Patent EA-003777-B1 (EA)
Title: Erythropoietin conjugates, their use, pharmaceutical compositions and methods of treatment
Inventor: BAILON PASCAL SEBASTIAN
IPC: A61K47/48
Filed: 2000-07-03
Abstract: 1. A conjugate, said conjugate comprising 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 -CO of each polyethylene glycol) group forming an amide bond with one of said amino groups; wherein R is lower alkyl; x is 2 or 3; m is from 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. 2. The conjugate of claim 1, of the formula: P-[NHCO-(CH2)x-(OCH2CH2)m-OR]n (I) wherein x, m, n and R are as defined in claim 1, 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 any preceding claim, wherein R is methyl. 4. The conjugate of any preceding claim, wherein m is from about 650 to about 750. 5. The conjugate of any preceding claim, wherein n is 1. 6. The conjugate of any preceding claim, wherein R is methyl; m is from about 650 to about 750; and n is 1. 7. The conjugate of any preceding claim having the formula (CH3O(CH2CH2O)m CH2CH2CH2CO-NH]n-P wherein m is from 650 to 750, n is 1 and P is as defined in claim 1. 8. The conjugate of any preceding claim, wherein the glycoprotein is a human erythropoietin. 9. The conjugate according to any of claims 1 to 7, wherein the human erythropoietin glycoprotein is expressed by endogenous gene activation. 10. The conjugate according to any of claims 1 to 9, wherein the glycoprotein has the sequence SEQ ID NO:1. 11. The conjugate according to any of claims 1 to 8, wherein the glycoprotein has the sequence of human erythropoietin modified by the addition of from 1 to 6 glycosylation sites. 12. The conjugate according to any of claims 1 to 11, wherein the glycoprotein has the sequence of human erythropoietin modified by a modification selected from the group consisting of Asn<30>Thr<32>; Asn<51>Thr<53>, sn<57>Thr<59>; Asn<69>; Asn<69>Thr<71>; Ser<68>Asn<69>Thr<71>; Val<87>Asn<88>Thr<90>; Ser<87>Asn<88>Thr<90>; Ser<87>Asn<88>Gly<89>Thr<90>; Ser<87>Asn<88>Thr<90>Thr<92>; Ser<87>Asn<88>Thr<90>Ala<162>; Asn<69>Thr<71>Ser<87>Asn<88>Thr<90>; Asn<30>Thr<32>Val<87>Asn<88>Thr<90>; Asn<89>Ile<90>Thr<91>; Ser<87>Asn<89>Ile<90>Thr<91>; Asn<136>Thr<138>; Asn<138>Thr<140>; Thr<125>; and Pro<124>Thr<125>. 13. The conjugate according to any of claims 1 to 12, wherein the glycoprotein has a sequence comprising the sequence of human erythropoietin and a second sequence at the carboxy terminus of the human erythropoietin sequence, wherein the second sequence contains at least one glycosylation site. 14. The conjugate of claim 13, wherein the second sequences comprises a sequence derived from the carboxy terminal sequence of human chorionic gonadotropin. 15. The conjugate of claim 13, wherein the glycoprotein has a sequence selected from the group consisting of (a) the sequence of human erythropoietin and the sequence SEQ ID NO:3 at the carboxy terminus of the human erythropoietin sequence; (b) the sequence in (a) modified by Ser<87> Asn<88> Thr<90>; and (c) the sequence in (a) modified by Asn<30> Thr<32> Val<87> Asn<88> Thr<90>. 16. The conjugate according to any of claims 1 to 7, wherein the glycoprotein has the sequence of human erythropoietin modified by a rearrangement of at least one glycosylation site. 17. The conjugate of claim 16, 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. 18. The conjugate of claim 17, wherein the glycoprotein has the sequence of human erythropoietin modified by a modification selected from the group consisting of: Gln<24> Ser<87> Asn<88> Thr<90>; Gln<38> Ser<87> Asn<88> Thr<90>; and Gln<83> Ser<87> Asn<88> Thr<90>. 19. A composition comprising conjugates, each of said conjugates comprising 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; the glycoprotein in each said conjugate being covalently linked to "n" polyethylene glycol) groups of the formula -CO-(CH2)X (OCH2CH2)m OR with the -CO of each polyethylene glycol) group forming an amide bond with one of said amino groups; where in each of said conjugates R is lower alkyl; x is 2 or 3; m is from about 450 to about 900; n is from 1 to 3; n and m are chosen so that the molecular weight of each conjugate minus the erythropoietin glycoprotein is from 20 kilodaltons to 100 kilodaltons; the percentage of conjugates where n is 1 is at least 90%. 20. The composition comprising conjugates as defined in any of claims 1 to 18 wherein the percentage of conjugates where n is 1 is at least 90%. 21. The composition of according to claims 19 or 20 wherein the percentage of conjugates where n is 1 is at least 92%. 22. The composition of claim 21 wherein the percentage of conjugates where n is 1 is at least 96%. 23. The composition of claim 19 or 20 wherein the percentage of conjugates where n is 1 is from ninety percent to 96%. 24. A pharmaceutical composition comprising a conjugate or a composition according to any of claims 1 to 23 and a pharmaceutically acceptable excipient. 25. Use of a conjugate or a composition according to any of claims 1 to 23 for the preparation of medicaments for the treatment or prophylaxis of diseases correlated with anemia in chronic renal failure patients (CRF), AIDS and for the treatment of cancer patients undergoing chemotherapy. 26. A method for the prophylactic and/or therapeutic treatment of disorders involving anemia in chronic renal failure patients (CRF), AIDS and cancer patients undergoing chemotherapy comprising the step of administering to a patient a composition as claimed in any one of claims 1 to 23. 27. A process for the preparation of compounds according to any of claims 1 to 23 which process comprises condensing the compound of Formula II with a erythropoietin glycoprotein and in which R, m and x are as defined in any of claims 1 to 6. 28. Compounds according to any of claims 1 to 3, whenever prepared by the process of claim 27. 29. Compounds according to any of claims 1 to 23 for the treatment of diseases which are associated with anemia in chronic renal failure patients (CRF), AIDS and cancer patients undergoing chemotherapy.