an integer of 1 to 16, or Z may also be polar heads hydrophilic neutral, sugar or polysaccharide, and their addition salts with a pharmaceutically acceptable acid or base.
• the invention also extends to polymerized micelles consisting of amphiphilic molecules whose neutral hydrophilic polar heads are crown ethers.
• the invention relates to polymerized micelles whose polar head Z is functionalized.
• polymerized micelles polymerized micelles modified with molecular recognition ligands to evolve towards intelligent and selective vectors in order to specifically identify antigens or receptors overexpressed on the surface of target cells such as cancerous or infectious cells.
• the polymerized micelles must be functionalized by specific ligands whose attachment to the surface of the vector is by a chemical coupling.
• the ligands attached to the surface of the polymerized micelles are: fluorophores or nuclear imaging agents ( 99 Tc,
• 111 In, 125 I, 18 F, 64 Cu or optical (cyanine, fluorescein, luciferase, quantum dots) or magnetic (iron oxide particles), folic acid, mannose, galactose, antibodies, ligands of the RGD type.
• the polymerized micelles according to the invention are functionalized at the level of the polar head Z with a folic acid.
• the polymerized micelles are functionalized according to a method as described in Example 5.
• the surface functionalization of the polymerized micelles thus renders said polymerized micelles stealthy and allows the targeting of specific cells.
• the invention also extends to polymerized micelles including one or more hydrophobic compounds within the polymerized amphiphiles according to the invention.
• hydrophobic compounds means small molecules having a low water solubility of less than 1 g per liter in all or part of the pH zone or proteins or nucleic acids having problems of solubility or stability in water. aqueous media.
• the polymerized micelles according to the invention consist of polymerized amphiphilic molecules obtained from the amphiphilic molecule II-4 of formula
• the polymerized micelles consist of polymerized amphiphilic molecules obtained from the amphiphilic molecule II-23 of formula
• the polymerized micelles according to the invention consist of polymerized amphiphilic molecules obtained from the amphiphilic molecule of formula
• Polymerized micelles are also formed from polymerized amphiphilic molecules obtained from the amphiphilic molecule of formula
• the polymerized micelles according to the invention consist of polymerized amphiphilic molecules obtained from the amphiphilic molecule of formula
• the polymerized micelles are formed from the polymerized amphiphilic molecules obtained from the amphiphilic molecule of formula
• the polymerized micelles according to the invention consist of polymerized amphiphilic molecules obtained from the amphiphilic molecule of formula
• the subject of the present invention is also the process for preparing the compounds according to the invention, characterized in that:
• R ' Cl, Br 1 OCOCH 3 , or
• R " AXBL with polar head
• compositions comprising polymerized micelles according to the invention.
• these pharmaceutical compositions comprise one or more pharmaceutically acceptable excipients, i.e. one or more inert, non-toxic and suitable vehicles or excipients.
• binders for example, binders, diluents, disintegrants, stabilizers, preservatives, lubricants, odorants, flavoring agents or sweeteners.
• compositions according to the invention those which are suitable for oral, parenteral and especially intravenous, per-or transcutaneous, nasal, rectal, perlingual, ocular, respiratory and more specifically single or coated tablets, sublingual tablets, capsules, glossettes, capsules, lozenges, injectables, aerosols, eye or nasal drops, suppositories, creams, ointments or dermal gels.
• the preferred route of administration is the intravenous route and the corresponding pharmaceutical compositions may allow instantaneous or delayed release of the active ingredients.
• the present invention also relates to the use of the polymerized micefles according to the invention as vectors of hydrophobic molecules.
• the invention also extends to the use of polymerized micelles as vectors of hydrophobic active ingredients.
• vector any molecule or assembly of molecules that can transport an active ingredient, a protein or a nucleic acid to its site of action by targeting specific ligands or passively by solubilization or stabilization of the species in question.
• active principle means any molecule, protein or nucleic acid having a therapeutic effect.
• the inclusion methods according to the invention are:
• DCM evaporation which consists of the inclusion by adding the active ingredient solubilized in dichloromethane (DCM) to an aqueous solution of polymerized miceiles heated to about 50 ° C.
• the solution is filtered to remove the excess of active ingredient not included.
• the invention relates, preferably, to a method of inclusion by magnetic stirring for 12 hours at 50 ° C. of a solution of polymerized miceiles and of active principle.
