WO2018041750A1 - Sulfated polysaccharide extracted from a red alga for the use thereof as an anticoagulant - Google Patents

Sulfated polysaccharide extracted from a red alga for the use thereof as an anticoagulant Download PDF

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WO2018041750A1
WO2018041750A1 PCT/EP2017/071491 EP2017071491W WO2018041750A1 WO 2018041750 A1 WO2018041750 A1 WO 2018041750A1 EP 2017071491 W EP2017071491 W EP 2017071491W WO 2018041750 A1 WO2018041750 A1 WO 2018041750A1
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polysaccharide
pharmaceutical composition
kda
composition according
molecular weight
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PCT/EP2017/071491
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French (fr)
Inventor
Cédric DELATTRE
Philippe Michaud
Guillaume Pierre
Nicolas BRIDIAU
Thierry MAUGARD
Taratra Andrée FENORADOSOA
Hernas Martial RAKOTOARISOA
Original Assignee
Universite Clermont Auvergne
Sigma Clermont
Centre National De La Recherche Scientifique
Universite De La Rochelle
Universite D'antsiranana
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Publication of WO2018041750A1 publication Critical patent/WO2018041750A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/02—Algae
    • A61K36/04—Rhodophycota or rhodophyta (red algae), e.g. Porphyra
    • 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
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • 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

Definitions

  • the present invention relates to a sulfated polysaccharide extracted from a red algae for use as an anticoagulant.
  • Blood coagulation is performed in cascading steps involving different proenzymes and procofactors in the blood that are converted, via proteolytic enzymes, into their activated form.
  • This succession of steps or cascade of coagulation is carried out according to three coagulation systems, respectively called extrinsic coagulation pathway, intrinsic pathway of coagulation and common pathway, leading to the transformation of prothrombin (Thread) into thrombin ( Fila).
  • the extrinsic pathway involves the intervention of factor VII present in the blood. However, this requires activation (factor VIIa) to initiate this coagulation cascade.
  • Factor VIIa exhibits low enzymatic activity until complexed with tissue factors of phospholipidic nature released after tissue injury.
  • factor VIIa transforms factor X (FX) into factor Xa (FXa) in the presence of calcium ions.
  • Factor Xa in turn transforms prothrombin into thrombin, which activates factor V (Factor Va).
  • Thrombin also activates factor XIII (factor XI Ia).
  • Thrombin in the presence of calcium, tissue factors, factor Va, acts on fibrinogen by transforming it into fibrin.
  • factor XI 11a allows the formation of a solid and adherent meshed fibrin clot which is progressively and slowly resorbed with the installation of the healing scar tissue, to which the network serves as a weft.
  • This crosslinked fibrin is insoluble and is not attackable by fibrinolytic enzymes, at least during the time of placement of scar tissue.
  • the intrinsic pathway of coagulation also involves the factor VIIa (FVIIa).
  • This pathway comprises a cascade of reactions resulting in the activation of thrombin via factor XII (FXII).
  • FXII factor VIIa
  • factor Xla - FXIa factor XI
  • factor IXa - FIXa phospholipid tissue factors
  • Factor IXa participates in factor Xa factor X activation in the presence of Villa factor, tissue factors and calcium ions. This then leads to the transformation of prothrombin (Fil) into thrombin (Fila).
  • Factor Xa Factor Xa adsorbed on the surface of phospholipids of tissue and platelet origin will, in the presence of the factor Go, make up the prothrombinase.
  • Factor Va is derived from thrombin-activated factor V (Thread).
  • Prothrombinase is therefore an enzymatic complex involving FXa, FVa, calcium and phospholipids. There is therefore a similarity with the FX activator complex.
  • Prothrombinase allows the formation of thrombin (Fila) from prothrombin (Fil).
  • Vascular thrombosis is defined as the obstruction of a vein or artery by a thrombus (or clot).
  • thrombosis There are two types of thrombosis: venous thrombosis or phlebitis: it can be superficial affecting a deep vein or deep, affecting a deep vein, especially in the thigh.
  • the main treatment used to prevent the risk of vascular thrombosis consists in the administration of anticoagulants.
  • An anticoagulant is a synthetic or natural substance with the property of inhibiting the natural coagulability of blood. The anticoagulant therefore has the mission of avoiding the formation of fibrin clot by inhibiting one or more factors of the cascade of coagulation.
  • Anticoagulants available on the market today are divided into two broad categories: oral anticoagulants and injectable anticoagulants.
  • Oral anticoagulants include vitamin K antagonists (AVK), direct thrombin inhibitors (anti-ILa) and direct factor Xa inhibitors.
  • AVK vitamin K antagonists
  • anti-ILa direct thrombin inhibitors
  • Xa inhibitors direct factor Xa inhibitors.
  • the vitamin K antagonists currently on the market in oral form are coumarinics and derivatives of indanedione.
  • Coumarinic products include acenocoumarol, marketed under the name Sintrom® and Minisintrom®, and warfarin marketed under the name Coumadine®.
  • Derivatives of indanedione include fluindione, marketed under the name Previscan®.
  • These vitamin K antagonists are indicated in the prevention of thromboembolic complications of emboligenic heart disease and complicated myocardial infarction, as well as in the treatment of deep vein thromboses and pulmonary embolisms, as well as in the prevention of their recurrence.
  • these oral anticoagulants have many side effects, such as risk of cerebral, abdominal and intra-articular hemorrhages, digestive disorders, risk of cutaneous necrosis and allergic rashes.
  • Injectable anticoagulants include unfractionated standard heparins and low molecular weight heparins.
  • Heparin is a sulfated polysaccharide consisting of 1-4-linked glucosamine and uronic acid units, in which the sulfate groups are present on the amino function of glucosamine and / or on the alcohol functions of glucosamine and uronic acid. This polysaccharide, whose anticoagulant properties are well known, is currently widely used in the treatment of thrombotic events. Heparin, however, has very significant side effects (bleeding, risk of immuno-allergic thrombocytopenia) and is very effective in arterial thrombosis.
  • LMWHs are currently on the market including: Enoxaparin sodium, marketed in particular under the name of Lovenox®, Dalteparin sodium, marketed in particular under the name of Fragmine®, Nadroparin sodium, sold in particular under the name of Fraxiparine® or Fraxodi®, Tinzaparin, sold in particular under the name Innohep®, Reviparin, Parnaparin, etc.
  • heparins have the same, if not higher, efficacy as unfractionated heparin.
  • LMWHs low molecular weight heparins
  • heparins are industrially extracted from pork intestine (Europe) or from beef lung. Offal of other farmed mammals may be used as secondary sources. Because of its animal origin, health risks can not be excluded.
  • novel anticoagulants of natural and non-animal origin capable of being administered orally and / or injectably, and capable of preventing and / or effectively treating vascular thromboses while reducing the risks. side effects and eliminating the risk of contamination.
  • the inventors have thus surprisingly shown that the sulphated polysaccharides extracted from red algae, and in particular the sulphated polysaccharides extracted from the red marine algae of the species Haliptilon subulatum, had anticoagulant activity similar to the standard heparin no fractionated or even higher than standard unfractionated heparin and low molecular weight heparins.
  • the international application WO2014 / 76261 describes a composition comprising at least one sulphated polysaccharide and at least one food ingredient for the treatment or prevention of an infection caused by at least one microsporidium in humans or animals. These sulfated polysaccharides are extracted from green algae, browns, reds and cyanobacteria.
  • the international application WO 01/15654 describes a sulphated polysaccharide of molar mass less than or equal to 10 kDa, obtainable by radical depolymerization of a fucan derived from Pheophyceae, in order to obtain a medicament active against arterial thrombosis. and against arterial restenosis. This sulphated polysaccharide is extracted from brown algae.
  • None of these documents relates to sulfated polysaccharides extracted from red algae of the species Haliptilon subulatum for the prevention and / or treatment of vascular thromboses, or even the prevention and / or treatment of ischemia, pulmonary embolism, pulmonary embolism, unstable angina, myocardial infarction. Moreover, none of these documents relates to the prevention of anticoagulation of extracorporeal circulation circuits and / or extra-renal purification.
  • the invention thus relates to a novel sulfated polysaccharide-based anticoagulant extracted from a red alga of the species Haliptilon subulatum or a pharmaceutically acceptable salt thereof.
  • Polysaccharides from red algae are constructed on the basis of a linear sequence of 3- / 3-galactopyranose and 4-a-galactopyranose units alternating regularly.
  • the ⁇ -galactopyranose unit is always of D configuration, whereas the ⁇ -galactopyranose unit is of D configuration in carrageenans and L in agarocolloids.
  • some of the 4-a-galactopyranose residues may exist under the form of 3,6-anhydrogalactopyranose.
  • the 3,6-anhydrogalactopyranose form is obtained by removal of the sulfate ester carried by the carbon 6 of the 4-linked ⁇ -galactose unit, under the action of galactose-6-sulfurylases during the biosynthesis or an alkaline treatment.
  • the subject of the invention is therefore a sulphated polysaccharide extracted from a red alga of the Haliptilon subulatum species or a pharmaceutically acceptable salt thereof, in which the sulphate level of said polysaccharide is less than or equal to 20% by weight. mass of the polysaccharide for its use as an anticoagulant.
  • the sulfation rate as well as the distribution of the sulphates on the polysaccharides can have a critical effect on the interaction between the proteases, the inhibitors and the activators of the coagulation cascade, and in particular on the activity or anticoagulant polysaccharides (see Fonseca et al, Thrombosis and Haemostasis, 2008, Vol 99 (3), pages 539-45).
  • polysaccharides extracted from a red alga of the Haliptilon subulatum species and having a sulphate level of less than or equal to 20% exhibit anticoagulant activity similar to unfractionated standard heparin, even higher than standard unfractionated heparin and low molecular weight heparins,
  • the sulphated polysaccharide is extracted from a red alga, advantageously from a red marine alga, advantageously a red marine alga from the class of Floridaophyceaea, even more advantageously from a red marine alga of the species Haliptilon subulatum.
  • the sulphated polysaccharide is extracted from a red algae of the species Haliptilon subulatum.
  • the sulphated polysaccharide according to the invention, in which the sulphate level of said polysaccharide is less than or equal to 20% by mass of the polysaccharide corresponds to formula (I):
  • unit A is a 3 ⁇ -D-galactopyranose, in which the free hydroxyl functions are substituted with one or more groups chosen from XA 2 , XA 4 , XA 6 , unit B is selected from the group consisting of residue B1 and residue B2:
  • residue B1 being a 4-aD / L-galactopyranose, in which the free hydroxyl functions are substituted with one or more groups chosen from X B 2, XB 3 and X B e and,
  • residue B2 being a 4-a-3,6-anhydrogalactopyranose, in which the free hydroxyl functions of 4-a-3,6-anhydrogalactopyranose are substituted with an XB2 group and,
  • residues B1 and B2 being distributed randomly in the polysaccharide and the residue B2 representing at most 5% by weight of the polysaccharide
  • unit A is connected to unit B by an O-glycosidic bond between the carbon at position 1 of unit A and the carbon at position 4 of unit B and,
  • unit B is connected to unit A by an O-glycosidic bond between the carbon at position 1 of unit B and the carbon at position 3 of unit A,
  • XA2, XA4, XA6, XB2, XB3 and XB6 are independently selected from each other and independently for each A and / or B unit in the group consisting of:
  • a pyruvate group (-COO-CO-CH3), said pyruvate group being linked to the XA group 4 by its carbon in position 1 and to the group XA6 by its carbon in position 3;
  • a saccharide unit linked to unit A or B by an O-glycoside type bond at position 1 (Ci) of the saccharide unit the saccharide unit being chosen from a galactose (or T-galactose), a xylose (or T-xylose), an arabinose (or T-arabinose) and a glucuronic acid (or T-glucuronic acid); and
  • a (C1-C6) alkoxyl group a (C1-C6) alkylcarbonyl group, a (C1-C6) alkoxycarbonyl group, a (C1-C6) acyloxy group, a group derived from a diacid, a phosphate group;
  • Xa represents a hydrogen atom, a hydroxyl group, a (C1-C6) alkoxyl group, a (C1-C6) acyloxy group, a sulfate group;
  • - n is an integer between 10 and 3000.
  • polysaccharide is understood to mean either a polysaccharide of high molecular mass or a polysaccharide of low molecular weight.
  • high molecular weight polysaccharide is meant a polysaccharide having a molecular weight between 100 and 1000 kDa.
  • low molecular weight polysaccharide is meant a polysaccharide having a molecular mass of between 5 and 100 kDa.
  • n represents an integer between 10 and 3000, advantageously between 10 and 2000, advantageously between 10 and 1000, advantageously between 10 and 900, advantageously between 10 and 800 advantageously included Between 10 and 700.
  • n is between 10 and 700, advantageously between 50 and 700, more advantageously between 70 and 650.
  • T-saccharide unit means a saccharide unit linked to unit A or unit B by an O-glycoside type bond at position 1 (C 1) of the saccharide unit. .
  • T-galactose means a galactose linked to unit A or B by an O-glycosidic type bond.
  • (Ci-Ce) alkoxyl or (C 1 -C 6) alkoxy or (C 1 -C 6) alkyloxy group means the groups -OR-, R being a C 1 -C 6 alkyl group, that is to say a straight or branched chain comprising from 1 to 6 carbon atoms.
  • alkyls mention may be made of methyl, ethyl, propyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl and isohexyl groups.
  • Examples of (C1-C10) alkoxyls include methoxy (OCH3), ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentoxy, isopentoxy, tert-pentoxy, hexoxy, isohexoxy.
  • (C 1 -C 6) alkylcarbonyl group means -COR groups, R being a C 1 -C 6 alkyl group as defined above. Mention may be made, by way of example, of methylcarbonyl, ethylcarbonyl, n-propylcarbonyl, iso-propylcarbonyl, n-butylcarbonyl, isobutylcarbonyl, sec-butylcarbonyl, tert-butylcarbonyl, n-pentylcarbonyl, isopentylcarbonyl, neopentylcarbonyl, tert-pentylcarbonyl n-hexylcarbonyl, isohexylcarbonyl.
  • (C 1 -C 6) alkyloxycarbonyl group means COOR groups, R being a C 1 -C 6 alkyl group as defined above.
  • R being a C 1 -C 6 alkyl group as defined above.
  • methoxycarbonyl (-COOCH 3) ethoxycarbonyl
  • propoxycarbonyl isopropoxycarbonyl
  • butoxycarbonyl isobutoxycarbonyl
  • tert-butoxycarbonyl tert-butoxycarbonyl
  • pentoxycarbonyl isopentoxycarbonyl, tert-pentoxycarbonyl, hexoxycarbonyl and isohexoxycarbonyl groups.
  • (C 1 -C 6) acyloxy group means the groups - OCOR where R is a C 1 -C 6 alkyl group as defined above.
  • R is a C 1 -C 6 alkyl group as defined above.
  • group derived from a diacid means a group corresponding to the formula -COO- (CH 2) -COOH, where p is between 0 and 4.
  • diacid from which these groups are derived the groups oxalate, malonate, succinate, glutarate.
  • sulfate group a group of type (- SO3H) or -SO3 kind "within the meaning of the invention, the term” phosphate group ", a group of type (. -PO3H2).
  • the XA2 groups XA 4 XA6, XB2, XB3 and XB6 are selected independently of each other and independently for each unit A and / or B from the group comprising:
  • a pyruvate group said pyruvate group being linked to the group XA 4 by its carbon in position 2 and to the group XA6 by its carbon in position 2,
  • XA2, XB2 and XB3 are chosen from a hydrogen atom and a sulfate group
  • XA4 is chosen from a hydrogen atom, a sulfate group and a pyruvate group, said pyruvate group being bonded to the XA group 4 by its carbon in the 2-position and to the XA6 group by its carbon in the 2-position ;
  • XA6 is chosen from a hydrogen atom, a sulfate group, a T-galactose saccharide unit linked to unit A by an O-glycosidic type bond, a T-xylose saccharide unit linked to unit A by a O-glycosidic type binding, a unit-linked T-arabinose saccharide unit with an O-glycosidic type bond and a unit-linked glucuronic T-glucuronic unit with an O-glycosidic type bond, and
  • XB6 is chosen from a hydrogen atom, a sulfate group, a T-galactose saccharide unit linked by an O-glycosidic type bond to residue B1, a T-xylose saccharide unit linked by an O-glycosidic type bond to residue B1, a saccharide unit T-arabinose linked by an O-glycosidic bond to residue B1, and a saccharide unit T-glucuronic acid linked by an O-glycosidic bond to residue B1.
  • pharmaceutically acceptable salt means any addition salt with a mineral or organic acid by the action of such an acid in an organic or aqueous solvent such as an alcohol, a ketone or an ether, and which is acceptable from a pharmaceutical point of view.
  • salts By way of example of such salts, mention may be made of the following salts: a sodium salt of SC Na type, a potassium salt of SO 3 K type, benzenesulphonate, hydrobromide, hydrochloride, citrate, ethanesulphonate, fumarate, gluconate , iodate, isethionate, maleate, methanesulfonate, methylene-bis-b-oxynaphthoate, nitrate, oxalate, palmoate, phosphate, salicylate, sulfate, tartrate, theophyllinacetate, and the like. toluenesulfonate.
  • the pharmaceutically acceptable salt is a sodium salt (SC Na) or a potassium salt (SO3K).
  • the sulfated polysaccharides of the present invention have many advantages over commercially available heparins.
  • the sulphated polysaccharides according to the invention and in particular those extracted from a red alga of the Haliptilon subulatum species, exhibit an activity identical to or greater than the standard unfractionated heparin or the low molecular weight heparins. Lovenox® type on thrombosis models.
  • the sulphated polysaccharides according to the invention have a similar activity to the unfractionated standard heparin and a better activity than the heparins of low molecular weight Lovenox® type on the endogenous and exogenous pathways of the coagulation cascade, thereby preventing and treating the risks associated with vascular thrombosis.
  • these sulfated polysaccharides are obtained from algae, unlike commercially available heparins, which are obtained from animals, thus reducing production costs and the risk of viral and / or prion contamination.
  • the sulphated polysaccharide has a molecular mass less than or equal to 1000 kDa.
  • the sulphated polysaccharide according to the invention has a molecular mass less than or equal to 450 kDa, advantageously less than or equal to 400 kDa, advantageously less than or equal to 350 kDa, advantageously less than or equal to 300 kDa, advantageously less than or equal to at 250 kDa, advantageously less than or equal to 200 kDa.
  • the sulfated polysaccharide according to the invention has a molecular weight between 10 kDa and 500 kDa, advantageously between 10 kDa and 400 kDa, advantageously between 10 kDa and 300 kDa, advantageously between 10 kDa and 250 kDa.
  • the sulphated polysaccharide has a sulphate level of less than or equal to 20% by weight of the polysaccharide.
  • the sulfates content of the sulphated polysaccharide is less than or equal to 19%, advantageously less than or equal to 18%, advantageously less than or equal to 17%, advantageously less than or equal to 16%.
