Classifications

• • 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
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

• the present invention relates to low molecular weight heparins, more generally mixtures of heparinoid-derived polysaccharides, which have at least one covalent bond with biotin or a biotin derivative, 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), used in particular for its anticoagulant and antithrombotic properties. Heparin, however, has drawbacks that limit the conditions of its use. In particular, its important anticoagulant activity (especially its high anti-Ha factor activity) can cause haemorrhages (Seminars in Thrombosis and Hemostasis, Vol 5, sup 3, 1999).
• Heparins of low molecular weight for example between 3000 and 7000 Da and more particularly between 3500 and 5500 Daltons, especially obtained by basic depolymerization of heparin esters and currently marketed, such as enoxaparin, also exhibit anti-inflammatory activity. -factor lia important. Heparin derivatives are known for these undesirable hemorrhagic side effects.
• the goal is to restore or maintain the fluidity of the blood while avoiding causing hemorrhage. It is well known that, for any accidental cause, hemorrhage may occur in a patient under treatment. It may also be necessary to intervene surgically in a patient under antithrombotic treatment.
• anticoagulants can be used in high doses to prevent blood clotting, and it would be useful to be able to neutralize them at the end of the procedure.
• the need is felt to have agents Neutralizable antithrombotic agents to stop anticoagulant activity at any time.
• Neutralizable antithrombotic agents such as biotinylated synthetic polysaccharides
• 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.
• 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.
• 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 Applicant therefore aims to provide new low molecular weight heparins neutralizable by avidin or streptavidin and which have biological properties, including anti-factor Xa and anti-factor IIa activities, comparable to heparins. low molecular weight starting.
• biotinylated low molecular weight heparins characterized in that they have an average molecular weight of between 3000 and 7000 Da and that their Constitutive polysaccharides are covalently bound with biotin or a derivative of biotin at their reducing end.
• biotin or a biotin derivative at the reducing end of the polysaccharide chains does not alter the pharmacological activity of low molecular weight heparins.
• novel biotinylated low molecular weight heparins, object of the invention have anti-thrombotic activities comparable to native low molecular weight heparins, that is to say before biotinylation.
• low molecular weight heparins refers to mixtures of sulphated polysaccharides which have the general structure of the polysaccharides constitutive of heparin, which have an average molecular weight of 3000 to 7000 Da and which are obtained by depolymerization of heparin.
• low molecular weight heparins or
• “native low molecular weight heparins” refers to polysaccharide mixtures prior to biotinylation, in contrast to the term “biotinylated low molecular weight heparins”, which refers to compounds according to the invention, comprising a covalent bond with biotin or one of its derivatives.
• reducing end is understood to mean the end of the polysaccharide chain in which glucosamine or terminal mannosamine (mannosamine resulting from a basic epimerization of glucosamine) has a cyclic hemi-acetal function, corresponding to the formula ( II) below:
• 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 (below: glucosamine, above: mannosamine).
• low molecular weight heparins that can be used in the present invention, some of them may be such that at least 75% of their polysaccharide chains contain at their reducing end a glucosamine in hemiacetal form; these are functionalisable polysaccharides of the mixture.
• Some polysaccharide chains present in the mixture can be in the form 1, 6-anhydro, at a content less than or equal to 25%; such polysaccharides are not functionalizable with biotin or the biotin derivative.
• 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).
• biotinylated low molecular weight heparins according to the invention are advantageously characterized in that their constituent polysaccharides correspond to the general formula (I):
• i is equal to 0 or 1
• R1 represents a sequence of formula (a) or (b):
• 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
• PE represents a polysaccharide chain having the general structure of the constituent polysaccharides of heparin
• X represents H or SO 3 Na
• Y represents SO 3 Na or COCH 3
• the wavy line denotes a bond located either below or above the plane of the pyranosic ring to which it is attached, as well as their pharmaceutically acceptable salts.
• Biot is a radical derived from hexahydro-2-oxo-1H-thieno [3,4-d] imidazole-4-pentanoic acid.
