Anionic polysaccharides functionalized by a hydrophobic acid derivative

This is a Continuation of International Application No. PCT/IB2009/007899 filed Dec. 23, 2009, and also claims the benefit of U.S. Provisional Application No. 61/282,836, filed Apr. 7, 2010 and French Patent Application No. 10/01439, filed Apr. 7, 2010.

The present invention relates to novel biocompatible polymers based on anionic polysaccharides functionalized by a hydrophobic acid derivative which can be of use in particular for the administration of active principle(s) (AP) to man or to animals for a therapeutic and/or prophylactic purpose.

Anionic polysaccharides functionalized by a hydrophobic acid derivative are, due to their structure and their biocompatibility, particularly advantageous in the pharmaceutical field and more particularly the field of the stabilization of protein active principles by the formation of complexes.

Hydrophobic acids are advantageous in the formulation of pharmaceutical active principles, in particular, due to their hydrophobic nature making it possible to adjust the hydrophobicity of the polymers onto which they may be grafted.

Their biocompatibility is excellent insofar as they play a role in numerous biochemical processes and are present in esterified form in the majority of tissues.

Techniques are known for grafting hydrophobic acids directly to the hydroxyl functions of dextran. These techniques result in the formation of a hydrophobic acid ester directly on the backbone of the polysaccharide. This has been carried out with activated derivatives of fatty acids, such as anhydrides (Novak L. J. and Tyree J. T. (1960), U.S. Pat. No. 2,954,372; Mooroka, T. et al., J. Appl. Polym. Sci., 1984, 29, 3981-3990), acyl chlorides (Ehrhardt S. et al. (1997), EP 792 888; Aburto, J. et al., J. Appl. Polym. Sci., 1999, 74, 1440-1451), N-acylureas (Nichifor, Marieta et al., Eur. Polym. J., 1999, 35, 2125-2129), and the like. Hydrophobic acids have also been grafted by transesterification starting from their ester derivatives (Laletin, A. J. et al., Vysokomol Soedin Ser A, 1968, 10, 652) and by activation techniques with tosyl chloride for example (Heinze, T. et al., Cellulose 2003, 10, 283-296). These methods have only been employed with neutral polysaccharides since they are not compatible with the presence of carboxylate functions on the polysaccharide.

The present invention relates to novel anionic polysaccharides functionalized by at least one hydrophobic acid derivative. These novel anionic polysaccharides comprising hydrophobic groups have good biocompatibility and their hydrophobicity can be easily adjusted without detrimentally affecting the biocompatibility or the stability.

It also relates to a method of synthesis which makes it possible to solve the abovementioned problems of synthesis. This method has made it possible to obtain anionic polysaccharides functionalized by hydrophobic acids.

The invention thus relates to anionic polysaccharides chosen from polysaccharides comprising carboxyl functions of formula I, said polysaccharides being monofunctionalized:

in which n represents the degree of functionalization of the saccharide units of the polysaccharide by -M-Ach and is between 0.01 and 0.7,

-M being:

either a sequence denoted —F 1 —R 1 -G comprising a connecting arm R 1 said connecting arm R 1 being bonded to the poly-saccharide via a bond F 1 resulting from the coupling between a reactive function of the precursor of the connecting arm R 1 ′and and a carboxyl function of the anionic polysaccharide and said hydrophobic radical (Ach) being bonded to the connecting arm R 1 via a function G resulting from the coupling between the acid function of the hydrophobic acid and a reactive function of the precursor of the connecting arm R 1 ′, or a sequence denoted —F 2 —R 2 -G comprising a connecting arm R 2 , said connecting arm R 2 being bonded to the polysaccharide via a bond F 2 resulting from the coupling between a reactive function of the precursor of the connecting arm R 2 ′ and a hydroxyl function of the anionic polysaccharide and said hydrophobic radical (Ach) being bonded to the connecting arm R 2 via a function G resulting from the coupling between the acid function of the hydrophobic acid and a reactive function of the precursor of the linking arm R 2 ′,

F 1 being either an amide function or an ester function or a thioester function,

F2 being a carbamate function,

the unfunctionalized carboxyl functions of the anionic polysaccharide being in the cation carboxylate form, the cation preferably being that of an alkali metal, such as Na + or K + ,