  • the sulphate content of the sulphated polysaccharide is between 5% and 20%, advantageously between 6% and 20%, advantageously between 7% and 19%, advantageously between 8% and 19%, advantageously between 9% and 18%. %, advantageously between 10% and 16%, advantageously between 13% and 16%.
  • the sulfated polysaccharide has a residue level of 3,6-anhydrogalactopyranose (or B2 residue) less than or equal to 5% by weight of the polysaccharide.
  • the sulphated polysaccharide has a residue level of 3,6-anhydrogalactopyranose (or residue B2) of less than or equal to 4%, advantageously less than or equal to 3%, advantageously less than or equal to 2%, advantageously less than or equal to 1, 5%.
  • the sulfated polysaccharide has a residue level of 3,6-anhydrogalactopyranose (or residue B2) less than or equal to 1, 4%.
  • the sulphated polysaccharide is of natural origin.
  • Another subject of the invention relates to a pharmaceutical composition for its use as an anticoagulant comprising at least one sulphated polysaccharide extracted from a red alga, in particular from a red algae of the species Haliptilon subulatum or a pharmaceutically acceptable salt thereof, wherein the sulfate level of said polysaccharide is less than or equal to 20% by weight of the polysaccharide, or a pharmaceutically acceptable salt thereof as defined above.
  • this pharmaceutical composition comprises at least one sulphated polysaccharide according to the invention, in combination with one or more other agents chosen from pharmaceutically acceptable excipients, active agents and food ingredients.
  • the pharmaceutical composition may be in liquid form, in particular in solution or suspension form or in gel form or in powder form.
  • pharmaceutically acceptable excipient any substance other than the active substance, intended to provide consistency, taste, color to a drug, while avoiding any interaction with the active ingredient.
  • the pharmaceutically acceptable excipient according to the invention will be chosen according to the pharmaceutical form and the desired mode of administration, from the usual excipients which are known to those skilled in the art, for administration to humans or animals.
  • Active agents means any substance other than the active substance and having a therapeutic effect. Examples that may be mentioned include anticoagulants, antibiotics, antithrombotics, anticancer agents, anti-inflammatories, antihistamines, cardiovascular drugs, antihypertensives, the present list not being limiting.
  • Food ingredients means any substance other than the active substance intended to be used in a food composition intended for humans or animals. This food ingredient responds in particular to the conditions of safety and non-toxicity associated with such use.
  • the pharmaceutical compositions comprising the sulfated polysaccharide may be administered orally, sublingually, subcutaneously, parenterally, intramuscularly, intravenously, topically, locally, intratracheally, intranasally, ocularly, intraperitoneally, by endoparietal, transdermal or rectal diffusion, or by any other route of administration.
  • Suitable forms of administration include oral forms such as tablets, soft or hard capsules, powders, granules and oral solutions or suspensions, sublingual, oral, intratracheal, intraocular, intranasal, by inhalation, topical, transdermal, subcutaneous, intramuscular or intravenous administration forms, rectal administration forms and implants.
  • the compounds according to the invention can be used in creams, oil-in-water or water-in-oil emulsions, gels, ointments, patches, solutions or lotions.
  • the pharmaceutical composition comprising at least one sulfated polysaccharide is particularly useful for preventing, reducing and treating vascular thromboses.
  • prevent or “prevention” or “prophylaxis” or “preventive treatment” or “prophylactic treatment” includes both treatment leading to the prevention of a disease and treatment that reduces and / or delays the incidence of an illness or the risk of it happening.
  • treat or “treatment” or “curative treatment” is defined as treatment leading to a cure or treatment that alleviates, improves and / or eliminates, reduces and / or stabilizes the symptoms of an illness or suffering that she provokes.
  • "Vascular thromboses” means arterial thromboses and deep vein thromboses or not.
  • the pharmaceutical composition comprising at least one sulphated polysaccharide according to the invention is particularly useful for preventing, reducing and treating arterial thromboses, deep vein thromboses or not.
  • the pharmaceutical composition comprising the sulphated polysaccharide may be administered to the patient at a daily dose of between 50 and 1000 mg, preferably between 100 and 1000 mg to prevent, reduce and treat vascular thromboses, in particular to prevent, reduce and treat arterial thromboses, deep vein thrombosis or not.
  • the pharmaceutical composition comprising the sulphated polysaccharide is administered to the patient at a daily dose of preferably between 100 and 950 mg, preferably between 100 and 900 mg, preferably between 100 and 850 mg, of preferably between 100 to 800 mg, preferably between 100 to 750 mg, preferably between 100 to 700 mg, preferably between 100 to 650 mg, preferably between 100 to 600 mg to prevent, reduce and treat the Vascular thromboses, especially to prevent, reduce and treat arterial thromboses, deep vein thrombosis or not.
  • the pharmaceutical composition comprising the sulfated polysaccharide is administered to the patient at a daily dose of between 200 to 600 mg to prevent, reduce and treat vascular thromboses, in particular to prevent, reduce and treat arterial thromboses, deep venous thromboses or not. It is, however, of course understood that those skilled in the art will adapt these doses according to the age, the weight and the pathology of the patient, particularly as a function of the thrombogenic risk.
  • the pharmaceutical composition comprising the sulfated polysaccharide can be administered to the patient by a single daily dose of between 50 and 1000 mg, preferably between 100 and 1000 mg to prevent, reduce and treat vascular thromboses, in particular to prevent, reduce and treat arterial thromboses, deep vein thrombosis or not.
  • the pharmaceutical composition comprising the sulphated polysaccharide is administered to the patient in a single daily dose, preferably in the range 100 to 950 mg, preferably in the range 100 to 900 mg, preferably in the range 100 to 850 mg.
  • the pharmaceutical composition comprising the sulfated polysaccharide is administered to the patient in a single daily dose of 200 to 600 mg to prevent, reduce and treat vascular thromboses, particularly to prevent, reduce and treat arterial thromboses. Deep venous thromboses or not.
  • the pharmaceutical composition comprising at least one sulfated polysaccharide is particularly useful for preventing, reducing and treating ischemia, pulmonary embolism, unstable angina and myocardial infarction.
  • the pharmaceutical composition comprising at least one sulphated polysaccharide is particularly useful for preventing coagulation in extracorporeal circulation and / or extra-renal purification circuits.
  • Another aspect of the invention relates to a method of treating vascular thromboses comprising administering to a patient suffering from vascular thromboses a pharmaceutical composition comprising at least one sulfated polysaccharide extracted from a marine red algae, in particular extracted from of a red alga of the species Haliptilon subulatum.
  • Another aspect of the invention relates to a method of treating ischemia, pulmonary embolism, unstable angina and myocardial infarction, comprising administering to a patient by requiring a pharmaceutical composition comprising at least one a sulphated polysaccharide extracted from a red alga, in particular extracted from a red alga of the species Haliptilon subulatum.
  • a pharmaceutical composition comprising at least one sulphated polysaccharide extracted from a red alga, in particular extracted from a red alga of the species Haliptilon subulatum.
  • administration is meant any act enabling the patient to be absorbed or introducing into the extracorporeal circulation and / or extra-renal purification circuit, the pharmaceutical composition according to the invention by any route, form or form. or mode of administration.
  • Another aspect of the present invention relates to a process for obtaining the sulfated polysaccharide.
  • the sulfated polysaccharide according to the invention can be obtained by methods that are well known to those skilled in the art.
  • the extraction of a sulfated polysaccharide of high molecular weight can in particular be obtained by a process comprising the steps described below: a) Dispersion in water of a red algae powder, in particular a powder Haliptilon subulatum previously dried;
  • the alcoholic precipitation step b) is carried out using ethanol or isopropanol.
  • the step of drying the precipitate (step c) can be carried out by lyophilization or with the aid of an oven, in particular at a temperature of between 40 ° C. and 75 ° C., advantageously 50 ° C. C overnight.
  • the method of obtaining according to the invention may be repeated several times in order to obtain a degree of purity of the satisfactory polysaccharide.
  • the extraction process according to the present invention makes it possible to obtain polysaccharides in the form of a fine creamy white powder with a production yield of the order of 10-20% relative to the dry weight of Haliptilum seaweed powder. subulatum used.
  • the low molecular weight sulfated polysaccharides are prepared by acid degradation of high molecular weight polysaccharides.
  • Sulfated polysaccharides of low molecular weight can also be obtained by depolymerization techniques well known to those skilled in the art, such as radical or enzymatic depolymerizations.
  • the process for extracting a low molecular weight sulphated polysaccharide from Haliptilon subulatum alga comprises the following steps:
  • the aqueous solution used in the dispersion step (step a) is a solution of hydrochloric acid (HCI).
  • HCI hydrochloric acid
  • the hydrochloric acid solution has a concentration of between 1M and 5M, advantageously 2M, at a temperature ranging from 50 ° C. to 100 ° C. and with stirring for 30 to 60 minutes.
  • the alcoholic precipitation step b) is carried out using ethanol or isopropanol.
  • the step of drying the precipitate (step c) is carried out by lyophilization or with the aid of an oven, in particular at a temperature of between 40 ° C. and 75 ° C., advantageously 50 ° C. for one night.
  • the sulphated polysaccharides extracted from red algae according to the invention have the advantage of not having the problems of contamination and safety, unlike the heparins available on the market which are obtained from pigs.
  • these sulfated polysaccharides offer an economic advantage.
  • the approximate yield of sulfated polysaccharides of the present invention is about 10% to 20% based on the initial dry weight of algae from which it was extracted. This high yield means a reduction in the final cost of the product compared to commercially available heparins.
  • red algae, especially of the class Floridaophyceae, especially the species Haliptilon subulatum are easy to grow which also contributes to the low cost of the final product.
  • Figure 1 Determination of the residual activity of Factor Xa according to Example 6, expressed as a percentage (%) as a function of the concentration of heparin, Lovenox®, sulfated high polysaccharide (PSHM) (213.5 kDa) and low (PSFM) (36.6 kDa) molecular weight according to the invention (PSHM and PSFM) expressed in ⁇ g mL.
  • PSHM sulfated high polysaccharide
  • PSFM low molecular weight according to the invention
  • FIG. 2 Determination of the residual activity of Factor IIa according to Example 6, expressed as a percentage (%) as a function of the concentration of heparin, Lovenox®, sulfated high polysaccharide (PSHM) (213.5 kDa) and low (PSFM) (36.6 kDa) molecular weight according to the invention expressed in ⁇ g mL.
  • FIG. 3 Determination of activated partial thromboplastin time (aPTT), according to Example 6, expressed in second (s) as a function of the concentration of heparin in Lovenox®, carrageenan Iota and sulfated high polysaccharide (PSHM). (213.5 kDa) and low (PSFM) (36.6 kDa) molecular weight according to the invention expressed in ⁇ g mL.
  • aPTT activated partial thromboplastin time
  • FIG. 4 Determination of the Time of Quick Time (PT), according to Example 6, expressed in second (s) as a function of the concentration of Lovenox® heparin, Iota carrageenan and sulfated high polysaccharide (PSHM) ( 213.5 kDa) and low (PSFM) (36.6 kDa) molecular weight according to the invention expressed in ⁇ g mL.
  • PSHM high polysaccharide
  • PSFM low
  • FIG. 5 Determination of the Thrombin Time (TT) time, according to Example 6, expressed in second (s) as a function of the concentration of Lovenox® heparin, Iota carrageenan and high sulphated polysaccharide (PSHM) ( 213.5 kDa) and low (PSFM) (36.6 kDa) molecular weight according to the invention expressed in ⁇ g mL.
  • TT Thrombin Time
  • high molecular weight polysaccharide or "PSHM” is meant a polysaccharide having a molar mass of between 100 and 1000 kDa. Extraction of the high molecular weight polysaccharides is carried out by dispersing 100 grams of Haliptilon subulatum seaweed powder in 1 liter of water at 90 ° C. with vigorous stirring (500 rpm) for 4 hours. The mixture is then filtered hot on diatom (100 g) on a sintered glass (porosity 1, more precisely 100 to 160 ⁇ ). The filtrate is then centrifuged (10000 g, 30 minutes) at room temperature to obtain the polysaccharide-enriched algal extract. The Haliptilon subulatum extract is then precipitated in 3 volumes of 96 ° ethanol (at 4 ° C.) with stirring (500 rpm) for 2 hours.
  • the precipitate is recovered by filtration on sintered glass (porosity 1 or 2, more precisely 100 to 160 ⁇ or 40 to 100 ⁇ respectively) or centrifugation (10,000 g, 30 minutes) at room temperature and then washed with acetone (50 to 100 ⁇ ). 100 mL). Then, the precipitate is recovered by filtration on sintered glass (porosity 2, more precisely 40 to 100 ⁇ ) or centrifugation (10,000 g, 30 minutes) at room temperature and then dried in an oven at 50 ° C overnight. Finally, the precipitate is ground (Blender) to obtain a fine powder of high molecular weight polysaccharides extracted from Haliptilon subulatum.
  • the yield of polysaccharides of high molecular mass thus obtained is of the order of 10 to 20% relative to the dry weight of algae powder Haliptilum subulatum used.
  • low molecular weight polysaccharide or "PSFM” is meant a polysaccharide having a molecular mass of between 5 and 100 KDa.
  • the production of low molecular weight polysaccharides is carried out by dispersing 2.5 grams of high molecular weight polysaccharide powder (Haliptilon subulatum extracts) in 125 ml of HCl (2M) at 100 ° C. with vigorous stirring (500 rpm). / min) for 1 hour. The mixture is then cooled to ambient temperature and then neutralized with sodium hydroxide (5M). The medium is precipitated in 7 volumes of ethanol 96 ° (at 4 ° C.) with stirring (500 rpm) for 2 hours.
  • high molecular weight polysaccharide powder Hydroxide
  • the precipitate is recovered by filtration on sintered glass (porosity 1 or 2, more precisely 100 to 160 ⁇ or 40 to 100 ⁇ respectively) or centrifugation (10000 g, 30 minutes) at room temperature and then washed with acetone (50 mL). Then, the precipitate is recovered by filtration on sintered glass (porosity 2, more precisely 40 to 100 ⁇ ) or centrifugation (10000 g, 30 minutes) at room temperature and then dried in an oven at 50 ° C overnight. Finally, the precipitate is ground (Blender) to obtain a fine powder of low molecular weight polysaccharides extracted from Haliptilon subulatum.
  • the yield of polysaccharides of low molecular weight thus obtained is of the order of 70% relative to the dry mass of high molecular weight polysaccharide powder and 14% relative to the dry weight of Haliptilum subulatum seaweed powder used.
  • High and low molecular weight polysaccharides are prepared according to previously described conditions (Examples 1 and 2).
  • the molecular masses of the sulphated polysaccharides are determined according to the following protocol:
  • Table 1 Summary of the weight and number average molecular weights observed for PSHM and PSFM.
  • PSFM 12840 36640 2.854 It emerges from this example that the molecular weight of polysaccharides of high molecular weight is of the order of 214 kDa and that the molecular weight of polysaccharides of low molecular weight is of the order of 37 kDa.
  • the sulphate ions released during the hydrolysis of the polysaccharides will form, in the presence of barium chloride (BaC, 2H2O) and gelatin, a precipitate of barium sulphate whose appearance is measured at 550 nm, as described in the publication by Dodson & Price (Dodgson & Price, 1962, Biochemical Journal 84: 106-1-10).
  • barium chloride BaC, 2H2O
  • 150 mg of gelatin are dissolved in 50 ml of milli-Q water at 70 ° C. After cooling for 16 hours at 4 ° C., 0.5 g of BaC is added to the gelatin solution.
  • 120 mg of freeze-dried polysaccharide are hydrolysed with 3 ml of 2 M HCl for 2 h at 100 ° C. The mixture is centrifuged at 13,000 g for 30 minutes. 1 ml of supernatant is mixed with 9 ml of milli-Q water, 1 ml of 0.5 M HCl and 0.5 ml of BaC reagent / gelatin. After 30 min at room temperature, the mixture is stirred and the absorbance read immediately at 550 nm. The standard range is performed using a stock solution of K2SO4 at 3 mg / mL.
  • the amount of sulfates was determined by the use of the colorimetric assay method developed by Jaques et al. (Jaques LB et al., 1968, Canadian Journal of Physiology and Pharmacology 46, pp. 351-360).
  • Jaques et al. Jaques LB et al., 1968, Canadian Journal of Physiology and Pharmacology 46, pp. 351-360.
  • 3-amino-7- (dimethylamino) phenothiazin-5-ium chloride (Azure A) complexes the sulphates that may be present, in particular within the polysaccharides composing the SPE fractions.
  • the assay is semi-quantitative and gives an order of magnitude ( ⁇ mg) of the sulfate concentration of a sample.
  • ⁇ mg the sulfate concentration of a sample.
  • plastic tanks for spectrophotometer are introduced 200 ⁇ of solution to be assayed.
  • 2 mL of Azar A aqueous solution at 10 mg / L are added and the sample is then stirred.
  • the quantification of sulphates is determined from the calibration range of dextran sulphate (17% sulphated) and correction of the degree of sulphation of the latter (17 mg of sulphates per 100 mg of dextran sulphate).
  • the sulfates content of polysaccharides of high molecular mass is of the order of 13.5% and that the sulfates content of low molecular weight polysaccharides is of the order of 15.7%.
  • the assay 50 to 100 ⁇ l of the polysaccharide solution to be assayed) are introduced into glass tubes. The volume is completed to 200 ⁇ using milli-Q water.
  • the resorcinol reagent is prepared extemporaneously by adding to 100 mL of 10 M HCl, 9 mL of the resorcinol solution and 1 mL of the 1/25 diluted acetaldehyde solution. This reagent is stable only 3 hours away from light.
  • the tubes 200 ml of the polysaccharide solution to be determined are added 1 ml of the resorcinol reagent. After stirring, the tubes are allowed to stand for 4 minutes and then placed in a water bath at 80 ° C. for 10 minutes. They are then transferred to an ice bath for 1 minute 30 minutes. The absorbance should be read within 15 minutes at 555 nm.
  • D-fructose (10 to 70 g / mL solutions) is used as a standard. Indeed, it has been shown that the absorbance curves at 555 nm as a function of the monosaccharide concentration of D-fructose and of 3,6-anhydrogalactose are identical (see Yaphe & Arsenaut, 1965, Analytical Biochemistry 13, pages 143-148). Table 3. Summary of 3.6-anhydroalactopyranose residue levels for PSHM.
  • the samples are evaporated under nitrogen jet, and the trimethylsilyl-O-glycoside residues are taken up in 500 ⁇ l of dichloromethane. At this stage, it is possible to dilute more or less the sample.
  • the standards (L-Rha, L-Fuc, L-Ara, D-Xyl, D-Man, D-Gal, D-GIc, D-GIcA, D-GalA) are prepared under the same conditions, at least three times. different concentrations.
  • the trimethylsilyl derivatives are analyzed by gas phase chromatography coupled to mass spectrometry, on an OPTIMA-1 MS column (30 m, 0.32 mm, 0.25 ⁇ ) with a helium flow rate of 2.3 mL / min. (up to 3 mL / min).
  • the helium pressure is set at 8.8 psi or 60673.9 Pa and the injection ratio at 25: 1 (or 50: 1).