• the Biot group in the general formula (I) according to the invention corresponds to formula (c):
• biotin derivatives are commercially available (Tierce catalog "Biotin-avidin products, 2005, pp. 7-11) or can be prepared using standard methods known to those skilled in the art. biotin mentioned in the patent application WO 02/24754.
• 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 level of the reducing end of the polysaccharide chains.
• 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.
• 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-.
• the biotinylated low molecular weight heparins according to the present invention are such that at least 60%, advantageously at least 80%, even more advantageously at least 90% of their constituent polysaccharides have at their reducing end a covalent bond with biotin or a biotin derivative.
• the low molecular weight heparins used in the present invention may be chosen for example from enoxaparin, ardeparin, bemiparin, parnaparin and tinzaparin.
• the low molecular weight heparins used in the present invention may especially be such that:
• said low molecular weight heparins exhibit better bioavailability and antithrombotic activity than heparin and have an average molecular weight between approximately 3500 and 5500 Da.
• the invention encompasses biotinylated low molecular weight 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 low molecular weight heparins mentioned above, characterized in that: a) a reductive amination is carried out in the presence of an amine salt and an agent reducing agent and at a temperature of between 20 and 80 ° C., on a heparin of low molecular weight as defined above, b) then acylation with a - (R 1) r Biot activated group, 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 organic medium.
• the steps of the above preparation method can be controlled by an analytical HPLC monitoring, in particular of the SAX type, for example using the method described in the patent application WO 2004/027087, or possibly by LC-MS, using by for example the method described by Robert J. Linhardt in J. Biol. Chem., 2004, 279 (4), p. 2608-2615.
• Biotinylated low molecular weight heparins can also be analyzed and characterized by supported monomeric avidin affinity chromatography, marketed by Pierce, according to the assay conditions described by the supplier.
• the overall yield of the process for preparing low molecular weight heparins biotinylated according to the invention is therefore at least 80%, advantageously at least 90%.
• the amine salt may be a quaternary amine salt; it is advantageously a salt ammonium halide compound having the formula NH4Z, wherein Z represents a halogen atom, such as a chlorine, fluorine, bromine or iodine atom.
• the reducing agent may be a borohydride salt, for example a cyanohydride salt.
• the temperature is advantageously between 50 and 80 ° C.
• 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.
• step b) of acylation of the above preparation process the term "organic medium” is understood to mean, for example, dichloromethane or dimethylformamide.
• the process for the preparation of biotinylated low molecular weight heparins advantageously comprises the following steps: a) a reductive amination is carried out on a low molecular weight heparin in the presence of an ammonium halide salt and a borohydride salt, at a temperature of between 50 and 80 ° C., then an acylation is carried out with a - (R 1) i-Biot group as defined previously in the form of an activated ester, in the presence of a base in aqueous medium.
• Biotinylated derivatives - (R 1) r Biot as defined above can be involved in the acylation reaction directly in the form of activated esters, preformed or generated in situ using standard coupling conditions known to humans. art.
• activated esters in the form of N-hydroxy-succinimide derivatives or of 3-sulfo-N-hydroxy-succinimide derivatives.
• the low molecular weight heparin is subjected to reductive amination to provide the derivative A, 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 derivative can then be acylated to provide the biotinylated derivative B, by reaction with an activated derivative of biotin - (R 1) r Biot, as defined above, in the presence of a base.
• HPLC High Performance Liquid Chromatography
• QSP "Sufficient Quantity For”
• LC “Long Chain", corresponding to the 6-amino-hexanoyl linkage
• LC-LC represents two LC sequences and corresponds to the sequence amido-hexanoyl-6-amino-hexanoyl
• Sulfo-NHS sodium salt of the 3-sulfosuccinimidyl ester
• Heparinase 1 enzyme heparin lyase I (EC 4.2.2.7) of Flavobacterium heparinum.
• FIG. 1 illustrates the reaction by HPLC SAX of the conversion of enoxaparin according to Example 1.
