EP2120970A1

EP2120970A1 - Heparins including at least one covalent bond with biotin or a biotin derivative, method for preparing same and use thereof

Info

Publication number: EP2120970A1
Application number: EP08761872A
Authority: EP
Inventors: Philippe Hubert; Pierre Mourier; Christian Viskov
Original assignee: Sanofi Aventis France
Current assignee: Sanofi SA
Priority date: 2007-02-14
Filing date: 2008-02-12
Publication date: 2009-11-25
Also published as: RU2009134160A; US20100075922A1; MX2009008025A; FR2912408A1; KR20090109105A; CA2678166A1; AR065328A1; CN101605549A; JP2010518251A; WO2008113918A1; IL199880A0; TW200902036A; AU2008228161A1; BRPI0808070A2

Abstract

The invention relates to biotinylated heparins, characterised in that the polysaccharides constituting said heparins have, at their reducing end, a covalent bond with a -(R1),-Biot and correspond to the general formula (I) in which i is = or 1, R1 is a chain of the formula -CO- (CH2)j-NH- or -CO-(CH2)jNH-CO-(CH2)k-NH where j and k are integers having any value from 1 to 10, Biot is a biotin or biotin derivative group, n is an integer having an average value of about 25 for a heparin with an average molecular mass of 15 000 Da, X is H or SO3Na, Y is COCH3 or SO3Na, the undulated line representing a bond under or above the pyrasonic cycle to which it is bonded. The invention also relates to a method for preparing the same and to their use in therapy.

Description

HEPARINS COMPRISING AT LEAST ONE COVALENT BINDING WITH BIOTIN OR A BIOTIN DERIVATIVE, PROCESS FOR PREPARING THEM AND USE THEREOF

The present invention relates to heparins having at least one covalent bond with biotin or a derivative of biotin, as well as their method of preparation, pharmaceutical compositions containing them and their use in therapy.

Heparin is a mixture of sulfated mucopolysaccharides of animal origin with a molecular weight of around 15,000 Dalton (Da). Heparin or heparins will be discussed in the following: the structures, average molecular masses and polydispersity of the heparin polysaccharide chains may indeed vary according to the animal species and the organ of origin of the heparin (examples heparin from pork mucus, beef intestine, etc.).

Heparin catalyzes, particularly via antithrombin III (ATIII), the inhibition of two enzymes involved in the blood coagulation cascade, namely factor Xa and factor Ma (or thrombin). It is thus used therapeutically for its anticoagulant and anti-thrombotic properties. Heparin, however, has drawbacks that limit the conditions of its use. In particular, its important anticoagulant activity (in particular its high anti-factor IIa activity) can cause haemorrhages (Seminars in Thrombosis and Hemostasis, Vol 5, sup 3, 1999). Heparins are known for these unwanted hemorrhagic side effects.

In the field of the treatment of thrombosis, the goal is to restore or maintain the fluidity of the blood while avoiding causing haemorrhage. It is well known that, for any accidental cause, hemorrhage may occur in a patient undergoing treatment. It may also be necessary to intervene surgically in a patient under anti-thrombotic treatment. In addition, during certain surgical procedures, anticoagulants can be used in high doses to prevent blood clotting, and it is desirable to neutralize them at the end of the procedure. The need is therefore felt to have neutralizing antithrombotic agents to stop the anticoagulant activity at any time. Neutralizable antithrombotic agents, such as biotinylated synthetic polysaccharides, have been described in patent applications WO 02/24754 and WO 06/030104. Their synthesis, including in particular the grafting of biotin or the biotin derivative carried out on protected equivalents of the polysaccharides mentioned above and not on these polysaccharides themselves, is not applicable to the compounds of the present invention. Indeed, it is desired to carry out the biotinylation on finished products, which are mixtures of polysaccharides and are therefore heterogeneous products, on which the biotin grafting as described in the aforementioned patent applications would not make it possible to induce a regrowth. sufficient selectivity of the grafting position and would not allow the biotinylation of all functionalizable polysaccharide chains of heparins.

The Osmond et al. described in Analytical Biochemistry, 31 (2002) 199-207, several techniques for the biotinylation of porcine heparin, one of which is described as involving a coupling of biotin to the reducing end of a heparin by reductive amination followed by coupling with biotin. However, the operating conditions described in this document do not make it possible to obtain a complete and reproducible manner of biotinylated heparins: they do not take into account the structural diversity of the heparins and the actual structure of the polysaccharide chains as they are present in commercially available heparins. The latter largely comprise polysaccharide chains which possess at their reducing end a degraded glycoserine, which can not be functionalized with biotin according to the protocol described by Osmond et al. Thus, the operating conditions described in this publication for the biotinylation of porcine heparin do not make it possible to obtain a complete and reproducible manner of biotinylated heparins with expected characteristics, such as a biotinylation rate sufficient to allow effective neutralization.

