Erythropoietin conjugates

• US 20030120045A1
• Filed: 11/14/2002
• Published: 06/26/2003
• Est. Priority Date: 07/02/1999
• Status: Abandoned Application

• Current Assignee: Pascal Sebastian Bailon
• Original Assignee: Pascal Sebastian Bailon
• Inventors: Bailon, Pascal Sebastian
• Application Number:
  US10/293,551
• Publication Number:
  US 20030120045A1
• Time in Patent Office: Days
• US Class Current: 530/397
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61K 47/62 the modifying agent being a protein, peptide or polyamino acid
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 25/18 Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C07K 17/08 the carrier being a synthetic polymer

Abstract

Conjugates of erythropoietin with poly(ethylene glycol) comprise an erythropoietin glycoprotein having at least one free amino group and having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells and selected from the group consisting of human erythropoietin and analogs thereof which have sequence of human erythropoietin modified by the addition of from 1 to 6 glycosylation sites or a rearrangement of at least one glycosylation site; said glycoprotein being covalently linked to “n” poly(ethylene glycol) groups of the formula —CO—(CH₂)_x(OCH₂CH₂)_m—OR with the carbonyl of each poly(ethylene glycol) group forming an amide bond with one of said amino groups; wherein R is lower alkyl; x is 2 or 3; m is about 450 to about 900; n is from 1 to 3; and n and m are chosen so that the molecular weight of the conjugate minus the erythropoietin glycoprotein is from 20 kilodaltons to 100 kilodaltons.

Claims

1. A conjugate comprising an erythropoietin glycoprotein having a free amino group and having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells and selected from the group consisting of human erythropoietin and analogs thereof which have sequence of human erythropoietin modified by the addition of from 1 to 6 glycosylation sites or a rearrangement of at least one glycosylation site;

said glycoprotein being covalently linked to “

n”

poly(ethylene glycol) groups of the formula —

CO—

(CH₂)_x—

(OCH₂CH₂)_m—

OR by the —

CO of said poly(ethylene glycol) group forming an amide bond with said amino groups;

wherein R is lower alkyl;

x is 2 or 3;

m is from about 450 to about 900; and

m is chosen so that the molecular weight of the conjugates minus the erythropoietin glycoprotein is from 20 kilodaltons to 100 kilodaltons.

2. The conjugate of claim 1, of the formula:

P—

[NHCO—

(CH₂)_x—

(OCH₂CH₂)_m—

OR]_n 

 

(I)

wherein m, n and R are as above, and P is the residue of the glycoprotein without the n amino group(s) which form amide linkage(s) with the poly(ethylene glycol) group(s).

3. The conjugate of claim 2 wherein x is 3.

4. The conjugate of claim 3 wherein n is 1.

5. The conjugate of claim 4 wherein said molecular weight is from about 20 kDa to about 40 kDa.

6. The conjugate of claim 5 wherein said molecular weight is about 30 kDa.

7. The conjugate of claim 2, wherein the glycoprotein has the sequence of human erythropoietin modified by the addition of from 1 to 6 glycosylation sites.

8. The conjugate of claim 2, wherein the glycoprotein has the sequence of human erythropoietin modified by the rearrangement of at least one glycosylation site.

9. The conjugate of claim 8 wherein n is 1.

10. The conjugate of claim 2 wherein the glycoprotein is human erythropoietin.

11. The conjugate of claim 8, wherein the human erythropoietin glycoprotein is expressed by endogenous gene activation.

12. The conjugate of claim 10, wherein the glycoprotein has the sequence SEQ ID NO:

1.

13. The conjugate of claim 12, wherein R is methyl.

14. The congjugate of claim 7 wherein the glycopprotein has the sequence of human erythropoietin modified by a modification selected from the group consisting of:

Asn³⁰Thr³²;

Asn⁵¹Thr⁵³,

Asn⁵⁷Thr⁵⁹;

Asn⁶⁹;

Asn⁶⁹Thr⁷¹;

Ser⁶⁸Asn⁶⁹Thr⁷¹;

Val⁸⁷Asn⁸⁸Thr⁹⁰;

Ser⁸⁷Asn⁸⁸Thr⁹⁰;

Ser⁸⁷Asn⁸⁸Gly⁸⁹Thr⁹⁰;

Ser⁸⁷Asn⁸⁸Thr⁹⁰Thr⁹²;

Ser⁸⁷Asn⁸⁸Thr⁹⁰ Ala¹⁶²;

Asn⁶⁹Thr⁷¹Ser⁸⁷ Asn⁸⁸Thr⁹⁰;

Asn³⁰Thr³²Val⁸⁷ Asn⁸⁸Thr⁹⁰;

Asn⁸⁹Ile⁹⁰Thr⁹¹;

Ser⁸⁷ Asn⁸⁹Ile⁹⁰Thr⁹¹;

Asn¹³⁶Thr¹³⁸;

Asn¹³⁸Thr¹⁴⁰;

Thr¹²⁵; and

Pro¹²⁴Thr¹²⁵.

15. The conjugate of claim 7, wherein the rearrangement comprises deletion of any of the N-linked glycosylation sites in human erythropoietin and addition of an N-linked glycosylation site at position 88 of the sequence of human erythropoietin.

16. The conjugate of claim 2, wherein the glycoprotein has the sequence of human erythropoietin modified by a modification selected from the group consisting of:

Gln²⁴ Ser⁸⁷ Asn⁸⁸ Thr⁹⁰;

Gln³⁸ Ser⁸⁷ Asn⁸⁸ Thr⁹⁰; and

Gln⁸³ Ser⁸⁷ Asn⁸⁸ Thr⁹⁰.

17. The conjugate of claim 16, wherein R is methyl.