WO2005061552A1 - Derive amphiphile d’heparine forme par couplage de l’heparine avec un acide biliaire - Google Patents
Derive amphiphile d’heparine forme par couplage de l’heparine avec un acide biliaire Download PDFInfo
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- WO2005061552A1 WO2005061552A1 PCT/FR2004/003285 FR2004003285W WO2005061552A1 WO 2005061552 A1 WO2005061552 A1 WO 2005061552A1 FR 2004003285 W FR2004003285 W FR 2004003285W WO 2005061552 A1 WO2005061552 A1 WO 2005061552A1
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- heparin
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- bile acid
- amphiphilic
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
Definitions
- the present invention relates to an amphiphilic heparin derivative formed by coupling heparin with a bile acid, having the capacity to form nanoparticles spontaneously in an aqueous medium.
- the nanoparticles formed from this heparin derivative can be used as a vector for active principles for oral administration. They make it possible in particular to dissolve and to transport hydrophobic active ingredients through the intestinal mucus to close contact with the intestinal mucosa, to release said active ingredients gradually and to promote their absorption.
- the present invention further relates to the process for the synthesis of this amphiphilic heparin derivative.
- the present invention finally relates to the uses which can be made of such a vector, in particular in the therapeutic field for the administration of active principles by oral route, and more particularly as a vector of active principles weakly absorbed by the intestinal mucosa.
- the present invention also relates to the use of said vector according to the invention for the transport of active principles in association with a conventional galenic support used for the administration of active principles by oral route such as granules, microgranules, capsules or drinkable solutions in particular.
- a conventional galenic support used for the administration of active principles by oral route
- hydrophobic means any compound which is insoluble in water or which has very little affinity for water, and by amphiphilic any compound which exhibits both a certain affinity for the aqueous phases and a certain affinity for the organic phases.
- any particle of size less than or equal to a micrometer is designated under the name of nanoparticle, and under that of microparticle, any particle of size between 1 and 1000 ⁇ m.
- Promotion of absorption is understood to mean any means capable of being used in order to increase the quantity of active principle which crosses the intestinal mucosa.
- Mucus is a clear, stringy product of normal secretion of the mucous glands of which mucin is the main constituent and which contains water, salts and scaled cells. It plays a protective role vis-à-vis the mucous membranes that it covers, both from the mechanical point of view and from the chemical point of view.
- Bile acids are derivatives of cholesterol made by the liver and secreted in the bile where they are stored.
- bioavailability is understood to mean the fraction of molecule of active principle found in the circulation after oral administration. The bioavailability is measured by evaluating the plasma level of the active ingredient observed compared to the plasma level of this same active ingredient administered intravenously.
- a significant part of the therapeutic active ingredients administered orally is absorbed in the small intestine, and more particularly in the upper part of the small intestine: the duodenum.
- This absorption firstly involves the passage of the molecule of active principle through the plasma membrane of the cells of the intestinal epithelium, the enterocytes, then the crossing of the vascular endothelium of the blood vessels.
- Such absorption is dependent on many parameters which influence the absorption efficiency of the active ingredient considered and more particularly on the crossing of the intestinal mucosa.
- three main parameters must be considered to explain the low bioavailability of certain active ingredients administered orally.
- the solubility of the active ingredient in the gastrointestinal environment is sometimes reduced, or even zero for certain active ingredients of a particularly hydrophobic nature.
- the gastrointestinal environment can be considered as a mainly aqueous medium, since it is partly composed of chyme and partly of gastric juices. Therefore, the oral administration of these active ingredients is not possible if they have not previously been the subject of a very fine dispersion or dissolution in an organic phase consisting of l 'assembly of hydrophobic parts of a micelle of surfactants or the heart of a nanoparticle.
- certain active ingredients although water-soluble, are subject to an "absorption window", that is to say that their absorption is only possible at the level of a defined zone of the intestine, such as the duodenum for example.
- the association of these active principles with a transporter or vector, which has an affinity for the intestinal mucosa makes it possible to increase the residence time of these compounds in an environment close to their absorption site.
- the active ingredient must have, in the gastrointestinal, therefore aqueous, medium, an external structure which allows it to approach the intestinal membrane so that it can be absorbed by the enterocytes.
- the epithelial cells of the intestine and more particularly of the duodenum are covered with dense mucus constituted by an entanglement of glycoproteins.
- This molecular network constitutes a physical barrier of the hydrophilic gel type that the molecules of hydrophobic active principles must cross in order to be in contact with the plasma membrane of the enterocytes (Larhed et al.
- the molecule of active principle must have a physical configuration allowing it to approach this membrane.
- the active principle or the vector / active principle complex must have a sufficiently small size (less than a micrometer) allowing it to diffuse within the network of glycoproteins.
- some authors such as Park and Robinson (1984) have found that the phenomenon of bioadhesion to the gastrointestinal mucus was improved for molecules having surface charges, compared to uncharged molecules.
- the constitution of vectors for active principles weakly absorbed by the intestinal mucosa must therefore make it possible to reduce the influence of these parameters to promote the crossing of the active ingredient across the intestinal barrier and therefore the bioavailability of these compounds.