G being either an amide function or an ester function or a thioester function

Ach being a radical resulting from the coupling between the acid function of a hydrophobic acid and a reactive function of the precursor of the connecting arm R 1 ′ or R 2 ′, Ach being composed of a chain comprising between 4 and 50 carbons which is optionally branched and/or unsaturated, which optionally comprises one or more heteroatoms, such as O, N and/or 5, and which optionally comprises one or more saturated, unsaturated or aromatic rings or heterocycles,

R 1 being a divalent radical composed of a chain comprising between 1 and 15 carbons which is optionally branched and/or unsaturated, which optionally comprises one or more heteroatoms, such as O, N and/or S, and which optionally comprises one or more saturated, unsaturated or aromatic rings or heterocycles and resulting from the reaction of a precursor R 1 ′ having at least two identical or different reactive functions chosen from the group consisting of alcohol, amine and thiol functions,

R2 being a divalent radical composed of a chain comprising between 1 and 15 carbons which is optionally branched and/or unsaturated, which optionally comprises one or more heteroatoms, such as O, N and/or S, and which optionally comprises one or more saturated, unsaturated or aromatic rings or heterocycles and resulting from the reaction of a precursor R2′ having at least two reactive functions, one being an amine and the other being chosen from the group consisting of alcohol, amine and thiol functions.

In one embodiment, the precursors R 1 ′ and R 2 ′ comprise an additional reactive function which is not functionalized.

When the unfunctionalized reactive function is an acid function, it is in the cation carboxylate form, the cation preferably being that of an alkali metal.

When the unfunctionalized reactive function is an amine function, it is in the form of an anion salt, the anion preferably being a halide.

In one embodiment, n is between 0.02 and 0.6.

In one embodiment, n is between 0.05 and 0.5.

In one embodiment, n is between 0.1 and 0.4.

The term “anionic” is understood to mean a polysaccharide which comprises unfunctionalized and salifiable carboxyl functions.

The term “monofunctionalized” is understood to mean an anionic polysaccharide which is functionalized by a single hydrophobic group and which does not comprise other substituents.

The term “degree of functionalization” is understood to mean the number of M-Ach functions per saccharide unit or, in other words, the total number of M-Ach functions with respect to the total number of saccharide units. This notion can also be expressed as molar fraction of the hydroxyl functions of the polysaccharide functionalized by M-Ach.

The term “degree of conversion” is understood to mean the number of hydroxyl functions converted to carboxylates per saccharide unit or, in other words, the total number of hydroxyl functions converted to carboxylates with respect to the total number of saccharide units. This notion can also be expressed as molar fraction. For example, polysaccharides for which the degree of conversion of the hydroxyl functions to carboxylates per saccharide unit is equal to or greater than 0.15 are polysaccharides for which at least 15 carboxyl functions per 100 saccharide units have been grafted.

The term “degree of polymerization m” is understood to mean the mean number of repeat units (monomers) per polymer chain. It is calculated by dividing the number-average molar mass by the mean weight of the repeat unit.

The term “number-average molar mass (M n )” is understood to mean the arithmetic mean of the weights of each of the polymer chains. Thus, for a number n i of chains i with the molar mass M i , M n =(Σ i n i M i )/(Σ i n i ).

The weight-average molar mass (M w ) is obtained with M w =(Σ i n i M i 2 )/(Σ i n i M i ), n i being the number of chains of polymer i with the molar mass M i .

The polymers can also be characterized by the distribution of chain lengths, also known as polydispersity index (PI), which is equal to M w divided by M n .

In one embodiment, the anionic polysaccharides are polysaccharides which naturally carry carboxyl functions and are chosen from the group consisting of alginate, hyaluronan and galacturonan.

