Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • 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
  • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
  • A61K31/727—Heparin; Heparan
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • 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
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
  • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
  • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
  • C08B37/0078—Degradation products

Definitions

• the present invention relates to the field of low molecular weight polysaccharides. More particularly, it relates to oosaccharide compositions exhibiting excellent pharmacological and antifhrombotic properties.
• antithrombotic treatments use two main categories of agents, namely anticoagulants and antiplatelet agents.
• anticoagulant agents the anti-vitamin K compounds constitute a very important family. These compounds being active orally, they are used in many indications. However, their use is still limited by certain drawbacks and in particular the risks of hemorrhage they cause - and the difficulty of adapting the dosage to a long-term treatment.
• Heparin is the second category of anticoagulant agents. They are biological substances of extraction from the family of glycosaminoglycans composed of oligosaccharides having chain lengths and varying degrees of sulfation. Heparin is used in various types of thrombosis, in particular in the treatment or prevention of venous thrombosis, possibly associated with other therapies.
• heparins lie in their high anticoagulant activity which can cause bleeding, and in their sensitivity to certain serum factors, such as pf4, which requires the use of relatively large doses. Furthermore, heparins are very heterogeneous products. It is therefore difficult to assess their mechanism of action, to assess the contribution of each of the components in the overall activity of heparin, and, therefore, to increase the antimrombotic activity without increasing the side effects. .
• heparins are obtained by fragmentation (depolymerization) of the oligosaccharide chains using chemical or enzymatic agents.
• depolymerization has been described by treatment of a heparin ester in the presence of a strong base (EP 40144). It can also be carried out by treatment of heparin in the presence of nitrous acid, or by the action of a heparinase (EP 64452).
• patent EP 27089 indicates that mixtures of oligosaccharides derived from heparin not containing more than 8 saccharide units have a specific antithrombotic activity greater than heparin.
• hexasaccharides were prepared and their antithrombotic properties studied (EP 64452).
• the present invention more specifically results from the identification of monodispersed heparin fractions and having an average molecular weight close to 6 kD, possessed a high antithrombin activity.
• An object of the invention resides in a mixture of sulfated oligosaccharides having the general structure of the oligosaccharides constituting heparin, characterized in that it has a weight-average molecular mass of 6 + 0.6 kD and a near polydispersity of 1, and in that it has the capacity to inhibit the generation of thrombin.
• the polydispersity corresponds to the ratio of the average molecular weight of the mixture to its number average molecular weight. She reports on the molecular homogeneity of the mixture. The closer this value is to 1, the more homogeneous the mixture.
• the mixtures of the invention have particularly advantageous pha ⁇ nacokinetic properties.
• the mixtures of the invention have a lower sensitivity to serum factors such as ⁇ f4, which increases their therapeutic potential.
• mixtures of the invention lie in particular in their excellent bioavailability and plasma half-life.
• the properties set out above allow particularly effective pharmaoological use, in particular in the prophylaxis and treatment of venous or arterial thromboses.
• they should make it possible to use larger doses in vivo without increasing the risk of bleeding.
• the mixtures of the invention are more particularly fractions of depolymerized heparin.
• the depolymerized heparin can be obtained by any chemical, enzymatic or other technique known to those skilled in the art making it possible to fragment the oligosaccharide chains of heparin.
• EP 337327 are suitable for the invention.
• the mixtures of the invention consist of oligosaccharides having a 2-Q-sulfo 4 enopyranosuronic acid at one of their ends.
• a particularly advantageous mixture consists of a fraction of heparin depolymerized by the action of a base on a heparin ester.
• the antithrombin activity of the mixtures of the invention can be demonstrated in a test in which the generation of thrombin is triggered in the presence of human thromboplasm (extrinsic route) or by contact (intrinsic route).
• a test has been described previously (Hemker et aL, Thromb. Haemostas. 56, 9-17,
• This activity can be expressed quantitatively, by the amount of product necessary for the inhibition of 25% of the generation of thrombin.
• the increase in activity of the mixtures of the invention appears clearly since, in vitro, their specific antithrombin activity is, surprisingly, increased by a factor greater than 100% compared to l heparin used at the start -
• this increase in specific activity is even greater in vivo.
• the mixtures of the invention make it possible, in a teist performed on plasma poor in platelets, to inhibit 25% of the generation of thrombin at concentrations below 300 ng ml.
• Another subject of the invention relates to a process for the preparation of a mixture as defined above, characterized in that a heparin or a heparin depolymerized by gel-filtration is fractionated.
• the process of the invention involves several parameters, the control of which makes it possible to calibrate the molecular mass of the final mixture, and to fix its polydispersity. These parameters are in particular the ionic strength of the eluent and the nature of the support used. More preferably, the fractionation is characterized in that the steps consisting in (i) putting the starting heparin or depolymerized heparin in solution in the eluent are carried out successively (ii) passing the solution thus obtained to through at least one column containing the solid support for gei-filtration previously balanced with the same eluent, and (iii) recovering the fractions of desired molecular weight.
