Derivatisation of Granulocyte Colony-Stimulating Factor

Abstract

The present invention relates to a compound which is a polysaccharide derivative of GCSF, or of a GCSF like protein, wherein the polysaccharide is anionic and comprises between 2 and 200 saccharide units. The present invention also relates to pharmaceutical compositions comprising the novel compounds, and methods for making the novel compounds.

1 . A method for stimulating neutrophils, increasing the number of hematopoietic stem cells in the blood, or producing white blood cells comprising administering to a patient in need thereof a composition, wherein the composition comprises an amino-terminal polysaccharide derivative of granulocyte colony-stimulating factor (GCSF), wherein the polysaccharide is attached to the amino terminus of a protein consisting of amino acids 30-207 of SEQ ID NO: 1, or a protein having at least 90% sequence homology thereto, wherein the polysaccharide comprises between 2 and 200 polysialic acid units. 
     
     
         2 . The method according to  claim 1 , wherein the GCSF is derivatised by the polysaccharide at the reducing terminal unit of the polysaccharide. 
     
     
         3 . The method according to  claim 1 , wherein the protein has at least 95% identity or similarity at the amino acid level to a protein consisting of amino acids 30-207 of SEQ ID NO: 1. 
     
     
         4 . The method according to  claim 1 , wherein the protein has at least 99% identity or similarity at the amino acid level to a protein consisting of amino acids 30-207 of SEQ ID NO: 1. 
     
     
         5 . The method according to  claim 1 , wherein the protein possesses at least fifty percent (50%) human GCSF activity as measured by a bioassay in comparison to the World Health Organization International Standard for human granulocyte colony stimulating factor (Human, rDNA-derived). 
     
     
         7 . The composition according to  claim 1 , wherein the polysaccharide comprises at least 5 polysialic acid units. 
     
     
         8 . The composition according to  claim 1 , wherein the polysaccharide comprises at least 10 polysialic acid units. 
     
     
         9 . The composition according to  claim 1 , wherein the polysaccharide comprises at least 50 polysialic acid units. 
     
     
         10 . The composition according to  claim 1 , wherein the polysaccharide comprises 80-180 sialic acid units. 
     
     
         11 . The composition according to  claim 1 , wherein the polysaccharide comprises 100-150 sialic acid units. 
     
     
         12 . The composition according to  claim 1 , wherein the polysaccharide comprises 130-140 sialic acid units. 
     
     
         13 . A method for stimulating neutrophils, increasing the number of hematopoietic stem cells in the blood, or producing white blood cells comprising administering to a patient in need thereof a pharmaceutical composition comprising a compound of formula (I) 
       
         
           
           
               
               
           
         
         wherein m is at least one; 
         XB is B—XH wherein B is GCSF and XH is NH 2  and is the N-terminal amine of the GCSF; 
         L is a bond or a linking group; 
         GlyO is an anionic saccharide unit; 
         wherein the linking group, if present, is of the formula
   —Y—C(O)—R 1 —C(O)—;
 
 
         wherein R 1  is a difunctional organic radical selected from the group consisting of alkanediyl, arylene, alkarylene, heteroarylene and alkylheteroarylene, any of which may substituted by and/or interrupted by carbonyl, ester, sulfide, ether, amide and/or amine linkages; and 
         Y is NR 2  or NR 2 —NR 2  wherein R 2  is H or C 1-6  alkyl. 
       
     
     
         19 . The method according to claim  18  wherein L is a bond or is 
       
         
           
           
               
               
           
         
       
     
     
         19 . The method according to claim  15 , wherein the administration is once a day.