- such vectors must be able not only to transport the active ingredients to their specific site of intestinal absorption, but also to promote their solubility in the gastrointestinal environment and / or to increase their intestinal transit time.
- the present invention therefore proposes to provide a new type of vector intended to improve the absorption of active principles having a low bioavailability by the oral route, that is to say being little or not absorbed by the intestinal mucosa.
- liposome-type vesicles have been developed for the purpose of transporting such compounds.
- These nanoparticles consist of a membrane composed of a double layer of phospholipids, whose internal and external surfaces are hydrophilic, which membrane delimits within it an aqueous compartment.
- the liposomes thus make it possible to convey water-soluble active principles incorporated within this central cavity.
- the document WO 9308802 describes, for example, liposomes intended for the transport and the release of active principles of the tricyclic immunosuppressive type.
- Such liposomes allow stable transport of these active ingredients in various aqueous media and in particular physiological liquids, while maintaining the stability of the active ingredients in injectable solutions such as glucose solutions for example.
- this type of vector has two major drawbacks.
- the stability of the liposomes in the digestive environment is poor. Indeed, these structures can be easily destabilized by surfactants such as bile salts.
- the lipids constituting these vectors are rapidly degraded, in particular by digestive enzymes, such as lipases, which reduces the absorption efficiency of the active principle.
- Patent JP58049311 discloses a means of solving the problem of the instability of liposomes by grafting fatty acid esters of polysaccharides on their surface.
- Chitosan, Pullulan or Dextran derivatives comprise at least one monosaccharide residue substituted with a nonionic hydrophilic compound and at least one monosaccharide residue substituted with a hydrophobic group.
- This hydrophobic group is of the long chain alkyl, alkenyl, alkynyl or acyl type. Training vesicles is then induced in the presence of cholesterol. Due to the hydrophobization of the hydrophilic polymer backbone, the complex precipitates in water and requires the presence of cholesterol and / or a steric stabilizer in order to obtain the vector in vesicular form. In this model, vesicular formation is induced, which implies an additional formulation step in order to trigger the passage into particulate form.
- a hydrophobized polysaccharide is used as a stabilizer for emulsions of fatty acids.
- the fatty vesicles formed are used for the purpose of encompassing various lipophilic substances and in particular active principles.
- the polysaccharide preferably used in this case is Pullulan, and the hydrophobic groups used to hydrophobize this polymer are cholesterol or certain fatty acids.
- the complex called CHP for the abbreviation of Cholesterol-Pullulan is the complex preferably used.
- the number of hydrophobic groups grafted onto the polysaccharide is variable.
- the modified polymer is not the majority component at the origin of the lipid particles formed, it only constitutes a stabilizer.
- Other work on molecules related to CHP has led to the use of these multimolecular clusters as vectors of active principles.
- These complexes are formed spontaneously in an aqueous medium and are in the form of nanoparticles having several internal hydrophobic domains into which a compound of hydrophobic nature can be introduced.
- Patent JP 7097333 thus describes a supramolecular complex composed of a hydrophobized polysaccharide intended to serve as a transporter for certain cytokines. This complex corresponds to the association of sterol-type residues, preferably cholesterol, with a polysaccharide molecule, preferably Pullulan.
- the hydrophobic residue is grafted in this case by means of a spacer arm.
- This type of transporter is used to increase the plasma lifespan of these active ingredients administered intramuscularly or percutaneously.
- these vesicles allow the encapsulation of liposoluble active principles dissolved within their internal hydrophobic domains.
- These polymers hydrophobized therefore present an important interest for the administration of such compounds.
- such types of vectors do not allow the transport of active principles at their absorption site on the intestinal mucosa.
- such vectors do not allow the improvement of the intestinal absorption of the active ingredients which they carry.
- heparin is capable of forming aggregates in an aqueous medium if certain hydrophobic residues are grafted onto its saccharide chain.
- heparin is not used as a carrier of active ingredients, but as the active ingredient itself.
- the objective of this study is to pass heparin through the intestinal mucosa and to check if it retains its anticoagulant properties after being hydrophobized by different hydrophobic residues.
- the object of the present invention is to provide a new type of vector of active principles which ordinarily are poorly or poorly absorbed by the intestinal mucosa, said vector making it possible to specifically increase this absorption by transporting the active principles through the intestinal mucus, then by gradually releasing them in close contact with the intestinal wall, but without the vector crossing this wall.
- the vector according to the invention also proposes to dissolve active principles of hydrophobic nature, while being of simple design, including only constituent elements derived from digestible metabolites or prometabolites and which can be loaded with active principle easily.
- the present invention also proposes to provide a vector of non-toxic and biocompatible active principles, having the capacity to pass spontaneously into an aqueous medium in the form of nanoparticles with an average diameter less than a micrometer.
- Said nanoparticles being both stable over time, much more stable than liposomes and other micellar associations of amphiphiles, including the micelles of di-block amphiphilic copolymers, and capable of gradually releasing their contents on contact with the intestinal membrane.
