Oligosaccharide mixtures derived from heparin, preparation thereof and pharmaceutical compositions containing them

Abstract

Oligosaccharide mixtures comprising anti-Xa activities of from 190 IU/mg to 450 IU/mg and anti-IIa activities of less than 0.2 IU/mg; and having constituent oligosaccharides with an average molecular weight of from 1800 to 2400 Daltons, constituent oligosaccharides comprise from 2 to 16 saccharide units, a 4,5-unsaturated uronic acid 2-O-sulfate unit at one end, and comprising at least one ΔIIa-IIs-Is hexasaccharide sequence are described. Processes for preparing the oligosaccharide mixtures, pharmaceutical compositions and articles of manufacture comprising the oligosaccharide mixtures are described. Methods of treatment and prevention comprising administering the oligosaccharide mixtures are described.

1 . An oligosaccharide mixture, wherein:
 the oligosaccharide mixture comprises an anti-Xa activity of from 190 μl/mg to 450 IU/mg;   the oligosaccharide mixture comprises an anti-IIa activity of less than 0.2 IU/mg;   the constituent oligosaccharides of the mixture have an average molecular weight of from 1800 to 2400 Daltons;   the constituent oligosaccharides of the mixture comprise from 2 to 16 saccharide units;   the constituent oligosaccharides of the mixture have a 4,5-unsaturated uronic acid 2-O-sulfate unit at one end; and   the constituent oligosaccharides of the mixture comprise oligosaccharides comprising a hexasaccharide of the following formula:   
       
         
           
           
               
               
           
         
         wherein the constituent oligosaccharides of the mixture are in the form of at least one metal salt chosen from alkali metal and alkaline-earth metal salts. 
       
     
     
         2 - 13 . (canceled) 
     
     
         14 . An oligosaccharide mixture, wherein:
 the mixture comprises an anti-Xa activity of from 190 IU/mg to 450 IU/mg;   the mixture comprises an anti-IIa activity of less than 0.2 IU/mg;   the constituent oligosaccharides of the mixture have an average molecular weight of from 1800 to 2400 Daltons;   the constituent oligosaccharides of the mixture comprise from 2 to 16 saccharide units; and   the constituent oligosaccharides of the mixture have a 4,5-unsaturated uronic acid 2-O-sulfate unit at one end;   wherein:   from 20% to 100% of the oligosaccharides of the mixture are hexasaccharides;   from 20% to 70% of the hexasaccharides have the sequence:   
       
         
           
           
               
               
           
         
       
       and
 the constituent oligosaccharides of the mixture are in the form of at least one metal salt chosen from alkali metal and alkaline-earth metal salts. 
 
     
     
         15 . A process for preparing an oligosaccharide mixture of  claim 1 , comprising:
 providing a quaternary ammonium salt of a low molecular weight heparin benzyl ester having an anti-IIa activity of from 0.2 to less than 5 IU/mg and an average molecular weight of from 1500 to 4,000 Daltons; and   depolymerizing said quaternary ammonium salt of a low molecular weight heparin benzyl ester by means of a strong organic base with a pKa value of greater than 20, so as to obtain a depolymerized low molecular weight heparin.   
     
     
         16 - 32 . (canceled) 
     
     
         33 . A process for preparing an oligosaccharide mixture, comprising:
 providing a LMWH having an anti-IIa activity of from 0.2 to less than 51 U/mg, and an average molecular weight of from 1,500 Daltons to 4,000 Daltons; and   subjecting the LMWH to the following chemical reactions:
 a) transsalification by the action of benzethonium chloride to obtain benzethonium heparinate; 
 b) esterification of the benzethonium heparinate, and treatment to obtain a sodium salt of the benzyl ester of the LMWH; 
 c) transsalification of the sodium salt of the benzyl ester and production of a quaternary ammonium salt; 
 d) depolymerization by means of a strong organic base, so as to obtain a depolymerized LMWH; 
 e) conversion of the quaternary ammonium salt of the depolymerized low molecular weight heparin into at least one metal salt chosen from alkali metal and alkaline-earth metal salts salt; and 
 f) saponification of the residual esters and optional purification; 
   wherein:   the oligosaccharide mixture comprises an anti-Xa activity of from 190 IU/mg to 450 IU/mg;   the oligosaccharide mixture comprises an anti-IIa activity of less than 0.2 IU/mg;   the constituent oligosaccharides of the mixture have an average molecular weight of from 1800 to 2400 Daltons;   the constituent oligosaccharides of the mixture comprise from 2 to 16 saccharide units;   the constituent oligosaccharides of the mixture have a 4,5-unsaturated uronic acid 2-O-sulfate unit at one end; and   the constituent oligosaccharides of the mixture comprise oligosaccharides comprising a hexasaccharide of the following formula:   
       
         
           
           
               
               
           
         
         wherein the constituent oligosaccharides of the mixture are in the form of at least one metal salt chosen from alkali metal and alkaline-earth metal salts. 
       
     
     
         34 . An oligosaccharide mixture prepared by the process of  claim 33 . 
     
