Glycosaminoglycan-antithrombin III/heparin cofactor II conjugates

Abstract

Novel conjugates of glycosaminoglycans, particularly heparin and dermatan sulfate, and amine containing species and therapeutic uses thereof are described. In particular, mild methods of conjugating heparins to proteins, such as antithrombin III and heparin cofactor II, which provide covalent conjugates which retain maximal biological activity are described. Uses of these conjugates to prevent thrombogenesis, in particular in lung airways, such as found in infant and adult respiratory distress syndrome are also described.

What is claimed is:  
     
         1 . A covalent conjugate comprising a glycosaminoglycan linked to a species by a covalent linkage wherein said species comprises at least one primary amino group, wherein said species is directly covalently linked via said amino group to a terminal aldose residue of said glycosaminoglycan, and pharmaceutically acceptable salts thereof.  
     
     
         2 . The conjugate of  claim 1  wherein said covalent linkage is selected from the group consisting of: 
 (a) a —HC═N— group formed between said amino group and the C1 carbonyl group of said terminal aldose residue; and  
 (b) a —CH 2 —NH— group between said amino group and the C1 carbonyl group of said terminal aldose residue.  
 
     
     
         3 . The compound of  claim 1  wherein said glycosaminoglycan is heparin, dermatan sulfate, heparan sulfate, chondroitin-6-sulfate, chondroitin-4-sulfate, chondroitin, keratan sulfate, hyaluronic acid, or a fragment thereof.  
     
     
         4 . The conjugate of  claim 1  wherein said molecule is a protein.  
     
     
         5 . The conjugate of  claim 4 , wherein said protein is a serine protease inhibitor.  
     
     
         6 . The conjugate of  claim 5 , wherein said protease inhibitor is selected from the group consisting of antithrombin III and heparin cofactor II.  
     
     
         7 . The conjugate of  claim 6  wherein, said glycosaminoglycan is heparin, dermatan sulfate, heparan sulfate, chondroitin-6-sulfate, chondroitin-4-sulfate, chondroitin, keratan sulfate, hyaluronic acid, or a fragment thereof.  
     
     
         8 . A covalent conjugate comprising a glycosaminoglycan and a molecule comprising at least one amino group, wherein said amino group is directly covalently linked to said glycosaminoglycan, wherein said conjugate is made by the process comprising: 
 (a) incubating said glycosaminoglycan with said compound under conditions which allow imine formation between said amino group and a terminal aldose residue of said glycosaminoglycan;    (b) optionally reducing said imine to an amine; and    (c) isolating said conjugate; and pharmaceutically acceptable salts thereof.    
     
     
         9 . The conjugate of  claim 8 , wherein said glycosaminoglycan is heparin, dermatan sulfate or a fragment thereof of either, and said molecule is antithrombin III or heparin cofactor II.  
     
     
         10 . A process for conjugating a molecule comprising at least one primary amino group to an glycosaminoglycan, said process comprising: 
 (a) incubating said glycosaminoglycan with said molecule under conditions which allow formation of an imine between said amino group and a terminal aldose residue of said glycosaminoglycan;    (b) optionally reducing said imine to an amine; and    (c) isolating said conjugate.    
     
     
         11 . A process for conjugating a molecule comprising at least one primary amino group to an glycosaminoglycan, said process comprising: 
 (a) incubating said glycosaminoglycan with said molecule under conditions which allow formation of an imine between said amino group and a terminal aldose residue of said glycosaminoglycan and subsequent rearrangement of said imine to a α-carbonyl amine; and    (b) isolating said conjugate.    
     
     
         12 . The process of  claim 10  or  11 , wherein said molecule is a protein.  
     
     
         13 . The process of  claim 10  or  11 , wherein said glycosaminoglycan is heparin, dermatan sulfate or a fragment thereof of either, and said molecule is a protein.  
     
     
         14 . The process of  claim 13 , wherein said protein is antithrombin III or heparin cofactor II.  
     
     
         15 . A pharmaceutical preparation, containing a conjugate as in  claim 3  as active ingredient in association with a pharmaceutically acceptable carrier.  
     
     
         16 . A pharmaceutical preparation as described in  claim 15 , in the form of an aqueous solution for injection or in the form of an ointment or in the form of an aerosol.  
     
     
         17 . The use of a compound as described in  claim 3  for the prophylactic and therapeutic treatment of ailments relating to high coagulation activity of blood in a mammal.  
     
     
         18 . The use-as described in  claim 17 , wherein said mammal is man.  
     
     
         19 . The use of a compound as described in  claim 3 , for the prevention and treatment of thrombosis.  
     
     
         20 . The use of a compound of  claim 3  for the treatment of infant or adult respiratory distress syndrome.  
     
     
         21 . The use as in  claim 20 , wherein the compound of  claim 3  is delivered directly to the airways of the lung.  
     
     
         22 . The use as in  claim 21 , wherein a lung surfactant, an anti-inflammatory steroid, an anti-asthmatic drug, or a bronchodilator is concurrently administered.  
     
     
         23 . The use of a compound of  claim 3  for the prevention of fibrin deposition in the lung alveoli.