- This ability to approach the intestinal membrane closely being due to their particular amphiphilic structure combining charged constituent elements and lipophilic elements, allows them to diffuse easily within the network of glycoproteins that constitute the intestinal mucus.
- the nanoparticles according to the invention have the advantage of significantly increasing the residence time of the active principle transported since they interact favorably with the glycoproteins of the intestinal mucus or even destabilize therein to release the active locally as a result of interactions with the macromolecules constituting mucus.
- This interaction resulting from a prolonged mucoadhesion phenomenon promotes the absorption of the active ingredients, in particular for those which have an absorption "window”.
- the present invention has the advantage of being simple to develop and inexpensive, being composed exclusively of readily available natural molecules and assembled together by simple chemical reactions.
- the vector of active principles in accordance with the invention can be easily associated or incorporated within a conventional galenical support used for the oral administration of medicaments.
- Such a vector can therefore be included in a simple manner in the composition of medicaments intended to be administered orally.
- Small particles (micro and nanoparticles) arriving in contact with the intestinal mucosa will present three types of destinies, closely related to their size and their chemical nature. These particles can first of all be captured by the lymphoid tissue associated with the intestine, according to a phenomenon endocytosis in the cells of Peyer's plaques. Furthermore, these particles can also be retained at the mucus level by a phenomenon of mucoadhesion. Finally, these particles can be directly eliminated in the faeces. Several authors have thus found that particles larger than a micrometer were easily eliminated during intestinal transit, due to their low rate of penetration into the intestinal mucus (Ponchel et al. 1997). The mucous network is indeed too dense for particles of such size to be able to diffuse there.
- the endocytosis phenomenon observed in the lymphoid tissue is a phenomenon which relates more particularly to particles having an uncharged surface and of hydrophobic nature (Desai et al. 1996; O'Hagan 1996; Florence 1997).
- the extent of this endocytosis phenomenon is, however, minimal since Peyer's patches represent only a tiny surface of the total surface of the intestinal mucosa.
- the vector of active principles in accordance with the present invention consists of the spontaneous assembly in aqueous medium of several heparin molecules onto which at least one hydrophobic residue derived from at least one bile acid has been grafted so as to produce a polymer amphiphilic in nature.
- This polymer makes it possible to promote to the maximum the phenomenon of mucoadhesion of the nanoparticles formed in accordance with the present invention while creating a vector capable of transporting in dissolved form a lipophilic active principle.
- the vector of active principles in accordance with the invention meets several criteria of size and structure which allow it to be particularly adapted to the environment of the intestine.
- the present invention relates to an amphiphilic heparin derivative formed from an at least partially N-desulfated heparin and at least one bile acid, comprising one or more bile acid molecules grafted on the heparin molecule by an amide bond formed between the terminal carboxylic acid function of bile acid and a primary amino function of heparin, originally present in heparin or resulting from N-desulfation, in which the number of bile acid molecules grafted for 100 units heparin disaccharides is between about 15 and about 80, preferably between about 20 and about 60.
- Heparin is a complex macromolecule made up of an assembly of saccharide units from the class of glycosaminoglycans.
- the polymer chain is mainly made up of an acid sugar (uronic acid) and an amino sugar (glucosamine) arranged in regular alternation.
- the corresponding disaccharide motif is multisulfated in well-defined positions.
- the acid functions are in the carboxylate and sulfate form.
- the nitrogen of the amino sugar is essentially in the N-sulfate form (in more than 80% of the units), but may also be in the N-acetyl form (approximately 15% of the units); the patterns comprising the free amine form are very little represented (1 to 2% at the chain end).
- the amino functions which are mainly in the N-sulfate form are not available for the chemical coupling reactions. It is therefore preferable to have heparin having numerous primary amine functions on which the coupling of hydrophobic groups can be carried out. In the present invention, it is an amidation reaction: the carboxylic acid function of the bile acid will have to react with the amine function of the polymer to form an amide bond.
- Heparin therefore constitutes a polyelectrolyte essentially negatively charged by sulphate (O-SO 3 " or NH-SO 3 " ) and carboxylate (COO " ) groups in their natural state.
- Bile acid is advantageously chosen from acid cholic acid, deoxycholic acid, lithocholic acid, cholanic acid and chenodeoxycholic acid, and mixtures thereof.
- cholic acid has the following chemical formula:
- Cholic acid which is part of the composition of bile salts, is a steroid derived from cholesterol which is predominantly hydrophobic in nature.
- the hydroxyl functions carried by this residue give the cholic acid molecule a certain hydrophilicity.
- the hydrophilic groups carried by cholic acid decrease the aggregation force of the hydrophobic groups together.
- the lipophilic core of these supramolecular complexes will therefore be looser than if it had been made up solely of cholesterol residues for example (which is a much less hydrophilic molecule). This phenomenon will contribute to increasing the diameter of the nanoparticles formed by the assembly of these complexes and will also determine the affinity of the active principles for the hydrophobic domains.
- amphiphilic heparin derivative which is the subject of the present invention can advantageously be prepared in the form of a calcium, magnesium or sodium salt.
- the subject of the present invention is also the nanoparticles which can be formed from the amphiphilic heparin derivative as defined above.
