Patent EA-004685-B1 (EA)
Title: Gcsf conjugates
Inventor: BAILON PASCAL SEBASTIAN
IPC: A61K47/48
Filed: 2000-01-19
Abstract: 1. A physiologically active conjugate having the formula wherein G is a granulocyte colony stimulating factor less the amino group or groups thereof which form an amide linkage with a polyethylene glycol moiety in the conjugate; R is alkyl with a straight or branched chain, having 1 to 6 carbon atoms, n is an integer from 420 to 550; and m is an integer from 1 to 5. 2. The conjugate of claim 1 wherein R is methyl. 3. The conjugate of claim 2 wherein n is from 450 to 490. 4. The conjugate of claim 2 wherein m is an integer from 1 to 4. 5. The conjugate of claim 4 wherein m is 2. 6. The conjugate of claim 1 wherein the granulocyte colony stimulating factor is GCSF Mutein having the sequence shown in Figure 1. 7. The conjugate of claim 1 wherein n is from 450 to 490. 8. The conjugate of claim 1 wherein m is an integer from 1 to 4. 9. The conjugate of claim 8 wherein m is 2. 10. The conjugate of claim 1, which has a longer circulating half life and greater in vivo granulopoietic activity than the corresponding unconjugated granulocyte colony stimulating factor. 11. The conjugate of claim 10 wherein the granulocyte colony stimulating factor is GCSF Mutein having the sequence shown in Figure 1. 12. A physiologically active conjugate having the formula wherein R is methyl; n is an integer from 450 to 490; m is 2; and G is GCSF Mutein having the sequence shown in Figure 1 less the amino groups thereof which form an amide linkage with a polyethylene glycol moiety in the conjugate. 13. A composition comprising physiologically active conjugates having the formula wherein G in each of the conjugates is a granulocyte colony stimulating factor less the amino group or groups thereof which form an amide linkage with a polyethylene glycol moiety in the conjugates; R in each of the conjugates is independently alkyl with a straight or branched chain, having 1 to 6 carbon atoms; n in each of the conjugates is independently an integer from 420 to 550; in each of the conjugates m is independently an integer from 1 to 4; the percentage of conjugates where m is 1 is from eighteen to twenty-five percent; the percentage of conjugates where m is 2 is from fifty to sixty-six percent; the percentage of conjugates where m is 3 is from twelve to sixteen percent; and the percentage of conjugates where m is 4 is up to five percent. 14. The composition of claim 13, wherein R is methyl in each of the conjugates. 15. The composition of claim 13, wherein n and R are the same in each of the conjugates. 16. The composition of claim 13 wherein n is from 450 to 490. 17. The composition of claim 13 where in each of the conjugates the granulocyte colony stimulating factor is GCSF Mutein having the sequence shown in Figure 1. 18. A composition comprising physiologically active conjugates having the formula wherein R is methyl; n in each of the conjugates is the same and is an integer from 450 to 490; G is GCSF Mutein having the sequence shown in Figure 1 less the amino groups thereof which form an amide linkage with a polyethylene glycol moiety in the conjugate; in each of the conjugates m is independently an integer from 1 to 4; the percentage of conjugates where m is 1 is from eighteen to twenty-five percent; the percentage of conjugates where m is 2 is from fifty to sixty six percent; the percentage of conjugates where m is 3 is from twelve to sixteen percent; and the percentage of conjugates where m is 4 is up to five percent. 19. A method for producing a "PEG-GCSF" conjugate having a longer circulating half life and greater in vivo granulopoietic activity than the corresponding unconjugated GCSF, which method consists of covalently linking a reagent of the formula to the GCSF to produce said conjugate. 20. The method of claim 21, wherein the GCSF is GCSF Mutein having the sequence shown in Figure 1.