• FIGS. 2, 3 and 4 illustrate the SAX HPLC analyzes of the biotinylated and non-biotinylated fractions obtained after passing over a supported monomeric avidin column of the products obtained according to Examples 1, 2 and 3, respectively.
• Enoxaparin is a low molecular weight heparin obtained according to the method described in US Patent RE38,743. It is converted into a biotinylated derivative according to the reaction sequence described in scheme 2: the enoxaparin is converted by a reductive amination reaction to compound 1 having an amino function on its reducing end, and then this derivative is converted into biotinylated compound 2 by reaction with 3-sulfosuccinmidyl 6-biotinamido hexanoate, sodium salt.
• the reaction medium obtained is diluted with water (QSP 200 ml), filtered through a 0.45 ⁇ m membrane and then desalted on a Sephadex G 10 column.
• 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 fraction harvested is desalted on a column of Sephadex G10.
• the product obtained is again purified by passage over a Q-Sepharose column and desalting on Sephadex G10.
• the final fraction harvested is lyophilized. 190 mg of a white lyophilisate are obtained. The yield observed is 87%.
• FIG. 1 illustrates the reaction by HPLC SAX of the conversion of enoxaparin by a reductive amination reaction in derivative 1 having an amino function on its reducing end (see diagram 2).
• This derivative is then converted into biotinylated derivative 2 by reaction with 3-sulfosuccinmidyl 6-biotinamido hexanoate, sodium salt.
• the method of analysis employed is described in patent application WO 2004/027087.
• FIG. 1 shows that the species having a functionalizable glucosamine are converted into derivatives having an amino function on their reducing end with a degree of conversion of greater than 90% to provide the enoxaparin 1-amino.
• FIG. 1 shows that the species having a functionalizable glucosamine are converted into derivatives having an amino function on their reducing end with a degree of conversion of greater than 90% to provide the enoxaparin 1-amino.
• FIG. 1 indicates the peaks corresponding to the main compounds present in the oligosaccharide mixtures obtained according to Example 1, the structure of which is represented below (the nomenclature used corresponds to that of the patent application WO 2004 / 027,087).
• Example 2 illustrates the SAX HPLC analysis of biotinylated and non-biotinylated fractions obtained after passage through the supported monomeric avidin column.
• Figure 2 shows that species with functionalizable glucosamine were converted to corresponding biotinylated species with a conversational level greater than 90%.
• the non-affine fraction consists mainly of 1,6-anhydro derivatives which, by their nature, can not be converted into biotinylated derivatives.
• the structures of some of the majority peaks are provided by way of example to characterize the product obtained (see structures illustrated above).
• Enoxaparin a low molecular weight heparin obtained according to the method described in US Pat. No. RE38,743, is converted into a biotinylated derivative according to the reaction sequence described in Scheme 3: the enoxaparin is converted by a reductive amination reaction into compound 1 having an amino function on its reducing end, then this derivative is converted to biotinylated compound 3 by reaction with the ester of biotinoyl-3-sulfosuccinimidyl, sodium salt
• the product is eluted with water and then with a gradient of sodium perchlorate.
• the fraction harvested is desalted on a Sephadex G10 column.
• the fraction harvested is freeze-dried. 190 mg of a white lyophilisate are obtained.
• the observed yield is about 90%.
• the product obtained is controlled by HPLC SAX (see FIG. 3, drawing 3/4, "Global” graph) and it is confirmed that the species having an amino function on their reducing end are converted into a biotinylated derivative by reaction with the ester. of biotinoyl-3-sulfosuccinimidyl, sodium salt, with a conversion of greater than 90%.
• Example 2 The product obtained according to Example 2 is injected onto a supported monomeric avidin column. The elution is carried out according to the conditions described by the supplier Pierce. Biotinylated (avidin-affine) and non-biotinylated fractions
• the non-affine fraction consists mainly of 1,6-anhydro derivatives which, by their nature, can not be converted into biotinylated derivatives.
• FIG. 3 describes the structure of certain main compounds of the oligosaccharide mixture.