The team of Tseng et al. described in Biomaterials, 27 (2006), 2627-2636, a technique for immobilizing heparin on films by interaction with avidin, following the functionalization of heparin with biotin. Biotinylation of heparin is accomplished by oxidation with iodine followed by of a lactone and then coupling with a 2- (4-aminophenyl) ethylamine derivative of biotin. The operating conditions presented by Tseng et al. do not presume, however, the complete and reproducible production of biotinylated heparins at the reducing end: indeed, there is no indication that the oxidation step can be selective at the reducing end, nor that the biological activity of heparin is preserved after such treatment.

The team of Kett ef al. described in Biochimica and Biophysica Acta, 1620 (2003), 225-234, affinity studies between avidin and various glycosaminoglycans and demonstrates that avidin has a high affinity for heparin. In these affinity studies, heparin can be derivatized with biotin, but there is no indication of the site of biotinylation in the heparin polysaccharide chains.

The Applicant therefore aims to provide new neutralizable heparins by avidin or streptavidin and have biological properties comparable to native heparins.

The present invention relates to novel modified heparins, hereinafter called "biotinylated heparins", characterized in that the constituent polysaccharides of said heparins have at their reducing end a covalent bond with a group - (R 1) _r Biot and meet the general formula (I):

in which:

i is equal to 0 or 1,

R1 represents a sequence of formula (a) or (b):

(at)

in which j and k, which may be identical or different, are integers which can take any value from 1 to 10, - Biot represents a biotin group or a biotin derivative,

n represents an integer having an average value of the order of 25 for a heparin with an average molecular mass of 15,000 Da,

X represents H or SO ₃ Na,

Y represents COCH ₃ or SO ₃ Na, the wavy line denotes a bond situated either below or above the plane of the pyranosic ring to which it is attached, as well as their pharmaceutically acceptable salts.

Surprisingly, the introduction of biotin or a biotin derivative at the reducing end of the polysaccharide chains does not modify the pharmacological activity of the heparins. Indeed, the novel biotinylated heparins, object of the invention, have anti-thrombotic activities comparable to native heparins, that is to say before biotinylation.

They have a considerable advantage over native heparins: they can be quickly neutralized by a specific antidote, in an emergency situation. This specific antidote is avidin, in tetrameric or monomeric form, or streptavidin, with respective masses of about 66,000, 16,400 and 60,000 Da (The Merck Index, Twelfth edition, 1996, MN 920, pages 151-152). Review Pierce Avidin-Biotin Handbook). They also have the advantage of being useful in therapeutic indications for which the doses used are higher, while reducing the risk of haemorrhage; they can thus be useful in therapeutics in the arterial field.

The biotin group (Biot) mentioned above is a radical derived from hexahydro-2-oxo-1H-thieno [3,4-d] imidazole-4-pentanoic acid. Advantageously, the Biot group in the general formula (I) according to the invention corresponds to formula (c):

The biotin derivatives are commercially available ("Pierce" Biotin-avidin products catalog, 2005, pp. 7-11) or can be prepared using conventional methods known to those skilled in the art. Mention may in particular be made of the biotin derivatives mentioned in the patent application WO 02/24754.

In the biotinylated heparins according to the invention, the index i may be equal to 0, in which case the binding with the biotin or the biotin derivative is carried out directly on the amine function carried by the saccharide unit at the level of the reducing end of the polysaccharide chains.

Alternatively, i may be equal to 1 and the bond with the biotin or biotin derivative group may consist, for example, of a sequence of formula (a) above in which j is equal to 5, or a sequence of formula (b ) in which j and k are identical and are equal to 5. Thus, in the formula (I) above, R1 may for example represent a sequence of formula -CO- (CH ₂ ) ₅ -NH or - CO- (CH ₂ ) ₅ -NH-CO- (CH ₂ ) ₅ -NH-.

In the context of the present invention, the term "reducing end" is understood to mean the end of the polysaccharide chain in which glucosamine or terminal mannosamine (mannosamine resulting from an epimerization in a basic medium of glucosamine) has a hemi-functional function. cyclic acetal, corresponding to formula (II) below:

(H)

in which:

X represents H or SO ₃ Na,

Y represents COCH ₃ or SO ₃ Na, and the wavy line denotes a bond located either below or above the plane of the pyranosic ring to which it is attached (below: glucosamine, above: mannosamine) .

