Polysaccharides comprising two antithrombin III-binding sites, preparation thereof and use thereof as antithrombotic medicaments

• US 9,346,894 B2
• Filed: 04/11/2012
• Issued: 05/24/2016
• Est. Priority Date: 04/11/2011
• Status: Active Grant

• Current Assignee: Sanofi
• Original Assignee: Sanofi
• Inventors: Mourier, Pierre, Viskov, Christian
• Primary Examiner(s): Lau, Jonathan S
• Application Number:
  US14/111,300
• Publication Number:
  US 20140031316A1
• Time in Patent Office: 1,504 Days
• US Class Current: 1/1
• CPC Class Codes: A61K 31/727 Heparin; Heparan
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: B01D 15/1878 for multi-dimensional chromatography
• CPC Class Codes: B01D 15/34 Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
• CPC Class Codes: B01D 15/363 Anion-exchange
• CPC Class Codes: B01D 15/3823 of other types, e.g. avidin, streptavidin, biotin
• CPC Class Codes: C08B 37/0075 Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The invention relates to sulphated polysaccharides which have the general structure of the constituent polysaccharides of heparin and which have a molecular weight of less than 8000 Daltons, comprising two antithrombin III-binding hexasaccharide sequences corresponding to formula (I) in which: R1 represents an OH group when the hexasaccharide of formula (I) is located at the reducing end of the polysaccharide, or else R1 represents a bond with another saccharide unit of said polysaccharide; R2 represents a hydrogen atom when the hexasaccharide of formula (I) is located at the non-reducing end of the polysaccharide, in which case the bond between carbon atoms 4 and 5 of the first saccharide unit of said hexasaccharide is a double bond, or else R2 represents a bond with another saccharide unit of said polysaccharide, in which case the bond between carbon atoms 4 and 5 of the first saccharide unit is a single bond. Preparation thereof, use thereof as antithrombotic medicaments, and also use thereof in a method for analyzing a sample of a heparin derivative.

Claims

1. A sulfonated polysaccharide having a polysaccharide of heparin which has a molecular weight of less than 8000 Daltons, said sulfonated polysaccharide comprising two antithrombin III-affinity sites, wherein said polysaacharide is in isolated form.

2. The polysaccharide according to claim 1, comprising two antithrombin III-binding hexasaccharide sequences.

3. The polysaccharide according to claim 2, wherein said polysaccharide twice comprises the antithrombin III-binding hexasaccharide sequence corresponding to formula (I):

4. The polysaccharide according to claim 1 comprising between 12 and 22 saccharide units.

5. The polysaccharide according to claim 1 corresponding to formula

(A)_d-(Formula(I))-(B)_f-(Formula(I))-(C)_g 

 

(II)

in which;

the A, B and C units, which may be identical to or different from one another, represent disaccharide sequences,

the units of formula (I) represent a hexasaccharide sequence corresponding to Formula (I);

6. The polysaccharide according to claim 1 corresponding to formula (II):

(A)_n-(Formula(I))-(B)_m-(Formula(I))-(C)_k 

 

(II)

in which;

the A, B and C units, which may be identical to or different from one another, represent disaccharide sequences,

the units of formula (I) represent a hexasaccharide sequence corresponding to formula (I);

7. The polysaccharide according to claim 1 comprising 12 saccharide units and corresponding to formula (III):

8. The polysaccharide according to claim 1 comprising 14 saccharide units.

9. The polysaccharide according to claim 8, corresponding to formulae (IV), (V) or (VI):

10. The polysaccharide according to claim 1, wherein said polysaccharide is in the form of a sodium salt.

11. A pharmaceutical composition, comprising the polysaccharide according to claim 1, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

12. A method for treating thrombosis, comprising administering to a patient in need thereof a therapeutically effective amount of the polysaccharide of claim 1 or a pharmaceutically acceptable salt thereof.

13. The method of claim 12, wherein the thrombosis is selected from vein and arterial thromboses, deep vein thrombosis and pulmonary embolism.

14. A process for preparing the polysaccharide according to claim 1 comprising orthogonally seperating said polysaccharide from a low-molecular-weight or ultra-low-molecular-weight heparin, said separating comprising performing gel permeation, high performance liquid chromatography and AM affinity chromatography, combined with one another.

15. The process according to claim 14, wherein said process comprises the following steps:

gel permeation, then

ATIII-affinity chromatography, then

high performance liquid chromatography on a CTA-SAX column, then

purification by anion exchange chromatography and desalting by gel filtration.

16. The process according to claim 14, comprising the use of an ultra-low-molecular-weight heparin.

17. The process according to claim 16, wherein the ultra-low-molecular-weight heparin is semuloparin.

18. A method for analysing a sample of a heparin derivative comprising the following steps:

a) subjecting a test sample to orthogonal separation comprising orthogonally separating polysaccharides from the heparin, further comprising a step of isolating a dodecasaccharide fraction and/or the tetradecasaccharide fraction of said sample, and

b) quantifying the amount of one or more sulphated polysaccharides of formula (III), (IV), (V) or (VI)

19. The method of claim 18, wherein the heparin derivative comprises semuloparin.

20. A method for analysing a sample of a heparin derivative, comprising the following steps:

a) subjecting a test sample to orthogonal separation comprising a step of orthogonally separating polysaccharides from the heparin, further comprising a step of isolating a dodecasaccharide fraction of said sample, and

b) quantifying the amount of the sulphated polysaccharide of formula (III)

21. The method of claim 20, wherein the heparin derivative comprises semuloparin.