  • the temperature rise is 8 ° C / min up to 100 ° C for 3 min.
  • the ionization is carried out by Impact Electronique (El, 70 eV), the temperature of the trap is fixed at 150 ° C and the targeted ions between 40 and 800 m / z. Table 4. Composition in monosaccharides of the PSHM sample.
  • the anticoagulant activity was studied in terms of inhibition of two key enzymes involved in coagulation: Xa and IIa factors, but also by measuring the time of Quick (PT), the thrombin time (TT) and the time of Activated cephalin TCA.
  • PT time of Quick
  • TT thrombin time
  • aPTT Activated Partial Thromboplastin Time
  • aPTT TCA
  • aPTT Activated Partial Thromboplastin Time
  • aPTT TCA
  • Activated Partial Thromboplastin Time test measures the clotting time of a dislodged citrated plasma supplemented with a particulate activator of contact factors (silica, kaolin, ellagic acid) and partial thromboplastin replacement. platelet factor III.
  • the normal aPTT varies according to the activators, commercial cephalins and devices used, usually 30 to 40 sec.
  • This semi-global test explores the endogenous pathway of coagulation (prekallikrein, high molecular weight kininogen, FXII, FXI, FVIII and FIX) and, to a lesser extent, the common final pathway (FX, FV, Fil, FI).
  • the PT (Prothrombin Time) test measures the coagulation time of the decalcified plasma, recalcified in vitro in the presence of tissue thromboplastin. It allows the global exploration of the exogenous pathway of coagulation (tissue factor pathway): thromboplastinformation, thrombinoformation and fibrinoformation. This concerns factor VII and to a lesser extent the common final path: FX, FV, Fil, FI.
  • TT Thrombin time
  • 50 ⁇ l of sulphated polysaccharide samples obtained according to the preceding examples are added to 50 ⁇ l of antithrombin III (0.125 for the test).
  • anti-Xa and 0.625 ⁇ s / ⁇ l. for the anti-11a test are added to 50 ⁇ l of antithrombin III (0.125 for the test).
  • anti-Xa and 0.625 ⁇ s / ⁇ l. for the anti-11a test were added according to the test (final concentration of 11.25 nKat / ml) before a second incubation of 30 seconds at 37 ° C.
  • Positive and negative controls are water respectively (wherein samples are diluted) and heparin non-depolymerized (Heparin, sodium salt, INTERCHIM, CAS 9041 - 08-1, batch 201274, DP av in 14.01 KDa 163 U / mg).
  • the anticoagulant activity of the sulfated polysaccharide is compared to that of a low molecular weight heparin available on the market: Lovenox® and a carrageenan iota.
  • the STart 4 Coagulometer, Test and Calibration Reagents, Positive and Negative Controls and Plasma come from the Stago Diagnostica brand.
  • the procedure for coagulation measurements is as follows:

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Abstract

The invention relates to a sulfated polysaccharide extracted from a red alga or a pharmaceutically acceptable salt thereof, in which the sulfate content is less than or equal to 20 mass % of the polysaccharide. The invention also relates to the use thereof as an anticoagulant, and to a pharmaceutical composition comprising same.

Description

Polysaccharide sulfaté extrait d'une algue rouge pour son utilisation en tant qu'anticoagulant  Sulphated polysaccharide extracted from a red algae for use as an anticoagulant
La présente invention a pour objet un polysaccharide sulfaté extrait d'une algue rouge pour son utilisation en tant qu'anticoagulant. The present invention relates to a sulfated polysaccharide extracted from a red algae for use as an anticoagulant.
La coagulation sanguine est réalisée selon des étapes en cascade impliquant différentes proenzymes et procofacteurs présents dans le sang qui sont convertis, par l'intermédiaire d'enzymes protéolytiques, en leur forme activée. Cette succession d'étapes ou cascade de la coagulation est effectuée selon trois systèmes de la coagulation, appelés respectivement, voie extrinsèque de la coagulation, voie intrinsèque de la coagulation et voie commune, conduisant à la transformation de la prothrombine (Fil) en thrombine (Fila). La voie extrinsèque implique l'intervention du facteur VII présent dans le sang. Toutefois, celui-ci requiert une activation (facteur Vlla) pour initier cette cascade de coagulation. Le facteur Vlla présente une faible activité enzymatique jusqu'à ce qu'il se complexe aux facteurs tissulaires de nature phospholipidique libérés après lésion tissulaire. Le facteur Vlla ainsi complexé transforme le facteur X (FX) en facteur Xa (FXa) en présence d'ions calcium. Le facteur Xa à son tour transforme la prothrombine en thrombine, laquelle active le facteur V (Facteur Va). La thrombine active également le facteur XIII (facteur XI I la). La thrombine, en présence de calcium, de facteurs tissulaires, de facteur Va, agit sur le fibrinogène en le transformant en fibrine. La présence de facteur XI lia permet la formation d'un caillot de fibrine à réseau maillé solide et adhérent qui est progressivement et lentement résorbé avec l'installation du tissu cicatriciel de consolidation, auquel le réseau sert de trame. Cette fibrine réticulée est insoluble et n'est pas attaquable par les enzymes fibrinolytiques, au moins durant le temps de mise en place du tissu cicatriciel. La voie intrinsèque de la coagulation implique également le facteur Vlla (FVIIa). Cette voie comprend une cascade de réactions aboutissant à l'activation de la thrombine par l'intermédiaire du facteur XII (FXII). Celui-ci active le facteur XI (Facteur Xla - FXIa) qui active le facteur IX (facteur IXa - FIXa) en présence de facteurs tissulaires phospholipidiques (FT). Le facteur IXa participe à l'activation du facteur X en facteur Xa en présence de facteur Villa, des facteurs tissulaires et d'ions calcium. Ceci conduit ensuite à la transformation de la prothrombine (Fil) en thrombine (Fila). Blood coagulation is performed in cascading steps involving different proenzymes and procofactors in the blood that are converted, via proteolytic enzymes, into their activated form. This succession of steps or cascade of coagulation is carried out according to three coagulation systems, respectively called extrinsic coagulation pathway, intrinsic pathway of coagulation and common pathway, leading to the transformation of prothrombin (Thread) into thrombin ( Fila). The extrinsic pathway involves the intervention of factor VII present in the blood. However, this requires activation (factor VIIa) to initiate this coagulation cascade. Factor VIIa exhibits low enzymatic activity until complexed with tissue factors of phospholipidic nature released after tissue injury. The thus complexed factor VIIa transforms factor X (FX) into factor Xa (FXa) in the presence of calcium ions. Factor Xa in turn transforms prothrombin into thrombin, which activates factor V (Factor Va). Thrombin also activates factor XIII (factor XI Ia). Thrombin, in the presence of calcium, tissue factors, factor Va, acts on fibrinogen by transforming it into fibrin. The presence of factor XI 11a allows the formation of a solid and adherent meshed fibrin clot which is progressively and slowly resorbed with the installation of the healing scar tissue, to which the network serves as a weft. This crosslinked fibrin is insoluble and is not attackable by fibrinolytic enzymes, at least during the time of placement of scar tissue. The intrinsic pathway of coagulation also involves the factor VIIa (FVIIa). This pathway comprises a cascade of reactions resulting in the activation of thrombin via factor XII (FXII). This activates factor XI (factor Xla - FXIa) which activates factor IX (factor IXa - FIXa) in the presence of phospholipid tissue factors (TF). Factor IXa participates in factor Xa factor X activation in the presence of Villa factor, tissue factors and calcium ions. This then leads to the transformation of prothrombin (Fil) into thrombin (Fila).
La voie intrinsèque de la coagulation implique le facteur Xa (FXa). Le facteur Xa adsorbé à la surface des phospholipides d'origine tissulaire et plaquettaire va, en présence du facteur Va, constituer la prothrombinase. Le facteur Va provient du facteur V activé par la thrombine (Fil). La prothrombinase est donc un complexe enzymatique faisant intervenir le FXa, le FVa, le calcium et des phospholipides. Il existe donc une similitude avec le complexe activateur du FX. La prothrombinase permet la formation de thrombine (Fila) à partir de prothrombine (Fil). The intrinsic pathway of coagulation involves factor Xa (FXa). Factor Xa adsorbed on the surface of phospholipids of tissue and platelet origin will, in the presence of the factor Go, make up the prothrombinase. Factor Va is derived from thrombin-activated factor V (Thread). Prothrombinase is therefore an enzymatic complex involving FXa, FVa, calcium and phospholipids. There is therefore a similarity with the FX activator complex. Prothrombinase allows the formation of thrombin (Fila) from prothrombin (Fil).
Il en résulte donc que les facteurs X et II sont les facteurs essentiels de la cascade de la coagulation, dans la mesure où ces deux facteurs de la coagulation conduisent à la formation du caillot de fibrine.  It follows therefore that factors X and II are the essential factors of the coagulation cascade, insofar as these two factors of coagulation lead to the formation of the fibrin clot.
La thrombose vasculaire se définit par l'obstruction d'une veine ou d'une artère par un thrombus (ou caillot). On distingue deux types de thrombose : la thrombose veineuse ou phlébite : elle peut être superficielle affectant une veine de surface ou profonde, affectant une veine profonde, notamment au niveau de la cuisse. Vascular thrombosis is defined as the obstruction of a vein or artery by a thrombus (or clot). There are two types of thrombosis: venous thrombosis or phlebitis: it can be superficial affecting a deep vein or deep, affecting a deep vein, especially in the thigh.
la thrombose artérielle obstruant une artère, qui se révèle plus grave si l'artère en cause est la seule à irriguer une zone précise du corps.  Arterial thrombosis obstructing an artery, which is more serious if the artery in question is the only one to irrigate a specific area of the body.
Le traitement principalement utilisé pour éviter les risques de thromboses vasculaires, consiste en l'administration d'anticoagulants. The main treatment used to prevent the risk of vascular thrombosis consists in the administration of anticoagulants.
Un anticoagulant est une substance synthétique ou naturelle ayant la propriété d'inhiber la coagulabilité naturelle du sang. L'anticoagulant a donc pour mission d'éviter la formation de caillot de fibrine en inhibant un ou plusieurs facteurs de la cascade de la coagulation. Les anticoagulants disponibles sur le marché aujourd'hui sont scindés en deux grandes catégories : les anticoagulants oraux et les anticoagulants injectables. Parmi les anticoagulants oraux, on trouve notamment les antivitamines K (AVK), les inhibiteurs directs de la thrombine (anti-lla) et les inhibiteurs directs du facteur Xa. Les antivitamines K actuellement sur le marché sous forme orales sont les coumariniques et les dérivés de l'indanedione. Les coumariniques regroupent l'acénocoumarol, commercialisé sous le nom de Sintrom® et Minisintrom® et la warfarine commercialisée sous le nom de Coumadine®. Les dérivés de l'indanedione comprennent notamment la fluindione, commercialisée sous le nom de Previscan®. Ces antivitamines K sont indiquées dans la prévention des complications thrombo-emboliques des cardiopathies emboligènes et des infarctus du myocarde compliqués, ainsi que dans le traitement de thromboses veineuses profondes et des embolies pulmonaires, ainsi que dans la prévention de leurs récidives. Toutefois, ces anticoagulants oraux présentent de nombreux effets secondaires, comme notamment des risques d'hémorragies cérébrales, abdominales et intra-articulaires, des troubles digestifs, des risques de nécroses cutanées et d'éruptions cutanées allergiques. An anticoagulant is a synthetic or natural substance with the property of inhibiting the natural coagulability of blood. The anticoagulant therefore has the mission of avoiding the formation of fibrin clot by inhibiting one or more factors of the cascade of coagulation. Anticoagulants available on the market today are divided into two broad categories: oral anticoagulants and injectable anticoagulants. Oral anticoagulants include vitamin K antagonists (AVK), direct thrombin inhibitors (anti-ILa) and direct factor Xa inhibitors. The vitamin K antagonists currently on the market in oral form are coumarinics and derivatives of indanedione. Coumarinic products include acenocoumarol, marketed under the name Sintrom® and Minisintrom®, and warfarin marketed under the name Coumadine®. Derivatives of indanedione include fluindione, marketed under the name Previscan®. These vitamin K antagonists are indicated in the prevention of thromboembolic complications of emboligenic heart disease and complicated myocardial infarction, as well as in the treatment of deep vein thromboses and pulmonary embolisms, as well as in the prevention of their recurrence. However, these oral anticoagulants have many side effects, such as risk of cerebral, abdominal and intra-articular hemorrhages, digestive disorders, risk of cutaneous necrosis and allergic rashes.
Parmi les anticoagulants injectables, on trouve notamment les héparines standards non fractionnées et les héparines de faibles masses moléculaires. L'héparine est un polysaccharide sulfaté constitué d'unités de glucosamine et d'acides uroniques liées en 1 - 4, dans lesquelles les groupes sulfates sont présents sur la fonction aminée de la glucosamine et/ou sur des fonctions alcools de la glucosamine et de l'acide uronique. Ce polysaccharide, dont les propriétés anticoagulantes sont bien connues, est actuellement largement utilisé dans le traitement des accidents thrombotiques. L'héparine présente toutefois des effets secondaires très importants (saignements, risques de thrombopénies immuno-allergiques) et elle est très peu efficace en thrombose artérielle. De plus, l'origine animale de ce produit est susceptible d'entraîner un risque potentiel de contamination par des agents infectieux non conventionnels. Des techniques de dépolymérisations chimique ou enzymatique ont permis d'obtenir, à partir d'héparine Non Fractionnée (HNF), dont la masse moléculaire est d'environ 15 kDa, des chaînes polysaccharidiques de faibles masses moléculaires, à savoir de masses moléculaire comprise entre 2 et 10 kDa, nommées Héparines de faibles masses moléculaires ou « Héparines de Bas Poids Moléculaire » (HBPM). De nombreuses HBPM sont actuellement sur le marché notamment : Enoxaparine sodique, commercialisée notamment sous le nom de Lovenox®, Dalteparine sodique, commercialisée notamment sous le nom de Fragmine®, Nadroparine sodique, commercialisée notamment sous le nom de Fraxiparine® ou de Fraxodi®, Tinzaparine, commercialisée notamment sous le nom d'Innohep®, Reviparine, Parnaparine, etc. Injectable anticoagulants include unfractionated standard heparins and low molecular weight heparins. Heparin is a sulfated polysaccharide consisting of 1-4-linked glucosamine and uronic acid units, in which the sulfate groups are present on the amino function of glucosamine and / or on the alcohol functions of glucosamine and uronic acid. This polysaccharide, whose anticoagulant properties are well known, is currently widely used in the treatment of thrombotic events. Heparin, however, has very significant side effects (bleeding, risk of immuno-allergic thrombocytopenia) and is very effective in arterial thrombosis. In addition, the animal origin of this product is likely to result in a potential risk of contamination by unconventional infectious agents. Chemical or enzymatic depolymerization techniques have made it possible to obtain, from unfractionated heparin (UFH), the molecular mass of which is approximately 15 kDa, polysaccharide chains of low molecular weight, namely molecular masses between 2 and 10 kDa, called Low Molecular Weight Heparins or Low Molecular Weight Heparins (LMWH). Many LMWHs are currently on the market including: Enoxaparin sodium, marketed in particular under the name of Lovenox®, Dalteparin sodium, marketed in particular under the name of Fragmine®, Nadroparin sodium, sold in particular under the name of Fraxiparine® or Fraxodi®, Tinzaparin, sold in particular under the name Innohep®, Reviparin, Parnaparin, etc.
Des études cliniques ont montré que, dans la prophylaxie des accidents thrombo- emboliques veineux, les héparines de faibles masses moléculaires possèdent une efficacité identique, sinon supérieure, à celle de l'héparine non fractionnée. Toutefois, les héparines de faible masse moléculaire (HBPM) n'abolissent pas le risque hémorragique et peuvent entraîner, au même titre que l'héparine non fractionnée, quoique avec une moindre fréquence, une thrombopénie immuno-allergique et de l'ostéoporose. De plus, les héparines sont extraites industriellement d'intestin de porc (Europe) ou de poumon de bœuf. Les abats d'autres mammifères d'élevage peuvent être utilisés comme sources secondaires. Du fait de son origine animale, les risques sanitaires ne peuvent pas être exclus. Par conséquent, il apparaît nécessaire de disposer de nouveaux anticoagulants d'origine naturelle et non animale, susceptibles d'être administrés par voie orale et/ou injectable, et capables de prévenir et/ou de traiter efficacement les thromboses vasculaires tout en diminuant les risques d'effets secondaires et en éliminant les risques de contamination. Les inventeurs ont ainsi montré de manière surprenante que les polysaccharides sulfatés extraits d'algues rouges, et en particulier les polysaccharides sulfatés extraits à partir de l'algue marine rouge de l'espèce Haliptilon subulatum avaient une activité anticoagulante similaire à l'héparine standard non fractionnée, voire même supérieure à l'héparine standard non fractionnée et aux héparines de faibles masses moléculaires. La demande internationale WO2014/76261 décrit une composition comprenant au moins un polysaccharide sulfaté et au moins un ingrédient alimentaire pour le traitement ou la prévention d'une infection provoquée par au moins une microsporidie chez l'Homme ou l'animal. Ces polysaccharides sulfatés sont extraits d'algues vertes, brunes, rouges et de cyanobactéries. La demande internationale WO 01/15654 décrit un polysaccharide sulfaté de masse molaire inférieure ou égale à 10 kDa, susceptible d'être obtenu par dépolymérisation radicalaire d'un fucane issu de Phéophycées, pour l'obtention d'un médicament actif contre la thrombose artérielle et contre la resténose artérielle. Ce polysaccharide sulfaté est extrait d'algues brunes. Aucun de ces documents ne concerne de polysaccharides sulfatés extraits d'algues rouges de l'espèce Haliptilon subulatum pour la prévention et/ou le traitement des thromboses vasculaires, ni même la prévention et/ou le traitement des ischémies, des embolies pulmonaires, de l'angor instable, de l'infarctus du myocarde. De plus, aucun de ces documents ne concerne la prévention de l'anticoagulation des circuits de circulation extracorporelle et/ou d'épuration extra-rénale. Clinical studies have shown that in the prophylaxis of venous thromboembolic events, low molecular weight heparins have the same, if not higher, efficacy as unfractionated heparin. However, low molecular weight heparins (LMWHs) do not abolish the risk of bleeding and may, as with unfractionated heparin, but with lesser frequency, lead to immunoallergic thrombocytopenia and osteoporosis. In addition, heparins are industrially extracted from pork intestine (Europe) or from beef lung. Offal of other farmed mammals may be used as secondary sources. Because of its animal origin, health risks can not be excluded. Therefore, it appears necessary to have novel anticoagulants of natural and non-animal origin, capable of being administered orally and / or injectably, and capable of preventing and / or effectively treating vascular thromboses while reducing the risks. side effects and eliminating the risk of contamination. The inventors have thus surprisingly shown that the sulphated polysaccharides extracted from red algae, and in particular the sulphated polysaccharides extracted from the red marine algae of the species Haliptilon subulatum, had anticoagulant activity similar to the standard heparin no fractionated or even higher than standard unfractionated heparin and low molecular weight heparins. The international application WO2014 / 76261 describes a composition comprising at least one sulphated polysaccharide and at least one food ingredient for the treatment or prevention of an infection caused by at least one microsporidium in humans or animals. These sulfated polysaccharides are extracted from green algae, browns, reds and cyanobacteria. The international application WO 01/15654 describes a sulphated polysaccharide of molar mass less than or equal to 10 kDa, obtainable by radical depolymerization of a fucan derived from Pheophyceae, in order to obtain a medicament active against arterial thrombosis. and against arterial restenosis. This sulphated polysaccharide is extracted from brown algae. None of these documents relates to sulfated polysaccharides extracted from red algae of the species Haliptilon subulatum for the prevention and / or treatment of vascular thromboses, or even the prevention and / or treatment of ischemia, pulmonary embolism, pulmonary embolism, unstable angina, myocardial infarction. Moreover, none of these documents relates to the prevention of anticoagulation of extracorporeal circulation circuits and / or extra-renal purification.