In one embodiment, the anionic polysaccharides are synthetic polysaccharides obtained from polysaccharides naturally comprising carboxyl functions or from neutral polysaccharides, for which the degree of conversion of the hydroxyl functions to carboxylates per saccharide unit is equal to or greater than 0.15, of general formula II,

the natural polysaccharides being chosen from the group of the polysaccharides predominantly composed of monomers bonded via glycoside bonds of (1,6) and/or (1,4) and/or (1,3) and/or (1,2) type,

L being a bond resulting from the coupling between a precursor of the connecting arm Q and an —OH function of the polysaccharide and being either an ester, carbamate or ether function,

i represents the degree of conversion of the hydroxyl functions to L-Q sequences per saccharide unit of the polysaccharide,

Q being chosen from the radicals of general formula III:

in which:

1≦a+b+c≦6, 0≦a≦3, 0≦b≦3 and 0≦c≦3,

R 3 and R 4 , which are identical or different, are chosen from the group consisting of —H, linear or branched C 1 to C 3 alkyl, —COOH and the radical

of general formula IV in which:

1≦d≦3 and

R′ 3 and R′ 4 , which are identical or different, are chosen from the group consisting of —H and a linear or branched C 1 to C 3 alkyl group.

In one embodiment, a+b+c≦5

In one embodiment, a+b+c≦4.

In one embodiment, i is between 0.15 and 3.

In one embodiment, i is between 0.3 and 25.

In one embodiment, i is between 0.5 and 1.7.

In one embodiment, i is between 0.8 and 1.5.

In one embodiment, the polysaccharide is predominantly composed of monomers bonded via glycoside bonds of (1,6) type.

In one embodiment, the polysaccharide predominantly composed of monomers bonded via glycoside bonds of (1,6) type is dextran.

In one embodiment, the polysaccharide is predominantly composed of monomers bonded via glycoside bonds of (1,4) type.

In one embodiment, the polysaccharide predominantly composed of monomers bonded via glycoside bonds of (1,4) type is chosen from the group consisting of pullulan, alginate, hyaluronan, xylan, galacturonan and a water-soluble cellulose.

in one embodiment, the polysaccharide is a pullulan.

In one embodiment, the polysaccharide is an alginate.

In one embodiment, the polysaccharide is a hyaluronan.

In one embodiment, the polysaccharide is a xylan.

In one embodiment, the polysaccharide is a galacturonan.

In one embodiment, the polysaccharide is a water-soluble cellulose.

In one embodiment, the polysaccharide is predominantly composed of monomers bonded via glycoside bonds of (1,3) type.

In one embodiment, the polysaccharide predominantly composed of monomers bonded via glycoside bonds of (1,3) type is a curdlan.

In one embodiment, the polysaccharide is predominantly composed of monomers bonded via glycoside bonds of (1,2) type.

In one embodiment, the polysaccharide predominantly composed of monomers bonded via glycoside bonds of (1,2) type is an inulin.

In one embodiment, the polysaccharide is predominantly composed of monomers bonded via glycoside bonds of (1,4) and (1,3) type.

In one embodiment, the polysaccharide predominantly composed of monomers bonded via glycoside bonds of (1,4) and (1,3) type is a glucan.

In one embodiment, the polysaccharide is predominantly composed of monomers bonded via glycoside bonds of (1,4) and (1,3) and (1,2) type.

In one embodiment, the polysaccharide predominantly composed of monomers bonded via glycoside bonds of (1,4) and (1,3) and (1,2) type is mannan.

In one embodiment, the polysaccharide according to the invention is characterized in that the L-Q sequence is chosen from the group consisting of the following sequences, L having the meaning given above:

In one embodiment, the polysaccharide according to the invention is characterized in that the L-Q sequence is chosen from the group consisting of the following sequences, L having the meaning given above:

In one embodiment, the polysaccharide according to the invention is characterized in that the L-Q sequence is chosen from the group consisting of the following sequences, L having the meaning given above:

In one embodiment, the polysaccharide according to the invention is characterized in that the precursor of Q is chosen from the group of the amino acids consisting of alanine, leucine, isoleucine, phenylalanine, valine and glycine.

In one embodiment, said anionic polysaccharide monofunctionalized by a hydrophobic acid derivative is chosen from the polysaccharides comprising carboxyl functions of general formula V:

in which n represents the degree of functionalization of the carboxyl functions of the polysaccharide by an F 1 —R 1 -G-Ach sequence and is between 0.01 and 0.7,

F 1 being either an amide function or an ester function or a thioester function, G being either an ester function or an amide function or a thioester function, R 1 being a divalent radical composed of a chain comprising between 1 and 15 carbon atoms which is optionally branched and/or unsaturated, which optionally comprises one or more heteroatoms, such as O, N and/or S, and which optionally comprises one or more saturated, unsaturated or aromatic rings or heterocycles and resulting from the reaction of a precursor R 1 ′ having at least two identical or different reactive functions chosen from the group consisting of alcohol, amine and thiol function, Ach being a radical which is the product of the coupling between the carboxyl function of the hydrophobic acid and at least one reactive functional group carried by the precursor R 1 ′ of the divalent radical R 1 ,

and, when the carboxyl function of the polysaccharide is not functionalized by F 1 —R 1 -G-Ach, then the carboxyl function or functions of the polysaccharide are cation carboxylates, the cation preferably being that of an alkali metal, such as Na + or K + .

