WO2002039979A1 - Copolymere a structure sequencee compose d'un segment saccharidique lie a au moins un segment hydrophobe bioerodable, et particules correspondantes - Google Patents
Copolymere a structure sequencee compose d'un segment saccharidique lie a au moins un segment hydrophobe bioerodable, et particules correspondantes Download PDFInfo
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- WO2002039979A1 WO2002039979A1 PCT/FR2001/003619 FR0103619W WO0239979A1 WO 2002039979 A1 WO2002039979 A1 WO 2002039979A1 FR 0103619 W FR0103619 W FR 0103619W WO 0239979 A1 WO0239979 A1 WO 0239979A1
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F251/00—Macromolecular compounds obtained by polymerising monomers on to polysaccharides or derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
Definitions
- the invention relates to a new family of bioerodible copolymers based on a polymer of alkyl cyanoacrylate type, or related and of poly- or oligosaccharides, particularly useful in the pharmaceutical, veterinary, food and cosmetic fields, in particular as vectors and / or excipients. It also proposes a process for preparing these copolymers.
- Bioerodible polymers generally formulated in the form of liposomes, microemulsions, nanospheres, nanocapsules, microspheres, microcapsules, microparticles and nanoparticles constitute effective delivery systems for active ingredients.
- micro- and nanoparticles based on poly (alkyl cyanoacrylate) are particularly advantageous given their rapid bioerosion compared to other biodegradable polymers, such as poly (lactic acid) / poly ( ⁇ -caprolactone) for example.
- these particles based on poly (alkyl cyanoacrylate) are not entirely satisfactory.
- these particles have the disadvantage of being rapidly captured by the macrophages of the System of
- Mononuclear phagocytes (PMS). Their lifespan, in vivo, is therefore very short.
- the present invention aims precisely to overcome the aforementioned drawbacks and to propose a new material for particles whose polymer structure derives from the association with a polymer. related to poly (alkyl cyanoacrylate), of a segment of poly- or oligosaccharide nature, such as dextran for example.
- Nanoparticles based on amphiphilic block copolymers comprising dextran and poly (alkyl cyanoacrylate) segments have already been described (S.J. Douglas et al.; Journal of Controlled Release (1986) 15-23).
- these copolymers which are derived from the anionic polymerization of cyanoacrylate monomers in the presence of dextran have grafted structures.
- the anionic polymerization of alkyl cyanocrylates in the presence of dextran leads to the grafting of several poly (cyanoacrylate) chains on the dextran polymer chain without it being moreover possible to control the number, size and localization of these poly (cyanoacrylate) chains.
- the particles thus formed have an interfacial layer of dextran on the surface, the overall structure of which leads to recognition by the complement system and by the macrophages of the SPM.
- the present invention aims for its part to provide copolymers derived from cyanoacrylate or equivalent monomers and from oligo- or polysaccharides but having a completely different structure.
- the copolymers are in a block form, as opposed to the graft forms described above. This sequenced form is in fact inaccessible by the anionic polymerization path discussed above.
- the present invention also aims to propose a synthetic route for preparing this type of block copolymer.
- the inventors have thus demonstrated that it is possible to effectively polymerize, by the radical route, molecules with a high charge density, of cyanoacrylate type, in the presence of poly- or oligosaccharides, this despite the fact that the energy of activation required for this radical polymerization is much higher than that required by anionic polymerization.
- the cyanoacrylate or equivalent type monomers are naturally inclined to generate their anionic form when they are placed in the presence of a nucleophilic agent such as, for example, OH anions " and therefore to polymerize anionically.
- the first object of the present invention is a copolymer with a block structure composed of a hydrophilic segment of saccharide nature, at least one of the ends of which is linked to a
- - X represents a CN or CONHR radical
- - Y represents a COOR 'or CONHR "radical, with R, R' and R" representing, independently of one another, an atom
- Hydrogen a linear or branched C 20 to C 20 alkyl group, a linear or branched C 1 to C 20 alkoxy group, an amino acid radical, a mono- or poly-hydroxylated acid radical or an aryl radical or C 5 to C 12 heteroaryl, with said segment of saccharide nature being linked either by one of its
- X preferably represents a CN radical. More preferably, Y represents a radical COOR 'with R' as defined above.