The term "polysaccharides constitutive of heparin" means polysaccharides characterized by the repetition of a disaccharide unit containing a uronic acid residue (D-glucuronic acid or L-iduronic acid) and a D-glucosamine, which can be N-sulfated or N-acetylated. The disaccharide unit may also be O-sulfated at the C6 and / or C3 positions of D-glucosamine and at the C2 position of uronic acid (Heparin-binding proteins, H. Edward Conrad, 1998, p.1).

As indicated above, heparin is a mixture of sulfated mucopolysaccharides of animal origin. Native heparins, ie starting heparins before biotinylation, are referred to as "heparins". The heparins used in the present invention may especially be of bovine, ovine or porcine origin; more specifically, they may be derived from the lungs of oxen, intestinal mucosa of beef, intestinal mucous membranes of pigs or intestinal lining of sheep. Advantageously, the heparins used in the present invention are of porcine origin, for example derived from intestinal pig mucosa.

The biotinylated heparins according to the present invention are such that at least 60%, advantageously at least 64%, of the constituent polysaccharides of said heparins have at their reducing end a covalent bond with a group - (R 1) _r Biot and correspond to the formula (I ) as defined above, and whatever the original structure of heparin; advantageously, at least 80% of the constituent polysaccharides of said heparins have at their reducing end a covalent bond with a - (R 1) -Biot group.

The invention encompasses biotinylated heparins in the form of any of their pharmaceutically acceptable salts.

The subject of the present invention is also a process for the preparation of the biotinylated heparins mentioned above, characterized in that: a) the heparin is treated with heparinase 3, b) and then a reductive amination is carried out on the product obtained previously in the presence of an amine salt and a reducing agent, at a temperature between 20 and 80 ^° C. ₁ c) finally acylation is carried out with a - (R 1) - group, activated Biot, where R 1, i and Biot are as defined in relation to formula (I) above, in the presence of a base in an aqueous medium or in an organic medium.

The steps of the above preparation process can be controlled by an analytical HPLC monitoring, in particular of the SAX type, and more particularly of the CTA-SAX type, after depolymerization of the constituents of the heparin-derived mixture in the presence of a mixture of heparinases 1, 2 and 3. Such analytical monitoring may for example be carried out using the method described in the patent application US2005 / 0186679 A1.

In particular, it is verified, after the reductive amination step b), that at least 80%, advantageously at least 90%, of the constituent polysaccharides of said heparins carry at their reducing end an -NH ₂ function (amino-reduced polysaccharides). In particular, it is verified after the acylation step c) that at least 80%, advantageously at least 90%, of said amino-reduced polysaccharides are biotinylated.

The overall yield of the process for preparing the biotinylated heparins according to the invention is therefore at least 60%, advantageously at least 64%. Advantageously, this yield is at least 80%. We hear by :

heparinase 1: the enzyme heparin lyase I (EC 4.2.2.7) of Flavobacterium heparinum,

heparinase 2: the heparin lyase II enzyme of Flavobacterium heparinum, heparinase 3: the heparin lyase III enzyme (EC 4.2.2.8) of Flavobacterium heparinum.

Heparinase 3 makes it possible to eliminate the protein binding zone (glycoserine) in the heparin polysaccharide chains and to obtain chains whose reducing ends are free of glycoserine residues. Heparinases 1 and 2 allow the cleavage of polysaccharide chains into low molecular weight fragments (heparin depolymerization reactions).

The process for the preparation of the compounds according to the invention uses as heparins of origin ("native" heparins) heparins prepared as previously reported in the literature. Reference is made in particular to the book "Heparin, manufacture, structure, properties, analyzes", JP Duclos, Ed. Masson, 1984. Heparins may in particular be prepared according to the process described in patent application US 2005/0215519. A1.

In the reductive amination step b) of the above preparation method, the amine salt may be a quaternary amine salt; it is advantageously an ammonium halide salt having the formula NH ₄ Z, where Z represents a halogen atom, such as a chlorine, fluorine, bromine or iodine atom.

In the reductive amination step b) of the above preparation process, the reducing agent may be a borohydride salt, for example a cyanohydride salt.

In step b) of reductive amination of the above preparation process, the temperature is advantageously between 50 and 80 ^° C.