In one embodiment, F 1 is an amide function, G is an ester function, R 1 ′ is an alcohol amine and Ach results from a hydrophobic acid.

In one embodiment, F 1 is an amide function, G is a thioester function, R 1 ′ is an aminothioalcohol and Ach results from a hydrophobic acid.

In one embodiment, F 1 is an amide function, G is an amide function, R 1 ′ is a diamine and Ach results from a hydrophobic acid.

In one embodiment, F 1 is an ester function, G is an amide function, R 1 ′ is an alcohol amine and Ach results from a hydrophobic acid.

In one embodiment, F 1 is an ester function, G is an ester function, R 1 ′ is a dialcohol and Ach results from a hydrophobic acid.

In one embodiment, F 1 is an ester function, G is a thioester function, R 1 ′ is a hydroxythioalcohol and Ach results from a hydrophobic acid.

In one embodiment, F 1 is a thioester function, G is an amide function, R 1 ′ is an aminothioalcohol and Ach results from a hydrophobic acid.

In one embodiment, F 1 is a thioester function, G is an ester function, R 1 ′ is a hydroxythioalcohol and Ach results from a hydrophobic acid.

In one embodiment, F 1 is a thioester function, G is a thioester function, R 1 ′ is a dithioalcohol and Ach results from a hydrophobic acid.

In one embodiment, said anionic polysaccharide monofunctionalized by a hydrophobic acid derivative is chosen from the polysaccharides comprising carboxyl functions of general formula VI:

in which n represents the degree of functionalization of the hydroxyl functions of the polysaccharide by an F 2 —R 2 -G-Ach sequence and is between 0.01 and 0.7,

F 2 being a carbamate function,

G being either an ester function or an amide function or a thioester function,

R 2 being a divalent radical composed of a chain comprising between 1 and 15 carbons which is optionally branched and/or unsaturated, which optionally comprises one or more heteroatoms, such as O, N and/or S, and which optionally comprises one or more saturated, unsaturated or aromatic rings or heterocycles and resulting from the reaction of a precursor R 2 ′ having at least two reactive functions, one being an amine function and the other being chosen from the group consisting of alcohol, amine and thiol functions,

Ach being a radical which is a product of the coupling between the carboxyl function of the hydrophobic acid and at least one reactive function carried by the precursor R 2 ′ of the divalent radical R 2 ,

and the carboxyl functions of the polysaccharide being in the form of cation carboxylates, the cation preferably being that of an alkali metal, such as Na + or K + .

In one embodiment, F 2 is a carbamate function, G is an ester function, R 2 ′ is an alcohol amine and Ach results from a hydrophobic acid.

In one embodiment, F 2 is a carbamate function, G is a thioester function, R 2 ′ is an aminothioalcohol and Ach results from a hydrophobic acid.

In one embodiment, F 2 is a carbamate function, G is an amide function, R 2 ′ is a diamine and Ach results from a hydrophobic acid.

In one embodiment, the precursor of the group R 1 , R 1 ′, is chosen from dialcohols.

in one embodiment, the dialcohols are chosen from the group consisting of glycerol, diglycerol and triglycerol.

In one embodiment, the dialcohol is triethanolamine.

In one embodiment, the dialcohols are chosen from the group consisting of diethylene glycol and triethylene glycol.

In one embodiment, the dialcohols are chosen from the group consisting of polyethylene glycols.

In one embodiment, the precursors of the groups R 1 and R 2 , R 1 ′ and R 2 ′, are chosen from diamines.

In one embodiment, the diamines are chosen from the group consisting of ethylenediamine and lysine and its derivatives.

In one embodiment, the diamines are chosen from the group consisting of diethylene glycol diamine and triethylene glycol diamine.