• the referenced structures are shown below.
• Enoxaparin a low molecular weight heparin obtained according to the process described in US Pat. No. RE38,743, can also be converted into a biotinylated derivative according to the reaction sequence described in scheme 4: the enoxaparin is converted by an amination reaction. reducing agent in compound 1 having a amino function on its reducing end, then this derivative is converted to biotinylated compound 4 by reaction with the ester of 6-biotinamidohexanoyl hexanoate 3-sulfosuccinimidyl, sodium salt.
• the product is eluted with water and then with a gradient of sodium perchlorate.
• the fraction harvested is desalted on a column of Sephadex G10.
• the harvested fraction is lyophilized. 210 mg of a white lyophilisate are obtained.
• the yield observed is about 92%.
• the product obtained is monitored by HPLC SAX (see FIG. 4, drawing 4/4, "Global" graph) and it is confirmed that the species having an amino function on their reducing end are converted into a biotinylated derivative by reaction with the 6- 3-sulfosuccinimidyl biotinamidohexanoyl hexanoate, sodium salt with a conversion of more than 90%.
• the product obtained according to Example 3 is injected onto a supported monomeric avidin column.
• the elution is carried out according to the conditions described by the supplier Pierce.
• the biotinylated (avidin affine) and non-biotinylated (non-avidin affinity) fractions obtained are then injected onto HPLC SAX (see FIG. 4).
• the non-affine fraction consists mainly of 1,6-anhydro derivatives which, by their nature, can not be converted into biotinylated derivatives.
• the structure of the main compounds is confirmed by LC-MS coupling.
• FIG. 4 describes the structure of certain main compounds of the oligosaccharide mixture.
• the referenced structures are shown below.
• LC-MS analysis makes it possible to confirm the structure of the above compounds by the mass spectra corresponding to the products in acid form: ⁇ lslS
• d1 _ 6 has nhyd ra m / z 1056; ⁇ lslS
• d 1 , d 1 , 6-an hydro m / z 1633; ⁇ lslS
• d 1, 6-a ⁇ hydro m / z 2210; ⁇ lslS
• Tinzaparin a low molecular weight heparin of approximately 6000 Daltons obtained by treatment with heparinase 1
• the compound can be controlled by HPLC SAX, using the method described previously in Example 1.
• the product is engaged as in the biotinylation step.
• the suspension is further diluted with 1 ml of 0.5M solution of sodium hydrogencarbonate and 47 mg of Sulfo-NHS-LC-Biotin are added again.
• the reaction mixture is stirred for 20 hours.
• the suspension is again diluted with 6.5 ml of 0.5 M sodium hydrogen carbonate solution and 47 mg of Sulfo-NHS-LC-Biotin are added again.
• the reaction mixture is stirred for 22 hours and is then diluted with water (QSP 100 ml), filtered through a 0.45 ⁇ m membrane and injected onto a Q-Sepharose column.
• the product is eluted with water and then with a gradient of sodium perchlorate.
• the fraction harvested is desalted on a column of Sephadex G10.
• the final fraction harvested is lyophilized. 110 mg of a white lyophilisate are obtained.
• the observed yield is quantitative.
• the tinzaparin 1-amino and tinzaparin NH LC Biotinoyl compounds obtained can also be characterized by the SAX HPLC methods previously used in Example 1.
• This HPLC control shows that the species having a functionalizable glucosamine are converted into derivatives having an amino function on their reducing end with a conversion rate greater than 90% to provide tinzaparin 1-amino. It also shows that the species having an amino function on their reducing end are converted into a biotinylated derivative by reaction with 3-sulfosuccinimidyl 6-biotinamido hexanoate, sodium salt with a conversion level greater than 90% to provide tinzaparin NH LC. biotinoyl.
• the structures of the main compounds can be confirmed by LC-MS analysis.
• the product obtained can also be injected onto a supported monomeric avidin column.
• the elution is carried out according to the conditions described by the supplier Pierce.