- the amphiphilic heparin derivatives according to the invention indeed have the capacity to assemble in an aqueous medium to form a stable colloidal suspension of nanoparticles with a diameter between 10 nanometers and 1 micrometer.
- the average diameter observed is however particularly homogeneous, of the order of 300 nanometers.
- the nanoparticles which are the subject of the present invention have a hydrophilic external surface and one or more hydrophobic internal domains.
- the hydrophobic residues of the polymer come together and form non-covalent crosslinking points, which are at the origin of the formation of amphiphilic nanoparticles, allowing the constitution of internal domains.
- hydrophobic in which lipophilic active ingredients can be dissolved and therefore transported.
- the heparins hydrophobized by bile acid exhibit a behavior in aqueous solution different from that of native heparin. The more the polymer is hydrophobized, the more it is hardly soluble in water.
- the aqueous solutions are opalescent and stable. These solutions are colored orange after the addition of Yellow OB, a lipophilic marker which colors the solutions in orange when it is dissolved in an organic phase. The orange coloration then makes it possible to prove the presence of hydrophobic domains in the molecule synthesized in accordance with the present invention, since this marker does not dissolve either in water or in a solution of non-hydrophobized heparin.
- the present invention also relates to said nanoparticles further containing one or more hydrophobic active principles dissolved in their hydrophobic internal domains.
- Said active ingredients preferably carry one or more polar groups. They are preferably chosen from anti-inflammatory drugs, antifungal agents, calcium channel blockers and anti-cancer agents.
- the present invention also relates to said nanoparticles as vectors of active principles which can be administered orally.
- the present invention also relates to said nanoparticles as vectors of active principles making it possible to increase the absorption of these by the intestinal mucosa, and / or allowing the gradual release of these at the level of the intestinal mucosa.
- the nanoparticles which are the subject of the present invention have the property of being able to reach and also remain in contact or in an environment close to the intestinal membrane.
- the amphiphilic heparin derivative which is the subject of the present invention has all the qualities necessary for good diffusion within the mucus and for good muco-adhesion.
- the charged groups carried by the vector according to the invention interacting favorably with the groups carried by the glycoproteins of the mucus, make it possible to increase their transit time in contact with the mucosa intestinal by diffusing mucus within the glycoprotein network.
- This potentiation of the muco-adhesion phenomenon by increasing the contact time of the active ingredient with the intestinal membrane, promotes its absorption.
- the choice of heparin as the polysaccharide backbone is therefore essential since this polymer has both many ionized functions in aqueous medium (polyelectrolyte with high charge density) and also primary amine functions which will be able to be easily released, making it possible to coupling of the hydrophobic residue.
- Nanoparticles are formed by spontaneous self-assembly in aqueous media and do not require the addition of surfactants or steric stabilizers.
- heparin has advantageously been chosen because it constitutes a natural polymer absolutely well tolerated by the body, and moreover commonly used, parenterally, in therapy in humans as an anti-coagulant agent.
- a bile acid as hydrophobizing agent is also one of the essential characteristics of the invention since this natural compound will allow the modified polymer to assemble in the form of stable nanoparticles in the intestinal medium but also to carry out intermolecular interactions which not only ensure the cohesion of the system but the solubilization of active principles of hydrophobic nature. These non-covalent interactions subsequently make it possible to release the content of active principles of the nanoparticles in the vicinity of the lipid membranes of the intestinal cells.
- the association of heparin with at least one bile acid allows the constitution of nanoparticles sufficiently stable in the intestinal environment to remain intact until close contact with the intestinal mucosa.
- the nanoparticles in accordance with the invention are sufficiently labile and biodegradable to then gradually release the active principle which they contain in the mucous environment near the lipid membrane of the intestinal cells, without crossing the intestinal mucosa.
- the nanoparticles which are the subject of the present invention have numerous advantageous properties in terms of size, stability and capacity for incorporating active principles.
- the present invention also relates to the colloidal suspension in an aqueous medium containing said nanoparticles. This suspension can for example be used to prepare an oral suspension or else be sprayed on neutral supports to prepare granules.
- the present invention also relates to the pharmaceutical composition comprising said nanoparticles associated with at least one pharmaceutically acceptable excipient.
- the excipient is advantageously chosen to allow administration of active principles by oral route.
- Said pharmaceutical composition can be in the form of granules, microgranules, tablets, capsules or oral solutions.
- the present invention also relates to the process for the preparation of the amphiphilic heparin derivative, which comprises at least partial N-desulfation of a heparin, then a coupling step which consists in reacting at least one primary amino function of the heparin, originally present or resulting from N-desulfation, with the terminal carboxylic acid function, optionally in activated form, of at least one bile acid.
- the preparation of the nanoparticles can be followed by a lyophilization step in order to be able to store them more easily.
- the active principle can be incorporated into the nanoparticles by direct dissolution with stirring, by dialysis, by oil / water emulsion, or by solvent evaporation.
- Preparation process As explained above, it is preferable to have heparin having many primary amine functions on which the coupling of the hydrophobic groups can be carried out.
- the primary amine functions will be released by selective hydrolysis of the N-Sulfate functions according to a method allowing precise control of the N-Desulfation rate.