Ainsi, l'invention porte donc sur un nouvel anticoagulant à base de polysaccharide sulfaté extrait d'une algue rouge de l'espèce Haliptilon subulatum ou d'un sel pharmaceutiquement acceptable de celui-ci. Thus, the invention thus relates to a novel sulfated polysaccharide-based anticoagulant extracted from a red alga of the species Haliptilon subulatum or a pharmaceutically acceptable salt thereof.
Les polysaccharides issus des algues rouges sont construits sur la base d'un enchaînement linéaires d'unités 3-/3-galactopyranose et 4-a-galactopyranose alternant régulièrement. L'unité /3-galactopyranose est toujours de configuration D, alors que l'unité a- galactopyranose est de configuration D chez les carraghénanes et L chez les agarocolloïdes. Par ailleurs, une partie des résidus 4-a-galactopyranoses peut exister sous la forme de 3,6-anhydrogalactopyranose. La forme 3,6-anhydrogalactopyranose est obtenue par une élimination de l'ester sulfate porté par le carbone 6 de l'unité a-galactose liée en 4, sous l'action de galactose-6-sulfurylases lors de la biosynthèse ou d'un traitement alcalin. L'invention a donc pour objet un polysaccharide sulfaté extrait à partir d'une algue rouge de l'espèce Haliptilon subulatum ou un sel pharmaceutiquement acceptable de celui-ci, dans lequel le taux de sulfate dudit polysaccharide est inférieur ou égal à 20% en masse du polysaccharide, pour son utilisation en tant qu'anticoagulant. Polysaccharides from red algae are constructed on the basis of a linear sequence of 3- / 3-galactopyranose and 4-a-galactopyranose units alternating regularly. The β-galactopyranose unit is always of D configuration, whereas the α-galactopyranose unit is of D configuration in carrageenans and L in agarocolloids. In addition, some of the 4-a-galactopyranose residues may exist under the form of 3,6-anhydrogalactopyranose. The 3,6-anhydrogalactopyranose form is obtained by removal of the sulfate ester carried by the carbon 6 of the 4-linked α-galactose unit, under the action of galactose-6-sulfurylases during the biosynthesis or an alkaline treatment. The subject of the invention is therefore a sulphated polysaccharide extracted from a red alga of the Haliptilon subulatum species or a pharmaceutically acceptable salt thereof, in which the sulphate level of said polysaccharide is less than or equal to 20% by weight. mass of the polysaccharide for its use as an anticoagulant.
Il est connu que le taux de sulfatation ainsi que la distribution des sulfates sur les polysaccharides peuvent avoir un effet critique sur l'interaction entre les protéases, les inhibiteurs et les activateurs de la cascade de la coagulation, et notamment sur l'activité pro- ou anticoagulante des polysaccharides (voir Fonseca et al, Thrombosis and Haemostasis, 2008, vol 99(3), pages 539-45). It is known that the sulfation rate as well as the distribution of the sulphates on the polysaccharides can have a critical effect on the interaction between the proteases, the inhibitors and the activators of the coagulation cascade, and in particular on the activity or anticoagulant polysaccharides (see Fonseca et al, Thrombosis and Haemostasis, 2008, Vol 99 (3), pages 539-45).
Les inventeurs ont donc montré de manière surprenante que les polysaccharides extraits à partir d'une algue rouge de l'espèce Haliptilon subulatum et ayant un taux de sulfate inférieur ou égal à 20% présentent une activité anticoagulante similaire à l'héparine standard non fractionnée, voire même supérieure à l'héparine standard non fractionnée et aux héparines de faibles masses moléculaires, The inventors have thus surprisingly shown that polysaccharides extracted from a red alga of the Haliptilon subulatum species and having a sulphate level of less than or equal to 20% exhibit anticoagulant activity similar to unfractionated standard heparin, even higher than standard unfractionated heparin and low molecular weight heparins,
Dans un mode de réalisation particulier de l'invention, le polysaccharide sulfaté est extrait à partir d'une algue rouge, avantageusement à partir d'une algue marine rouge, avantageusement une algue marine rouge de la classe des Florideophyceaea, encore plus avantageusement d'une algue marine rouge de l'espèce Haliptilon subulatum. Dans un mode de réalisation particulièrement avantageux de l'invention, le polysaccharide sulfaté est extrait à partir d'une algue rouge de l'espèce Haliptilon subulatum. Dans un mode de réalisation particulier, le polysaccharide sulfaté selon l'invention, dans lequel le taux de sulfate dudit polysaccharide est inférieur ou égal à 20% en poids masse du polysaccharide, répond à la formule (I) : In a particular embodiment of the invention, the sulphated polysaccharide is extracted from a red alga, advantageously from a red marine alga, advantageously a red marine alga from the class of Floridaophyceaea, even more advantageously from a red marine alga of the species Haliptilon subulatum. In a particularly advantageous embodiment of the invention, the sulphated polysaccharide is extracted from a red algae of the species Haliptilon subulatum. In a particular embodiment, the sulphated polysaccharide according to the invention, in which the sulphate level of said polysaccharide is less than or equal to 20% by mass of the polysaccharide, corresponds to formula (I):
[(unité A)-(unité B)]n (I), dans laquelle : [(unit A) - (unit B)] n (I), wherein:
- l'unité A est un 3-/3-D-galactopyranose, dans lequel les fonctions hydroxyles libres sont substituées par un ou plusieurs groupements choisis parmi XA2, XA4, XA6, l'unité B est choisie dans le groupe constitué du résidu B1 et du résidu B2 :the unit A is a 3β-D-galactopyranose, in which the free hydroxyl functions are substituted with one or more groups chosen from XA 2 , XA 4 , XA 6 , unit B is selected from the group consisting of residue B1 and residue B2:
- le résidu B1 étant un 4-a-D/L-galactopyranose, dans lequel les fonctions hydroxyles libres sont substituées par un ou plusieurs groupements choisis parmi, XB2, XB3 et XBe et, the residue B1 being a 4-aD / L-galactopyranose, in which the free hydroxyl functions are substituted with one or more groups chosen from X B 2, XB 3 and X B e and,
- le résidu B2 étant un 4-a-3,6-anhydrogalactopyranose, dans lequel les fonctions hydroxyles libres du 4-a-3,6-anhydrogalactopyranose sont substituées par un groupement XB2 et,  the residue B2 being a 4-a-3,6-anhydrogalactopyranose, in which the free hydroxyl functions of 4-a-3,6-anhydrogalactopyranose are substituted with an XB2 group and,
- les résidus B1 et B2 étant distribués de manière aléatoire dans le polysaccharide et le résidu B2 représentant au plus 5% en masse du polysaccharide,  the residues B1 and B2 being distributed randomly in the polysaccharide and the residue B2 representing at most 5% by weight of the polysaccharide,
- l'unité A est reliée à l'unité B par une liaison O-glycosidique entre le carbone en position 1 de l'unité A et le carbone en position 4 de l'unité B et,  unit A is connected to unit B by an O-glycosidic bond between the carbon at position 1 of unit A and the carbon at position 4 of unit B and,
- l'unité B est reliée à l'unité A par une liaison O-glycosidique entre le carbone en position 1 de l'unité B et le carbone en position 3 de l'unité A,  unit B is connected to unit A by an O-glycosidic bond between the carbon at position 1 of unit B and the carbon at position 3 of unit A,
- XA2, XA4, XA6, XB2, XB3 et XB6 sont choisis indépendamment les uns des autres et indépendamment pour chaque unité A et/ou B dans le groupe comprenant :  XA2, XA4, XA6, XB2, XB3 and XB6 are independently selected from each other and independently for each A and / or B unit in the group consisting of:
- un atome d'hydrogène;  a hydrogen atom;
- un groupement sulfate,  a sulphate group,
- un groupement pyruvate (-COO-CO-CH3), ledit groupement pyruvate étant lié au groupement XA4 par son carbone en position 1 et au groupement XA6 par son carbone en position 3; a pyruvate group (-COO-CO-CH3), said pyruvate group being linked to the XA group 4 by its carbon in position 1 and to the group XA6 by its carbon in position 3;
- une unité saccharidique liée à l'unité A ou B par une liaison de type O- glycosidique en position 1 (Ci ) de l'unité saccharidique, l'unité saccharidique étant choisie parmi un galactose (ou T-galactose), un xylose (ou T-xylose), un arabinose (ou T-arabinose) et un acide glucuronique (ou T- acide glucuronique); et  a saccharide unit linked to unit A or B by an O-glycoside type bond at position 1 (Ci) of the saccharide unit, the saccharide unit being chosen from a galactose (or T-galactose), a xylose (or T-xylose), an arabinose (or T-arabinose) and a glucuronic acid (or T-glucuronic acid); and
- un groupe (C1-C6) alcoxyle, un groupe (C1-C6) alkylcarbonyle, un groupe (C1-C6) alkoxycarbonyle, un groupe (C1-C6) acyloxy, un groupement issu d'un diacide, un groupement phosphate ;  a (C1-C6) alkoxyl group, a (C1-C6) alkylcarbonyl group, a (C1-C6) alkoxycarbonyl group, a (C1-C6) acyloxy group, a group derived from a diacid, a phosphate group;
- un groupement -ChbXa, dans lequel Xa représente un atome d'hydrogène, un groupe hydroxy, un groupe (C1-C6) alcoxyle, un groupe (C1-C6) acyloxy, un groupement sulfate ;  a group -ChbXa, in which Xa represents a hydrogen atom, a hydroxyl group, a (C1-C6) alkoxyl group, a (C1-C6) acyloxy group, a sulfate group;
- n est un entier compris entre 10 et 3000.  - n is an integer between 10 and 3000.
Les formules (la) et (Ib) ci-dessous illustrent respectivement les motifs (unité A) - (résidu B1 ) et (unité A) - (résidu B2) : (la) The formulas (Ia) and (Ib) below respectively illustrate the units (unit A) - (residue B1) and (unit A) - (residue B2): (the)
Figure imgf000008_0001
Figure imgf000008_0001
Au sens de la présente invention, on entend par « polysaccharide » aussi bien un polysaccharide de haute masse moléculaire ou un polysaccharide de faible masse moléculaire. Par «polysaccharide de haute masse moléculaire», on entend un polysaccharide ayant une masse moléculaire comprise entre 100 et 1 000 kDa. Par «polysaccharide de faible masse moléculaire», on entend un polysaccharide ayant une masse moléculaire comprise entre 5 et 100 kDa. For the purposes of the present invention, the term "polysaccharide" is understood to mean either a polysaccharide of high molecular mass or a polysaccharide of low molecular weight. By "high molecular weight polysaccharide" is meant a polysaccharide having a molecular weight between 100 and 1000 kDa. By "low molecular weight polysaccharide" is meant a polysaccharide having a molecular mass of between 5 and 100 kDa.
Au sens de la présente invention, « n » représente un nombre entier compris entre 10 et 3000, avantageusement compris entre 10 et 2000, avantageusement compris entre 10 et à 1000, avantageusement compris entre 10 et 900, avantageusement compris entre 10 et 800 avantageusement compris entre 10 et 700. De manière avantageuse, « n » est compris entre 10 et 700, avantageusement entre 50 et 700, de manière plus avantageuse entre 70 et 650. Au sens de la présente invention, on entend par « T-unité saccharidique », une unité saccharidique liée à l'unité A ou l'unité B par une liaison de type O-glycosidique en position 1 (Ci) de l'unité saccharidique. Ainsi, on entend respectivement par « T-galactose », « T- xylose », « T-arabinose » et « T-acide glucuronique », un galactose lié à l'unité A ou B par une liaison de type O-glycosidique en position 1 (Ci) du galactose, un xylose lié à l'unité A ou B par une liaison de type O-glycosidique en position 1 (Ci) du xylose, un arabinose lié à l'unité A ou B par une liaison de type O-glycosidique en position 1 (Ci) de l'arabinose et un acide glucuronique lié à l'unité A ou B par une liaison de type O-glycosidique en position 1 (Ci) de l'acide glucuronique. For the purposes of the present invention, "n" represents an integer between 10 and 3000, advantageously between 10 and 2000, advantageously between 10 and 1000, advantageously between 10 and 900, advantageously between 10 and 800 advantageously included Between 10 and 700. Advantageously, "n" is between 10 and 700, advantageously between 50 and 700, more advantageously between 70 and 650. For the purposes of the present invention, the term "T-saccharide unit" means a saccharide unit linked to unit A or unit B by an O-glycoside type bond at position 1 (C 1) of the saccharide unit. . Thus, the term "T-galactose", "T-xylose", "T-arabinose" and "T-glucuronic acid" respectively means a galactose linked to unit A or B by an O-glycosidic type bond. position 1 (Ci) of galactose, a xylose linked to unit A or B by an O-glycoside type bond in position 1 (Ci) of xylose, an arabinose linked to unit A or B by a binding of type O-glycoside in position 1 (Ci) of arabinose and a glucuronic acid linked to unit A or B by an O-glycoside bond at position 1 (Ci) of glucuronic acid.
Au sens de la présente invention, on entend par groupe (Ci-Ce) alcoxyle ou (Ci-Ce) aikoxyle ou (Ci-Ce) alkyloxy, les groupes -OR-, R étant un groupe alkyle en C1-C6, c'est-à-dire une chaîne droite ou ramifiée comprenant de 1 à 6 atomes de carbone. A titre d'exemples d'alkyles, on peut citer les groupes méthyle, éthyle, propyle, butyle, isobutyle, tert-butyle, pentyle, isopentyle, tert-pentyle, hexyle et isohexyle. On peut citer à titre d'exemples de (Ci-Ce) alcoxyles, les groupes méthoxy (OCH3), éthoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentoxy, isopentoxy, tert-pentoxy, hexoxy, isohexoxy. For the purposes of the present invention, the term "(Ci-Ce) alkoxyl or (C 1 -C 6) alkoxy or (C 1 -C 6) alkyloxy group means the groups -OR-, R being a C 1 -C 6 alkyl group, that is to say a straight or branched chain comprising from 1 to 6 carbon atoms. As examples of alkyls, mention may be made of methyl, ethyl, propyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl and isohexyl groups. Examples of (C1-C10) alkoxyls include methoxy (OCH3), ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentoxy, isopentoxy, tert-pentoxy, hexoxy, isohexoxy.
Au sens de la présente invention on entend par groupe (C1-C6) alkylcarbonyle, les groupes -COR, R étant un groupe alkyle en Ci-Ce tel que défini précédemment. On peut citer à titre d'exemples les groupes methylcarbonyle, ethylcarbonyle, n-propylcarbonyle, iso-propylcarbonyle, n-butylcarbonyle, iso-butylcarbonyle, sec-butylcarbonyle, tert- butylcarbonyle, n-pentylcarbonyle, iso-pentylcarbonyle, neo-pentylcarbonyle, tert- pentylcarbonyle n-hexylcarbonyle, iso-hexylcarbonyle. Au sens de la présente invention, on entend par groupe (C1-C6) aikoxycarbonyle, les groupes COOR, R étant un groupe alkyle en Ci-Ce tel que défini précédemment. On peut citer à titre d'exemples les groupes méthoxycarbonyle (-COOCH3), éthoxycarbonyle, propoxycarbonyle, isopropoxycarbonyle, butoxycarbonyle, isobutoxycarbonyle, tert- butoxycarbonyle, pentoxycarbonyle, isopentoxycarbonyle, tert-pentoxycarbonyle, hexoxycarbonyle, isohexoxycarbonyle. For the purposes of the present invention, the term "(C 1 -C 6) alkylcarbonyl group" means -COR groups, R being a C 1 -C 6 alkyl group as defined above. Mention may be made, by way of example, of methylcarbonyl, ethylcarbonyl, n-propylcarbonyl, iso-propylcarbonyl, n-butylcarbonyl, isobutylcarbonyl, sec-butylcarbonyl, tert-butylcarbonyl, n-pentylcarbonyl, isopentylcarbonyl, neopentylcarbonyl, tert-pentylcarbonyl n-hexylcarbonyl, isohexylcarbonyl. For the purposes of the present invention, the term "(C 1 -C 6) alkyloxycarbonyl group" means COOR groups, R being a C 1 -C 6 alkyl group as defined above. By way of example, mention may be made of methoxycarbonyl (-COOCH 3), ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl, pentoxycarbonyl, isopentoxycarbonyl, tert-pentoxycarbonyl, hexoxycarbonyl and isohexoxycarbonyl groups.
Au sens de la présente invention, on entend par groupe (C1-C6) acyloxy, les groupes - OCOR où R est un groupe alkyle en Ci-Ce tel que défini précédemment. On peut citer à titre d'exemple les groupes acétyloxy (-OCOCH3), propionyloxy. Au sens de la présente invention, on entend par groupement issu d'un diacide, un groupement répondant à la formule -COO -(CH2) -COOH, où p est compris entre 0 et 4. On peut citer à titre d'exemple de diacide dont sont issus ces groupements : les groupes oxalate, malonate, succinate, glutarate. For the purposes of the present invention, the term "(C 1 -C 6) acyloxy group" means the groups - OCOR where R is a C 1 -C 6 alkyl group as defined above. By way of example, mention may be made of acetyloxy (-OCOCH 3) and propionyloxy groups. For the purposes of the present invention, the term "group derived from a diacid" means a group corresponding to the formula -COO- (CH 2) -COOH, where p is between 0 and 4. By way of example, diacid from which these groups are derived: the groups oxalate, malonate, succinate, glutarate.
Au sens de l'invention, on entend par « groupement sulfate », un groupement du type (- SO3H) ou de type -SO3". Au sens de l'invention, on entend par « groupement phosphate », un groupement du type (-PO3H2). For the purposes of the invention, the term "sulfate group", a group of type (- SO3H) or -SO3 kind "within the meaning of the invention, the term" phosphate group ", a group of type (. -PO3H2).