In step c) of acylation of the above preparation process, the base may be a carbonate or hydrogencarbonate salt, especially in the form of a sodium or potassium salt, or any other water-soluble or soluble organic base. in an organic medium known to those skilled in the art. In the acylation step c) of the above preparation process, the expression "organic medium" is understood to mean, for example, dichloromethane or dimethylformamide.

The process for preparing the biotinylated heparins according to the invention advantageously comprises the following steps: a) heparin is treated with heparinase 3, b) and then reductive amination is carried out on the product obtained previously in the presence of a sodium salt; ammonium halide and a borohydride salt, at a temperature between 50 and 80 ⁰ C, c) finally acylation with a - (R1) - Biot group as defined previously in the form of activated ester, in the presence of a base in an aqueous medium.

Biotinylated - (R1) - Biot derivatives as defined above can be involved in the acylation reaction directly in the form of activated esters, preformed or generated in situ using conventional coupling conditions known from the Man of the art. In particular, it is possible to use activated esters in the form of N-hydroxy-succinimide derivatives or of 3-sulfo-N-hydroxy-succinimide derivatives.

The preparation process according to the invention is illustrated in Scheme 1.

Diagram 1

Heparinase 3

Heparin

Compound 3

According to Scheme 1, heparin is treated with heparinase 3 to remove the remaining protein binding (i.e., native or degraded glycoserines) to obtain a compound 1 having a glycoserine residue-free reducing end. . This heparin (compound 1) can then be subjected to a reductive amination to provide compound 2, having a free amine function at the reducing end, in the presence of an amine salt and a reducing agent such as a borohydride salt.

This compound can then be acylated to provide the biotinylated compound 3, by reaction with an activated derivative of biotin - (R 1) _r Biot, as defined above, in the presence of a base. This reaction may for example be carried out with the sodium salt of the ester of 6-biotinamidohexanoyl hexanoate 3-sulfosuccinimidyl when R1 is -CO- chain (CH ₂₎ _S -NH-CO- (CH _Z) ₅ - NI-I-, or with the sodium salt of 3-sulfosuccinimidyl 6-biotinamido hexanoate ester when R1 represents the -CO- (CH ₂ ) ₅ -NH- sequence, or alternatively with the sodium salt of the biotinoyl-3-sulfosuccinimidyl ester when R1 is not present (i = O). In Scheme 1, it is understood that compounds 1, 2 and 3 are a theoretical representation since they are actually, as heparin derivatives, mixtures of polysaccharide chains.

In the following, examples of synthesis of biotinylated heparins according to the invention and of various intermediates useful for obtaining them are detailed by way of illustration.

The following abbreviations are used:

Heparin EPB: heparin marketed by Bioibérica;

HPLC: "High Performance Liquid Chromatography";

SAX: "Strong anion exchange chromatography"; CTA: "Cetyl Trimethyl Ammonium";

Q.S.P .: "Sufficient Quantity For";

LC: "Long Chain" (corresponds to the sequence 6-amino-hexanoyl);

Sulfo-NHS: sodium salt of the 3-sulfosuccinimidyl ester;

Heparinase 1: heparin lyase I enzyme (EC 4.2.2.7) from Flavobacterium heparinum;

Heparinase 2: heparin lyase II enzyme of Flavobacterium heparinum;

Heparinase 3: heparin lyase III enzyme (EC 4.2.2.8) of Flavobacterium heparinum.

Example 1

1.1 Heparin EPB purified by depolymerization with heparinase 3: At a temperature close to 20 ^° C., 1 g of crude heparin EPB is dissolved in 15 ml of the aqueous solution of 5 mM sodium phosphate adjusted to pH = 7.0 ± 0.1, 20 mM sodium chloride and 1 mg / ml BSA. 0.5 IU heparinase 3 is added to the heparin solution. The reaction mixture obtained is stirred for 5 days. 1 g of sodium chloride and 45 ml of methanol are added to the reaction mixture. The suspension obtained is filtered through a 0.45 μm membrane. The cake is washed with methanol, with diethyl ether, then dried under vacuum. 0.98 g of a white solid are obtained. The observed yield is 98%.

The product can be controlled by depolymerization using a mixture of heparinases 1, 2 and 3 and analyzed by HPLC-SAX using the method described in US2005 / 0186679 A1. The results show a disappearance of at least 80% of the glycoserine species present in the starting heparin.