- the repeating unit of isobutyl cyanoacrylate may be more particularly cited by way of illustration of a unit capable of composing a segment of general formula (I).
- sequenced structure is intended to denote according to the invention a structure which derives from the establishment of a covalent bond between at least one of the ends of the segment of saccharide nature and one of the ends of the polymer chain of general formula (I).
- copolymer structures comprising either a single segment of general formula (I) linked to one end of the segment of saccharide nature, or two segments of general formula (I), identical or different, linked respectively on either side of the saccharide segment.
- the claimed copolymers do not have side branch (s) of saccharide nature on the hydrophobic segment, nor side branch (s) of hydrophobic nature on the saccharide segment .
- the covalent bond, established between the two types of segment, is generally of C-C or C-O-C nature.
- it is derived from the radical polymerization of at least one molecule of a compound of formula (II):
- This radical polymerization is preferably carried out under the conditions set out below for the process claimed. In particular, it is carried out under conditions of pH and atmosphere unfavorable to the presence and or to the generation of anions in the reaction medium and in the presence of a sufficient amount of a suitable radical initiator Redox.
- the saccharide-type segment is derived from an oligo- or polysaccharide of natural or synthetic origin, modified or not.
- Modified polysaccharide is understood to mean any polysaccharide which has undergone a change on its skeleton, such as for example the introduction of reactive functions, the grafting of chemical entities (molecules, aliphatic links, PEG chains, etc.).
- chemical entities molecules, aliphatic links, PEG chains, etc.
- PEG polysaccharides modified by grafting biotin, fluorescent compounds, etc.
- Other polysaccharides grafted with hydrophilic chains eg PEG
- the oligo- or polysaccharide used according to the invention may already in itself have biological properties and / or activities. For example, it can confer anticoagulant, vaccinating, targeting or even mask properties to avoid capture by PMS macrophages. This is how it can be:
- oligo- or polysaccharides with biological activity such as, for example, heparin, heparan sulfate, dermatan sulfate, dextran sulfate and pentosan sulfate, dextran substituted with carboxylic groups and sulfates or sulfonates , polysaccharides sulfates extracted from algae (fucans and fucoidans), poly (sialic acids), sulfated hyaluronic acid which have anticoagulant, anti-inflammatory activities to varying degrees, and / or
- oligo- or polysaccharides involved in cell recognition and cell signaling processes such as, for example, poly (sialic acids), heparan sulfate, blood group antigens, polysaccharides and lipopolysaccharides of various strains bacterial, oligosaccharide chains of membrane and / or circulating glycoproteins, and oligosaccharide chains of glycolipids.
- the copolymers derived from this type of polysaccharides are of course particularly advantageous in terms of therapeutic use, insofar as they naturally have their own biological activity and therefore can be used as such for this reason.
- the polysaccharides which are particularly suitable for the invention are or are derived from D-glucose (cellulose, starch, dextran, cyclodextrin), D-galactose, D-mannose, D-fructose (galactosane, manane, fructosan), fucose (fucan).
- D-glucose cellulose, starch, dextran, cyclodextrin
- D-galactose D-mannose
- D-fructose galactosane, manane, fructosan
- fucose fucose
- hyaluronic acid (composed of N-acetyl glucosamine and glucuronic acid units), poly (sialic acid) also called colominic acid or poly (N-acetyinaminic acid), chitosan, chitin, heparin or the orosomucoid contains nitrogen, while the agar, a polysaccharide extracted from seaweed, contains sulfur in the form of acid sulfate (> CH-O-SO 3 H). Chondroitin-sulfuric acid and heparin simultaneously contain sulfur and nitrogen.
- the polysaccharide has a molecular weight greater than or equal to 6000 g / mole.
- n varies between 10 and 620 and preferably between 33 and 220.
- the molar mass varies between 5 10 3 and 5 10 6 g / mole, preferably between 5 10 4 and 2 10 6 g / mole.
