Erythropoietin composition

• US 20040147431A1
• Filed: 02/17/2004
• Published: 07/29/2004
• Est. Priority Date: 05/15/2000
• Status: Active Grant

• Current Assignee: Apollon Papadimitriou
• Original Assignee: Apollon Papadimitriou
• Inventors: Papadimitriou, Apollon
• Granted Patent: US 7,202,208 B2
• Time in Patent Office: Days
• US Class Current: 514/8
• CPC Class Codes: A61K 38/1816 Erythropoietin [EPO]
• CPC Class Codes: A61K 47/02 Inorganic compounds
• CPC Class Codes: A61K 47/10 Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• CPC Class Codes: A61K 47/183 Amino acids, e.g. glycine, EDTA or aspartame
• CPC Class Codes: A61K 47/20 containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
• CPC Class Codes: A61K 47/26 Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• CPC Class Codes: A61K 9/0019 Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: A61K 9/08 Solutions composition of solutions A61K47/00
• CPC Class Codes: A61P 13/00 Drugs for disorders of the urinary system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/06 Antianaemics

Abstract

The present invention relates to a liquid pharmaceutical composition consisting essentially of an erythropoietin protein, a multiple charged inorganic anion in a pharmaceutically acceptable buffer suitable to keep the solution pH in the range from about 5.5 to about 7.0, and optionally one or more pharmaceutically acceptable excipients. This composition is especially useful for the prophylaxis and treatment of diseases related to erythropoiesis.

Claims

1. A liquid pharmaceutical composition in the form of an aqueous solution consisting essentially of an erythropoietin glycoprotein product having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells, a multipli-charged inorganic anion, a pharmaceutically acceptable buffer, said anion and said buffer being present in said solution in an amount to provide the solution with a pH of from 5.5 to about 7.0, said product being present in said solution in a sufficient amount to provide a therapeutically effective amount of said product when the solution is administered to a patient.

2. The composition of claim 1 wherein said solution is an isotonic solution.

3. The composition of claim 1 wherein the anion is an anion of a multipli-charged strong inorganic acid.

4. The composition of claim 3 wherein the anion is selected from the group consisting of sulfate, citrate or phosphate.

5. The composition of claim 4 wherein the anion is a sulfate anion.

6. The composition of claim 1 wherein the pH is 5.8 to 6.7

7. The composition of claim 6 wherein the pH is 6.0 to 6.5

8. The composition of claim 7 wherein the pH is about 6.2.

9. The composition of claim 1 wherein the buffer is selected form the group consisting of a phosphate or arginine/H₂SO₄/Na₂SO₄ buffers.

10. The composition of claim 9 wherein the buffer is a phosphate buffer in an amount of from 10 to 50 mmol per liter of said solution.

11. The composition of claim 1 wherein the product is a human erythropoietin.

12. The composition of claim 11 wherein the erythropoietin has the amino acid sequence of SEQ ID NO:

1 or SEQ ID NO;

2.

13. The composition of claim 12 wherein the amino acid sequence of the erythropoietin is modified by the addition of from 1 to 6 glycosylation sites.

15. The composition of claim 12, wherein the erythropoietin has the sequence of human erythropoietin modified by a rearrangement of at least one glycosylation site.

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

17. The composition of claim 16, wherein the sequence modification is selected from the group consisting of

18. The composition of claim 1, wherein the glycoprotein product is a pegylated erythropoietin.

14. The composition of claim 14 wherein the sequence modification is selected from the group consisting of:

19. A liquid pharmaceutical composition in the form of an aqueous solution consisting essentially of a pegylated erythropoietin glycoprotein conjugate, said glycoprotein having at least one free amino group and having the sequence SEQ ID NO:

1 or SEQ ID NO;

2 or having said sequence modified by the addition of from 1 to 6 glycosylation sites;

said glycoprotein being covalently linked to from one to three lower-alkoxy poly(ethylene glycol) groups with each poly(ethylene glycol) group being covalently linked to the glycoprotein via a linker of the formula —

C(O)—

X—

S—

Y—

with the C(O) of the linker forming an amide bond with one of said amino groups;

X is —

(CH₂)_k—

or —

CH₂(O—

CH₂—

CH₂)_k—

;

k is from 1 to 10;

Y is selected from

20. The composition of claim 19 wherein said conjugate has the formula:

21. The composition of claim 19 wherein the conjugate has the formula:

22. A liquid pharmaceutical composition in the form of an aqueous solution consisting essentially of from 10 μ

g to 10,000 μ

g per ml of said solution of an erythropoietin glycoprotein product having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells, from 10 to 200 mmol per liter of said solution of a multipli-charged inorganic anion and from 10 to 50 mmol per liter of said solution of a pharmaceutically acceptable buffer, said anion and said buffer being present in said solution in an amount to provide the solution with a pH of from 5.5 to about 7.0;

said liquid pharmaceutical composition being stable at room temperature.