• the inclusion of active ingredient in the polymerized miceiles is controlled by the stirring energy and the temperature. Incorporation rate is improved by more vigorous agitation, longer contact time, and higher temperature.
• micellar solutions obtained at the end of the inclusion step are sterilized by filtration on a 0.22 ⁇ m filter for intravenous injection without loss of concentration of active ingredient.
• miceiles demonstrated the effect of micelle polymerization on the ability to incorporate active ingredient.
• the miceiles must be polymerized so that this nanovector has the capacity to include a very large amount of active ingredients.
• a rate 48% incorporation of S39625 is obtained for this transporter, ie a rate 5 times greater than that obtained with the nanobagging formulation.
• the inclusion study on different active ingredients confirmed the ability of nanovectors to solubilize different types of hydrophobic molecules with high inclusion rates.
• the work done on a reference molecule, paclitaxel made it possible to compare the values of incorporation rates with data from the literature.
• the polymerized micelles of the H-4 amphiphile made it possible to include paclitaxel at an inclusion rate of 33%.
• inclusion rates of paclitaxel between 6.7% and 14.3% have been reached with different types of vesicles, incorporation rates between 0.2% and 27% with nanoparticles, and inclusion rates between 0.2 and 25% with polymer micelles.
• Polymerized micelles thus appear as a nanovector with a remarkable solubilization power which made it possible to solubilize active ingredients of different structures and molecular weights.
• the present invention finally relates to a process for obtaining polymerized micelles according to the invention.
• This method of obtaining comprises the following steps:
• the lipid compounds to be polymerized according to the invention are self-assembled into spherical micelles
• the self-assembled spherical micelles are polymerized.
• self-assembly of amphiphilic molecules is meant the spontaneous organization into spherical micelles of amphiphilic molecules in aqueous media at a concentration greater than the critical micelle concentration or CMC.
• the critical micellar concentration of the amphiphilic molecule II-4 as described above was determined experimentally at 0.082 mg / ml.
• the invention also extends to the process for obtaining polymerized micelles in which the polymerization step is of the light irradiation or photopolymerization type.
• the photopolymerization is a polymerization method particularly well suited to the polymerization of diacetylenic units.
• Photopolymerization is a "clean" method using 254 nm light irradiation and no external chemical agents.
• the photopolymerization of the diacetylenic units involves the formation of diradical intermediates: the first step is to form the diradical species by photon excitation; the second step is the propagation reaction of the radical to a new polymerizable unit in the vicinity, thereby increasing the polymer chain; the last step is a termination step by coupling two radicals.
• the invention also extends to the process for obtaining polymerized micelles in which the polymerization step is of the radical polymerization type.
• the vinyl and acrylate units are generally polymerized by radical polymerization. This polymerization route is known and commonly used.
• the initiation of the radical polymerization can be carried out using a radical initiator generated by thermal and homolytic dissociation, oxidation-reduction reaction or by irradiations.
• the invention also extends to the process for obtaining polymerized micelles in which the polymerization step comprises several successive types of polymerization, for example a photopolymerization and then a radical initiator polymerization.
• the polymerization step comprises several successive types of polymerization, for example a photopolymerization and then a radical initiator polymerization.
• the present invention is illustrated without being limited by the following figures and examples:
• Figure 1 Chemical structure of S39625
• Figure 2 Method of inclusion of S39625 in polymerized and unpolymerized micelles
• Figure 3 (1) chromatogram of S39625 in acetonitrile, (2) chromatogram of unpolymerized / polymerized micelles, (3) chromatogram of S39625 included in unpolymerized micelles, (4) chromatogram of S39625 included in micelles polymerized;
• Figure 4 (1) chromatogram of S39625 solubilized in acetonitrile, (2) chromatogram of S39625 included in the polymerized micelles;
• Figure 5 Structures of the hydrophobic active ingredients (1) S44563, (2) 42909, (3) paclitaxel and (4) pyrene;
• Figure 6 Chemical structure of 4-amino-3-hydroxy-1-sulfonatenaphthalene;
• Figure 7 Fluorescence spectra of 4-amino-3-hydroxy-1-sulfonatenaphthalene, nanobagues and polymerized micelles functionalized by the fluorophore and of the control sample (excitation at 345 nm);
• Figure 8 (A) absorbance spectra of amine III-9, polymerized micelles and polymerized micelles functionalized by amine III-9 (B) fluorescence spectra of amine III-9, polymerized micelles functionalized by amine III-9 and control sample (excitation at 280 nm).
