Stabilization of somatotropins and other proteins by modification of cysteine residues

• US 5,951,972 A
• Filed: 02/06/1995
• Issued: 09/14/1999
• Est. Priority Date: 05/04/1990
• Status: Expired due to Fees

• Current Assignee: American Cyanamid Co (Pfizer Inc.)
• Original Assignee: American Cyanamid Co (Pfizer Inc.)
• Inventors: Bohlen, Peter, Shieh, Hong-Ming, Cady, Susan Mancini, Buckwalter, Brian Lee, Seddon, Andrew P., Daley, Michael Joseph
• Primary Examiner(s): Ulm, John
• Assistant Examiner(s): SAOUD, CHRISTINE J
• Application Number:
  US08/383,621
• Time in Patent Office: 1,681 Days
• Field of Search: 514/2, 514/12, 435/69.1, 435/69.4, 435/69.5, 435/69.51, 435/69.52, 530/345, 530/350, 530/351, 530/381, 530/399, 530/402, 530/410, 930/120, 930/140, 930/141, 930/142, 424/85.2, 424/85.4
• US Class Current: 424/85.2
• 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: C07K 14/50 Fibroblast growth factor [FGF]
• CPC Class Codes: C07K 14/503 basic FGF [bFGF]

Abstract

The present invention relates to physiologically-active derivatized natural and recombinant mammalian and human proteins and polypeptides. The invention provides chemical methods for derivatizing natural and recombinantly-derived proteins or polypeptides containing cysteine residues, either naturally or through site specific mutageneses. The pharmaceutical compositions containing the derivatized proteins and/or polypeptides are formulated to provide stable, long-acting compositions of such proteins and/or polypeptides, previously difficult to achieve.

Claims

1. A derivatized animal somatotropin protein or polypeptide wherein at least one cysteine residue corresponding to positions 183 or 191 of the bovine somatotropin is derivatized with a substituent selected from the group consisting of:

CH₂ CO₂ R₁, CH(COR₂)(CH₂)_x (COR₃), CH₂ CONHCH(COR₂)(CH₂)_x (COR₃) and CH₂ CONH(CH₂)_x (COR₄);

wherein R₁ is CH₂ CH₂ (OCH₂ CH₂)_y OMe;

R₂ and R₃ are OH or R₄ ;

R₄ is NHCH₂ CH₂ (OCH₂ CH₂)_y OMe or OCH₂ CH₂ (OCH₂ CH₂)_y OMe;

x is an integer of from 1 to 3 and y is an integer of from 10 to 300;

with the proviso that R₂ and R₃ cannot simultaneously be OH; and

further provided that, when two derivatized cysteine residues of said protein or polypeptide form a disulfide linkage prior to derivatization, both cysteines will bear the same sulfhydryl derivative.

2. The protein or polypeptide according to claim 1, wherein said protein or polypeptide contains at least one cysteine residue in its natural form.

3. The protein according to claim 2, wherein said protein is Cys¹⁰²,112 Glu¹⁸³,191 pST or Cys¹¹²,122 Glu¹⁸³,191 pST.

4. The protein or polypeptide according to claim 1, wherein said protein or polypeptide has an additional or substituted cysteine residue in the natural form of the protein or polypeptide.

5. A pharmaceutical composition comprising a pharmacologically effective amount of the derivatized protein or polypeptide of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable solid or liquid carrier.

6. The composition according to claim 5, wherein said composition contains a liquid carrier which is suitable for parenteral administration to an animal.

7. A pharmaceutical composition comprising a pharmacologically effective amount of the derivatized protein or polypeptide of claim 2 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable solid or liquid carrier.

8. A pharmaceutical composition comprising a pharmacologically effective amount of the derivatized protein or polypeptide of claim 4 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable solid or liquid carrier.

9. A derivatized protein or polypeptide selected from the group consisting of an interleukin, an interferon, a prourokinase, IGF-1, IGF-2 and antithrombin III wherein at least one cysteine residue of said protein or polypeptide is derivatized with a substituent selected from the group consisting of:

CH₂ CO₂ R₁, CH₂ CONHR₁, CH(COR₂)_x (CH₂)(COR₃), CH₂ CONHCH(COR₂)(CH₂)_x (COR₃) and CH₂ CONH(CH₂)_x (COR₄);

wherein R₁ is CH₂ CH₂ (OCH₂ CH₂)_y OMe;

R₂ and R₃ are OH or R₄ ;

R₄ is NHCH₂ CH₂ (OCH₂ CH₂)_y OMe or OCH₂ CH₂ (OCH₂ CH₂)_y OMe;

x is an integer of from 1 to 3 and y is an integer of from 10 to 300;

with the proviso that R₂ and R₃ cannot simultaneously be OH; and

further provided that, when two derivatized cysteine residues of said protein or polypeptide form a disulfide linkage prior to derivatization, both cysteines will bear the same sulfhydryl derivative.

10. The protein or polypeptide according to claim 9, wherein said protein or polypeptide contains at least one cysteine residue in its natural form.

11. The protein or polypeptide according to claim 9, wherein said protein or polypeptide has an additional or substituted cysteine residue in the natural form of the protein or polypeptide.

12. The protein according to claim 10, wherein said protein is interleukin-2.

13. The protein according to claim 11, wherein said protein is interleukin-2.

14. A pharmaceutical composition comprising a pharmacologically effective amount of the derivatized protein or polypeptide of claim 9 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable solid or liquid carrier.

15. The composition according to claim 14, wherein said composition contains a liquid carrier which is suitable for parenteral administration to an animal.

16. A pharmaceutical composition comprising a pharmacologically effective amount of the derivatized protein or polypeptide of claim 10 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable solid or liquid carrier.

17. A pharmaceutical composition comprising a pharmacologically effective amount of the derivatized protein or polypeptide of claim 11 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable solid or liquid carrier.