- this step is followed by a step of forming a cetyltrimethylammonium salt of the desulfated polysaccharide molecule, so as to give it solubility in organic medium, before applying the method of coupling the cholic acid residues on the amino functions released. This salt is subsequently removed at the end of the coupling step.
- N-Desulfation Desulfation is preferably selective on the N-sulfate groups so as not to hydrolyze the O-sulfates which would result in a reduction in the number of ionized groups and therefore a loss of solubility.
- Two methods can be used: hydrolysis by autocatalysis of heparinic acid which corresponds to the traditionally used method and hydrolysis in a solvent medium or "solvolysis" of heparinic acid.
- the main disadvantage of hydrolysis by autocatalysis is the time required to obtain products with acceptable degrees of N-desulfation. Indeed, such a reaction takes between a week and a month.
- solvolysis makes it possible to obtain, within a few hours, heparin derivatives with a high proportion of primary amine functions.
- this method makes it possible, for fixed concentration and temperature parameters, to obtain reproducibly derivatives having the desired content of primary amine function, by stopping the hydrolysis reaction at a given time.
- the selective hydrolysis of the N-sulfate groups can be obtained by placing heparinic acid salified with pyridine in a mixture of DMSO and water (or DMSO / methanol) whose proportion in water does not exceed not 10%.
- N-desulfated samples without causing depolymerization or alteration of the structure (Nagasawa and Inoue 1974; Inoue and Nagasawa 1976).
- the speed of this reaction can be controlled by temperature for a concentration in given heparin.
- samples are obtained within a few hours with the desired degrees of N-desulfation.
- the disaccharide units of the heparin molecule are in the N-sulfate form for slightly more than 80% of them.
- the heparin molecule is desulfated at a rate of between 10 and 65%.
- N-desulfation can be carried out as follows. First of all, heparin is purified by dialysis (or ultrafiltration). Then, the heparin solution is percolated at 4 ° C on a cation exchange resin in H form. A heparinic acid solution is then obtained. The concentration of the solution obtained is then adjusted so that the proportion of residual water represents 5% of the final total volume when the DMSO is added.
- the heparinic acid solution can be lyophilized after the concentration step or concentrated to dryness before adding the desired amount of water.
- the solution is then transferred to a large-volume flask for the reaction.
- a sufficient amount of pyridine representing as many equivalents as acid functions. These functions are then in the form of a pyridinium salt.
- a volume of DMSO is then added until the following concentration is reached: DMSO / H 2 O (95/5 v / v).
- the heparin concentration in the solution is 2% (m / v).
- This solution is then preferably brought to 40 ° C., but it is also possible to place it at different temperatures depending on the rate of hydrolysis that one wishes to have). Samples are taken at different times.
- N-desulfated heparin is found in a solution comprising released sulfate ions, pyridinium salts, DMSO.
- the solution is dialyzed several times against water so as to remove these impurities, then it undergoes a concentration step and finally, a lyophilization step. Purified N-desulfated heparin is therefore obtained in dry form, the percentage of N-desulfation can then be determined.
- a solution of cetyl trimethylammonium bromide is added. The latter compound forms an insoluble salt with heparin which precipitates in an aqueous medium. It is then separated by filtration, and rinsed several times with hot water so as to remove the sulfate ions and the other molecules soluble in water. The product is then dried.
- the choice of temperature is based on what you want and the reaction control. Thus, by placing it at 20 ° C it is easier to control the production of weakly N-desulfated heparins while at 40 ° C the rate of hydrolysis is too rapid at the start, which is not suitable for recovering heparins. weakly N-desulfated but rather for heparins having 20 to 60% of N-desulfation. By placing itself at a temperature greater than or equal to 50 ° C., a completely N-desulfated sample is obtained in 24 hours.
- hydrophobized heparins comprising an N-desulfation rate of between 8 and 65% will be used for the production of nanoparticles in accordance with the present invention and well suited to the transport of active principles.
- Method for assaying the primary amine functions of non-hydrophobized heparin N-desulphated This method is based on the colorimetric method for assaying amines developed by Snyder and Sobocinski (1975). The dosage is based on the determination the optical density at the wavelength of 420 nm of the chromophore formed by the covalent bond of 2,4,6-trinitrobenzene sulfonic acid (TNBS) to the free amino functions.
- TNBS 2,4,6-trinitrobenzene sulfonic acid
- N-desulfation rates are determined by taking into account the kinetic parameters of the samples (extrapolation of the curves and determination of the parameters for a reaction at 100 % of completion) and the values given by the standard at 100% N-desulfation, as in Example 1.
- the above method applies to samples of non-hydrophobized N-desulfated heparins in the form sodium salt (not in the form of cetyltrimethylammonium salt).
- acyl chlorides R - CO - Cl
- acid anhydrides R - CO - O - CO - R
- Another way is to go through activated complexes.
- the chemistry of activating groups is thus widely used in peptide synthesis.
- a coupling agent is preferably used to activate the terminal carboxylic function of bile acid.