23. The composition of claim 22 wherein said solution is an isotonic solution.

24. The composition of claim 22 wherein the anion is an anion of a multipli-charged strong inorganic acid.

25. The composition of claim 24 wherein the anion is selected from the group consisting of sulfate, citrate or phosphate.

26. The composition of claim 25 wherein the anion is a sulfate anion.

27. The composition of claim 26 wherein the pH is 5.8 to 6.7

28. The composition of claim 26 wherein the pH is 6.0 to 6.5

29. The composition of claim 26 wherein the pH is about 6.2.

30. The composition of claim 22 wherein the buffer is selected from the group consisting of phosphate or arginine/H₂SO₄/Na₂SO₄ buffers.

31. The composition of claim 30 wherein the buffer is a phosphate buffer in an amount of from 10 to 50 mmol per liter of said solution.

32. The composition of claim 22 wherein the product is a human erythropoietin.

33. The composition of claim 32 wherein the erythropoietin has the amino acid sequence SEQ ID NO:

1 or SEQ ID NO;

2.

34. The composition of claim 22 wherein said erythropoietin glycoprotein product has the sequence SEQ ID NO:

1 or SEQ ID NO;

2 that is modified by the addition of from 1 to 6 glycosylation sites.

35. The composition of claim 34 wherein the sequence modification is selected from the group consisting of:

36. The composition of claim 22 wherein said erythropoietin glycoprotein product has the sequence SEQ ID NO:

1 or SEQ ID NO;

2 that is modified by a rearrangement of at least one glycosylation site.

37. The composition of claim 36, wherein the rearrangement comprises deletion of any of the N-linked glycosylation sites in human erythropoietin with the addition of an N-linked glycosylation site at position 88 of the sequence of human erythropoietin.

38. The composition of claim 37, wherein the sequence modification is selected from the group consisting of:

39. The composition of claim 22, wherein said glycoprotein product is a pegylated erythropoietin glycoprotein product.

40. The composition of claim 39, wherein the pegylated erythropoietin glycoprotein product is a conjugate of an erythropoietin glycoprotein having at least one free amino group, said glycoprotein having the sequence SEQ ID NO:

1 or SEQ ID NO;

2 or having said sequence modified by the addition of from 1 to 6 glycosylation sites;

said glycoprotein being covalently linked to from one to three lower-alkoxy poly(ethylene glycol) groups with each poly(ethylene glycol) group being covalently linked to the glycoprotein via a linker of the formula —

C(O)—

X—

S—

Y—

with the C(O) of the linker forming an amide bond with one of said amino groups, X is —

(CH₂)_k—

or —

CH₂(O—

CH₂—

CH₂)_k—

; and

k is from 1 to 10;

Y is selected from

41. The composition of claim 40 wherein said conjugate has the formula:

42. The composition of claim 41 wherein said conjugate has the formula:

43. The composition of claim 22 wherein said solution contains 10 μ

g to 10000 μ

g erythropoietin protein per ml of solution, from 10 to 200 mmol/liter of solution of a sulfate as the multipli-charged inorganic anion, and 10 to 50 mmol/liter of solution of a phosphate as the pharmaceutically acceptable buffer, said solution having a pH of from about 6.0 to about 6.5.

44. The composition of claim 43 further containing up to 20 mM of methionine and 1-5% of a polyol (w/v).

45. The composition of claim 44 consisting essentially of 10 μ

g to 10000 μ

g erythropoietin protein per ml of solution, 40 mmol/liter of solution of the sulfate, 10 mmol/liter of said solution of the phosphate, 10 mM methionine, said composition having a pH of about 6.2, and wherein the polyol is mannitol which is present in the solution at 3% (w/v).

46. The composition of claim 22 wherein the solution contains 10 μ

g to 10000 μ

g erythropoietin protein per ml of solution, the buffer is phosphate which is present at 10 to 50 mmol/liter of solution, said solution further containing NaCl which is present at 10 to 100 mmol/liter of solution and having a pH of from about 6.0 to about 7.0.