¹ H NMR spectrum of the polysaccharide mixture in D ₂ O (25 ° C, δ in ppm), main signals: 2.05 (CH ₃ CO, s), 3.28 (CH, m), 3.65 (CH 3) ₁ m), 3.77 (CH, m), 4.05 (CH, m), 4.10 (CH, s), 4.20 (CH, s), 4.25 (CH, d, 12Hz) , 4.35 (CH, s), 4.42 (CH, m), 4.88 (CH, s), 5.22 (CH, s), 5.42 (CH, s).

1.2 Heparin depolymerized with 3 amino-reduced heparinase:

0.5 g of heparin purified by heparinase 3 are dissolved in a 5 M aqueous ammonium chloride solution. 0.5 g of sodium cyanoborohydride are added to the heparin solution. The mixture is heated at 70 ^° C. for 24 hours.

The solution is brought to a temperature close to 20 ⁰ C, diluted with water (QSP

50 ml), then desalted on a column of Sephadex G10. The fraction obtained is injected onto a Q-Sepharose column. The product is eluted with water and then with a gradient of sodium perchlorate. The product obtained is desalted on a Sephadex G10 column and then lyophilized. 444 mg of a white lyophilisate are obtained. The yield observed is 89%. The product is engaged as in the next acylation step.

¹ H NMR spectrum of the polysaccharide mixture in D ₂ O (25 ^° C., δ in ppm), main signals: 2.05 (CH ₃ CO, s), 3.28 (CH ₁ m), 3.65 (CH 2) , m), 3.77 (CH, m),

4.05 (CH, m), 4.10 (CH, s), 4.20 (CH, s), 4.25 (CH, d, 12Hz), 4.35 (CH, s), 4.42

(CH, m), 4.88 (CH, s), 5.22 (CH, s), 5.42 (CH, s).

Heparin depolymerized with heparinase 3, amino-reduced and biotinylated: At a temperature in the region of 20 ^° C., 200 mg of amino-reduced depolymerized heparin are dissolved in 2 ml of 0.5M sodium hydrogen carbonate solution. 37 mg of Sulfo-NHS-LC-Biotin are added to the solution obtained. The solution obtained is stirred at a temperature close to 20 ^° C. for 1 hour. The solution is diluted with 4 ml of 0.5M sodium hydrogen carbonate solution. 37 mg of Sulfo-NHS-LC-Biotin are added and the mixture is stirred for 2 hours. 37 mg of Sulfo-NHS-LC-Biotin are added again and the reaction mixture is stirred for 16 hours. The reaction medium obtained is diluted with water (QSP 200 ml), filtered through a 0.45 μm membrane and injected onto a column of Q-Sepharose. The product is eluted with water and then with a gradient of sodium perchlorate. The product obtained is desalted on a Sephadex G10 column and then lyophilized. 188 mg of a white lyophilisate are obtained. The yield observed is 94%. The product can be controlled by depolymerization using a mixture of heparinases 1, 2 and 3 and analyzed by HPLC-SAX, using the method described in US2005 / 0186679 A1.

¹ H NMR spectrum of the polysaccharide mixture in D ₂ O (25 ° C, δ in ppm), main signals: between 1, 3 and 1, 8 (CH ₂ biotin, m), 2.05 (CH ₃ CO, s ), 2.25 (CH ₂ CO biotin, t, 7Hz), 2.79 (1H, d, 12Hz), 3.00 (1H, dd, 12 and 5Hz), 3.20 (NCH ₂ biotin, m), 3.30 (CH, m), 3.68 (CH, m), 3.80 (CH, m), 4.05 (CH, m), 4.10 (CH, s), 4, (CH, s), 4.25 (CH, m), 4.35 (CH, s), 4.45 (CH, m), 4.63 (Biotin NCH, m), 4.85 (CH, s), 5.22 (CH, s), 5.42 (CH ₁ s).

Example 2

2.1 Heparin obtained according to US 2005/0215519 A1, depolymerization with heparinase 3:

At a temperature close to 20 ^° C., 1 g of heparin according to US 2005/0215519 A1 is dissolved in 15 ml of the aqueous solution of 5 mM sodium phosphate adjusted to pH = 7.0 ± 0.1, 20 mM sodium chloride and 1 mg / ml BSA. 0.5 IU heparinase 3 is added to the heparin solution. The reaction mixture obtained is stirred for 7 days. 1 g of sodium chloride and 45 ml of methanol are added to the reaction mixture. The suspension obtained is filtered through a 0.45 μm membrane. The cake is washed with methanol, diethyl ether and then dried under vacuum. 0.96 g of a white solid are obtained. The yield observed is 96%.

The product can be controlled by depolymerization using a mixture of heparinases 1, 2 and 3 and analyzed by HPLC-SAX using the method described in US patent application 2005/0186679 A1. The results show a at least 80% disappearance of the glycoserine species present in the starting heparin.