- the molar mass varies between 6 10 3 and 6 10 5 g / mole, preferably between 6 10 3 and 15 10 4 g / mole.
- polydextroses such as dextran, chitosan, pullulan, starch, amylose, cyclodextrins, hyaluronic acid, heparin, amy
- the copolymers have a controlled oligo- or polysaccharide content.
- the claimed copolymer can be in a soluble form or in the form of a precipitate, micelles or particles. According to an advantageous aspect of the invention, it takes the form of particles. They can be micro- or nanoparticles. In the particular case of particles and micelles, it is likely that the copolymer has a structure organized as follows: chains of the same nature, that is to say saccharide or hydrophobic, are grouped together, either to constitute the structure heart of the micelle or particle, or the brush crown around this heart structure. Their distribution between the core structure and the crown will of course depend on the nature, aqueous or organic, of the solvent in which the particles or micelles are dispersed.
- brush crown is intended to denote a structure in which the segments constituting the crown are linked by one of their ends to the segments constituting the heart. Their free ends constitute the periphery of the crown.
- the hydrophobic segments are grouped so as to constitute the heart and the segments of a saccharide nature are arranged in a brush crown all around this heart.
- the heart is hydrophilic in nature and therefore consists of the saccharidic segments
- the brush crown is hydrophobic in nature and therefore constituted by the segments of formula general (I).
- this brush crown structure cannot exist in an aqueous medium, insofar as several hydrophobic segments are linked by covalent bond to a single chain of saccharide nature.
- a second aspect of the invention relates to particles consisting of a copolymer in accordance with the invention.
- - isohexyl cyanoacrylate isobutyl cyanoacrylate, N-butyl cyanoacrylate, N-propyl cyanoacrylate, ethyl cyanoacrylate or 2-methoxyethyl cyanoacrylate in the presence of dextran
- - isobutyl cyanoacrylate in the presence of heparin, chitosan, pectin, hyaluronic acid, dextran sulfate or ⁇ -cyclodextrin isohexyl cyanoacrylate, isobutyl cyanoacrylate, N-butyl cyanoacrylate, N-propyl cyanoacrylate, ethyl cyanoacrylate or 2-methoxyethyl cyanoacrylate in the presence of dextran, - isobutyl cyanoacrylate in the presence of heparin, chitosan, pectin, hyaluronic acid,
- the claimed particles can have a size between 1 nm and 1 mm and preferably between 60 nm and 100 ⁇ m.
- the particles having a size between 1 and 1000 nm are then called nanoparticles.
- Particles varying in size from 1 to several thousand microns refer to microparticles. These particles may in certain cases be in an aggregated or micellar form.
- the particles resulting from the polymerization of isobutyl cyanoacrylate in the presence of ⁇ -cyclodextrin have an aggregated appearance.
- These particles can have a biological activity either because of the nature of the polysaccharide which constitutes them, or because they incorporate in addition a biological or pharmaceutical active material.
- biological active materials mention may more particularly be made of peptides, proteins, carbohydrates, nucleic acids, lipids, polysaccharides or their mixtures. It can also be synthetic organic or inorganic molecules which, administered in vivo to an animal or to a patient, are capable of inducing a biological effect and / or of manifesting a therapeutic activity. It can thus be antigens, enzymes, hormones, receptors, peptides, vitamins, minerals and / or steroids.
- the particles can thus include magnetic particles, radio-opaque materials (such as, for example, air or barium) or fluorescent compounds.
- fluorescent compounds such as rhodamine or Nile red can be included in particles with a hydrophobic core.
- gamma emitters for example Indium or Technetium
- Hydrophilic fluorescent compounds can also be loaded into the particles, but with a lower yield compared to hydrophobic compounds, because of their reduced affinity with the matrix.
- these particles can be combined with peptides / proteins capable of helping them to diffuse through biological membranes such as the TAT peptide, or compounds such as the 2OT protein (Zonula Occludens Toxin) and zonulin or equivalents, or any other absorption promoter.
- biological membranes such as the TAT peptide, or compounds such as the 2OT protein (Zonula Occludens Toxin) and zonulin or equivalents, or any other absorption promoter.