- the coupling agent used to activate the terminal carboxylic function of the bile acid is preferably chosen from benzotriazolyl-oxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP), benzotriazolyl-oxy-tris-pyrrolidino hexafluorophosphate -phosphonium (PyBOP) and bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP).
- BOP comprises a good nucleofuge group (leaving group), HOBT (1-hydroxybenzotriazole), which has the advantage of accelerating the coupling reaction and of suppressing parasitic reactions; the oxyphosphonium salt constitutes the "coupling agent” part and binds to the carboxylate to activate the carbonyl (Evin 1978).
- the chemical formula for BOP is as follows:
- the coupling agent makes it possible to obtain the hydrophobized polysaccharide in a single step and thus avoids having to synthesize and isolate beforehand an activated ester of cholic acid which are two long and costly steps.
- the coupling reaction between N-desulfated heparin and bile acid is preferably carried out in an organic medium.
- Heparin is a very polymer strongly ionized and is therefore very soluble in water but insoluble in organic solvents.
- Cetyl trimethylammonium bromide has a long hydrocarbon chain of 16 carbon atoms and has surfactant properties due to its cationic polar head (quaternary ammonium) which gives it its solubility in water.
- Phosphate buffer pH 7.4 is added thereto: the calcium binds to the phosphate ions to form an insoluble precipitate of calcium phosphate.
- the hydrophobized heparin is then in sodium form.
- the preferred coupling rate is that making it possible to obtain a heparin where the number of bile acid molecules grafted per 100 disaccharide units of heparin is between approximately 15 and approximately 80, preferably between approximately 20 and approximately 60. With the use of an agent making it possible to activate the carboxylic function of the bile acid, one can generally reach a rate of amidation between 80% and 95%.
- the coupling rate (or the number of bile acid molecules grafted per 100 disaccharide units of heparin) can be calculated by determining the level of residual NH 2 on a hydrophobized heparin according to the invention. For that, it is necessary beforehand, on a larger sample, to carry out the coupling of TNBS on all the NH 2 of heparin (one works with a higher concentration in TNBS). Then the picric acid and the borate buffer are removed by dialysis, then a concentration and lyophilization step is carried out. The residual NH 2 level is determined by measuring the optical density at 420 nm of a solution of hydrophobized heparin coupled to TNBS.
- the quantity of active principle dissolved in the hydrophobized heparin solutions can be determined by UN / Nisible spectroscopy or by HPLC. However, the active principle can also be incorporated into the nanoparticles by dialysis, oil / water emulsion, or solvent evaporation.
- FIGURES Figure 1 Evolution of the blanks (samples without heparin) during an assay of the amino functions by the T ⁇ BS.
- Figure 2 Kinetics of the amino function assay reaction for a 100% ⁇ -desulfated heparin by T ⁇ BS (experimental values and calculated values).
- Figure 4 Concentrations of active ingredient in water and in hydrophobic domains.
- Figure 5 partition coefficient between the hydrophobic domains and the aqueous phase.
- Figure 6 Incorporation of ⁇ ifedipine in various samples of modified heparins (Cp # 8 mg / ml) after 4 days. Influence of the degree of hydrophobization and the nature of the counterion.
- Figure 7 Absorption of édifedipine by the spilled intestinal sac for 90 minutes. Effect of vectorization with hydrophobized heparin (HEP30CHO) compared to a control solution of édifedipine (water / DMSO).
- Example 1 Determination of the percentage of N-desulfation of two samples of hydrophobized heparin.
- TNBS assay method N-desulfated heparin solutions are produced in TB borate buffer (Na 2 BO 7 -10H 2 O 0.1 M, pH 10) (the quantities to be used are calculated in order to have a priori about 1.6 to 2.4 mM NH 2 ).
- the TNBS solution is diluted in water so as to obtain a concentration of 0.08 M.
- the solutions are distributed in tubes; each heparin sample is duplicated with a sample to which a dilution factor of 0.75 is applied (Table 1).
- Blanks samples without heparin
- the assays always include the reference samples ND 10 o-HEP (totally N-desulfated heparin).
- the time is counted as soon as TNBS is added (additions are made at regular intervals).
- the reaction takes place at room temperature.
- Amounts of 100 ⁇ l are taken at various times which are immediately diluted by adding 800 ⁇ l of TB.
- the OD is read at 420 nm and the values are multiplied by 9 for the calculations.
- the OD values obtained for the heparin samples are subtracted from the OD values of the blanks taken at the corresponding times.
- the evolution of the whites is linear ( Figure 1).
- the kinetic parameters of the heparins are calculated so as to be able to determine the theoretical OD (DOmax) for 100% "of completion of the reaction of dosage.
- the TNBS concentration being much higher than the NH 2 concentration, this first can be assimilated to a constant (we will express it by [TNBS]) and the kinetics is then of pseudo-order type 1.
- the speed V of the reaction can be considered to be directly proportional to the concentration of NH 2 :
- This adjustment is very easy to perform by entering the data in a model spreadsheet (Microsoft ® Excel spreadsheet).
- Example 2 Demonstration of the increase in the aqueous solubility of certain active principles insoluble in water
- the incorporation of active ingredients was carried out essentially by the dissolution method.