¹ H NMR spectrum of the polysaccharide mixture in D ₂ O (25 ° C, δ in ppm), main signals: 2.05 (CH ₃ CO, s), 3.28 (CH, m), 3.65 (CH 3) , m), 3.77 (CH, m), 4.05 (CH, m), 4.10 (CH, s), 4.20 (CH, s), 4.25 (CH, d, 12 Hz) , 4.35 (CH, s), 4.42 (CH, m), 4.88 (CH, s), 5.22 (CH, s), 5.42 (CH, s).

2.2 heparin according to US 2005/0215519 A1 depolymerized by heparinase 3 and amino-reduced: 0.5 g of heparin according to LJS 2005/0215519 A1 treated with heparinase 3 are dissolved in 20 ml of a 5 M aqueous solution of ammonium chloride. 0.5 g of sodium cyanoborohydride are added to the heparin solution. The mixture is heated at 70 ^° C. for 24 hours. The solution is brought to a temperature close to 20 ⁰ C, diluted with water (QSP 50 ml), then desalted on a column of Sephadex G10. The harvested fraction is lyophilized. 446 mg of a white lyophilisate are obtained. The yield observed is 89%. The product is engaged as in the next acylation step.

¹ H NMR spectrum of the polysaccharide mixture in D ₂ O (25 ° C, δ in ppm), main signals: 2.05 (CH ₃ CO, s), 3.28 (CH ₁ m), 3.65 (CH 3) , m), 3.77 (CH, m), 4.05 (CH, m), 4.10 (CH, s), 4.20 (CH, s), 4.25 (CH, d, 12 Hz) , 4.35 (CH, s), 4.42 (CH, m), 4.88 (CH, s), 5.22 (CH, s), 5.42 (CH, s).

2.3 Heparin according to US 2005/0215519 A1 depolymerized with heparinase 3, amino-reduced and biotinylated: At a temperature in the region of 20 ° C., 200 mg of amino-reduced depolymerized heparin are dissolved in 2 ml of 0.5 M solution. of sodium hydrogencarbonate. 37 mg of Sulfo-NHS-LC-Biotin are added to the solution obtained. The solution is stirred at a temperature close to 20 ° C for 1 hour. The suspension obtained is diluted with 4 ml of 0.5M sodium hydrogen carbonate solution. 37 mg of Sulfo-NHS-LC-Biotin are added and the mixture is stirred for 2 hours. 37 mg of Sulfo-NHS-LC-Biotin are added again and the reaction mixture is stirred for 16 hours. The reaction medium obtained is diluted with water (QSP 200 ml), filtered through a 0.45 μm membrane and injected onto a Q-Sepharose column. The product is eluted with water and then with a gradient in sodium perchlorate. The product obtained is desalted on a Sephadex G10 column and then lyophilized. 188 mg of a white lyophilisate are obtained. The yield observed is 94%.

The product can be controlled by depolymerization using a mixture of heparinases 1, 2 and 3 and analyzed by HPLC-SAX using the method described in US patent application 2005/0186679 A1.

¹ H NMR spectrum of the polysaccharide mixture in D ₂ O (25 ° C, δ in ppm), main signals: between 1.3 and 1.8 (CH ₂ biotin, m), 2.05 (CH ₃ CO, s) ), 2.25 (CH ₂ CO biotin, t, 7Hz), 2.79 (1H, d, 12Hz), 3.00 (1H, dd, 12 and 5Hz), 3.20 (NCH ₂ biotin, m ), 3.30 (CH, m), 3.68 (CH, m), 3.80 (CH, m), 4.05 (CH, m), 4.10 (CH, s), 4.20 (CH, s), 4.25 (CH, m), 4.35 (CH, s), 4.45 (CH, m), 4.63 (NCH biotin, m), 4.85 (CH, s). ), 5.22 (CH, s), 5.42 (CH, s).

The compounds according to the invention have been the subject of biochemical and pharmacological studies.

Measurement of anti-factor Ha activity and anti-factor Xa activity

The anti-factor IIa (anti-Flla) activity and the anti-factor Xa (anti-FXa) activity in human plasma or a buffer system are analyzed by chromogenic method: the anti-factor IIa activity is tested by means of of anti-factor IIa activity of heparin Actichrome kit (American Diagnostica) containing the chromogenic substrate S-2238, the α-thrombin and I ¹ human ATIII (antithrombin III). The anti-FXa activity is determined with the automated instrument of coagulation ACL 7000 (Instrumentation Laboratory) using the Heparin kit (Instrumentation

Laboratory) containing I ¹ ₁ ATHI factor Xa and the chromogenic substrate S-2765.

