Von Willebrand factor- and factor VIII-polymer conjugates having a releasable linkage
View Patent ↗The present invention provides von Willebrand Factor-polymer conjugates and Factor VIII-polymer conjugates, each having a releasable linkage. Methods of making conjugates, methods for administering conjugates, are also provided.
1. A method of preparing a compound, said method comprising contacting a polymeric reagent with an amine-containing biologically active agent that is either a von Willebrand Factor moiety or a Factor VIII moiety to form a reaction mixture under conditions suitable to form a releasable covalent attachment between the polymeric reagent and an amino-group of the biologically active agent, wherein the polymeric reagent has the following structure:
wherein:
POLY 1 is a first water-soluble polymer;
POLY 2 is a second water-soluble polymer;
X 1 is a first spacer moiety;
X 2 is a second spacer moiety;
H α is an ionizable hydrogen atom;
R 1 is H or an organic radical;
R 2 is H or an organic radical;
(a) is either zero or one;
(b) is either zero or one;
R e1 , when present, is a first electron altering group;
R e2 , when present, is a second electron altering group; and
(FG) is a functional group capable of reacting with an amino group of the biologically active agent to form a releasable linkage.
2. The method of claim 1 , wherein the releasable linkage is selected from the group consisting of a carbamate linkage, a thiocarbamate linkage and a dithiocarbamate linkage, and the product possesses the structure:
where POLY, POLY 2 , X 1 , X 2 , H α , R 1 , R 2 , (a), (b), R e1 , and R e2 have the values recited in claim 1 ,
Y 1 is O or S;
Y 2 is O or S; and
(vWF/F8) is a residue of the amine-containing biologically active agent that is either a von Willebrand Factor moiety or a Factor VIII moiety.
3. The method of claim 2 , wherein the releasable linkage is a carbamate linkage, and the compound possesses the structure:
4. The method of claim 1 , wherein the first water-soluble polymer is a poly(alkylene oxide) and the second water-soluble polymer is a poly(alkylene oxide).
5. The method of claim 4 , wherein the first water-soluble polymer has a weight-average molecular weight of between 10,000 Daltons to 85,000 Daltons and the second water-soluble polymer has a weight-average molecular weight of between 10,000 Daltons to 85,000 Daltons.
6. The method of claim 4 , wherein the polymeric reagent has a structure selected from the group consisting of:
wherein, for each structure and in each instance, (n) is independently an integer from 4 to 1500.
7. The method of claim 6 , wherein the amine-containing biologically active agent is a von Willebrand Factor moiety.
8. The method of claim 7 , wherein the von Willebrand Factor moiety is human recombinant von Willebrand Factor.
9. The method of claim 6 , wherein the amine-containing biologically active agent is a Factor VIII moiety.
10. The method of claim 9 , wherein the Factor VIII moiety is human recombinant B-domain deleted Factor VIII.
11. The method of claim 9 , wherein the Factor VIII moiety is human recombinant full length Factor VIII.
12. The method of claim 6 , wherein the molar ratio of polymeric agent to biologically active agent ranges from 1:1 to 10:1.
13. The method of claim 6 , wherein said contacting is carried out at room temperature.
14. The method of claim 6 , wherein said contacting is conducted at a pH of about 7.3.
15. The method of claim 6 , wherein the contacting has a duration of from 5 minutes to 24 hours.
16. The method of claim 6 , further comprising purifying the reaction mixture to isolate the compound.
17. The method of claim 16 , comprising further purifying the compound by gel filtration chromatography.
18. A method of administration comprising
administering a composition comprising a compound and a pharmaceutically acceptable excipient to a patient, the compound having the structure:
wherein POLY 1 is a first water-soluble polymer; POLY 2 is a second water-soluble polymer; X 1 is a first spacer moiety; X 2 is a second spacer moiety; H α , is an ionizable hydrogen atom; R 1 is H or an organic radical; R 2 is H or an organic radical; (a) is either zero or one; (b) is either zero or one; R e1 , when present, is a first electron altering group; R e2 , when present, is a second electron altering group; Y 1 is O or S; Y 2 is O or S; —Y 1 —C(Y 2 )—NH˜ is a releasable linkage selected from the group consisting of a carbamate linkage, a thiocarbamate linkage and a dithiocarbamate linkage, and (vWF/F8) is a residue of an amine-containing biologically active agent that is either a von Willebrand Factor moiety or a Factor VIII moiety.
19. The method of claim 18 , wherein said administering is by injection.
20. The method of claim 18 , wherein the releasable linkage is a carbamate linkage.