- this type of association can be achieved by chemical functionalization of the polysaccharide surface of the particles. It is thus possible to envisage covalently fixing, at the level of functions present on the skeleton of a saccharide nature, specific ligands, such as targeting agents, markers or more generally any compound capable of conferring on said particles an ability to react with a external species, as by example a function on a support or a biological entity present in a considered environment.
- specific ligands such as targeting agents, markers or more generally any compound capable of conferring on said particles an ability to react with a external species, as by example a function on a support or a biological entity present in a considered environment.
- the active material can be incorporated into these particles during their formation process or, on the contrary, be loaded at the level of the particles once they are obtained. It is thus possible to charge them by adsorption or by covalent grafting.
- the particles according to the invention can be administered in different ways, for example by the oral, parenteral, ocular, pulmonary, nasal, vaginal, cutaneous, oral, etc. routes.
- the non-invasive oral route is the route of choice.
- the present invention also relates to the use of the particles as a vector of pharmaceutical, cosmetic, agrifood or veterinary active principles.
- a third aspect of the present invention relates to a process for the preparation of the claimed copolymer. More specifically, the present invention relates to a process which is useful for the preparation of block copolymers composed of a hydrophilic segment of saccharide nature, at least one of the ends of which is linked to a hydrophobic segment, characterized in that it comprises polymerization by radical route of at least one molecule of a compound of general formula (11):
- - X represents a CN or CONHR radical
- - Y represents a COOR 'or CONHR "radical with R, R 'and R "representing independently of one another, a hydrogen atom, an alkyl group in C 1 -C 20 linear or branched alkoxy, C, to C 20 linear or branched , an amino acid radical, a mono- or poly-hydroxylated acid radical or a C 5 to C 12 aryl or heteroaryl radical, said radical polymerization being carried out in the presence of at least one molecule of a poly- or oligosaccharide , under pH and atmosphere conditions unfavorable to the presence and / or generation of anions in the reaction medium and in the presence of a sufficient amount of a suitable radical redox initiator.
- X represents a CN radical and / or more preferably Y represents a COOR radical.
- the pH of the reaction medium is preferably adjusted to a value less than 2 and more preferably less than 1.5. Surprisingly, it appeared that adjusting the pH to such a value was not detrimental to the rate of polymerization. Against all expectations and as is apparent from the examples present below, the radical polymerization initiated under these pH conditions is carried out on the contrary at a speed greater than that of an anionic polymerization. This is illustrated in particular in FIGS. 1 and 2.
- reaction is also carried out under inert atmosphere conditions.
- the solvent is advantageously chosen so that, while maintaining conditions favorable to radical polymerization and more particularly to the formation of the hydrophobic segment of formula (I), the solubility of the oligo or polysaccharide is complete in the medium defined by this solvent.
- the solvent is also chosen to be weakly or non-solubilizing with respect to the copolymer.
- the poly- or oligosaccharide molecule is chosen from dextran, heparin, poly (N-acetylneuraminic acid), amylose, chitosan, pectin and hyaluronic acid, and their derivatives. It is also of course chosen to remain inert with respect to the polymerization.
- such a solvent is preferably chosen from aqueous, hydroalcoholic or hydroacetonic solvents.
- the solvent chosen is acidified with an organic or inorganic acid and preferably a nitric acid to obtain an adequate pH during the course of the radical polymerization.
- oligo- or polysaccharide and monomer of general formula (II) can vary widely.
- the claimed process precisely has the advantage of allowing control of the structure of the copolymer which it is desired to prepare.
- the quantities of reagents introduced also depend on their respective molecular weights and their degrees of solubility in the reaction medium.
- Redox initiator these are generally mixtures of oxidizing and reducing agents, organic or inorganic, generating radicals during the electronic transfer step. This generation of radicals has the advantage of requiring a low activation energy unlike conventional radical initiators, which makes it possible to initiate radical polymerizations at relatively low temperatures (0-50 ° C).