Dans un mode de réalisation avantageux, les groupements XA2, XA4, XA6, XB2, XB3 et XB6 sont choisis indépendamment les uns des autres et indépendamment pour chaque unité A et/ou B dans le groupe comprenant : In an advantageous embodiment, the XA2 groups XA 4 XA6, XB2, XB3 and XB6 are selected independently of each other and independently for each unit A and / or B from the group comprising:
- un atome d'hydrogène,  a hydrogen atom,
- un groupement sulfate,  a sulphate group,
- un groupement pyruvate, ledit groupement pyruvate étant lié au groupement XA4 par son carbone en position 2 et au groupement XA6 par son carbone en position 2,a pyruvate group, said pyruvate group being linked to the group XA 4 by its carbon in position 2 and to the group XA6 by its carbon in position 2,
- une unité saccharidique liée à l'unité A ou B par une liaison de type O-glycosidique en position 1 (Ci) de l'unité saccharidique et choisie parmi un T-galactose, un T- xylose, un T-arabinose et un T-acide glucuronique. a saccharide unit linked to unit A or B by an O-glycoside type bond at position 1 (Ci) of the saccharide unit and chosen from a T-galactose, a T-xylose, a T-arabinose and a T-glucuronic acid.
Dans un mode de réalisation plus avantageux, In a more advantageous embodiment,
- XA2, XB2 et XB3 Sont choisis parmi un atome d'hydrogène et un groupement sulfate;XA2, XB2 and XB3 are chosen from a hydrogen atom and a sulfate group;
- XA4 est choisi parmi un atome d'hydrogène, un groupement sulfate et un groupement pyruvate, ledit groupement pyruvate étant lié au groupement XA4 par son carbone en position 2 et au groupement XA6 par son carbone en position 2; XA4 is chosen from a hydrogen atom, a sulfate group and a pyruvate group, said pyruvate group being bonded to the XA group 4 by its carbon in the 2-position and to the XA6 group by its carbon in the 2-position ;
- XA6 est choisi parmi un atome d'hydrogène, un groupement sulfate, une unité saccharidique T-galactose liée à l'unité A par une liaison de type O-glycosidique, une unité saccharidique T-xylose liée à l'unité A par une liaison de type O-glycosidique, une unité saccharidique T-arabinose liée à l'unité A par une liaison de type O-glycosidique et une unité saccharidique T-acide glucuronique liée à l'unité A par une liaison de type O- glycosidique, et  XA6 is chosen from a hydrogen atom, a sulfate group, a T-galactose saccharide unit linked to unit A by an O-glycosidic type bond, a T-xylose saccharide unit linked to unit A by a O-glycosidic type binding, a unit-linked T-arabinose saccharide unit with an O-glycosidic type bond and a unit-linked glucuronic T-glucuronic unit with an O-glycosidic type bond, and
- XB6 est choisi parmi un atome d'hydrogène, un groupement sulfate, une unité saccharidique T-galactose liée par une liaison de type O-glycosidique au résidu B1 , une unité saccharidique T-xylose liée par une liaison de type O-glycosidique au résidu B1 , une unité saccharidique T-arabinose liée par une liaison de type O-glycosidique au résidu B1 , et une unité saccharidique T-acide glucuronique liée par une liaison de type O-glycosidique au résidu B1 . XB6 is chosen from a hydrogen atom, a sulfate group, a T-galactose saccharide unit linked by an O-glycosidic type bond to residue B1, a T-xylose saccharide unit linked by an O-glycosidic type bond to residue B1, a saccharide unit T-arabinose linked by an O-glycosidic bond to residue B1, and a saccharide unit T-glucuronic acid linked by an O-glycosidic bond to residue B1.
On entend par « sel pharmaceutiquement acceptable » tout sel d'addition avec un acide minéral ou organique par action d'un tel acide au sein d'un solvant organique ou aqueux tel qu'un alcool, une cétone, un éther, et qui soit acceptable d'un point de vue pharmaceutique. A titre d'exemple de tels sels, on peut citer les sels suivants : un sel sodique de type SC Na, un sel potassique de type SO3K, le benzènesulfonate, le bromhydrate, le chlorhydrate, le citrate, éthanesulfonate, le fumarate, le gluconate, l'iodate, l'iséthionate, le maléate, méthanesulfonate, le méthylène-bis-b-oxynaphtoate, le nitrate, l'oxalate, le palmoate, le phosphate, le salicylate, le sulfate, le tartrate, le théophyllinacétate et le p-toluènesulfonate. Dans un mode de réalisation avantageux, le sel pharmaceutiquement acceptable est un sel sodique (SC Na) ou un sel potassique (SO3K). The term "pharmaceutically acceptable salt" means any addition salt with a mineral or organic acid by the action of such an acid in an organic or aqueous solvent such as an alcohol, a ketone or an ether, and which is acceptable from a pharmaceutical point of view. By way of example of such salts, mention may be made of the following salts: a sodium salt of SC Na type, a potassium salt of SO 3 K type, benzenesulphonate, hydrobromide, hydrochloride, citrate, ethanesulphonate, fumarate, gluconate , iodate, isethionate, maleate, methanesulfonate, methylene-bis-b-oxynaphthoate, nitrate, oxalate, palmoate, phosphate, salicylate, sulfate, tartrate, theophyllinacetate, and the like. toluenesulfonate. In an advantageous embodiment, the pharmaceutically acceptable salt is a sodium salt (SC Na) or a potassium salt (SO3K).
Les polysaccharides sulfatés de la présente invention présentent de nombreux avantages par rapport aux héparines disponibles sur le marché. En particulier, les polysaccharides sulfatés selon l'invention, et en particulier ceux extraits à partir d'une algue rouge de l'espèce Haliptilon subulatum, présentent une activité identique ou supérieure à l'héparine standard non fractionnée ou aux héparines de faibles masses moléculaires de type Lovenox® sur des modèles de thrombose. A faibles concentrations, les polysaccharides sulfatés selon l'invention, et en particulier ceux extraits à partir d'une algue rouge de l'espèce Haliptilon subulatum, ont une activité similaire à l'héparine standard non fractionnée et une activité meilleure que les héparines de faibles masses moléculaires de type Lovenox® sur les voies endogène et exogène de la cascade de la coagulation, permettant ainsi de prévenir et de traiter les risques associés à la thrombose vasculaire. De plus, ces polysaccharides sulfatés sont obtenus à partir d'algues, contrairement aux héparines disponibles sur le marché, qui elles sont obtenues à partir d'animaux, réduisant ainsi les coûts de production et les risques de contamination virale et/ou par prion. The sulfated polysaccharides of the present invention have many advantages over commercially available heparins. In particular, the sulphated polysaccharides according to the invention, and in particular those extracted from a red alga of the Haliptilon subulatum species, exhibit an activity identical to or greater than the standard unfractionated heparin or the low molecular weight heparins. Lovenox® type on thrombosis models. At low concentrations, the sulphated polysaccharides according to the invention, and in particular those extracted from a red alga of the species Haliptilon subulatum, have a similar activity to the unfractionated standard heparin and a better activity than the heparins of low molecular weight Lovenox® type on the endogenous and exogenous pathways of the coagulation cascade, thereby preventing and treating the risks associated with vascular thrombosis. In addition, these sulfated polysaccharides are obtained from algae, unlike commercially available heparins, which are obtained from animals, thus reducing production costs and the risk of viral and / or prion contamination.
Dans un mode de réalisation particulier de l'invention, le polysaccharide sulfaté possède une masse moléculaire inférieure ou égale à 1000 kDa. De manière avantageuse, le polysaccharide sulfaté selon l'invention possède une masse moléculaire inférieure ou égale à 450 kDa, avantageusement inférieure ou égale à 400 kDa, avantageusement inférieure ou égale à 350 kDa, avantageusement inférieure ou égal à 300 kDa, avantageusement inférieure ou égale à 250 kDa, avantageusement inférieure ou égale à 200 kDa. De manière avantageuse, le polysaccharide sulfaté selon l'invention possède une masse moléculaire comprise entre 10 kDa et 500 kDa, avantageusement comprise entre 10 kDa et 400 kDa, avantageusement comprise entre 10 kDa et 300 kDa, avantageusement compris entre 10 kDa et 250 kDa. In a particular embodiment of the invention, the sulphated polysaccharide has a molecular mass less than or equal to 1000 kDa. Advantageously, the sulphated polysaccharide according to the invention has a molecular mass less than or equal to 450 kDa, advantageously less than or equal to 400 kDa, advantageously less than or equal to 350 kDa, advantageously less than or equal to 300 kDa, advantageously less than or equal to at 250 kDa, advantageously less than or equal to 200 kDa. Advantageously, the sulfated polysaccharide according to the invention has a molecular weight between 10 kDa and 500 kDa, advantageously between 10 kDa and 400 kDa, advantageously between 10 kDa and 300 kDa, advantageously between 10 kDa and 250 kDa.
Dans un autre mode de réalisation particulier de l'invention, le polysaccharide sulfaté présente un taux de sulfates inférieur ou égal à 20% en masse du polysaccharide. De manière avantageuse, le taux de sulfates du polysaccharide sulfaté est inférieur ou égal à 19%, avantageusement inférieur ou égal à 18%, avantageusement inférieur ou égal à 17%, avantageusement inférieur ou égal à 16%. De manière avantageuse, le taux de sulfates du polysaccharide sulfaté est compris entre 5% et 20%, avantageusement entre 6% et 20%, avantageusement entre 7% et 19%, avantageusement entre 8% et 19%, avantageusement entre 9% et 18%, avantageusement entre 10% et 16%, avantageusement entre 13% et 16%. In another particular embodiment of the invention, the sulphated polysaccharide has a sulphate level of less than or equal to 20% by weight of the polysaccharide. Advantageously, the sulfates content of the sulphated polysaccharide is less than or equal to 19%, advantageously less than or equal to 18%, advantageously less than or equal to 17%, advantageously less than or equal to 16%. Advantageously, the sulphate content of the sulphated polysaccharide is between 5% and 20%, advantageously between 6% and 20%, advantageously between 7% and 19%, advantageously between 8% and 19%, advantageously between 9% and 18%. %, advantageously between 10% and 16%, advantageously between 13% and 16%.
Dans un autre mode de réalisation de l'invention, le polysaccharide sulfaté possède un taux de résidus 3,6-anhydrogalactopyranose (ou résidu B2) inférieur ou égal à 5% en masse du polysaccharide. Dans un mode de réalisation avantageuse, le polysaccharide sulfaté possède un taux de résidus 3,6-anhydrogalactopyranose (ou résidu B2) inférieur ou égal à 4%, avantageusement inférieur ou égal à 3%, avantageusement inférieur ou égal à 2%, avantageusement inférieur ou égal à 1 ,5%. De manière encore plus avantageuse, le polysaccharide sulfaté possède un taux de résidus 3,6-anhydrogalactopyranose (ou résidu B2) inférieur ou égal à 1 ,4%. In another embodiment of the invention, the sulfated polysaccharide has a residue level of 3,6-anhydrogalactopyranose (or B2 residue) less than or equal to 5% by weight of the polysaccharide. In one advantageous embodiment, the sulphated polysaccharide has a residue level of 3,6-anhydrogalactopyranose (or residue B2) of less than or equal to 4%, advantageously less than or equal to 3%, advantageously less than or equal to 2%, advantageously less than or equal to 1, 5%. Even more advantageously, the sulfated polysaccharide has a residue level of 3,6-anhydrogalactopyranose (or residue B2) less than or equal to 1, 4%.
Dans un autre mode de réalisation particulier de l'invention, le polysaccharide sulfaté est d'origine naturelle. In another particular embodiment of the invention, the sulphated polysaccharide is of natural origin.
Un autre objet de l'invention concerne une composition pharmaceutique pour son utilisation en tant qu'anticoagulant comprenant au moins un polysaccharide sulfaté extrait à partir d'une algue rouge, en particulier à partir d'une algue rouge de l'espèce Haliptilon subulatum ou un sel pharmaceutiquement acceptable de celui-ci, dans lequel le taux de sulfate dudit polysaccharide est inférieur ou égal à 20% en poids du polysaccharide, ou un sel pharmaceutiquement acceptable de celui-ci tel que défini précédemment. Another subject of the invention relates to a pharmaceutical composition for its use as an anticoagulant comprising at least one sulphated polysaccharide extracted from a red alga, in particular from a red algae of the species Haliptilon subulatum or a pharmaceutically acceptable salt thereof, wherein the sulfate level of said polysaccharide is less than or equal to 20% by weight of the polysaccharide, or a pharmaceutically acceptable salt thereof as defined above.
Dans un mode de réalisation particulier, cette composition pharmaceutique comprend au moins un polysaccharide sulfaté selon l'invention, en association avec un ou plusieurs autres agents choisis parmi les excipients pharmaceutiquement acceptables, les agents actifs et les ingrédients alimentaires. Conformément à la présente invention, la composition pharmaceutique peut se présenter sous forme liquide, en particulier sous forme de solution ou de suspension ou sous forme de gel ou sous forme de poudre. In a particular embodiment, this pharmaceutical composition comprises at least one sulphated polysaccharide according to the invention, in combination with one or more other agents chosen from pharmaceutically acceptable excipients, active agents and food ingredients. According to the present invention, the pharmaceutical composition may be in liquid form, in particular in solution or suspension form or in gel form or in powder form.
Par « excipient pharmaceutiquement acceptable », on entend toute substance autre que la substance active, destinée à apporter une consistance, un goût, une couleur à un médicament, tout en évitant toute interaction avec le principe actif. L'excipient pharmaceutiquement acceptable selon l'invention sera choisi selon la forme pharmaceutique et le mode d'administration souhaité, parmi les excipients habituels qui sont connus de l'homme du métier, en vue d'être administré à l'Homme ou aux animaux. Par « agents actifs », on entend toute substance autre que la substance active et ayant un effet thérapeutique. On peut notamment citer à titre d'exemples les anticoagulants, les antibiotiques, les anti-thrombotiques, les anticancéreux, les anti-inflammatoires, les antihistaminiques, les médicaments cardiovasculaires, les antihypertenseurs, la présente liste n'étant pas limitative. Par « ingrédients alimentaires », on entend toute substance autre que la substance active destinée à être utiliser dans une composition alimentaire à destination de l'Homme ou de l'animal. Cet ingrédient alimentaire répond notamment aux conditions de sécurité et de non- toxicité liées à une telle utilisation. A titre d'exemple, on peut notamment citer le sucre, en particulier le saccharose, le miel, les agents protéiques alimentaires, comme notamment le gluten, les protéines de soja, les protéines de blé, les vitamines, les oligonutriments comme le fer, le calcium, le magnésium, la présente liste n'étant pas limitative. By "pharmaceutically acceptable excipient" is meant any substance other than the active substance, intended to provide consistency, taste, color to a drug, while avoiding any interaction with the active ingredient. The pharmaceutically acceptable excipient according to the invention will be chosen according to the pharmaceutical form and the desired mode of administration, from the usual excipients which are known to those skilled in the art, for administration to humans or animals. . "Active agents" means any substance other than the active substance and having a therapeutic effect. Examples that may be mentioned include anticoagulants, antibiotics, antithrombotics, anticancer agents, anti-inflammatories, antihistamines, cardiovascular drugs, antihypertensives, the present list not being limiting. "Food ingredients" means any substance other than the active substance intended to be used in a food composition intended for humans or animals. This food ingredient responds in particular to the conditions of safety and non-toxicity associated with such use. By way of example, mention may in particular be made of sugar, in particular sucrose, honey, food protein agents, such as, in particular, gluten, soy proteins, wheat proteins, vitamins, oligonutrients such as iron, calcium, magnesium, this list is not limiting.
Dans un mode de réalisation particulier de l'invention, les compositions pharmaceutiques comprenant le polysaccharide sulfaté peuvent être administrées par voie orale, sublinguale, sous-cutanée, parentérale, intramusculaire, intraveineuse, topique, locale, intratrachéale, intranasale, oculaire, intrapéritonéale, par diffusion endopariétale, transdermique ou rectale, ou par toute autre voie d'administration. Les formes d'administration appropriées comprennent les formes par voie orale telles que les comprimés, les gélules molles ou dures, les poudres, les granules et les solutions ou suspensions orales, les formes d'administration sublinguale, buccale, intratrachéale, intraoculaire, intranasale, par inhalation, les formes d'administration topique, transdermique, sous-cutanée, intramusculaire ou intraveineuse, les formes d'administration rectale et les implants. Pour l'application topique, on peut utiliser les composés selon l'invention dans des crèmes, des émulsions huile dans l'eau ou eau dans l'huile, des gels, des pommades, des patches, des solutions ou des lotions. Selon l'invention, la composition pharmaceutique comprenant au moins un polysaccharide sulfaté est particulièrement utile pour prévenir, réduire et traiter les thromboses vasculaires. In a particular embodiment of the invention, the pharmaceutical compositions comprising the sulfated polysaccharide may be administered orally, sublingually, subcutaneously, parenterally, intramuscularly, intravenously, topically, locally, intratracheally, intranasally, ocularly, intraperitoneally, by endoparietal, transdermal or rectal diffusion, or by any other route of administration. Suitable forms of administration include oral forms such as tablets, soft or hard capsules, powders, granules and oral solutions or suspensions, sublingual, oral, intratracheal, intraocular, intranasal, by inhalation, topical, transdermal, subcutaneous, intramuscular or intravenous administration forms, rectal administration forms and implants. For topical application, the compounds according to the invention can be used in creams, oil-in-water or water-in-oil emulsions, gels, ointments, patches, solutions or lotions. According to the invention, the pharmaceutical composition comprising at least one sulfated polysaccharide is particularly useful for preventing, reducing and treating vascular thromboses.
Le terme « prévenir » ou «prévention » ou « prophylaxie » ou « traitement préventif » ou « traitement prophylactique » comprend aussi bien un traitement aboutissant à la prévention d'une maladie qu'un traitement réduisant et/ou retardant l'incidence d'une maladie ou le risque qu'elle survienne. The term "prevent" or "prevention" or "prophylaxis" or "preventive treatment" or "prophylactic treatment" includes both treatment leading to the prevention of a disease and treatment that reduces and / or delays the incidence of an illness or the risk of it happening.
Le terme «traiter » ou « traitement » ou « traitement curatif » est défini par un traitement aboutissant à une guérison ou un traitement allégeant, améliorant et/ou éliminant, réduisant et/ou stabilisant les symptômes d'une maladie ou la souffrance qu'elle provoque. Par « thromboses vasculaires », on entend les thromboses artérielles et les thromboses veineuses profondes ou non. Ainsi, la composition pharmaceutique comprenant au moins un polysaccharide sulfaté selon l'invention est particulièrement utile pour prévenir, réduire et traiter les thromboses artérielles, les thromboses veineuses profondes ou non. The term "treat" or "treatment" or "curative treatment" is defined as treatment leading to a cure or treatment that alleviates, improves and / or eliminates, reduces and / or stabilizes the symptoms of an illness or suffering that she provokes. "Vascular thromboses" means arterial thromboses and deep vein thromboses or not. Thus, the pharmaceutical composition comprising at least one sulphated polysaccharide according to the invention is particularly useful for preventing, reducing and treating arterial thromboses, deep vein thromboses or not.