- the compound to be incorporated is placed under solid form in a tube containing water or a colloidal solution of hydrophobized heparin, at the concentration Cp (mg / ml).
- the mixing is carried out with stirring at room temperature for different times.
- the compound to be incorporated remains in saturated concentration in the medium.
- the tubes are subjected to a centrifugation step and the supernatants are filtered through 0.45 ⁇ m filters and assayed. Control solutions (water or non-hydrophobized heparin) are treated in the same way.
- the amount of active ingredient incorporated in the various media is quantified by HPLC and / or by measuring the absorbance in ONE. Filtration and determination of the quantity dissolved in water then make it possible to access the quantity of active principle incorporated in the hydrophobic domains of the hydrophobized heparins according to the invention, and to the calculation of the
- the incorporation is expressed in milligrams of active ingredient per gram of polymer (mg / g P) or in micromoles of active ingredient per gram of polymer ( ⁇ mol / g P). This incorporation rate is calculated by making the ratio between the concentration of the active principle in solution and the concentration of the polymer. It is important to take into consideration the amount of active ingredient dissolved in controls such as water or a non-hydrophobized heparin solution.
- FIG. 3 represents the rate of incorporation of carbamazepine in the two control solutions (unmodified heparin and water) and in a 8 mg / ml solution of heparin hydrophobized by cholic acid, the rate of ⁇ -desulfation is 19% (HEP 19 CHO) according to the present invention.
- the concentration of the active ingredient in solution in water or in the presence of heparin is very much lower than the concentration in a solution comprising hydrophobized heparin according to the invention.
- the gain in solubility with respect to water exceeds 500%.
- the concentration of carbamazepine present in the hydrophobic domains (559.9 mg / ml) can be determined by subtracting from the concentration of carbamazepine in the HEP 19 CHO solution the concentration of the active ingredient present in the aqueous phase.
- the incorporation rate in the hydrophobic domains is then determined by dividing the concentration of active ingredient incorporated by the mass concentration of the polymer in solution.
- FIG. 4 represents the quantities of CBZ, NIF and ITR present in water and in the hydrophobic domains of a heparin hydrophobized by cholic acid at an N-desulfation rate of 19% (HEP 19 CHO) after 6 days of incorporation period.
- Example 5 Determination of the coupling efficiency between heparin and cholic acid (HEPCHO).
- HEPCHO heparin and cholic acid
- the number of residual amino functions was quantified at the end of the hydrophobization reaction with cholic acid.
- the Applicant has used a method for coupling TNBS to the amino functions of HEPCHOs, then isolating the compounds obtained and subsequently quantifying their absorption.
- HEPCHO heparin N-desulfated at a rate of 30% then hydrophobized with cholic acid
- HEP 63 CHO heparin N-desulfated at a rate of 63% then hydrophobized with cholic acid
- the procedure is very simple.
- the second method of evaluating the coupling efficiency of cholic acid involves the production of solutions from the four lyophilized samples, followed by a reading of the optical density at 420 nm.
- the glucosamine motif of our reference ND 100 -HEP is "100% NH" (it is on this basis that all the other% NH 2 of ND-HEP ⁇ are determined), that is to say approximately 15% of N functions -acetyl and 85% of NH functions.
- HEP 63 CHO and HEP 3 oCHO have the following theoretical structure (Table 8).
- Table 8 Structures of the glucosamine units of two heparins hydrophobized by cholic acid
- the percentage corresponding to -NH-cholyl represents the number of molecules of cholic acid grafted per 100 disaccharide units of heparin.
- Example 6 Demonstration of the increase in intestinal absorption in the presence of hydrophobized heparin.
- the Applicant has demonstrated on an animal model, the modification of the intestinal absorption of certain poorly water-soluble active principles in the presence of heparin hydrophobized by cholic acid, the heparin having been N-desulfated at 30%, and prepared in the form of magnesium salt (HEP30CHO).
- the model used is the everted rat intestinal sac. It is an ex vivo method on an isolated organ part (Barthe et al. 1998, 1999). To do this, the small intestine of an adult rat is removed and then turned over using a glass rod. Bags about 2 cm long are made by sealing the ends of the intestinal segments.
- Said bags then have the mucous side comprising the intestinal villi on the outside.
- These bags are incubated in a cell culture medium oxygenated at 37 ° C. and rich in vitamins and nutrients (TC 199) so as to increase the survival of the intestinal cells.
- TC 199 vitamins and nutrients
- the intestinal mucosa is physiologically functional since the cells consume glucose from the culture medium and produce abundant mucus during the experiment.
- the active ingredient whose absorption is to be measured is placed in a solution outside the bag.
- the bags are taken at different times and the quantity of active ingredient which is absorbed by the intestinal mucosa is quantified inside the bags by HPLC. Two experiments were carried out on this model in the presence of nifedipine vectorized by hydrophobized heparin according to the invention.
- nifedipine was made from nifedipine solubilized in DMSO (Dimethylsulfoxide) and added to the cell culture medium (ie 0.1% DMSO at most) so as to obtain a homogeneous solution of nifedipine.