47. The composition of claim 46 wherein the NaCl is present at 100 mmol/liter of solution, the phosphate is present at 10 mmol/l, said solution further containing 10 mM methionine and having a pH of about 7.0.

48. The composition of claim 22 wherein the amount of erythropoietin is 50, 100, 400, 800 or 2,500 μ

g/ml of solution.

59. The composition of claim 22 wherein the erythropoietin is present at about 25 to about 2,500 μ

g/ml, the buffer is sodium or potassium phosphate which is present in an amount of about 10 mM, said composition further containing NaCl which is present in an amount of about 100 mM—

and having a pH of about 7.0.

49. A liquid pharmaceutical composition in the form of an aqueous solution consisting essentially of a therapeutically effective amount of a pegylated erythropoietin glycoprotein product of formula

P—

(NHCO—

(CH₂)_x—

(OCH₂CH₂)_m—

OR)_n 

 

(I)

wherein

P is an erythropoietin glycoprotein having the sequence SEQ ID NO;

1, SEQ ID NO;

2, or either of these sequences modified by the addition of from 1 to 6 glycosylation sites or by rearrangement of at least one glycosylation site, minus the amino group of said glycoprotein which forms an amide linkage;

R is lower alkyl;

x is 2 or 3;

m is from about 450 to about 900;

n is from 1 to 3; and

wherein the values of n and m are such that the molecular weight of the conjugate minus the erythropoietin glycoprotein is from 20 kilodaltons to 100 kilodaltons; and

a multipli-charged inorganic anion and a pharmaceutically acceptable buffer, said anion and said buffer being present in said solution in an amount such that the pH of the solution is from about 5.5 to about 7.0.

50. The composition of claim 49 wherein x is 2, m is 650 to 750, n is 1, and R is methyl.

51. The liquid pharmaceutical composition of claim 49 wherein the pegylated erythropoietin glycoprotein product is present in an amount of from about 10 μ

g to about 10,000 μ

g per ml of said liquid composition, the multipli-charged inorganic anion is present in an amount of from 10 to 200 mmol per liter of said liquid composition, and the pharmaceutically acceptable buffer is present in an amount of from about 10 to about 50 mmol per liter of said liquid composition, said anion and said buffer being present in said liquid composition in an amount to provide a pH of from about 5.5 to about 7.0.

52. The composition of claim 51 wherein x is 2, m is 650 to 750, n is 1, and R is methyl.

53. The liquid pharmaceutical composition of claim 50 wherein the pegylated erythropoietin glycoprotein is present in an amount of about 100.0 μ

g/mL of solution, the multipli-charged inorganic anion is sodium sulphate which is present in an amount of about 5.68 mg/mL, the pharmaceutically acceptable buffer is sodium phosphate which is present in an amount of about 1.38 mg/mL, and wherein the pH of the solution is about 6.2^±

0.2.

54. The liquid pharmaceutical composition of claim 53 further comprising methionine in an amount of about 1.49 mg/mL, mannitol in an amount of about 30.0 mg/mL and poloxamers type 188 in an amount of 0.1 mg/mL.

55. The liquid pharmaceutical composition of claim 50 wherein the pegylated erythropoietin glycoprotein is present in an amount of about 400 μ

g/mL of solution, the multipli-charged inorganic anion is sodium sulphate which is present in an amount of about 5.68 mg/mL, the pharmaceutically acceptable buffer is sodium phosphate which is present in an amount of about 1.38 mg/mL, and wherein the pH of the solution is about 6.2^±

0.2.

56. The liquid pharmaceutical composition of claim 55 further containing methionine in an amount of about 1.49 mg/mL, mannitol in an amount of about 30.0 mg/mL and poloxamers type 188 in an amount of 0.1 mg/mL.

57. The liquid pharmaceutical composition of claim 50 wherein the pegylated erythropoietin glycoprotein is present in an amount of about 800.0 μ

g/mL of solution, the multipli-charged inorganic anion is sodium sulphate which is present in an amount of about 5.68 mg/mL, the pharmaceutically acceptable buffer is sodium phosphate which is present in an amount of about 1.38 mg/mL, and wherein the pH of the solution is about 6.2^±

0.2.

58. The liquid pharmaceutical composition of claim 57 further containing methionine in an amount of about 1.49 mg/mL, mannitol in an amount of about 30.0 mg/mL and poloxamers type 188 in an amount of 0.1 mg.