- the Redox initiator used preferably comprises at least one metal salt chosen from the salts of Ce 4+ , V 5+ , Cr 6+ , Mn 3+ . According to a preferred variant of the invention, it is Ce 4+ . It is generally introduced in the form of cerium and ammonium nitrate. The concentration of radical initiator is also capable of influencing the course of the radical polymerization.
- composition of the copolymer and the length of the respective blocks of the polysaccharide and of the polymer of general formula (I) can be adjusted as a function of the initiator concentration. Its adjustment is within the competence of a person skilled in the art.
- reaction temperature it is adjusted to a value compatible with the initiation of the polymerization.
- this temperature is between 0 and 50 ° C.
- the oligo- or polysaccharide is preferably dissolved in the chosen solvent, then the radical initiator Redox is added.
- the monomer of formula (H) is then introduced into the mixture.
- the copolymer can be obtained in a soluble form or in the form of micelles, powders or particles. Preferably, it is obtained directly in the form of particles.
- the particles can be loaded with active ingredients either after their preparation or during their preparation.
- the copolymer is obtained in the form of a powder, it is of course possible to formulate this powder in the form of particles, using suitable transformation techniques.
- suitable transformation techniques one can more particularly mention the techniques of emulsification-evaporation of solvent, emulsification-diffusion of solvent or nanoprecipitation.
- the particles meet the specificities set out above.
- the polymerization of the compound of general formula (II) is carried out in the presence of the active material to be loaded.
- the pH of the reaction medium can be neutralized if necessary. Preferably, this is adjusted to a value that remains less than or equal to 7.5.
- the copolymer is recovered by conventional techniques.
- the metal salt is complexed before the isolation of the copolymer resulting from the reaction of the radical initiator. This complexation, which is within the competence of a person skilled in the art, makes it possible to eliminate these metal salts.
- Figure 1 Kinetics of radical polymerizations according to the invention of isobutyl cyanoacrylate in the presence of dextran, chitosan or pectin.
- Figure 2 Reference kinetics of anionic polymerizations of isobutyl cyanoacrylate in the presence of dextran or chitosan.
- FIG. 3 Electronic paramagnetic resonance spectrum (EPR) of copolymers in accordance with the invention of dextran-poly (isobutyl cyanoacrylate) labeled with 4-aminoTEMPO.
- EPR Electronic paramagnetic resonance spectrum
- the size of the polymer particles (average hydrodynamic diameter) is determined using a nanosizer (Coulter N4 PLUS®) by quasi-elastic diffusion of laser radiation.
- the surface charge of the particles is determined using a
- the dextran-poly (isobutyl cyanoacrylate) and heparin-poly (isobutyl cyanoacrylate) suspensions are diluted 1/200 e and 1/30 e respectively in potassium chloride at 1 mmol / l.
- the purification of the copolymer particles is carried out as follows:
- Dialysis tubes (Spectra / Por ® CE MWCO: 100,000) are regenerated for 30 minutes with osmosis water, and the colloidal suspensions passed through a vortex and then introduced into the tubes.
- the lyophilization of the copolymer particles is carried out as follows:
- Lyophilizations are carried out without the addition of cryoprotective.
- the suspensions of colloidal particles are aliquoted in pillars and then frozen (-18 ° C). Lyophilization (Bioblock Scientific Christ alpha 1-4) is carried out for 48 hours. The lyophilisates (white powders) are stored in the refrigerator.
- a specific mass lyophilisate is dispersed in a known volume of water MilliQ ® so that the mass ratio of freeze-dried / water volume is 1%.
- the suspension is homogenized using a vortex at maximum speed and then by ultrasound for a few minutes using a sonicator (Branson 5200 ® ).
- dextran sulfate of molecular mass (6-8000, 10000, 40000, 50000 or 500000 g / mol); - 0.1375 g of ⁇ -cyclodextrin; or
- Example 3 The protocol of Example 1 is reproduced by substituting for isobutyl cyanoacrylate, one of the following monomers:
- Table 1 summarizes the particle sizes of the various particulate suspensions produced.
- the standard deviations represent the repeatability of the measurements.
