Low molecular weight heparin composition and uses thereof

Abstract

Preparations of low molecular weight heparins (LMWHs) having improved properties, e.g., properties that provide a clinical advantage, are provided herein. Methods of making and using such preparations as well as methods of analyzing starting materials, processing, intermediates and final products in the production of such LMWH preparations are provided.

1 . A low molecular weight heparin (LMWH) composition comprising:
 oligosaccharide chains that have the following structure:   
       
         
           
           
               
               
           
         
         and oligosaccharide chains that have the following structure: 
       
       
         
           
           
               
               
           
         
       
       wherein
 R is H or SO 3 Na; 
 R 1  is SO 3 Na or COCH 3 ; 
 n=2-50; 
 wherein the composition has the following properties: 
 (a) an average value for n of 9-16, 
 (b) 5 to 15% ΔUH NAc,6S GH NS,3S,6S  as measured by mole %, 
 a weight average molecular weight of 5500 to 8500 Da, 
 (d) anti-Xa activity of 120 to 380 IU/mg, 
 (e) an anti-Xa to anti-IIa ratio of 2:1 to 1:1, and 
 (f) the ratio of (e) is constant over a period of about 30 to 120 minutes after administration to a subject. 
 
     
     
         2 - 29 . (canceled) 
     
     
         30 . A pharmaceutical composition comprising a LMWH composition of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         31 . A method of treating a subject having or at risk of having a thrombolytic disorder, comprising administering a composition of  claim 1  to a subject, to thereby treat the disorder. 
     
     
         32 .- 47 . (canceled) 
     
     
         48 . A method of monitoring a subject treated with a LMWH composition of  claim 1  comprising:
 evaluating activated clotting time (ACT) or activated partial thromboplastin time (aPTT) in a subject who has been administered a LMWH composition of  claim 1     
     
     
         49 . A method of treating a subject who has been administered a LMWH composition of  claim 1  comprising:
 administering protamine sulfate to a subject to neutralize some or all of the anti-Xa activity, anti-IIa activity or both of a LMWH composition of  claim 1  in the subject.   
     
     
         50 . (canceled) 
     
     
         51 . A method of making a low molecular weight heparin (LMWH) composition, the method
 comprising
 providing a precursor LMWH composition having an average chain length of about 8 to 14 disaccharides obtained by salt precipitation and cleavage of a heparin sample with an enzyme which cleaves at unsulfated uronic acid linkages; and 
 subjecting the precursor LMWH composition to a size selection step to obtain a LMWH composition having an average chain length (n) of about 9 to 16 disaccharides, wherein the LMWH composition comprises: 
 oligosaccharide chains that have the following structure: 
   
       
         
           
           
               
               
           
         
         and oligosaccharide chains that have the following structure: 
       
       
         
           
           
               
               
           
         
       
       wherein
 R is H or SO 3 Na; 
 R 1  is SO 3 Na or COCH 3 ; 
 n=2-50; 
 and wherein the composition has the following properties: 
 (g) 5 to 15% ΔUH NAc,6S GH NS,3S,6S  as measured by mole %, 
 (h) weight average molecular weight of 5500 to 8500 Da, 
 (i) anti-Xa activity of 120 to 380 IU/mg, 
 (j) an anti-Xa to anti-IIa ratio of 2:1 to 1:1, and 
 (k) the ratio of (d) is constant over a period of about 30 to 120 minutes after administration to a subject. 
 
     
     
         52 .- 57 . (canceled) 
     
     
         58 . The method of  claim 51 , wherein the size selection step comprises chromatography or filtration. 
     
     
         59 . The method of  claim 58 , wherein the chromatography is size-selection chromatography or ion-exchange chromatography. 
     
     
         60 . The method of  claim 51 , wherein the enzyme is a heparinase III. 
     
     
         61 . The method of  claim 51 , wherein the enzyme is a modified heparinase III enzyme having a substitution of an alanine for a histidine at His225. 
     
     
         62 . The method of  claim 51 , wherein the enzyme is heparin sulfate glycosaminoglycan lyase III from  Bacteroides thetaiotaomicron.    
     
     
         63 . The method of  claim 51 , wherein the LMWH composition has an average molecular weight of about 5700 to 7900 Da. 
     
     
         64 . The method of  claim 51 , wherein the LMWH composition has an anti-Xa activity of about 170 to 330 IU/mg. 
     
     
         65 . The method of  claim 64 , wherein the LMWH composition has an anti-Xa activity of about 180 to 300 IU/mg. 
     
     
         66 . The method of  claim 51 , wherein the LMWH composition has an anti-IIa activity of about 130 to 190 IU/mg. 
     
     
         67 . The method of  claim 66 , wherein the LMWH composition has an anti-IIa activity of about 155 to 185 IU/mg. 
     
     
         68 . The method of  claim 51 , wherein the LMWH composition has less than 1000 ng/mg of a heparinase enzyme; less than 1% w/w methanol; less than 1% w/w ethanol; and less than 2000 ppm free sulfate. 
     
     
         69 . The method of  claim 51 , wherein 15 to 35% of the total number of chains in the composition have a Δ4,5 unsulfated uronic acid at the non-reducing end. 
     
     
         70 . The method of  claim 51 , wherein at least 60% of the chains in the composition have an N-acetylated hexosamine at the reducing end. 
     
     
         71 . The method of  claim 70 , wherein at least 80% of the chains of the composition have an N-acetylated hexosamine at the reducing end. 
     
     
         72 . The method of  claim 51 , further comprising
 precipitating the precursor LMWH composition having an average chain length of about 8 to 14 disaccharides prior to the size selection step.   
     
     
         73 . The method of  claim 51 , further comprising processing the LMWH composition into drug product.