Dans un mode de réalisation particulier, la composition pharmaceutique comprenant le polysaccharide sulfaté peut être administrée au patient à une dose journalière comprise entre 50 et 1000 mg, de préférence comprise entre 100 et 1000 mg pour prévenir, réduire et traiter les thromboses vasculaires, en particulier pour prévenir, réduire et traiter les thromboses artérielles, des thromboses veineuses profondes ou non. Dans un mode de réalisation particulier, la composition pharmaceutique comprenant le polysaccharide sulfaté est administrée au patient à une dose journalière de préférence comprise entre 100 et 950 mg, de préférence comprise entre 100 à 900 mg, de préférence comprise entre 100 à 850 mg, de préférence comprise entre 100 à 800 mg, de préférence comprise entre 100 à 750 mg, de préférence comprise entre 100 à 700 mg, de préférence comprise entre 100 à 650 mg, de préférence comprise entre 100 à 600 mg pour prévenir, réduire et traiter les thromboses vasculaires, en particulier pour prévenir, réduire et traiter les thromboses artérielles, des thromboses veineuses profondes ou non. Dans un mode de réalisation préféré, la composition pharmaceutique comprenant le polysaccharide sulfaté est administrée au patient à une dose journalière comprise entre 200 à 600 mg pour prévenir, réduire et traiter les thromboses vasculaires, en particulier pour prévenir, réduire et traiter les thromboses artérielles, des thromboses veineuses profondes ou non. Il est toutefois bien entendu que l'Homme de l'art adaptera ces doses en fonction de l'âge, du poids et de la pathologie du patient, notamment en fonction du risque thrombogène. Dans un mode de réalisation particulièrement avantageux de la présente invention, la composition pharmaceutique comprenant le polysaccharide sulfaté peut être administrée au patient en une unique dose journalière comprise entre 50 et 1000 mg, de préférence comprise entre 100 et 1000 mg pour prévenir, réduire et traiter les thromboses vasculaires, en particulier pour prévenir, réduire et traiter les thromboses artérielles, des thromboses veineuses profondes ou non. Dans un mode de réalisation particulier, la composition pharmaceutique comprenant le polysaccharide sulfaté est administrée au patient en une unique dose journalière, de préférence comprise entre 100 et 950 mg, de préférence comprise entre 100 à 900 mg, de préférence comprise entre 100 à 850 mg, de préférence comprise entre 100 à 800 mg, de préférence comprise entre 100 à 750 mg, de préférence comprise entre 100 à 700 mg, de préférence comprise entre 100 à 650 mg, de préférence comprise entre 100 à 600 mg pour prévenir, réduire et traiter les thromboses vasculaires, en particulier pour prévenir, réduire et traiter les thromboses artérielles, des thromboses veineuses profondes ou non. Dans un mode de réalisation préféré, la composition pharmaceutique comprenant le polysaccharide sulfaté est administrée au patient en une unique dose journalière comprise entre 200 à 600 mg pour prévenir, réduire et traiter les thromboses vasculaires, en particulier pour prévenir, réduire et traiter les thromboses artérielles, des thromboses veineuses profondes ou non. In a particular embodiment, the pharmaceutical composition comprising the sulphated polysaccharide may be administered to the patient at a daily dose of between 50 and 1000 mg, preferably between 100 and 1000 mg to prevent, reduce and treat vascular thromboses, in particular to prevent, reduce and treat arterial thromboses, deep vein thrombosis or not. In a particular embodiment, the pharmaceutical composition comprising the sulphated polysaccharide is administered to the patient at a daily dose of preferably between 100 and 950 mg, preferably between 100 and 900 mg, preferably between 100 and 850 mg, of preferably between 100 to 800 mg, preferably between 100 to 750 mg, preferably between 100 to 700 mg, preferably between 100 to 650 mg, preferably between 100 to 600 mg to prevent, reduce and treat the Vascular thromboses, especially to prevent, reduce and treat arterial thromboses, deep vein thrombosis or not. In a preferred embodiment, the pharmaceutical composition comprising the sulfated polysaccharide is administered to the patient at a daily dose of between 200 to 600 mg to prevent, reduce and treat vascular thromboses, in particular to prevent, reduce and treat arterial thromboses, deep venous thromboses or not. It is, however, of course understood that those skilled in the art will adapt these doses according to the age, the weight and the pathology of the patient, particularly as a function of the thrombogenic risk. In a particularly advantageous embodiment of the present invention, the pharmaceutical composition comprising the sulfated polysaccharide can be administered to the patient by a single daily dose of between 50 and 1000 mg, preferably between 100 and 1000 mg to prevent, reduce and treat vascular thromboses, in particular to prevent, reduce and treat arterial thromboses, deep vein thrombosis or not. In a particular embodiment, the pharmaceutical composition comprising the sulphated polysaccharide is administered to the patient in a single daily dose, preferably in the range 100 to 950 mg, preferably in the range 100 to 900 mg, preferably in the range 100 to 850 mg. , preferably between 100 to 800 mg, preferably between 100 to 750 mg, preferably between 100 to 700 mg, preferably between 100 to 650 mg, preferably between 100 to 600 mg to prevent, reduce and treat vascular thromboses, in particular to prevent, reduce and treat arterial thromboses, deep vein thrombosis or not. In a preferred embodiment, the pharmaceutical composition comprising the sulfated polysaccharide is administered to the patient in a single daily dose of 200 to 600 mg to prevent, reduce and treat vascular thromboses, particularly to prevent, reduce and treat arterial thromboses. Deep venous thromboses or not.
Dans un autre mode de réalisation particulier de l'invention, la composition pharmaceutique comprenant au moins un polysaccharide sulfaté est particulièrement utile pour prévenir, réduire et traiter les ischémies, les embolies pulmonaires, l'angor instable et l'infarctus du myocarde. In another particular embodiment of the invention, the pharmaceutical composition comprising at least one sulfated polysaccharide is particularly useful for preventing, reducing and treating ischemia, pulmonary embolism, unstable angina and myocardial infarction.
Dans un autre mode de réalisation particulier de l'invention, la composition pharmaceutique comprenant au moins un polysaccharide sulfaté est particulièrement utile pour prévenir la coagulation dans les circuits de circulation extracorporelle et/ou d'épuration extra-rénale. In another particular embodiment of the invention, the pharmaceutical composition comprising at least one sulphated polysaccharide is particularly useful for preventing coagulation in extracorporeal circulation and / or extra-renal purification circuits.
Un autre aspect de l'invention concerne une méthode de traitement des thromboses vasculaires comprenant l'administration à un patient souffrant de thromboses vasculaires d'une composition pharmaceutique comprenant au moins un polysaccharide sulfaté extrait d'une algue rouge marine, en particulier extrait à partir d'une algue rouge de l'espèce Haliptilon subulatum. Another aspect of the invention relates to a method of treating vascular thromboses comprising administering to a patient suffering from vascular thromboses a pharmaceutical composition comprising at least one sulfated polysaccharide extracted from a marine red algae, in particular extracted from of a red alga of the species Haliptilon subulatum.
Un autre aspect de l'invention concerne une méthode de traitement des ischémies, des embolies pulmonaires, de l'angor instable et de l'infarctus du myocarde, comprenant l'administration à un patient en nécessitant, d'une composition pharmaceutique comprenant au moins un polysaccharide sulfaté extrait d'une algue rouge, en particulier extrait à partir d'une algue rouge de l'espèce Haliptilon subulatum. Un autre aspect de l'invention concerne une méthode de prévention de la coagulation dans les circuits de circulation extracorporelle et/ou d'épuration extra-rénale, comprenant l'administration dans le circuit de circulation extracorporelle et/ou d'épuration extra-rénale, d'une composition pharmaceutique comprenant au moins un polysaccharide sulfaté extrait d'une algue rouge, en particulier extrait à partir d'une algue rouge de l'espèce Haliptilon subulatum. Another aspect of the invention relates to a method of treating ischemia, pulmonary embolism, unstable angina and myocardial infarction, comprising administering to a patient by requiring a pharmaceutical composition comprising at least one a sulphated polysaccharide extracted from a red alga, in particular extracted from a red alga of the species Haliptilon subulatum. Another aspect of the invention relates to a method of preventing coagulation in extracorporeal circulation circuits and / or extra-renal purification, including administration in the extracorporeal circulation circuit and / or extra-renal purification. , a pharmaceutical composition comprising at least one sulphated polysaccharide extracted from a red alga, in particular extracted from a red alga of the species Haliptilon subulatum.
Par « administration », on entend tout acte permettant de faire absorber au patient ou d'introduire dans le circuit de circulation extracorporelle et/ou d'épuration extra-rénale, la composition pharmaceutique selon l'invention par n'importe quelle voie, forme ou mode d'administration. By "administration" is meant any act enabling the patient to be absorbed or introducing into the extracorporeal circulation and / or extra-renal purification circuit, the pharmaceutical composition according to the invention by any route, form or form. or mode of administration.
Un autre aspect de la présente invention concerne un procédé d'obtention du polysaccharide sulfaté. Le polysaccharide sulfaté selon l'invention peut être obtenu par des procédés bien connus de l'Homme du métier. En particulier, l'extraction d'un polysaccharide sulfaté de haute masse moléculaire peut notamment être obtenu par un procédé comprenant les étapes décrites ci-après : a) Dispersion dans l'eau d'une poudre d'algue rouge, en particulier une poudre d' Haliptilon subulatum préalablement séchée ; Another aspect of the present invention relates to a process for obtaining the sulfated polysaccharide. The sulfated polysaccharide according to the invention can be obtained by methods that are well known to those skilled in the art. In particular, the extraction of a sulfated polysaccharide of high molecular weight can in particular be obtained by a process comprising the steps described below: a) Dispersion in water of a red algae powder, in particular a powder Haliptilon subulatum previously dried;
b) Précipitation par au moins un solvant polaire des polysaccharides de hautes masses moléculaires;  b) Precipitation with at least one polar solvent of high molecular weight polysaccharides;
c) Séchage du précipité contenant les polysaccharides de hautes masses moléculaires.  c) Drying of the precipitate containing the polysaccharides of high molecular masses.
Dans un mode de réalisation particulier, l'étape b) de précipitation alcoolique est réalisée en utilisant de l'éthanol ou de l'isopropanol. Dans un mode de réalisation particulier, l'étape de séchage du précipité (étape c) peut être réalisée par lyophilisation ou à l'aide d'une étuve, notamment à une température comprise entre 40°C et 75°C, avantageusement 50°C pendant une nuit. La méthode d'obtention selon l'invention peut être renouvelée plusieurs fois afin d'obtenir un degré de pureté du polysaccharide satisfaisant. In a particular embodiment, the alcoholic precipitation step b) is carried out using ethanol or isopropanol. In a particular embodiment, the step of drying the precipitate (step c) can be carried out by lyophilization or with the aid of an oven, in particular at a temperature of between 40 ° C. and 75 ° C., advantageously 50 ° C. C overnight. The method of obtaining according to the invention may be repeated several times in order to obtain a degree of purity of the satisfactory polysaccharide.
Le procédé d'extraction selon la présente invention permet l'obtention de polysaccharides sous forme d'une fine poudre blanc crème avec un rendement de production de l'ordre de 10-20% par rapport à la masse sèche de poudre d'algue Haliptilum subulatum utilisée. The extraction process according to the present invention makes it possible to obtain polysaccharides in the form of a fine creamy white powder with a production yield of the order of 10-20% relative to the dry weight of Haliptilum seaweed powder. subulatum used.
Dans un autre mode de réalisation particulier, les polysaccharides sulfatés de faibles masses moléculaires sont préparés par dégradation acide de polysaccharides de hautes masses moléculaires. Les polysaccharides sulfatés de faible masse moléculaire peuvent également être obtenus par des techniques de dépolymérisation bien connues de l'Homme du métier, telles que les dépolymérisations radicalaire ou enzymatique. In another particular embodiment, the low molecular weight sulfated polysaccharides are prepared by acid degradation of high molecular weight polysaccharides. Sulfated polysaccharides of low molecular weight can also be obtained by depolymerization techniques well known to those skilled in the art, such as radical or enzymatic depolymerizations.
Dans un mode de réalisation particulier, le procédé d'extraction d'un polysaccharide sulfaté de faible masse moléculaire à partir d'algue Haliptilon subulatum comprend les étapes suivantes : In a particular embodiment, the process for extracting a low molecular weight sulphated polysaccharide from Haliptilon subulatum alga comprises the following steps:
a) Dispersion de la poudre de polysaccharides de haute masse moléculaire dans une solution aqueuse à pH acide, en particulier un pH compris entre 0 et 6,5 ;  a) Dispersion of the polysaccharide powder of high molecular mass in an aqueous solution at acidic pH, in particular a pH of between 0 and 6.5;
b) Précipitation par au moins un solvant polaire des polysaccharides de faible masse moléculaire;  b) Precipitation with at least one polar solvent of low molecular weight polysaccharides;
c) Séchage du précipité contenant les polysaccharides de faibles masses moléculaires.  c) Drying of the precipitate containing polysaccharides of low molecular weight.
Dans un mode de réalisation particulier, la solution aqueuse utilisée dans l'étape de dispersion (étape a) est une solution d'acide chlorhydrique (HCI). Dans un mode de réalisation particulier, la solution d'acide chlorhydrique présente une concentration comprise entre 1 M et 5M, avantageusement 2M, à une température allant de 50°C à 100°C et sous agitation pendant 30 à 60 minutes. Dans un mode de réalisation particulier, l'étape b) de précipitation alcoolique est réalisée en utilisant de l'éthanol ou de l'isopropanol. Dans un mode de réalisation particulier, l'étape de séchage du précipité (étape c) est réalisée par lyophilisation ou à l'aide d'une étuve, notamment à une température comprise entre 40°C et 75°C, avantageusement 50°C pendant une nuit. In a particular embodiment, the aqueous solution used in the dispersion step (step a) is a solution of hydrochloric acid (HCI). In a particular embodiment, the hydrochloric acid solution has a concentration of between 1M and 5M, advantageously 2M, at a temperature ranging from 50 ° C. to 100 ° C. and with stirring for 30 to 60 minutes. In a particular embodiment, the alcoholic precipitation step b) is carried out using ethanol or isopropanol. In a particular embodiment, the step of drying the precipitate (step c) is carried out by lyophilization or with the aid of an oven, in particular at a temperature of between 40 ° C. and 75 ° C., advantageously 50 ° C. for one night.
Les polysaccharides sulfatés extraits à partir d'algues rouges selon l'invention présentent l'avantage de ne pas avoir les problèmes de contamination et de sécurité, contrairement aux héparines disponibles sur le marché qui sont obtenues à partir de porcs. De plus, ces polysaccharides sulfatés offrent un avantage économique. Le rendement approximatif de polysaccharides sulfatés de la présente invention est d'environ 10% à 20% par rapport au poids sec initial d'algues à partir desquelles il a été extrait. Ce rendement élevé signifie réduction du coût final du produit, par rapport aux héparines disponibles sur le marché. En outre, les algues rouges, en particulier de la classe des Florideophyceae, en particulier les espèces Haliptilon subulatum, sont faciles à cultiver ce qui contribue également au faible coût de revient du produit final. The sulphated polysaccharides extracted from red algae according to the invention have the advantage of not having the problems of contamination and safety, unlike the heparins available on the market which are obtained from pigs. In addition, these sulfated polysaccharides offer an economic advantage. The approximate yield of sulfated polysaccharides of the present invention is about 10% to 20% based on the initial dry weight of algae from which it was extracted. This high yield means a reduction in the final cost of the product compared to commercially available heparins. In addition, red algae, especially of the class Floridaophyceae, especially the species Haliptilon subulatum, are easy to grow which also contributes to the low cost of the final product.
Les figures 1 à 5 et les exemples 1 à 6 qui suivent illustrent l'invention sans toutefois la limiter. FIGURES Figures 1 to 5 and Examples 1 to 6 which follow illustrate the invention without limiting it. FIGURES
Figure 1 : Détermination de l'activité résiduelle du Facteur Xa selon l'exemple 6, exprimée en pourcentage (%) en fonction de la concentration en héparine, en Lovenox®, en polysaccharide sulfaté de haute (PSHM) (213,5 kDa) et faible (PSFM) (36,6 kDa) masse moléculaire selon l'invention (PSHM et PSFM) exprimée en μg mL. Figure 1: Determination of the residual activity of Factor Xa according to Example 6, expressed as a percentage (%) as a function of the concentration of heparin, Lovenox®, sulfated high polysaccharide (PSHM) (213.5 kDa) and low (PSFM) (36.6 kDa) molecular weight according to the invention (PSHM and PSFM) expressed in μg mL.
Figure 2 : Détermination de l'activité résiduelle du Facteur lia selon l'exemple 6, exprimée en pourcentage (%) en fonction de la concentration en héparine, en Lovenox®, en polysaccharide sulfaté de haute (PSHM) (213,5 kDa) et faible (PSFM) (36,6 kDa) masse moléculaire selon l'invention exprimée en μg mL. Figure 3 : Détermination du temps de Temps de Céphaline activée (aPTT), selon l'exemple 6, exprimée en seconde (s) en fonction de la concentration en héparine en Lovenox®, en carraghénane Iota et en polysaccharide sulfaté de haute (PSHM) (213,5 kDa) et faible (PSFM) (36,6 kDa) masse moléculaire selon l'invention exprimée en μg mL. Figure 2: Determination of the residual activity of Factor IIa according to Example 6, expressed as a percentage (%) as a function of the concentration of heparin, Lovenox®, sulfated high polysaccharide (PSHM) (213.5 kDa) and low (PSFM) (36.6 kDa) molecular weight according to the invention expressed in μg mL. FIG. 3: Determination of activated partial thromboplastin time (aPTT), according to Example 6, expressed in second (s) as a function of the concentration of heparin in Lovenox®, carrageenan Iota and sulfated high polysaccharide (PSHM). (213.5 kDa) and low (PSFM) (36.6 kDa) molecular weight according to the invention expressed in μg mL.
Figure 4 : Détermination du temps de Temps de Quick (PT), selon l'exemple 6, exprimée en seconde (s) en fonction de la concentration en héparine en Lovenox®, en carraghénane Iota et en polysaccharide sulfaté de haute (PSHM) (213,5 kDa) et faible (PSFM) (36,6 kDa) masse moléculaire selon l'invention exprimée en μg mL. FIG. 4: Determination of the Time of Quick Time (PT), according to Example 6, expressed in second (s) as a function of the concentration of Lovenox® heparin, Iota carrageenan and sulfated high polysaccharide (PSHM) ( 213.5 kDa) and low (PSFM) (36.6 kDa) molecular weight according to the invention expressed in μg mL.
Figure 5 : Détermination du temps de Temps de thrombine (TT), selon l'exemple 6, exprimée en seconde (s) en fonction de la concentration en héparine en Lovenox®, en carraghénane Iota et en polysaccharide sulfaté de haute (PSHM) (213,5 kDa) et faible (PSFM) (36,6 kDa) masse moléculaire selon l'invention exprimée en μg mL. FIG. 5: Determination of the Thrombin Time (TT) time, according to Example 6, expressed in second (s) as a function of the concentration of Lovenox® heparin, Iota carrageenan and high sulphated polysaccharide (PSHM) ( 213.5 kDa) and low (PSFM) (36.6 kDa) molecular weight according to the invention expressed in μg mL.