- the polymer content of the suspensions is determined by evaluating the weight of the dry residue obtained after lyophilization of a known quantity of suspension purified by dialysis. To do this, an aliquot of purified suspension prepared according to Example 1 or 2 is weighed with precision in a pill box and then frozen at (-18 ° C) before lyophilization for 48 hours in a Christ Alpha 1-4 lyophilizer (Bioblock Scientific). The mass of lyophilisate is weighed and then brought back to the mass of initial suspension.
- the suspension of dextran-poly copolymer (isobutyl cyanoacrylate) obtained according to Example 1 contains 3.1 ⁇ 0.4% of copolymer (mass / mass).
- the suspension of heparin-poly copolymer (isobutyl cyanoacrylate) obtained according to Example 2 contains 2.4 ⁇ 0.7% of copolymer (mass / mass).
- the composition of the copolymers is evaluated by elementary analysis of the powders obtained by lyophilization of the purified suspensions as indicated above.
- the dextran-poly (isobutyl cyanoacrylate) copolymer obtained according to Example 1 contains 20%
- PC2000 Plug-in type spectrometer (Ocean Optics Europe) inserted into a PC type computer, an HL-2000-LL light source (Ocean Optics Europe), Fiber optics (200 and 100 ⁇ m) (Top sensor Systems FC-UV, Ocean Optics Europe) and OOI Base V 1.5 Software (Ocean Optics Europe).
- the polymerization is carried out according to the protocol described in examples 1 to 3 in the 2 cm diameter glass tube placed in a water bath at 40 ° C. and on which is mounted the Teflon® ring supporting the optical fibers connected to the spectrometer and the light source. Argon bubbling is placed so as not to disturb the acquisition of the measurements. The background noise of the spectrometer is recorded before the introduction of the acid solution of cerium IV ions (8.10 "2 mol / l of cerium ammonium nitrate in HNO 3 at 0.2 mol / l).
- the reference is recorded after l addition of the acid solution of cerium IV ions (8.10 "2 mol / l of cerium ammonium nitrate in HNO 3 at 0.2 mol / l).
- the recording of the polymerization kinetics is started as soon as the 0.5 ml of monomer is added. It is carried out by the almost instantaneous acquisition of an absorbance spectrum over a wide wavelength range (400 - 800 nm) every 30 seconds for 50 min.
- the absorbances measured at the wavelength of 650 nm are used to draw absorbance curves as a function of time, thus reflecting the kinetics of polymerization.
- EXAMPLE 7 This example illustrates the synthesis of isobutyl dextran-polycyanoacrylate particles on a larger scale.
- 0.6875 g of dextran 70,000 g / mol are dissolved in 40 ml of 0.2 mol / l nitric acid with magnetic stirring, at 40 ° C. and under gentle bubbling of argon for 10 min.
- This example illustrates the synthesis of isobutyl dextran-polycyanoacrylate particles according to simplified experimental conditions.
- the anticoagulant activity of the heparin of the heparin-poly (isobutyl cyanoacrylate) copolymer is evaluated by measuring the activated partial thromboplastin time (TCA) or anti-Ila activity and by measuring the anti-Xa activity produced by the particles consisting of said copolymer synthesized according to Example 2.
- a calibration range of the method is carried out with the same heparin as that used for the synthesis of the copolymer.
- a stock solution at 1700 IU / ml is prepared in the TOK buffer and then diluted in this same buffer to give solutions at 0.17; 0.85; 1, 7; 4.25 and 8.5 IU / ml.
- 100 ⁇ l of each of the dilutions are themselves diluted in 900 ⁇ l of normal plasma.
- the suspensions copolymer particles are diluted in the TOK at 1/100 and 1/200 ⁇ .
- 100 ⁇ l of each of the dilutions of the suspension are themselves diluted in 900 ⁇ l of normal plasma.