• EXAMPLE 1 SYNTHESIS OF AMPHIPHILIC MOLECULES II-4 AND II-23
• the first step of this synthesis according to Scheme 1 is to prepare the hydrophilic part of the amphiphilic molecule (nitrilotriacetic acid derivative) from N-benzyloxycarbonyl-L-lysine (Z-L-lysine).
• the second step of the synthesis according to Scheme 2 consists in coupling the hydrophilic head 11-2 with the previously activated 10,12-pentacosadiynoic acid.
• Pentacosadiynoic acid is activated by N-hydroxysuccinimide in the presence of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide to give II-3 activated acid.
• Surfactant II-4 is obtained by the peptide coupling between the hydrophilic head NTA II-2 and the activated acid II-3.
• the pure product is obtained by precipitation in water by adding hydrochloric acid.
• the amphiphilic molecule II-23 is a surfactant comprising two types of polymerizable units: a diacetylene unit polymerizable by irradiation luminous at 254nm and an acrylate pattern crosslinkable by electron bombardment.
• the first step of the synthesis consists in preparing the hydrophilic part of the amphiphilic molecule (NTA head) from N-benzyloxycarbonyl-L-lysine (ZL-lysine) according to scheme 1 and then protecting the carboxylic acids in the form of ter-butyl esters.
• the hydrophilic protected tBu-NTA head 11-14 is obtained.
• Spherical micelles are amphiphilic arrangements obtained from critical micelle concentration (CMC). These spherical shaped organizations have a hydrophilic surface and a lipophilic or hydrophobic core.
• the analysis of the polymerized micelles of 11-4 amphiphiles is carried out by laser granulometry. This technique, based on the quasi-elastic light scattering and the photon correlation cross spectroscopy, makes it possible to determine the size distribution of a sample.
• the intensity analysis of a sample of micelles polymerized at 50 mg / ml by the Nanophox apparatus shows a main peak of population at 4.9 nm.
• the number analysis of the micelle sample polymerized at 1 mg / ml by the Malvern Zetasizer apparatus revealed the presence of a single population at 5.1 nm.
• the micelle polymerization induces a phenomenon of micelle contraction that can be explained by the fact that the polymerization freezes the assembly of the amphiphiles and limits the phenomena of swelling.
• S39625 was encapsulated in a solution of 10 mg / ml of unpolymerized miceiles and of micelles polymerized by the "stirring and heating at 50 ° C.” method as detailed in the description and represented in FIG. 2.
• the inclusion rate of S39625 is determined by the reverse phase HPLC method. This technique is based on the difference of interaction of the molecules between the mobile phase and the stationary phase. This difference affects the retention times of these molecules. For given HPLC conditions, the same molecule always has the same retention time.
• the reverse phase HPLC conditions for determining the inclusion rate of S39625 are an RP-18 C grafted silica column for the stationary phase and a water and acetonitrile gradient of 5% to 100% for the mobile phase. .
• the detection of S39625 at the column outlet is performed by a UV detector at a wavelength of 385 nm.
• a calibration curve of S39625 in acetonitrile was performed by reverse HPLC. Under these HPLC conditions, the active ingredient S39625 has a retention time of 25.8 minutes.
• the retention time of S39625 encapsulated in polymerized micelles was also determined by reverse phase HPLC for which the solvent gradient is replaced by an isocratic pure acetonitrile regime.
• Acetonitrile is one of the best solvents of S39625 and its use as an eluent makes it possible to ensure very fast release of the active ingredient and its elution as a free entity.
• the chromatogram of S39625 included in the unpolymerized micelles shows the three peaks at 3.2 minutes, 25.8 minutes and 31.9 minutes. In view of the peak area at 25.8 minutes, the incorporation of the active ingredient in the unpolymerized micelles is between 1.7 and 3.3%.
• the chromatogram of S39625 included in the polymerized micelles shows a broad peak at a very low retention time. This sharp decrease in retention time shows, on the one hand, that the hydrophobicity of the
• S39625 is strongly masked by the very hydrophilic surface of the polymerized micelle and secondly that the association S39625 / polymerized micelles is particularly robust.