Both analyzes are performed exactly according to the manufacturer's instructions.

The following standards are used to establish a standard calibration curve for measuring the in vitro activity of biotinylated heparin fractions in human plasma and the buffer system:

- 1st International Standard for Low Molecular Weight Heparins (National Institute for Biological Standards and Contrai, London, UK, established in 1987, Code No. 85/600), - 2nd international standard for low molecular weight heparins (National Institute for Biological Standards and Control, London, UK, established in 1987, code no. 01/608, used since June 2006),

- enoxaparin (Clexane ^®, sanofi-aventis, France) was used as internal reference.

For determinations of anti-Flla activity, 10 μl of sample or international standards of low molecular weight heparins are diluted 1: 16 with Pantithrombin in human plasma or the buffer system containing 0.05 M Tris HCl, NaCl 0.154M, pH 7.4. 10 μl of this solution are added to a 96-well microtiter plate. The measurement is reproduced in triplicate (on 3 wells). The microtiter plate is maintained at 37 ° C. with stirring at 300 rpm. 40 μl of thrombin are added to each of the wells and incubated for exactly 2 minutes. 40 μl of Spectrozyme are added. After 90 seconds, the reaction is stopped by adding 40 μl of acetic acid. Absorption is measured at 405 nm using a SpectraMax 340 (Molecular devices).

For anti-FXa activity measurements, the sample or international standards of low molecular weight heparins are diluted in human plasma or buffer system containing 0.05 M Tris HCl, 0.154 M NaCl, pH 7.4. The samples containing the heparinoids in plasma or buffer were further diluted 1:20 with a working buffer containing I ¹ ATIII, and placed in duplicate in the sample rotor. The factor Xa reagent and the chromogenic substrate are poured into the indicated reservoirs of the automated ACL 7000 coagulation instrument.

The measurement of the anti-FXa activity is carried out with the "heparin" protocol integrated in the ACL 7000 software. During the analysis, 50 μl of the sample (diluted with the working buffer) are mixed with 50 μl. μl of factor Xa reagent. After an incubation time of 60 seconds at 37 ^° C., 50 μl of chromogenic substrate with a concentration of 1.1 mM are added and the changes in absorption as a function of time are measured at the wavelength of 405 nM.

The results obtained are described in particular in the table below.

It thus appears that the anti-FXa activity of the biotinylated heparins according to the invention is preserved by comparison with the native starting heparins.

Biotinylated heparins according to the present invention can be used for the preparation of medicaments. They can especially be used as anti-thrombotic drugs. Thus, according to another of its aspects, the subject of the invention is medicaments which comprise a biotinylated heparin as defined above. These medicaments find use in therapeutics, in particular in the treatment and prevention of venous thromboses, arterial thrombotic accidents, particularly in the case of myocardial infarction or unstable angina, peripheral arterial thromboses, such as osteoarthritis of the arteries. lower limbs, cerebral arterial thromboses and strokes. They are also useful in the prevention and treatment of smooth muscle cell proliferation, angiogenesis, and as neuroprotective agents of atherosclerosis and arteriosclerosis. The present invention, according to another of its aspects, also relates to a method of treatment of the aforementioned pathologies which comprises the administration to a patient of an effective dose of a compound according to the invention, or one of its pharmaceutically acceptable salts. The use of biotinylated heparins as defined above for the treatment and prevention of the aforementioned pathologies is therefore part of the invention, as well as the use of said biotinylated heparins for the manufacture of a medicament for treating or preventing these pathologies.

According to another of its aspects, the subject of the present invention is a pharmaceutical composition comprising, as active ingredient, a biotinylated heparin according to the invention or a pharmaceutically acceptable salt thereof, as well as at least one pharmaceutically acceptable inert excipient . Said excipients are chosen according to the desired pharmaceutical form and mode of administration, for example the oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, transmucosal, local or rectal route.

In each dosage unit, the active ingredient is present in the amounts adapted to the daily doses envisaged in order to obtain the desired prophylactic or therapeutic effect. Each dosage unit may contain from 25 to 150 mg of active ingredient, advantageously from 30 to 100 mg. These doses of anticoagulant compounds may be neutralized by avidin or streptavidin. There may be special cases where higher or lower dosages are appropriate; such dosages are not outside the scope of the invention. According to the usual practice, the dosage appropriate to each patient is determined by the physician according to the mode of administration, the weight and the response of said patient.