- DMSO Dimethylsulfoxide
- the cell culture medium ie 0.1% DMSO at most
- an amount of polymer corresponding to 20-25 ⁇ g / ml of active principle is dissolved in water.
- the absorption of Nifedipine was quantified after 30, 60 and 90 minutes of incubation. The results of this experiment are represented in FIG. 7.
- FIG. 7 represents the absorption of Nifedipine by the intestinal mucosa according to its vectorization or not.
- the present invention makes it possible to provide a new type of vector making it possible to significantly increase the solubility and the intestinal absorption of lipophilic active ingredients usually weakly absorbed by the cells of the intestinal mucosa, such as drugs belonging to the class of anticancer or anti-inflammatory drugs for example.
- the nanoparticles according to the invention can be easily integrated into a galenical support traditionally used for the oral administration of drugs, such as granules, microgranules, tablets, capsules or oral solutions.
- Bioadhesive polymers as platforms for oral controlled drug delivery method to study bioadhesion.
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Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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US10/582,989 US20070141158A1 (en) | 2003-12-19 | 2004-12-17 | Amphiphilic heparin derivative formed by coupling a heparin with a bile acid |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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FR0315003A FR2864091B1 (fr) | 2003-12-19 | 2003-12-19 | Derive amphiphile d'heparine forme par couplage de l'heparine avec un acide biliaire |
FR0315003 | 2003-12-19 |
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WO2005061552A1 true WO2005061552A1 (fr) | 2005-07-07 |
WO2005061552A8 WO2005061552A8 (fr) | 2005-09-15 |
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PCT/FR2004/003285 WO2005061552A1 (fr) | 2003-12-19 | 2004-12-17 | Derive amphiphile d’heparine forme par couplage de l’heparine avec un acide biliaire |
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US (1) | US20070141158A1 (fr) |
FR (1) | FR2864091B1 (fr) |
WO (1) | WO2005061552A1 (fr) |
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WO2009005258A2 (fr) | 2007-06-29 | 2009-01-08 | Mediplex Corp. | Conjugués d'héparine et procédés associés |
KR101313894B1 (ko) | 2011-04-08 | 2013-10-01 | 경북대학교 산학협력단 | 신규한 탈황산화된 헤파린-담즙산 유도체를 포함하는 염증성 질환의 예방 및 치료용 조성물 |
US20190247312A1 (en) * | 2016-06-27 | 2019-08-15 | Tamarisk Technologies Group Llc | Pharmaceutical preparation for delivery of peptides and proteins |
CN109879977B (zh) * | 2019-01-30 | 2022-03-04 | 中山大学 | 一种含有胆固醇和植物凝集素基团的两亲性多糖衍生物及其制备方法和应用 |
CN112121177B (zh) * | 2020-03-25 | 2021-09-14 | 健进制药有限公司 | 负载羧酸抗肿瘤药物的peg化肝素纳米胶束及其制备方法 |
CN112898440B (zh) * | 2021-02-10 | 2022-05-24 | 杭州楠大环保科技有限公司 | 一种多孔材料及在微生物菌剂中的用途 |
Citations (2)
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WO1999061481A1 (fr) * | 1998-05-28 | 1999-12-02 | Mediplex Corporation, Korea | Derives de polysaccharide amphiphiles |
US20020013292A1 (en) * | 1998-05-28 | 2002-01-31 | Youngre Byun | Formulation of amphiphilic heparin derivatives for enhancing mucosal absorption |
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JPH0661455B2 (ja) * | 1988-11-25 | 1994-08-17 | 日本油脂株式会社 | 多糖誘導体によって安定化された脂肪乳剤 |
US5817333A (en) * | 1991-10-31 | 1998-10-06 | Fujisawa Pharmaceutical Co., Ltd. | Liposome preparation containing a tricyclic compound |
GB9706195D0 (en) * | 1997-03-25 | 1997-05-14 | Univ London Pharmacy | Particulate drug carriers |
-
2003
- 2003-12-19 FR FR0315003A patent/FR2864091B1/fr not_active Expired - Fee Related
-
2004
- 2004-12-17 US US10/582,989 patent/US20070141158A1/en not_active Abandoned
- 2004-12-17 WO PCT/FR2004/003285 patent/WO2005061552A1/fr active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999061481A1 (fr) * | 1998-05-28 | 1999-12-02 | Mediplex Corporation, Korea | Derives de polysaccharide amphiphiles |
US20020013292A1 (en) * | 1998-05-28 | 2002-01-31 | Youngre Byun | Formulation of amphiphilic heparin derivatives for enhancing mucosal absorption |
Non-Patent Citations (1)
Title |
---|
DIANCOURT F. ET AL.: "Chemical Modifications of Heparins. II. Hydrophobization of Partially N-Desulfated Heparin", JOURNAL OF BIOACTIVE AND COMPATIBLE POLYMERS, vol. 11, 1996, pages 203 - 218, XP008047382 * |
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US20070141158A1 (en) | 2007-06-21 |
WO2005061552A8 (fr) | 2005-09-15 |
FR2864091A1 (fr) | 2005-06-24 |
FR2864091B1 (fr) | 2006-04-07 |
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