• the chromatogram of S39625 solubilized in acetonitrile peaks at 5.2 minutes while S39625 included in the polymerized micelle shows two peaks at 4.0 and 4.9 minutes.
• the inclusion rate in the polymerized micelles of S39625 and its degradation products is estimated by integration of the two peaks. The incorporation rate of these molecules in the polymerized micelles is therefore 48%.
• the solubility and thus the encapsulation capacity of the polymerized micelles is improved by a factor of 5 with respect to the nanobagues and by a factor of 13 relative to the unpolymerized micelles.
• hydrophobic active ingredients S42909, S44563, paclitaxel a reference hydrophobic active molecule and pyrene a very hydrophobic molecule The chemical formulas of these hydrophobic active ingredients are shown in FIG. 5. The inclusion of these active ingredients was carried out with micelle solutions polymerized at 10 mg / m 2 by the heating method at 50 ° C. for 12 hours.
• miceiles are functionalized by a hydrophilic fluorophore, 4-amino-3-hydroxy-sulfonatenaphthalene described in FIG. 6.
• This fluorophore has an excitation wavelength of 340 nm. and an emission wavelength of 455 nm.
• DCC coupling agent dicyclohexylcarbodiimide
• the fluorescence spectra of the functionalized polymerized miceiles show a change in the peak of fluorescence of 4-amino-3-hydroxy-sulfonatenaphthalene with a bathochromic fluorescence shift of 492nm ( Figure 7).
• the control shows that the fluorescence shift of the polymerized micelles comes from the covalent grafting of the fluorophore on the surface of the nanovector.
• folic acid is widely used for the specific targeting of cancer cells in the brain, kidney, breast, ovaries and lungs.
• the folic acid has been modified in order to introduce an amine function for grafting.
• This grafting is carried out in a large excess of amino derivative of folic acid (50 equivalents) and coupling agent (100 equivalents).
• the sample was purified by filtration and then by size exclusion column.
• the absorption spectra of FIG. 8 show that the amine derivative of folic acid IH-9 is present on the surface of the polymerized micelles because of the presence of the peak at 285 nm.
• the fluorescence spectra of the functionalized polymerized micelles show an evolution of the fluorescence peaks of the amino derivative of folic acid at 360 nm and 450 nm.
• the potential phagocytosis of micelles of manometric size according to the invention by macrophages can be avoided by grafting on these micelles chains of PEG or polyethylene glycol.
• the presence of PEG on the surface of micelles creates an inert outer layer preventing the adhesion of opsonines, thus making said micelles stealthy vis-à-vis macrophages and thus increasing their circulation time in the blood compartment.
• Coupling of a methoxyPEG-amine 5000 to the surface of the polymerized micelle occurs at pH 12 with DCC as coupling agent. This grafting is carried out in a large excess of PEG derivative (25 equivalents) and coupling agent (100 equivalents). Derivatized micelles are purified by filtration followed by a size exclusion column.
• the distribution of polymerized micelles in the body is a paramount parameter to be tested in order to determine the mode of excretion and the mode of accumulation of said polymerized micelles.
• the amphiphilic II-4 is radiolabeled with carbon 14 in order to synthesize the radiolabeled polymerized micelle for an autoradiographic tracking study in the rat.
• the carbon-14 radiolabeled amphiphile is obtained with a specific activity of 7.6 ⁇ Ci / mg.
• the administration of the polymerized micelles in the rat is carried out at a dose of 4MBq / kg and 100mg / kg for an administration volume of 2.5ml / kg. To do this, a solution of 40 mg / ml is necessary with a specific activity of 7.6 ⁇ Ci / mg.
• the study of accumulation / elimination of polymerized micelles radiolabeled with carbon 14 was carried out on 3 male Wistar rats.
• a single dose of 100mg / kg of polymerised micelles [14 C] was administered by intravenous route (bolus) to each of the rats, and the animals were euthanized at 10 minutes, 24 hours and 48 hours.
• Urines from rats 24 hours and 48 hours were harvested to measure their activity by liquid scintillation. Quantification of the levels of radioactivity in the tissues was performed by radioluminography of specific sections of the rats, obtained by cryomicrotomy.

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