• the biotinylated (avidin affine) and non-biotinylated (non-avidin avidin) fractions obtained can be monitored by HPLC SAX.
• Bemiparin a low molecular weight heparin of approximately 3500 Daltons obtained by alkaline depolymerization, can also be converted into a biotinylated derivative according to the reaction sequence described in the following scheme 6: the bemiparin is converted by a reductive amination reaction to compound 7 having an amino function on its reducing end, then this derivative is converted to biotinylated compound 8 by reaction with 3-sulfosuccinimidyl 6-biotinamido hexanoate, sodium salt.
• the compound can be controlled by HPLC SAX, using the method previously discussed in Example 1.
• the reaction medium obtained is diluted with water (QSP 50 ml) and 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 fraction harvested is desalted on a column of Sephadex G10.
• the product obtained is again purified by passage over a column of Q-Sepharose and desalted on Sephadex G10.
• the final fraction harvested is lyophilized. 101 mg of a white lyophilisate are obtained. The yield observed is 92%.
• the obtained hemiparin-1-amino and b-hemiparin NH LC Biotinoyl compounds can also be characterized by the SAX HPLC methods previously used in Example 1.
• This HPLC control shows that the species having a functionalizable glucosamine are converted into derivatives having an amino function on their reducing end with a conversion rate greater than 90% to provide the bemiparin 1-amino. It also shows that the species having an amino function on their reducing end are converted into a biotinylated derivative by reaction with 3-sulfosuccinimidyl 6-biotinamido hexanoate, sodium salt with a conversion level greater than 90% to provide the bemiparin NH LC. biotinoyl.
• the structures of the main compounds can be confirmed by LC-MS analysis.
• the product obtained can also be injected onto a supported monomeric avidin column. The elution is carried out according to the conditions described by the supplier Pierce.
• the biotinylated (avidin affine) and non-biotinylated (non-avidin avidin) fractions obtained can be monitored by HPLC SAX.
• the compounds according to the invention have been the subject of biochemical and pharmacological studies.
• 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 Ha activity is tested by means of of anti-factor Ma heparin Actichrome kit (American Diagnostica) containing the chromogenic substrate S-2238, the ⁇ -thrombin and I 1 human ATIII (antithrombin III).
• the anti-FXa activity is determined with the automated ACL 7000 coagulation instrument (Instrumentation Laboratory) using the Heparin kit (Instrumentation Laboratory) containing ATIII, factor Xa and chromogenic substrate S-2765. Both analyzes are performed according to the manufacturer's instructions.
• 10 ⁇ l of sample or international standards of low molecular weight heparins are diluted 1: 16 with antithrombin in human plasma or Tris HCl buffer system. 0.05 M, 0.154 M NaCl, pH 7.4. 10 ⁇ l 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 ⁇ l of thrombin are added to each of the wells and incubated for exactly 2 minutes. 40 ⁇ l of Spectrozyme are added. After 90 seconds, the reaction is stopped by adding 40 ⁇ l of acetic acid. Absorption is measured at 405 nm using a SpectraMax 340 (Molecular devices).
• 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. Samples containing the heparinoids in the plasma or buffer are further diluted 1: 20 with working buffer containing I 1 ATIII 1 and placed in duplicate in the sampling rotor. The factor Xa reagent and the chromogenic substrate are poured into the indicated reservoirs of the automated LCD coagulation instrument
• the measurement of the anti-FXa activity is carried out with the "heparin" protocol integrated in the ACL 7000 software.
• 50 ⁇ l of the sample (diluted with the working buffer) are mixed with 50 ⁇ l. ⁇ l of factor Xa reagent.
• 50 ⁇ l of chromogenic substrate with a concentration of 1.1 mM are added and the changes of absorption as a function of time are measured at the wavelength of 405 nM.
• MM denotes the average molar mass (in Daltons) and the "corrected" activity makes it possible to correct, to the extent, the mass dilution effect.
• the corrected activity is calculated as follows:
• MM compound prepared theoretical average molecular weight of the compound prepared,.