- a coagulation control consists of 100 ⁇ l of TOK and 900 ⁇ l of normal plasma. Measurement of coagulation times:
- a bead is placed in each of the tanks of the ST4 coagulometer (Diagnostica Stago) then 100 ⁇ l of one of the samples prepared in the previous step and 100 ⁇ l of the APTT solution are introduced into the various tanks. After 300 seconds of incubation at 37 ° C, 100 ⁇ l of the calcium chloride solution are added. The coagulometer measures the coagulation times of the different samples in seconds. The results obtained for the heparin standard solutions make it possible to establish a calibration curve giving the activity of the heparin solution expressed in U.l./ml as a function of the coagulation time expressed in seconds. The heparin activity associated with the copolymer particles is evaluated on the calibration curve from the coagulation times measured for the suspensions.
- the suspension containing particles of heparin-poly copolymers (isobutyl cyanoacrylate) prepared according to Example 2 have an anti-lla activity of 329 + 28 U.l./ml.
- the copolymer particle suspension was diluted to 1/50 th, 1/100 th and 1/200 8 in TOK buffer then to 1/10 in the normal plasma thawed as described above.
- the coagulation times are automatically evaluated on an ST1 coagulomer
- a suspension not purified by dialysis of particles of copolymers is prepared according to Example 2 with dextran 15-20000 g / mol.
- the suspension is filtered on a filter 1, 2 .mu.m (Millipore ® SLA PO 2550) and then purified by dialysis 2 2 hours against 11 of osmosed water followed by dialysis for 2 hours against 11 of phosphate buffer (Sigma ref. P 3813) (dialysis membrane: Spectra / Por ® CE MWCO: 100,000 regenerated 30 min in osmosis water).
- the suspension was placed in dialysis tubing (Spectra / Por ® CE MWCO: 100000) previously regenerated with 30 min of reverse osmosis water and then dialyzed 3 times against 1 L of phosphate buffer for 2 hours.
- the suspensions of grafted particles are recovered and can be stored at (+ 4 ° C).
- the grafting of the marker can be demonstrated by electron paramagnetic resonance spectroscopy (EPR).
- EPR electron paramagnetic resonance spectroscopy
- the suspension obtained is placed in a measurement cell of a Varian E-4 EPR spectrometer.
- the spectrum obtained presented in FIG. 3 indicates that the 4-amino TEMPO has indeed been grafted onto the dextran chains of the copolymer forming the particles and that it is animated by slow movements for 81% and by rapid movements for 19% according to Kivelson's simulation (Kivelson DJ, Journal Chem. Phys; 1960; 33; 1107).
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Abstract
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
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AU2002220793A AU2002220793A1 (en) | 2000-11-17 | 2001-11-16 | Block-structure copolymer consisting of a saccharide segment bound to at least abiodegradable hydrophobic segment, and corresponding particles |
US10/416,840 US20040028635A1 (en) | 2000-11-17 | 2001-11-16 | Block-structure copolymer consisting of a saccharide segment bound to at least a biodegradable hydrophobic segment, and corresponding particles |
EP01996362A EP1355627A1 (fr) | 2000-11-17 | 2001-11-16 | Copolymere a structure sequencee compose d'un segment saccharidique lie a au moins un segment hydrophobe bioerodable, et particules correspondantes |
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FR00/14900 | 2000-11-17 | ||
FR0014900A FR2816949B1 (fr) | 2000-11-17 | 2000-11-17 | Copolymere a structure sequencee compose d'un segment saccharidique lie a au moins un segment hydrophobe bioerodable, et particules correspondantes |
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EP (1) | EP1355627A1 (fr) |
AU (1) | AU2002220793A1 (fr) |
