High molecular weight derivatives of vitamin K-dependent polypeptides
Modifications of vitamin K-dependent polypeptides that lead to enhanced protein function on a weight or molar basis and/or increase of protein lifetime in the circulation are described. Both objectives are important for using vitamin K-dependent polypeptides for pro- and anti-coagulation therapies, as well as for other uses in the circulation.
1. An isolated vitamin K-dependent polypeptide linked to a polyethylene glycol (PEG) polymer, wherein said polypeptide is wild-type factor VIIa, wherein said PEG polymer has a molecular weight of 10,000 to 40,000 Da and is covalently linked to said wild-type factor VIIa polypeptide such that said wild-type factor VIIa polypeptide is monosubstituted with said PEG polymer, wherein said wild-type factor VIIa polypeptide linked to PEG retains measurable coagulation activity to a level that is from 4% to 15% of the level of coagulation activity of wild-type factor VIIa that is not conjugated to a PEG polymer, and where said coagulation activity is measured using a thromboplastin assay.
2. The isolated vitamin K-dependent polypeptide of claim 1 , wherein said PEG polymer has a molecular weight of 10,000 Da.
3. The isolated vitamin K-dependent polypeptide of claim 1 , wherein said PEG polymer has a molecular weight of 20,000 Da.
4. The isolated vitamin K-dependent polypeptide of claim 1 , wherein said PEG polymer has a molecular weight of 40,000 Da.
5. A pharmaceutical composition comprising (a) an isolated vitamin K-dependent polypeptide linked to a PEG polymer, and (b) a pharmaceutically acceptable carrier, wherein said vitamin K-dependent polypeptide is wild-type factor VIIa, wherein said PEG polymer has a molecular weight of 10,000 to 40,000 Da and is covalently linked to said wild-type factor VIIa polypeptide such that said wild-type factor VIIa polypeptide is monosubstituted with said PEG polymer, wherein said wild-type factor VIIa polypeptide linked to PEG retains measurable coagulation activity to a level that is from 4% to 15% of the level of coagulation activity of wild-type factor VIIa that is not conjugated to a PEG polymer, and wherein said coagulation activity is measured using a thromboplastin assay.
6. The pharmaceutical composition of claim 5 , wherein said PEG polymer has a molecular weight of 10,000 Da.
7. The pharmaceutical composition of claim 5 , wherein said PEG polymer has a molecular weight of 20,000 Da.
8. The pharmaceutical composition of claim 5 , wherein said PEG polymer has a molecular weight of 40,000 Da.
9. A method for increasing blood clot formation in a subject in need thereof, comprising administering to said subject a wild-type factor VIIa polypeptide covalently linked to a PEG polymer having a molecular weight of 10,000 to 40,000 Da, wherein said wild-type factor VIIa polypeptide is monosubstituted with said PEG polymer, wherein said wild-type factor VIIa polypeptide linked to PEG retains measurable coagulation activity to a level that is from 4% to 15% of the level of coagulation activity of wild-type factor VIIa that is not conjugated to a PEG polymer, wherein said coagulation activity is measured using a thromboplastin assay, and wherein said polypeptide is administered in a concentration effective to increase blood clot formation.
10. The method of claim 9 , wherein said subject is diagnosed with hemophilia.