In one embodiment, the precursors of the groups R 1 and R 2 , R 1 ′ and R 2 ′, are chosen from alcohol amines.

In one embodiment, the alcohol amines are chosen from the group consisting of ethanolamine, amino-2-propanol, isopropanolamine, 3-amino-1,2-propanediol, diethanolamine, diisopropanolamine, tromethamine (Tris) and 2-(2-aminoethoxy)ethanol.

In one embodiment, the alcohol amines are chosen from the group consisting of reduced amino acids.

In one embodiment, the reduced amino acids are chosen from the group consisting of alaminol, valinol, leucinol, isoleucinol, prolinol and phenylalaminol.

In one embodiment, the alcohol amines are chosen from the group consisting of charged amino acids.

In one embodiment, the charged amino acids are chosen from the group consisting of serine and threonine.

In one embodiment, the hydrophobic acid is chosen from fatty acids.

In one embodiment, the fatty acids are chosen from the group consisting of the acids composed of a saturated or unsaturated and branched or unbranched alkyl chain comprising from 6 to 50 carbon atoms.

In one embodiment, the fatty acids are chosen from the group consisting of linear fatty acids.

In one embodiment, the linear fatty acids are chosen from the group consisting of caproic acid, oenanthic acid, caprylic acid, capric acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, tricosanoic acid, lignoceric acid, heptacosanoic acid, octacosanoic acid and melissic acid.

In one embodiment, the fatty acids are chosen from the group consisting of unsaturated fatty acids.

In one embodiment, the unsaturated fatty acids are chosen from the group consisting of myristoleic acid, palmitoleic acid, oleic acid, elaidic acid, linoleic acid, alpha-linoleic acid, arachidonic acid, eicosapentaenoic acid, erucic acid and docosahexaenoic acid.

In one embodiment, the fatty acids are chosen from the group consisting of bile acids and their derivatives.

In one embodiment, the bile acids and their derivatives are chosen from the group consisting of cholic acid, dehydrocholic acid, deoxycholic acid and chenodeoxycholic acid.

The polysaccharide can have a degree of polymerization m of between 5 and 10 000.

In one embodiment, it has a degree of polymerization m of between 10 and 1000.

In another embodiment, it has a degree of polymerization m of between 10 and 500.

The invention also relates to the synthesis of the polysaccharides according to the invention.

When the polysaccharides are chosen from the polysaccharides of general formula V, said synthesis comprises a step of producing an aminated intermediate Ach-G-R 1 —NH 2 or an ammonium salt Ach-G-R 1 —NH 3 + , the counterion of which is an anion chosen from halides, sulfates, sulfonates or carboxylates, and a step of grafting this aminated intermediate to a carboxyl function of a polysaccharide, R 1 , G and Ach corresponding to the definitions given above.

In one embodiment, a step of conversion of the hydroxyls of the polysaccharide to at least 15 carboxyl functions per 100 saccharide units is carried out by grafting compounds of formula Q-L′ to at least 15 hydroxyl functions per 100 saccharide units of the polysaccharide, Q-L′ being a precursor of the sequence Q-L, Q and L corresponding to the definitions given above.

In a preferred embodiment, the aminated intermediate of formula Ach-G-R 1 —NH 2 or Ach-G-R 1 —NH 3 + is obtained by reaction of a compound of formula G′-R 1 —NH 2 , G′ being an amine, thiol or alcohol function, with the acid function of the hydrophobic acid, R 1 , G and Ach corresponding to the definitions given above.

If necessary, in this step of producing the aminated intermediate, the protection or deprotection techniques well known to a person skilled in the art of peptide synthesis are used.

Preferably, the step of grafting the aminated intermediate to an acid function of the polysaccharide is carried out in an organic medium.

When the polysaccharides are chosen from the polysaccharides of general formula VI, said synthesis comprises a step of producing an aminated intermediate Ach-G-R 2 —NH 2 or an ammonium salt Ach-G-R 2 —NH 3 + , the counterion of which is an anion chosen from halides, sulfates, sulfonates or carboxylates, and a step of grafting this aminated intermediate to a hydroxyl function of a polysaccharide, R 2 , G and Ach corresponding to the definitions given above.