• heparin a depolymerized heparin.
• a heparin depolymerized by the action of a base on a heparin ester is made of.
• the depolymerization can be carried out in an aqueous medium or in an inert organic solvent, under the action an organic or inorganic base such as for example sodium or potassium hydroxide, an alkaline carbonate or a tertiary amine (triethylamine, triethylene diamine, etc.).
• an organic or inorganic base such as for example sodium or potassium hydroxide, an alkaline carbonate or a tertiary amine (triethylamine, triethylene diamine, etc.).
• the action of the base on the ester makes it possible to carry out a partial and controlled depolymerization of the heparin without altering its general structure.
• eluent which can be used in the process of the invention, mention may be made of different types of saline solutions, such as sodium chloride solutions.
• saline solutions such as sodium chloride solutions.
• the applicant has shown that, in order to obtain fractions having the best properties, it is particularly advantageous to carry out the fractionation using an eluent chosen from phosphate buffers, such as in particular potassium phosphate, sodium phosphate or NH 4 H2PO 4 . It is also possible to use NaClO 4 or NH .NOg solutions which make it possible to obtain mixtures having excellent characteristics.
• concentration of the eluent and therefore its ionic strength are adapted to the desired final mixture.
• concentration of the eluent is advantageously less than 1M, and even more preferably between 0.1 and 0.5 M.
• concentrations close to 0.2 M When using a phosphate buffer, it is particularly advantageous to operate at concentrations close to 0.2 M.
• the support used is generally chosen as a function of the average molecular weight of the starting mixture (native heparin, depolymerized, etc.), of the desired final product, and of the behavior of the mixture of departure in the eluent used.
• a polyacrylamide-agarose type gel is used as support.
• the solid support is distributed in several columns arranged in series.
• This variant of the invention makes it possible to use large final amounts of support for gel-filtration, without the drawbacks of the prior art, namely-essentially the packing phenomena. In this way, the separation is much clearer, including in the high molecular weights, in a single operation of fractionation, and the supports are more easily regenerable.
• the number of columns used is adapted by those skilled in the art according to the volume and the nature of the gel used, so as to obtain the best balance between the effectiveness of the separation and the harmful effect due to the packing of the gel. For practical reasons of implementation, it is generally preferred to use in the second step of the process a number of columns less than 20.
• 40 liters of AcA 202 gel can be divided into 10 columns of 4 liters.
• At least two types of support are used in the second stage of the fractionation, having -different separation characteristics.
• This variant of the invention makes it possible to obtain a final fractionation of better quality.
• the fractionation can be carried out on the following sequence of gels: AcA 202 - AcA 54 - AcA 202.
• the volume of gel must be adapted to the quantity of product to be separated, so as to obtain the best balance between separation and the effect of longitudinal diffusion.
• the starting heparin (g) / gel volume (1) ratio is less than 2, and even more preferably between 0.5 and 1.5.
• the invention also relates to a process for the preparation of mixtures of weakly dispersed oligosaccharides and of calibrated molecular weight by fractionation by gel-filtration on solid support of heparin or depolymerized heparin, characterized in that the solid support is divided into several columns arranged in series.
• Another subject of the invention relates to a pharmaceutical composition having as active ingredient a mixture as defined above.
• Such a composition can be used particularly advantageously in the prophylaxis or the treatment or prevention of thrombotic accidents. More specifically, it can be used: - in the prevention of venous thrombosis in risk situations, - in the prevention of arterial thrombotic accidents, in particular in the case of myocardial infarction,
• Example 1 Preparation of mixtures according to the invention.
• a solution containing 20 g of heparin depolymerized under the conditions described above is placed at the head of column (a) and eluted with a phase mobile consisting of a 0.33M NaCl solution, at a flow rate of 210 ml / hour.
• a solution containing 2 g of heparin depolymerized under the conditions of example 1 is loaded at the top of the device and eluted with a 0.2 M aqueous solution of H2PO4 at a flow rate of 0.42 ml / min. - from 9 p.m., 113 fractions of 12.6 ml are collected.
• Example 2 The procedure is as in Example 2: - 10 columns with an internal diameter of 10 cm and a height of 50 cm each containing 3 to 4 liters of AcA 202 gel are connected in series,
• Example 2 A solution containing 30 g of heparin depolymerized under the conditions of Example 1 is loaded at the top of the device and eluted with a 0.2 M aqueous solution of KH2PO4 at a flow rate of 6.8 ml / min. Fractions are obtained having the desired polydispersity characteristics.
• the antithrombin activity of the mixtures of the invention is measured on plasma stimulated by human thromboplastin (extrinsic route) or by contact (phospholipids + kaoline: intrinsic route) under the conditions described above (see Hemker et al. Cited above).
• the activity is estimated by the decrease the peak of the thrombin generation curve compared to a control carried out in the presence of buffer only.
• the results are expressed by the IC25: concentration necessary to obtain 25% inhibition of the generation of thrombin.
• thrombin is triggered by the addition of 1/4 of volume of diluted thromboplastin 1:40 in 0.1 M CaC12 (extrinsic system) or by 6 ⁇ M of phospholipids (20% phosphatidyl serine, 80 % phosphatidyl choline) and 0.15 mg / ml of kaoline in 0.1 M CaCl 2 (intrinsic system).
• the generation of thrombin is obtained by measuring at regular intervals (15-30 sec.) The amydolitic activity on the substrate S2238, chromogenic substrate at 405 nM specific for thrombin. Different concentrations of the samples are tested, in order to obtain 25% of control ihnibition.