EXEMPLES EXAMPLES
Exemple 1 : Extraction des polysaccharides de haute masse moléculaire (PSHM) Example 1 Extraction of High Molecular Weight Polysaccharides (PSHM)
Par «polysaccharide de haute masse moléculaire» ou «PSHM», on entend un polysaccharide ayant une masse molaire comprise entre 100 et 1000 kDa. L'extraction des polysaccharides de haute masse moléculaire est réalisée en dispersant 100 grammes de poudre d'algue Haliptilon subulatum dans 1 litre d'eau à 90 °C sous vive agitation (500 Tr/min) pendant 4H. Le mélange est ensuite filtré à chaud sur diatomée (100 g) sur un verre fritté (porosité 1 , plus précisément 100 à 160 μηη). Le filtrat est ensuite centrifugé (10000 g, 30 minutes) à température ambiante pour obtenir l'extrait d'algue enrichi en polysaccharides. L'extrait d'Haliptilon subulatum est ensuite précipité dans 3 volumes d'éthanol 96° (à 4°C) sous agitation (500 Tr/min) pendant 2 heures. By "high molecular weight polysaccharide" or "PSHM" is meant a polysaccharide having a molar mass of between 100 and 1000 kDa. Extraction of the high molecular weight polysaccharides is carried out by dispersing 100 grams of Haliptilon subulatum seaweed powder in 1 liter of water at 90 ° C. with vigorous stirring (500 rpm) for 4 hours. The mixture is then filtered hot on diatom (100 g) on a sintered glass (porosity 1, more precisely 100 to 160 μηη). The filtrate is then centrifuged (10000 g, 30 minutes) at room temperature to obtain the polysaccharide-enriched algal extract. The Haliptilon subulatum extract is then precipitated in 3 volumes of 96 ° ethanol (at 4 ° C.) with stirring (500 rpm) for 2 hours.
Le précipité est récupéré par filtration sur verre fritté (porosité 1 ou 2, plus précisément 100 à 160 μηη ou 40 à 100 μηη respectivement) ou centrifugation (10000 g, 30 minutes) à température ambiante puis lavé avec de l'acétone (50 à 100 mL). Ensuite, le précipité est récupéré par filtration sur verre fritté (porosité 2, plus précisément 40 à 100 μηη) ou centrifugation (10000 g, 30 minutes) à température ambiante puis séché à l'étuve à 50°C durant une nuit. Finalement, le précipité est broyé (au Blender) afin d'obtenir une fine poudre de polysaccharides de haute masse moléculaire extraits d'Haliptilon subulatum. The precipitate is recovered by filtration on sintered glass (porosity 1 or 2, more precisely 100 to 160 μηη or 40 to 100 μηη respectively) or centrifugation (10,000 g, 30 minutes) at room temperature and then washed with acetone (50 to 100 μηη). 100 mL). Then, the precipitate is recovered by filtration on sintered glass (porosity 2, more precisely 40 to 100 μηη) or centrifugation (10,000 g, 30 minutes) at room temperature and then dried in an oven at 50 ° C overnight. Finally, the precipitate is ground (Blender) to obtain a fine powder of high molecular weight polysaccharides extracted from Haliptilon subulatum.
Le rendement en polysaccharides de haute masse moléculaire ainsi obtenu est de l'ordre de 10 à 20% par rapport à la masse sèche de poudre d'algue Haliptilum subulatum utilisée. The yield of polysaccharides of high molecular mass thus obtained is of the order of 10 to 20% relative to the dry weight of algae powder Haliptilum subulatum used.
Exemple 2 : Extraction des polysaccharides de faible masse moléculaire (PSFM) Example 2 Extraction of Low Molecular Weight Polysaccharides (PSFM)
Par «polysaccharide de faible masse moléculaire» ou «PSFM», on entend un polysaccharide ayant une masse moléculaire comprise entre 5 et 100 KDa. La production de polysaccharides de faibles masses moléculaires est réalisée en dispersant 2,5 grammes de poudre de polysaccharides de haute masse moléculaire (extraits d'Haliptilon subulatum) dans 125 mL d'HCI (2M) à 100 °C sous vive agitation (500 Tr/min) pendant 1 heure. Le mélange est ensuite refroidi à température ambiante puis neutralisé avec de la soude (5 M). Le milieu est précipité dans 7 volumes d'éthanol 96° (à 4°C) sous agitation (500 Tr/min) pendant 2 heures. Le précipité est récupéré par filtration sur verre fritté (porosité 1 ou 2, plus précisément 100 à 160 μηη ou 40 à 100 μηη respectivement) ou centrifugation (10000 g, 30 minutes) à température ambiante puis lavé avec de l'acétone (50 mL). Ensuite, le précipité est récupéré par filtration sur verre fritté (porosité 2, plus précisément 40 à 100 μηι) ou centrifugation (10000 g, 30 minutes) à température ambiante puis séché à l'étuve à 50°C durant une nuit. Finalement, le précipité est broyé (au Blender) afin d'obtenir une fine poudre de polysaccharides de faible masse moléculaire extraits d'Haliptilon subulatum. By "low molecular weight polysaccharide" or "PSFM" is meant a polysaccharide having a molecular mass of between 5 and 100 KDa. The production of low molecular weight polysaccharides is carried out by dispersing 2.5 grams of high molecular weight polysaccharide powder (Haliptilon subulatum extracts) in 125 ml of HCl (2M) at 100 ° C. with vigorous stirring (500 rpm). / min) for 1 hour. The mixture is then cooled to ambient temperature and then neutralized with sodium hydroxide (5M). The medium is precipitated in 7 volumes of ethanol 96 ° (at 4 ° C.) with stirring (500 rpm) for 2 hours. The precipitate is recovered by filtration on sintered glass (porosity 1 or 2, more precisely 100 to 160 μηη or 40 to 100 μηη respectively) or centrifugation (10000 g, 30 minutes) at room temperature and then washed with acetone (50 mL). Then, the precipitate is recovered by filtration on sintered glass (porosity 2, more precisely 40 to 100 μηι) or centrifugation (10000 g, 30 minutes) at room temperature and then dried in an oven at 50 ° C overnight. Finally, the precipitate is ground (Blender) to obtain a fine powder of low molecular weight polysaccharides extracted from Haliptilon subulatum.
Le rendement en polysaccharides de faibles masses moléculaires ainsi obtenu est de l'ordre de 70% par rapport à la masse sèche de poudre de polysaccharides de hautes masses moléculaires et 14 % par rapport à la masse sèche de poudre d'algue Haliptilum subulatum utilisée.  The yield of polysaccharides of low molecular weight thus obtained is of the order of 70% relative to the dry mass of high molecular weight polysaccharide powder and 14% relative to the dry weight of Haliptilum subulatum seaweed powder used.
Exemple 3 : Détermination des masses moléculaires des polysaccharides de hautsExample 3 Determination of the Molecular Weights of High Polysaccharides
(PSHM) et de faibles (PSFM) masse molaires (PSHM) and low (PSFM) molar mass
Les polysaccharides de haute et faible masse moléculaire sont préparés selon des conditions décrites préalablement (exemples 1 et 2). Les masses moléculaires des polysaccharides sulfatés sont déterminées selon le protocole ci-après : High and low molecular weight polysaccharides are prepared according to previously described conditions (Examples 1 and 2). The molecular masses of the sulphated polysaccharides are determined according to the following protocol:
a) Solubilisation de la poudre de polysaccharides à hauteur de 0,5 à 10 g/L dans une solution aqueuse de qualité ultrapure à une température allant de 4°C à 60°C et sous agitation pendant 30 minutes à 48 heures ;  a) Solubilization of the polysaccharide powder at a level of 0.5 to 10 g / l in an aqueous solution of ultrapure quality at a temperature ranging from 4 ° C. to 60 ° C. and stirring for 30 minutes to 48 hours;
b) Filtration des échantillons sur membrane de porosité 0,45 μηη ;  b) Filtration of the samples on membrane of porosity 0.45 μηη;
c) Injection et analyse par chromatographie d'exclusion stérique couplée à la diffusion de la lumière (SEC/MALLS) ;  c) Injection and analysis by size exclusion chromatography coupled with light scattering (SEC / MALLS);
d) La technique permet d'accéder aux masses molaires moyennes en nombre (Mn) et en poids (Mw) et renseigne également sur la forme et la dimension des chaînes et la polydispersité (Ip).  d) The technique gives access to the number average molar masses (Mn) and weight (Mw) and also provides information on the shape and size of the chains and the polydispersity (Ip).
Tableau 1 : Récapitulatif des masses moléculaires moyennes en poids et en nombre observées pour PSHM et PSFM. Table 1: Summary of the weight and number average molecular weights observed for PSHM and PSFM.
Mn (g/mol) Mw (g/mol) Ip (Mw/Mn) Mn (g / mol) Mw (g / mol) Ip (Mw / Mn)
PSHM 133300 213500 1 ,602  PSHM 133300 213500 1, 602
PSFM 12840 36640 2,854 Il ressort de cet exemple que la masse moléculaire des polysaccharides de hautes masses moléculaire est de l'ordre de 214 kDa et que la masse moléculaire des polysaccharides de faible masse moléculaire est de l'ordre de 37 kDa. PSFM 12840 36640 2.854 It emerges from this example that the molecular weight of polysaccharides of high molecular weight is of the order of 214 kDa and that the molecular weight of polysaccharides of low molecular weight is of the order of 37 kDa.
Exemple 4 : Détermination du taux de sulfates et de résidus 3,6- anhydrogalactopyranoses des polysaccharides de l'invention EXAMPLE 4 Determination of the Level of Sulfates and Residues of 3,6-Anhydrogalactopyranoses of the Polysaccharides of the Invention
1 . Détermination du taux de sulfates 1. Determination of sulphate level
Dosage par turbidimétrie (BaC /gélatine) Turbidimetric determination (BaC / gelatin)
Les ions sulfates libérés lors de l'hydrolyse des polysaccharides vont former, en présence de chlorure de baryum (BaC , 2H2O) et de gélatine un précipité de sulfate de baryum dont l'apparition est mesurée à 550 nm, comme décrit dans la publication deDodgson & Price (Dodgson & Price, 1962, Biochemical Journal 84 : 106-1 10). The sulphate ions released during the hydrolysis of the polysaccharides will form, in the presence of barium chloride (BaC, 2H2O) and gelatin, a precipitate of barium sulphate whose appearance is measured at 550 nm, as described in the publication by Dodson & Price (Dodgson & Price, 1962, Biochemical Journal 84: 106-1-10).
150 mg de gélatine sont dissous dans 50 ml_ d'eau milli-Q à 70°C. Après refroidissement 16 h à 4°C, la solution de gélatine est additionnée de 0,5 g de BaC . 120 mg de polysaccharide lyophilisé sont hydrolysés par 3 ml_ d'HCI 2 M pendant 2 h à 100°C. Le mélange est centrifugé à 13 000 g pendant 30 min. 1 mL de surnageant est mélangé à 9 mL d'eau milli-Q, 1 mL d'HCI 0,5 M et 0,5 mL de réactif BaC /gélatine. Après 30 min à température ambiante, le mélange est agité et l'absorbance est lue immédiatement à 550 nm. La gamme étalon est réalisée à l'aide d'une solution mère de K2SO4 à 3 mg/mL.  150 mg of gelatin are dissolved in 50 ml of milli-Q water at 70 ° C. After cooling for 16 hours at 4 ° C., 0.5 g of BaC is added to the gelatin solution. 120 mg of freeze-dried polysaccharide are hydrolysed with 3 ml of 2 M HCl for 2 h at 100 ° C. The mixture is centrifuged at 13,000 g for 30 minutes. 1 ml of supernatant is mixed with 9 ml of milli-Q water, 1 ml of 0.5 M HCl and 0.5 ml of BaC reagent / gelatin. After 30 min at room temperature, the mixture is stirred and the absorbance read immediately at 550 nm. The standard range is performed using a stock solution of K2SO4 at 3 mg / mL.
Dosage à l'Azuré A Dosage with Azure A
La quantité de sulfates a été déterminée par l'utilisation de la méthode de dosage colorimétrique développée par Jaques et al. (Jaques L.B et al., 1968, Canadian Journal of Physiology and Pharmacology 46, pages : 351 -360). En phase aqueuse, le chlorure de 3- amino-7-(diméthylamino)phénothizin-5-ium (Azuré A) complexe les sulfates pouvant être présents, notamment au sein des polysaccharides composant les fractions de SPE. Le milieu développe alors une couleur rose-violet absorbant à λ=535 nm, due à la formation d'un chromophore en présence de sulfates. Le dosage est semi-quantitatif et donne un ordre de grandeur (~mg) de la concentration en sulfates d'un échantillon. Dans des cuves en plastique pour spectrophotomètre sont introduits 200 μί de solution à doser. 2 mL de solution aqueuse d'Azuré A à 10 mg/L sont ajoutés puis l'échantillon est agité. L'absorbance est mesurée à λ=535 nm. La quantification des sulfates est déterminée à partir de la gamme d'étalonnage de dextrane sulfate (17 % sulfaté) et correction du degré de sulfatation de ce dernier (17 mg de sulfates pour 100 mg de dextrane sulfate). The amount of sulfates was determined by the use of the colorimetric assay method developed by Jaques et al. (Jaques LB et al., 1968, Canadian Journal of Physiology and Pharmacology 46, pp. 351-360). In the aqueous phase, 3-amino-7- (dimethylamino) phenothiazin-5-ium chloride (Azure A) complexes the sulphates that may be present, in particular within the polysaccharides composing the SPE fractions. The medium then develops an absorbing pink-violet color at λ = 535 nm, due to the formation of a chromophore in the presence of sulphates. The assay is semi-quantitative and gives an order of magnitude (~ mg) of the sulfate concentration of a sample. In plastic tanks for spectrophotometer are introduced 200 μί of solution to be assayed. 2 mL of Azuré A aqueous solution at 10 mg / L are added and the sample is then stirred. The absorbance is measured at λ = 535 nm. The quantification of sulphates is determined from the calibration range of dextran sulphate (17% sulphated) and correction of the degree of sulphation of the latter (17 mg of sulphates per 100 mg of dextran sulphate).
Tableau 2. Récapitulatif des taux de sulfates pour PSHM et PSFM. Table 2. Summary of sulphate levels for PSHM and PSFM.
Taux de sulfates (% m/m) Ecart-type Sulphate rate (% m / m) Standard deviation
PSHM 13,5 0,09  PSHM 13.5 0.09
PSFM 15,7 0,26  PSFM 15.7 0.26
Il ressort de cet exemple que le taux de sulfates des polysaccharides de haute masse moléculaire est de l'ordre de 13,5% et que le taux de sulfates des polysaccharides de faible masse moléculaires est de l'ordre de 15,7%.  It emerges from this example that the sulfates content of polysaccharides of high molecular mass is of the order of 13.5% and that the sulfates content of low molecular weight polysaccharides is of the order of 15.7%.
2. Détermination du taux de résidus 3,6-anhvdrogalactopyranose 2. Determination of the Residue Level 3.6-Anhydrogalactopyranose
La méthode colorimétrique la plus reproductible pour doser les résidus 3,6- anhydrogalactoses est celle qui emploie un réactif à base de résorcinol (voir Yaphe & Arsenaut, 1965, Analytical Biochemistry 13, pages 143-148). La coloration rose qui se développe au cours de la réaction est suivie à 555 nm. Trois solutions sont nécessaires à la réalisation de ce dosage : (i) une solution d'acétaldéhyde préparée en diluant 1 mL d'acétaldéhyde dans 100 mL d'eau ultrapure (stable environ 1 mois) ; (ii) une solution de résorcinol préparée par dissolution de 150 mg de résorcinol dans 100 mL d'eau ultrapure (stable 7 jours, à l'abri de la lumière) et (iii) une solution d'HCI 10 M.  The most reproducible colorimetric method for assaying the 3,6-anhydrogalactose residues is that which employs a resorcinol-based reagent (see Yaphe & Arsenaut, 1965, Analytical Biochemistry 13, pages 143-148). The pink color that develops during the reaction is monitored at 555 nm. Three solutions are necessary to carry out this assay: (i) an acetaldehyde solution prepared by diluting 1 mL of acetaldehyde in 100 mL of ultrapure water (stable for about 1 month); (ii) a solution of resorcinol prepared by dissolving 150 mg of resorcinol in 100 ml of ultrapure water (stable for 7 days, protected from light) and (iii) 10 M HCl solution.
Pour le dosage, 50 à 100 μί de la solution de polysaccharide à doser) sont introduits dans des tubes en verre. Le volume est complété à 200 μί à l'aide d'eau milli-Q. Le réactif au résorcinol est préparé de façon extemporanée en ajoutant à 100 mL d'HCI 10 M, 9 mL de la solution de résorcinol et 1 mL de la solution d'acétaldéhyde diluée au 1/25. Ce réactif n'est stable que 3 h à l'abri de la lumière. For the assay, 50 to 100 μl of the polysaccharide solution to be assayed) are introduced into glass tubes. The volume is completed to 200 μί using milli-Q water. The resorcinol reagent is prepared extemporaneously by adding to 100 mL of 10 M HCl, 9 mL of the resorcinol solution and 1 mL of the 1/25 diluted acetaldehyde solution. This reagent is stable only 3 hours away from light.
A 200 L de la solution de polysaccharide à doser sont ajoutés 1 mL du réactif au résorcinol. Après agitation, les tubes sont laissés au repos pendant 4 min, puis placés dans un bain- marie à 80°C pendant 10 min. Ils sont ensuite transférés dans un bain de glace pendant 1 min 30. L'absorbance doit être lue dans les 15 min qui suivent à 555 nm.  200 ml of the polysaccharide solution to be determined are added 1 ml of the resorcinol reagent. After stirring, the tubes are allowed to stand for 4 minutes and then placed in a water bath at 80 ° C. for 10 minutes. They are then transferred to an ice bath for 1 minute 30 minutes. The absorbance should be read within 15 minutes at 555 nm.
Le D-fructose (solutions de 10 à 70 g/mL) est utilisé comme standard. En effet, il a été démontré que les courbes d'absorbance à 555 nm en fonction de la concentration en monosaccharide du D-fructose et du le 3,6-anhydrogalactose sont identiques (voir Yaphe & Arsenaut, 1965, Analytical Biochemistry 13, pages 143-148). Tableau 3. Récapitulatif du taux de résidus 3,6-anhydroaalactopyranose pour PSHM. D-fructose (10 to 70 g / mL solutions) is used as a standard. Indeed, it has been shown that the absorbance curves at 555 nm as a function of the monosaccharide concentration of D-fructose and of 3,6-anhydrogalactose are identical (see Yaphe & Arsenaut, 1965, Analytical Biochemistry 13, pages 143-148). Table 3. Summary of 3.6-anhydroalactopyranose residue levels for PSHM.