In a preferred embodiment, the aminated intermediate of formula Ach-G-R 2 —NH 2 or Ach-G-R 2 —NH 3 + is produced by reaction of a compound of formula G′—R 2 —NH 2 , G′ being an amine, thiol or alcohol function, with the acid function of the hydrophobic acid, R 2 , G and Ach corresponding to the definitions given above.

If necessary, in this step of producing the aminated intermediate, the protection or deprotection techniques well known to a person skilled in the art of peptide synthesis are used.

Preferably, the step of grafting the aminated intermediate to a hydroxyl function of the polysaccharide is carried out in an organic medium.

In one embodiment, the invention relates to a polysaccharide chosen from the group consisting of the following polysaccharides:

sodium dextranmethylcarboxylate modified by ethanolamine caprylate ester sodium dextranmethylcarboxylate modified by ethanolamine laurate ester (dextran 40 kDa) sodium dextranmethylcarboxylate modified by ethanolamine laurate ester (dextran 10 kDa) sodium dextranmethylcarboxylate modified by phenylalaninol caprylate ester sodium dextranmethylcarboxylate modified by N-(2-aminoethyl)octanamide sodium dextranmethylcarboxylate modified by N-(2-aminoethyl)decanamide sodium dextranmethylcarboxylate modified by N-(2-aminoethyl)dodecanamide (dextran 10 kDa) sodium dextranmethylcarboxylate modified by N-(2-aminoethyl)dodecanamide (dextran 40 kDa) sodium dextranmethylcarboxylate modified by 2-(2-[dodecanoylamino]ethoxy)ethylamine sodium dextranmethylcarboxylate modified by 2-(2-{2-[dodecanoylamino]ethoxy}ethoxy)ethylamine sodium dextranmethylcarboxylate modified by 2-(2-{2-[hexadecanoylamino]ethoxy}ethoxy)ethylamine sodium dextranmethylcarboxylate modified by 2-(2-aminoethoxy)ethyl octanoate sodium dextranmethylcarboxylate modified by 2-(2-aminoethoxy)ethyl dodecanoate sodium dextranmethylcarboxylate modified by 2-(2-aminoethoxy)ethyl hexadecanoate sodium dextranmethylcarboxylate modified by 2-[(2-octanoylamino-3-phenyl)propanoylamino]ethanamine sodium dextranmethylcarboxylate modified by 2-[(2-octanoylamino-3-phenyl)propanoylamino]ethanamine N-(sodium methylcarboxylate) dextran carbamate modified by N-(2-aminoethyl)dodecanamide sodium dextransuccinate modified by N-(2-aminoethyl)dodecanamide dextran modified by N-(sodium methylcarboxylate) carbamate and N-(2-aminoethyl)dodecanamide carbamate.

The invention also relates to the use of the functionalized polysaccharides according to the invention in the preparation of pharmaceutical compositions.

The invention also relates to a pharmaceutical composition comprising one of the polysaccharides according to the invention as described above and at least one active principle.

The invention also relates to a pharmaceutical composition, wherein the active principle is chosen from the group consisting of proteins, glycoproteins, peptides and nonpeptide therapeutic molecules.

The term “active principle” is understood to mean a product in the form of a single chemical entity or in the form of a combination having a physiological activity. Said active principle can be exogenous, that is to say that it is introduced by the composition according to the invention. It can also be endogenous, for example the growth factors which will be secreted in a wound during the first phase of healing and which can be retained on said wound by the composition according to the invention.

Depending on the pathologies targeted, it is intended for a local or systemic treatment.

In the case of local and systemic releases, the methods of administration envisaged are by the intravenous, subcutaneous, intradermal, transdermal, intramuscular, oral, nasal, vaginal, occular, buccal or pulmonary routes, and the like.

The pharmaceutical compositions according to the invention are either in the liquid form, in aqueous solution, or in a powder, implant or film form. They additionally comprise the conventional pharmaceutical excipients well known to a person skilled in the art.

Depending on the pathologies and methods of administration, the pharmaceutical compositions can advantageously comprise, in addition, excipients which make it possible to formulate them in the form of a gel, sponge, injectable solution, solution to be taken orally, lyophilized tablet, and the like.

The invention also relates to a pharmaceutical composition, which can be administered in the form of a stent, of a film or coating of implantable biomaterials, or of an implant.