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Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
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FR2839517A1 (fr) * | 2002-05-07 | 2003-11-14 | Centre Nat Rech Scient | Nouveaux agents tensioactifs biodegradables de type copolymeres amphiphiles constitues de segments hydrophobes et d'oligo et/ou polysaccharides |
FR2844512A1 (fr) * | 2002-09-17 | 2004-03-19 | Centre Nat Rech Scient | Nouveaux composes a base de copolymeres et leurs applications |
US8466232B2 (en) | 2004-05-05 | 2013-06-18 | Firmenich Sa | Biodegradable grafted copolymers |
US9486409B2 (en) | 2006-12-01 | 2016-11-08 | Anterios, Inc. | Peptide nanoparticles and uses therefor |
US9724299B2 (en) | 2006-12-01 | 2017-08-08 | Anterios, Inc. | Amphiphilic entity nanoparticles |
US10016451B2 (en) | 2007-05-31 | 2018-07-10 | Anterios, Inc. | Nucleic acid nanoparticles and uses therefor |
US10532019B2 (en) | 2005-12-01 | 2020-01-14 | University Of Massachusetts Lowell | Botulinum nanoemulsions |
US11311496B2 (en) | 2016-11-21 | 2022-04-26 | Eirion Therapeutics, Inc. | Transdermal delivery of large agents |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7763663B2 (en) * | 2001-12-19 | 2010-07-27 | University Of Massachusetts | Polysaccharide-containing block copolymer particles and uses thereof |
JP5618284B2 (ja) * | 2007-12-27 | 2014-11-05 | 昇一 城武 | グラム陽性細菌用抗菌剤 |
KR102080429B1 (ko) * | 2008-06-26 | 2020-02-21 | 안테리오스, 인코퍼레이티드 | 경피 운반 |
US9062151B1 (en) | 2012-09-21 | 2015-06-23 | The United States of America, as represented by The Secretary of Agiculture | Protein-cyanoacrylate nanoparticles that improve wetting property of materials |
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FR2839517A1 (fr) * | 2002-05-07 | 2003-11-14 | Centre Nat Rech Scient | Nouveaux agents tensioactifs biodegradables de type copolymeres amphiphiles constitues de segments hydrophobes et d'oligo et/ou polysaccharides |
WO2003095597A1 (fr) * | 2002-05-07 | 2003-11-20 | Centre National De La Recherche Scientifique (C.N.R.S.) | Nouveaux agents tensioactifs biodegradables de type copolymeres amphiphiles constitues de segments hydrophobes et d'oligo et/ou polysaccharides |
FR2844512A1 (fr) * | 2002-09-17 | 2004-03-19 | Centre Nat Rech Scient | Nouveaux composes a base de copolymeres et leurs applications |
WO2004026278A1 (fr) * | 2002-09-17 | 2004-04-01 | Centre National De La Recherche Scientifique (Cnrs) | Nouveaux composes a base de copolymere et d'hemoproteine et leurs applications |
US8466232B2 (en) | 2004-05-05 | 2013-06-18 | Firmenich Sa | Biodegradable grafted copolymers |
US10532019B2 (en) | 2005-12-01 | 2020-01-14 | University Of Massachusetts Lowell | Botulinum nanoemulsions |
US10576034B2 (en) | 2005-12-01 | 2020-03-03 | University Of Massachusetts Lowell | Botulinum nanoemulsions |
US9724299B2 (en) | 2006-12-01 | 2017-08-08 | Anterios, Inc. | Amphiphilic entity nanoparticles |
US10285941B2 (en) | 2006-12-01 | 2019-05-14 | Anterios, Inc. | Amphiphilic entity nanoparticles |
US9486409B2 (en) | 2006-12-01 | 2016-11-08 | Anterios, Inc. | Peptide nanoparticles and uses therefor |
US10758485B2 (en) | 2006-12-01 | 2020-09-01 | Anterios, Inc. | Amphiphilic entity nanoparticles |
US10905637B2 (en) | 2006-12-01 | 2021-02-02 | Anterios, Inc. | Peptide nanoparticles and uses therefor |
US10016451B2 (en) | 2007-05-31 | 2018-07-10 | Anterios, Inc. | Nucleic acid nanoparticles and uses therefor |
US11311496B2 (en) | 2016-11-21 | 2022-04-26 | Eirion Therapeutics, Inc. | Transdermal delivery of large agents |
Also Published As
Publication number | Publication date |
---|---|
US20040028635A1 (en) | 2004-02-12 |
FR2816949A1 (fr) | 2002-05-24 |
FR2816949B1 (fr) | 2003-11-28 |
AU2002220793A1 (en) | 2002-05-27 |
EP1355627A1 (fr) | 2003-10-29 |
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