The compounds according to the invention may also be used in combination with one or more other active ingredients useful for the desired therapy, such as antithrombotics, anticoagulants or antiplatelet agents.

The subject of the present invention is also a method employing avidin or streptavidin, characterized in that it makes it possible to neutralize the biotinylated heparins according to the invention. Thus, avidin or streptavidin may be used for the preparation of medicaments for neutralizing biotinylated heparins according to the present invention.

Claims

1. Biotinylated heparins, characterized in that the constituent polysaccharides of said heparins have at their reducing end a covalent bond with a group - (R 1), - Biot and correspond to the general formula (I):

in which: - i is equal to 0 or 1,

R1 represents a sequence of formula (a) or (b):

in which j and k, which are identical or different, are integers that can take any value from 1 to 10,

Biot represents a biotin or biotin derivative group,

X represents H or SO ₃ Na,

Y represents COCH ₃ or SO ₃ Na,

the wavy line denotes a bond situated either below or above the plane of the pyranosic ring to which it is attached, as well as their pharmaceutically acceptable salts.

2. Heparins according to claim 1, characterized in that i is equal to 0, and their pharmaceutically acceptable salts.

3. Heparins according to claim 1, characterized in that i is equal to 1 and R1 represents a sequence of formula (a) in which j is equal to 5, as well as their pharmaceutically acceptable salts.

4. Heparins according to claim 1, characterized in that i is equal to 1 and R1 represents a sequence of formula (b) in which j and k are identical and are equal to 5, and their pharmaceutically acceptable salts.

5. Heparins according to any one of claims 1 to 4, characterized in that Biot represents the biotin group of formula (c):

as well as their pharmaceutically acceptable salts.

6. Heparins according to any one of claims 1 to 5, characterized in that at least 64% of the constituent polysaccharides of said heparins have at their reducing end a covalent bond with a - (R 1) _r Biot group, as well as their salts. pharmaceutically acceptable.

7. Heparins according to claim 6, characterized in that at least 80% of the constituent polysaccharides of said heparins have at their reducing end a covalent bond with a group - (RI) ₁ -BiOt, as well as their pharmaceutically acceptable salts.

8. Heparins according to any one of claims 1 to 7, characterized in that said heparins are of porcine origin. θ. Heparins according to claim 8, characterized in that said heparins are derived from intestinal pig mucosa.

10. Process for the preparation of biotylated heparins as defined in one of claims 1 to 9, characterized in that it comprises the following steps: a) heparin is treated with heparinase 3, b) and then performs a reductive amination on the product obtained previously in the presence of an amine salt and a reducing agent, at a temperature of between 20 and 80 ^° C., and finally an acylation with a group - (R 1) is carried out , -Biotic activated, wherein R1, i and Biot are as defined in any one of claims 1 to 5, in the presence of a base in an aqueous medium or in an organic medium.

11. The method of claim 10, characterized in that it comprises the following steps: a) heparin is treated with heparinase 3, b) and reductive amination is carried out on the previously obtained product in the presence of a ammonium halide salt and a borohydride salt, at a temperature between 50 and 80 ° C, c) finally acylation is carried out with a - (RI) ₁ -BiOt group in the form of activated ester, in the presence of a base in an aqueous medium.

12. Medicament, characterized in that it comprises a biotinylated heparin according to any one of claims 1 to 9 or a pharmaceutically acceptable salt of said biotinylated heparins.

13. Pharmaceutical composition, characterized in that it comprises, as active principle, a biotinylated heparin according to any one of claims 1 to 9, or a pharmaceutically acceptable salt of said biotinylated heparins, as well as at least one pharmaceutically acceptable excipient. acceptable.

14. Use of a biotinylated heparin according to any one of claims 1 to 9 as antithrombotic drug.

15. Use according to claim 14 for the treatment and prevention of venous thromboses, arterial thrombotic accidents, particularly in the case of myocardial infarction or unstable angina, peripheral arterial thromboses, such as lower extremity arteriopathies, cerebral arterial thromboses, stroke, for the treatment and prevention of smooth muscle cell proliferation, angiogenesis, and as a neuroprotective agent for atherosclerosis and arteriosclerosis.

16. A method employing avidin or streptavidin, characterized in that it makes it possible to neutralize biotinylated heparins according to any one of claims 1 to 9.

17. Use of avidin or streptavidin for the preparation of medicaments for neutralizing biotinylated heparins according to any one of claims 1 to 9.