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Epidemiology (AREA)
• Molecular Biology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Dermatology (AREA)
• Polymers & Plastics (AREA)
• Materials Engineering (AREA)
• Biochemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Diabetes (AREA)
• Hematology (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Polysaccharides And Polysaccharide Derivatives (AREA)

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• 1991
  • 1991-04-23 FR FR919104991A patent/FR2675806B1/fr not_active Expired - Fee Related
• 1992
  • 1992-04-21 CA CA002108363A patent/CA2108363A1/fr not_active Abandoned
  • 1992-04-21 AU AU17485/92A patent/AU1748592A/en not_active Abandoned
  • 1992-04-21 WO PCT/FR1992/000352 patent/WO1992018544A1/fr not_active Application Discontinuation
  • 1992-04-21 EP EP92910133A patent/EP0581846A1/de not_active Ceased
  • 1992-04-21 ZA ZA922874A patent/ZA922874B/xx unknown
  • 1992-04-21 JP JP4509237A patent/JPH06506968A/ja active Pending
  • 1992-04-21 EP EP92401111A patent/EP0511075A1/de active Pending
  • 1992-04-22 IE IE128792A patent/IE921287A1/en not_active Application Discontinuation
  • 1992-04-22 NZ NZ242431A patent/NZ242431A/en unknown
  • 1992-04-22 MX MX9201846A patent/MX9201846A/es unknown

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