Taux de (3,6) Ecart-type Rate of (3.6) standard deviation
anhydrogalactopyranose (%  anhydrogalactopyranose (%
m/m)  m / m)
PSHM 1 ,32 0,013  PSHM 1, 32 0.013
Il ressort de cet exemple que le taux de 3,6-anhydrogalactopyranose des polysaccharides de haute masse moléculaire est de 1 ,32 %.  It emerges from this example that the 3,6-anhydrogalactopyranose level of polysaccharides of high molecular mass is 1.32%.
Exemple 5 : Détermination de la composition en monosaccharides et de la structure des polysaccharides selon l'invention EXAMPLE 5 Determination of the Monosaccharide Composition and the Structure of the Polysaccharides According to the Invention
10 mg de polysaccharides sont dissous dans 1 mL d'acide trifluoracétique à 2 M pendant 90 min à 120 °C, avec agitation manuelle toutes les 30 minutes. Les échantillons sont évaporés sous jet d'azote pour éliminer les traces d'acide en excès. 1 mL de méthanol est ajouté, puis l'échantillon est agité au vortex et évaporé sous jet d'azote. Cette étape est répétée 2 fois pour éliminer les traces résiduelles d'acides. La dérivatisation est réalisée via l'utilisation de BSTFA : TMCS (99 : 1 ). Pour 2 mg de monosaccharides, on ajoute 400 μί de pyridine et 400 μί de Λ/, O-bis(triméthylsilyl) trifluoroacétamide : triméthylchlosylane (BSTFA : TMCS) (99 : 1 ). Les échantillons sont ensuite mélangés puis placés à température ambiante pendant 2 heures sous agitation (450 rpm). 10 mg of polysaccharides are dissolved in 1 ml of 2 M trifluoroacetic acid for 90 min at 120 ° C., with manual stirring every 30 minutes. The samples are evaporated under a jet of nitrogen to remove traces of excess acid. 1 mL of methanol is added and the sample is vortexed and evaporated under a jet of nitrogen. This step is repeated twice to eliminate residual traces of acids. Derivatization is achieved via the use of BSTFA: TMCS (99: 1). For 2 mg of monosaccharides, 400 μl of pyridine and 400 μl of Λ /, O-bis (trimethylsilyl) trifluoroacetamide: trimethylchlosylane (BSTFA: TMCS) (99: 1) are added. The samples are then mixed and then placed at room temperature for 2 hours with stirring (450 rpm).
On évapore sous jet d'azote les échantillons, puis les résidus triméthylsilyl-O-glycosides sont repris par 500 μί de dichlorométhane. A cette étape, il est possible de diluer plus ou moins l'échantillon. Les standards (L-Rha, L-Fuc, L-Ara, D-Xyl, D-Man, D-Gal, D-GIc, D-GIcA, D-GalA) sont préparés dans les mêmes conditions, à au moins trois concentrations différentes.  The samples are evaporated under nitrogen jet, and the trimethylsilyl-O-glycoside residues are taken up in 500 μl of dichloromethane. At this stage, it is possible to dilute more or less the sample. The standards (L-Rha, L-Fuc, L-Ara, D-Xyl, D-Man, D-Gal, D-GIc, D-GIcA, D-GalA) are prepared under the same conditions, at least three times. different concentrations.
Les dérivés triméthylsilylés sont analysés par chromatographie phase gazeuse couplée à la spectrométrie de masse, sur une colonne OPTIMA-1 MS (30 m, 0,32 mm, 0,25 μηη) avec un débit d'hélium de 2,3 mL/min (jusqu'à 3 mL/min). La pression d'hélium est fixée à 8,8 psi soit 60673,9 Pa et le ratio d'injection à 25: 1 (ou 50 : 1 ). La montée de température est de 8°C/min jusqu'à 100°C, pendant 3 min. On programme une autre montée en température de 8°C/min jusqu'à 200°C, maintenue pendant 1 min. On termine par une montée en température de 5°C/min jusqu'à 250°C. L'ionisation est réalisée par Impact Electronique (El, 70 eV), la température de la trappe est fixée à 150 °C et les ions ciblés entre 40 et 800 m/z. Tableau 4. Composition en monosaccharides de l'échantillon PSHM. The trimethylsilyl derivatives are analyzed by gas phase chromatography coupled to mass spectrometry, on an OPTIMA-1 MS column (30 m, 0.32 mm, 0.25 μηη) with a helium flow rate of 2.3 mL / min. (up to 3 mL / min). The helium pressure is set at 8.8 psi or 60673.9 Pa and the injection ratio at 25: 1 (or 50: 1). The temperature rise is 8 ° C / min up to 100 ° C for 3 min. Another increase in temperature from 8 ° C./min up to 200 ° C., maintained for 1 min. It ends with a rise in temperature of 5 ° C / min to 250 ° C. The ionization is carried out by Impact Electronique (El, 70 eV), the temperature of the trap is fixed at 150 ° C and the targeted ions between 40 and 800 m / z. Table 4. Composition in monosaccharides of the PSHM sample.
Monosaccharides (mol %)* Monosaccharides (mol%) *
Gai Ara Xyl GlcA Glc  Gay Ara Xyl GlcA Glc
94,1 1 ,51 1 ,88 1 ,51 1 ,0  94.1 1, 51 1, 88 1, 51 1, 0
* Composition en monosaccharides estimées par CG/SM-IE. Gai: Galactose; Ara: Arabinose; Xyl: Xylose; GlcA: acide glucuronique, Glc: Glucose. * Composition in monosaccharides estimated by GC / MS-IE. Gay: Galactose; Ara: Arabinose; Xyl: Xylose; GlcA: glucuronic acid, Glc: Glucose.
Exemple 6 : Détermination des activités anticoagulantes des polysaccharides selon l'invention Example 6 Determination of anticoagulant activities of polysaccharides according to the invention
L'activité anticoagulante a été étudiée en termes d'inhibition de deux enzymes clés intervenant dans la coagulation : les facteurs Xa et lia, mais également en mesurant le temps de Quick (PT), le temps de thrombine (TT) et le temps de Céphaline activée TCA. Le test aPTT (Activated Partial Thromboplastin Time) ou Temps de Céphaline Activée TCA mesure le temps de coagulation d'un plasma citraté déplaquetté auquel on ajoute un activateur particulaire des facteurs contacts (silice, kaolin, acide ellagique) et de la céphaline, substitut du facteur III plaquettaire. L'aPTT normal varie suivant les activateurs, les céphalines commerciales et les appareils utilisés, de 30 à 40 s en général. Ce test semi- global explore la voie endogène de la coagulation (prékallicréine, kininogène de haute masse moléculaire, FXII, FXI, FVIII et FIX) et, dans une moindre mesure, la voie finale commune (FX, FV, Fil, FI). The anticoagulant activity was studied in terms of inhibition of two key enzymes involved in coagulation: Xa and IIa factors, but also by measuring the time of Quick (PT), the thrombin time (TT) and the time of Activated cephalin TCA. Activated Partial Thromboplastin Time (aPTT) or TCA Activated Partial Thromboplastin Time (aPTT) test measures the clotting time of a dislodged citrated plasma supplemented with a particulate activator of contact factors (silica, kaolin, ellagic acid) and partial thromboplastin replacement. platelet factor III. The normal aPTT varies according to the activators, commercial cephalins and devices used, usually 30 to 40 sec. This semi-global test explores the endogenous pathway of coagulation (prekallikrein, high molecular weight kininogen, FXII, FXI, FVIII and FIX) and, to a lesser extent, the common final pathway (FX, FV, Fil, FI).
Le test PT (Prothrombin Time) ou temps de Quick mesure le temps de coagulation du plasma décalcifié, recalcifié in vitro en présence de thromboplastine tissulaire. Il permet l'exploration globale de la voie exogène de la coagulation (voie du facteur tissulaire) : thromboplastinoformation, thrombinoformation et fibrinoformation. Cela concerne donc le facteur VII et dans une moindre mesure la voie finale commune : FX, FV, Fil, FI. The PT (Prothrombin Time) test measures the coagulation time of the decalcified plasma, recalcified in vitro in the presence of tissue thromboplastin. It allows the global exploration of the exogenous pathway of coagulation (tissue factor pathway): thromboplastinformation, thrombinoformation and fibrinoformation. This concerns factor VII and to a lesser extent the common final path: FX, FV, Fil, FI.
Le test TT (Thrombin time) ou temps de thrombine mesure le temps de coagulation du plasma décalcifié, recalcifié in vitro en présence de thrombine. Il explore la fibrinoformation dans la voie commune de la coagulation, sauf le facteur XIII. Cela correspond à la transformation du fibrinogène en fibrine. The Thrombin time (TT) test measures the coagulation time of the decalcified plasma, recalcified in vitro in the presence of thrombin. It explores fibrinoformation in the common pathway of coagulation, except factor XIII. This corresponds to the transformation of fibrinogen into fibrin.
Mode opératoire : Operating mode:
50 μί d'échantillons de polysaccharides sulfatés obtenus selon les exemples précédents (de 25 g/L à 25.10"4 g/L) sont ajoutés à 50 \\L d'antithrombine III (0,125 pour le test anti-Xa et 0,625 μς/μί. pour le test anti-lla) et incubés 30 secondes à 37°C dans une plaque 96 puits. Sont ensuite ajoutés 50 μΙ_ de facteur Xa ou lia selon le test (concentration finale de 1 1 .25 nKat/mL) avant une deuxième incubation de 30 secondes à 37°C. Pour finir, 50 μΙ_ de substrat (CBS 31.39; CH2S02-d-Leu-Gly-Arg-pNA, AcOH pour le test anti-Xa et CBS 61.50; EtM-SPro-Arg-pNA, AcOH pour le test lia) sont ajoutés dans le puits. Le milieu réactionnel est incubé 5 minutes et l'absorbance est lue à 405 nm toutes les 6 secondes. La vitesse initiale (v,) est calculée comme la pente du segment linéaire de la cinétique (5 premiers points de la courbe). La répétition a été réalisée sur une population n≥ 5. 50 μl of sulphated polysaccharide samples obtained according to the preceding examples (from 25 g / l to 25 × 10 -4 g / l) are added to 50 μl of antithrombin III (0.125 for the test). anti-Xa and 0.625 μs / μl. for the anti-11a test) and incubated 30 seconds at 37 ° C in a 96-well plate. Then 50 μΙ of Factor Xa or IIa were added according to the test (final concentration of 11.25 nKat / ml) before a second incubation of 30 seconds at 37 ° C. Finally, 50 μl of substrate (CBS 31.39, CH 2 S0 2 -d-Leu-Gly-Arg-pNA, AcOH for the anti-Xa test and CBS 61.50, EtM-SPro-Arg-pNA, AcOH for the test IIa ) are added to the well. The reaction medium is incubated for 5 minutes and the absorbance is read at 405 nm every 6 seconds. The initial velocity (v,) is calculated as the slope of the kinetic linear segment (first 5 points of the curve). The repetition was performed on a population n≥ 5.
Les contrôles négatif et positif sont respectivement l'eau (dans laquelle les échantillons sont dilués) et l'héparine non dépolymérisée (Héparine, sel de sodium, INTERCHIM, CAS 9041 - 08-1 , batch 201274, DPmoyen 14,01 KDa, 163 U/mg). Positive and negative controls are water respectively (wherein samples are diluted) and heparin non-depolymerized (Heparin, sodium salt, INTERCHIM, CAS 9041 - 08-1, batch 201274, DP av in 14.01 KDa 163 U / mg).
Dans cet exemple, l'activité anticoagulante du polysaccharide sulfaté est comparée à celle d'une héparine de faible masse moléculaire disponible sur le marché : Lovenox® et à un carraghénane iota.  In this example, the anticoagulant activity of the sulfated polysaccharide is compared to that of a low molecular weight heparin available on the market: Lovenox® and a carrageenan iota.
L'activité est calculée grâce à la formule suivante : The activity is calculated using the following formula:
Equation 1 : Activité (%) = 100 - ("' Μ»""" *100) Equation 1: Activity (%) = 100 - ("'Μ""""* 100 )
Le coagulomètre STart 4, les réactifs des tests et d'étalonnage, les contrôles positifs et négatifs et le plasma proviennent de l'enseigne Stago Diagnostica. Le mode opératoire des mesures de coagulation se déroule comme suit : The STart 4 Coagulometer, Test and Calibration Reagents, Positive and Negative Controls and Plasma come from the Stago Diagnostica brand. The procedure for coagulation measurements is as follows:
PT* aPTT*  PT * aPTT *
Préparation du plasma  Plasma preparation
1 bille d'agitation  1 ball of agitation
90 [}L de plasma  90 [} L of plasma
10 [}L de solution d'échantillon solubilisé dans 0,9% NaCI  10 μl of sample solution solubilized in 0.9% NaCl
100 μL de réactif aPTT  100 μL aPTT reagent
Incubation 37°C  Incubation 37 ° C
2 min 1 min 3 min  2 min 1 min 3 min
Ajout du réactif déclenchant la mesure Added reagent triggering measurement
Figure imgf000025_0001
Figure imgf000025_0001
200 μί de réactif PT 100 μί de réactif TT  200 μί PT reagent 100 μί TT reagent
0,025 M  0.025M
Mesure du temps de coagulation  Measurement of clotting time
*Temps de Quick (PT) ** Temps de thrombine (TT) * Quick time (PT) ** Thrombin time (TT)
*** Temps de Céphaline activée TCA (aPTT)  *** Activated cephalin time TCA (aPTT)
Les résultats ainsi obtenus sont présentés aux Figures 1 à 5. The results thus obtained are presented in FIGS. 1 to 5.
Les résultats de ces tests montrent que le polysaccharide sulfaté (PSHM et/ou PSFM) extrait à partir de l'algue Haliptilon subulatum possède : The results of these tests show that the sulfated polysaccharide (PSHM and / or PSFM) extracted from the algae Haliptilon subulatum has:
Une activité anti-FXa de PSHM à 70^g.mL"1, équivalente à celle de l'héparine ou duAn anti-FXa activity of PSHM at 70 μg.mL -1 , equivalent to that of heparin or
Lovenox® (Figure 1 ). Lovenox® (Figure 1).
Une activité anti-lla de PSHM à 8μg.mL-1 , équivalente à celle de l'héparine ou du Lovenox® (Figure 2).  An anti-lla activity of PSHM at 8μg.mL-1, equivalent to that of heparin or Lovenox® (Figure 2).
Une activité sur la voie endogène très proche de celle de l'héparine à faible concentration ainsi qu'une activité améliorée par rapport au Lovenox® (Figure 3). Une activité sur la voie exogène très proche de celle de l'héparine à faible concentration ainsi qu'une activité améliorée par rapport au Lovenox® (Figure 4). Une activité sur la voie commune (Figure 5).  Endogenous pathway activity very close to that of low concentration heparin and improved activity compared to Lovenox® (Figure 3). Activity on the exogenous pathway very close to that of low concentration heparin and improved activity compared to Lovenox® (Figure 4). An activity on the common path (Figure 5).

Claims

REVENDICATIONS
1 . Polysaccharide sulfaté extrait d'une algue rouge de l'espèce Haliptilon subulatum ou un sel pharmaceutiquement acceptable de celui-ci, caractérisé en ce que le taux de sulfate dudit polysaccharide est inférieur ou égal à 20% en masse du polysaccharide, pour son utilisation en tant qu'anticoagulant. 1. Sulfated polysaccharide extracted from a red algae of the species Haliptilon subulatum or a pharmaceutically acceptable salt thereof, characterized in that the sulfate level of said polysaccharide is less than or equal to 20% by weight of the polysaccharide, for its use in as anticoagulant.
2. Polysaccharide sulfaté selon la revendication 1 , caractérisé en ce que le polysaccharide sulfaté possède une masse moléculaire inférieure ou égale à 1000 kDa. Sulfated polysaccharide according to Claim 1, characterized in that the sulphated polysaccharide has a molecular mass less than or equal to 1000 kDa.
3. Polysaccharide sulfaté selon l'une des revendications 1 à 2, caractérisé en ce que le polysaccharide sulfaté possède une masse moléculaire comprise entre 10 kDa et 250 kDa.  3. Sulphated polysaccharide according to one of claims 1 to 2, characterized in that the sulphated polysaccharide has a molecular mass of between 10 kDa and 250 kDa.
4. Polysaccharide sulfaté selon l'une des revendications 1 à 3, caractérisé en ce qu'il est d'origine naturelle.  4. sulfated polysaccharide according to one of claims 1 to 3, characterized in that it is of natural origin.
5. Composition pharmaceutique pour son utilisation en tant qu'anticoagulant comprenant au moins un polysaccharide sulfaté tel que défini dans les revendications 1 à 4.  A pharmaceutical composition for use as an anticoagulant comprising at least one sulfated polysaccharide as defined in claims 1 to 4.
6. Composition pharmaceutique selon la revendication 5, comprenant en outre un ou plusieurs autres agents choisis parmi les excipients pharmaceutiquement acceptables, les agents actifs et les ingrédients alimentaires.  The pharmaceutical composition according to claim 5, further comprising one or more other agents selected from pharmaceutically acceptable excipients, active agents and food ingredients.
7. Composition pharmaceutique selon l'une des revendications 5 à 6, caractérisée en ce qu'elle est administrée par voie orale, topique ou injectable.  7. Pharmaceutical composition according to one of claims 5 to 6, characterized in that it is administered orally, topically or injectable.
8. Composition pharmaceutique selon l'une des revendications 5 à 7 destinée à être utilisée dans la prévention et/ou le traitement des thromboses vasculaires.  8. Pharmaceutical composition according to one of claims 5 to 7 for use in the prevention and / or treatment of vascular thromboses.
9. Composition pharmaceutique selon la revendication 8 destinée à être utilisée dans la prévention et/ou le traitement des thromboses artérielles, des thromboses veineuses profondes ou non.  9. Pharmaceutical composition according to claim 8 for use in the prevention and / or treatment of arterial thromboses, deep vein thrombosis or not.
10. Composition pharmaceutique selon l'une des revendications 8 à 9 caractérisé en ce que la composition comprenant le polysaccharide sulfaté est administrée au patient à une dose journalière comprise entre 50 mg et 1000 mg.  10. Pharmaceutical composition according to one of claims 8 to 9 characterized in that the composition comprising the sulfated polysaccharide is administered to the patient at a daily dose of between 50 mg and 1000 mg.
1 1 . Composition pharmaceutique selon l'une des revendications 5 à 7 destinée à être utilisée dans la prévention et/ou le traitement des ischémies, des embolies pulmonaires, de l'angor instable, de l'infarctus du myocarde. 1 1. Pharmaceutical composition according to one of claims 5 to 7 for use in the prevention and / or treatment of ischemia, pulmonary embolism, unstable angina, myocardial infarction.
12. Composition pharmaceutique selon l'une des revendications 5 à 7 destinée à être utilisée dans la prévention de la coagulation des circuits de circulation extracorporelle et/ou d'épuration extra-rénale. 12. Pharmaceutical composition according to one of claims 5 to 7 for use in the prevention of coagulation of extracorporeal circulation circuits and / or extra-renal purification.
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