     
         35 . A pharmaceutical composition comprising an oligosaccharide mixture according to  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         36 - 38 . (canceled) 
     
     
         39 . A pharmaceutical composition comprising an oligosaccharide mixture according to  claim 14 , and a pharmaceutically acceptable carrier. 
     
     
         40 - 46 . (canceled) 
     
     
         47 . A method of treating and/or preventing one or more of venous thrombosis, arterial thrombosis, deep vein thrombosis, pulmonary embolism, unstable angina, myocardial infarction, cardiac ischemia, occlusive diseases of the peripheral arteries and atrial fibrillation, smooth muscle cell proliferation, atherosclerosis, arteriosclerosis, cancer by modulating angiogenesis and growth factors, and also diabetic disorders such as diabetic retinopathy or diabetic nephropathy in a patient, the method comprising administering to a patient in need thereof a therapeutically effective amount of the oligosaccharide mixture according to  claim 1 . 
     
     
         48 . A method of treating and/or preventing one or more of venous thrombosis, arterial thrombosis, deep vein thrombosis, pulmonary embolism, unstable angina, myocardial infarction, cardiac ischemia, occlusive diseases of the peripheral arteries and atrial fibrillation, smooth muscle cell proliferation, atherosclerosis, arteriosclerosis, cancer by modulating angiogenesis and growth factors, and also diabetic disorders such as diabetic retinopathy or diabetic nephropathy in a patient, the method comprising administering to a patient in need thereof a therapeutically effective amount of the oligosaccharide mixture according to  claim 14 . 
     
     
         49 . (canceled) 
     
     
         50 . A method for treating and/or preventing thrombotic episodes in a patient, comprising administering to a patient in need thereof a therapeutically effective amount of the oligosaccharide mixture according to  claim 1 . 
     
     
         51 . A method for treating and/or preventing thrombotic episodes in a patient, comprising administering to a patient in need thereof a therapeutically effective amount of the oligosaccharide mixture according to  claim 14 . 
     
     
         52 - 61 . (canceled) 
     
     
         62 . A therapeutic method for controlling thrombosis and/or decreasing blood hypercoagulation and/or hemorrhaging risks in a human patient, comprising administering to a patient in need thereof a therapeutically effective amount of the oligosaccharide mixture according to  claim 1 . 
     
     
         63 . A therapeutic method for controlling thrombosis and/or decreasing blood hypercoagulation and/or hemorrhaging risks in a human patient, comprising administering to a patient in need thereof a therapeutically effective amount of the oligosaccharide mixture according to  claim 14 . 
     
     
         64 . (canceled) 
     
     
         65 . An article of manufacture for use in connection with the treatment and/or prevention of venous thrombosis, arterial thrombosis, deep vein thrombosis, pulmonary embolism, unstable angina, myocardial infarction, cardiac ischemia, occlusive diseases of the peripheral arteries and atrial fibrillation, smooth muscle cell proliferation, atherosclerosis, arteriosclerosis, cancer by modulating angiogenesis and growth factors, and also diabetic disorders such as diabetic retinopathy or diabetic nephropathy in a patient, comprising the oligosaccharide mixture according to  claim 1  and instructions for the administration thereof. 
     
     
         66 . An article of manufacture for use in connection with the treatment and/or prevention of venous thrombosis, arterial thrombosis, deep vein thrombosis, pulmonary embolism, unstable angina, myocardial infarction, cardiac ischemia, occlusive diseases of the peripheral arteries and atrial fibrillation, smooth muscle cell proliferation, atherosclerosis, arteriosclerosis, cancer by modulating angiogenesis and growth factors, and also diabetic disorders such as diabetic retinopathy or diabetic nephropathy in a patient, comprising the oligosaccharide mixture according to  claim 14  and instructions for the administration thereof. 
     
     
         67 . (canceled) 
     
     
         68 . An article of manufacture for use in connection with the treatment and/or prevention of thrombotic episodes in a patient, comprising the oligosaccharide mixture according to  claim 1 , and instructions for the administration thereof. 
     
     
         69 . An article of manufacture for use in connection with the treatment and/or prevention of thrombotic episodes in a patient, comprising the oligosaccharide mixture according to  claim 14 , and instructions for the administration thereof. 
     
     
         70 - 79 . (canceled) 
     
     
         80 . An article of manufacture for use in connection with controlling thrombosis and/or decreasing blood hypercoagulation and/or hemorrhaging risks in a human patient, comprising the oligosaccharide mixture according to  claim 1 , and instructions for the administration thereof. 
     
     
         81 . An article of manufacture for use in connection with controlling thrombosis and/or decreasing blood hypercoagulation and/or hemorrhaging risks in a human patient, comprising the oligosaccharide mixture according to  claim 14 , and instructions for the administration thereof. 
     
     
         82 . (canceled) 
     
     
         83 . A method for determining the anti-Xa activity of the oligosaccharide mixture as claimed in  claim 1 , wherein an amidolytic method is used on a chromogenic substrate in which the reconstitution buffer is Polyethylene Glycol 6000 (PEG 6000).