• MM starting material average molar mass of the starting low molecular weight heparin.
• biotinylated low molecular weight heparins according to the invention retain anti-factor Xa and anti-factor Ma activities. comparable to native low molecular weight heparins. The conservation of these biological properties makes them therefore usable in therapy.
• Antithrombin-dependent anti-FXa or anti-Flla activity is measured in the presence of increasing avidin concentration to measure the effect of avidin binding to biotin of the product on this activity.
• the products to be studied are dissolved in 1 mg / ml in water at 0.9% NaCl.
• the products are then diluted so as to obtain a concentration of product capable of inhibiting 50% of the factor Xa activity (Factor Xa, chromogenix Milan, Italy), or factor Ha (Factor IIa, blood laboratory, France). in the presence of antithrombin (human antithrombin Milan, Italy).
• This inhibition is then measured in the presence of a decreasing concentration of avidin (Avidin SIGMA of the egg white, Ref A-9275, to be diluted in NaCl): 300, 30, 3, 0.3, 0.03, 0.003, 0 ⁇ g / ml.
• factor Xa (or factor IIa) is done by adding a specific chromogenic substrate; S2222 (Chromogenix, Milan, Italy) for factor Xa, substrate S2238 (Chromogenix, Milan, Italy) for factor Ma.
• the reading of the optical density is at 405 nm.
• the bead solution is made to 1 mg / ml by diluting with the 20 mM Tris maleate wash buffer, 150 mM NaCl, pH 7.35.
• the solution is well stirred and 100 .mu.l of the solution containing the beads (1 mg / ml) are placed in an Eppendorf tube. 500 ⁇ l of buffer are added.
• the tubes are centrifuged at 12000 rpm for 5 minutes. After removal of the supernatant, the pellet is taken up in 500 .mu.l of buffer. After stirring a second centrifugation is performed and the supernatant is again removed.
• the product solutions are then placed in the presence of different solutions containing the beads so as to have the ratio product / beads expressed in ⁇ g product / ⁇ g avidin (Avidine SIGMA Ref A-9275, solution at about 3 mg / ml depending on the batch) of 1 , 0.1, 0.01 and 0.001.
• the mixtures are then stirred and allowed to stand for 1 hour before centrifugation at 12000 rpm for 5 minutes.
• the supernatant is then taken up to determine the anti-FXa activity in order to determine the concentration of product remaining in the supernatant.
• the assay of anti-FXa or anti-Flla activity is carried out following a modified method of that described by Teien A. N and Lie M., Thrombosis Research, 1977, 10, 399-410. The results obtained are described in particular in Table 2.
• Biotinylated low molecular weight heparins according to the present invention can be used for the preparation of medicaments. They can especially be used as anti-thrombotic drugs.
• the subject of the invention is medicaments which comprise a biotinylated low molecular weight heparin as defined above.
• These drugs find their use in therapy, particularly in the treatment and prevention of venous thromboses, accidents arterial thrombotic diseases, particularly in the case of myocardial infarction or unstable angina, peripheral arterial thromboses, such as lower limb arteriopathies, cerebral arterial thromboses and cerebrovascular accidents. 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 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.
• 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 subject of the present invention is a pharmaceutical composition
• a pharmaceutical composition comprising, as active ingredient, a biotinylated low molecular weight heparin according to the invention or a pharmaceutically acceptable salt thereof, as well as at least one excipient. inert, pharmaceutically acceptable.
• 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.
• 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 20 to 150 mg of active ingredient, advantageously from 40 to 100 mg.
• These doses of anticoagulant compounds can be neutralized by doses of avidin or streptavidin ranging from 0.2 g to 2 g by intravenous injection, bolus or infusion. There may be special cases where higher or lower dosages are appropriate; such dosages are not outside the scope of the invention.
• 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 low molecular weight heparins according to the invention.
• avidin or streptavidin can be used for the preparation of drugs for neutralizing biotinylated low molecular weight heparins according to the present invention.

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• 2007
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