Peptide dimers as agonists of the erythropoietin (EPO) receptor, and associated methods of synthesis and use

• US 7,459,522 B2
• Filed: 12/15/2003
• Issued: 12/02/2008
• Est. Priority Date: 11/24/1999
• Status: Expired due to Fees

• Current Assignee: Affymax Incorporated
• Original Assignee: Affymax Incorporated
• Inventors: Balu, Palani
• Primary Examiner(s): KAM, CHIH MIN
• Application Number:
  US10/737,245
• Publication Number:
  US 20060270831A1
• Time in Patent Office: 1,814 Days
• Field of Search: 530/300, 530/334, 530/345, 530/317, 514/2, 514/12
• US Class Current: 530/300
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 1/1072 by covalent attachment of residues or functional groups
• CPC Class Codes: C07K 1/1075 by covalent attachment of amino acids or peptide residues
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]

Abstract

Novel peptide dimers are provided that bind and activate the erythropoietin receptor (EPO-R) or otherwise act as an EPO agonist. The novel compounds have a first peptide chain R¹ and a second peptide chain R², wherein R¹ and R² may be the same or different, and are linked through a linking moiety. R¹ is approximately 10 to 40 amino acid residues in length and comprises the sequence X₃X₄X₅GPX₆TX₇X₈X₉ (SEQ ID NO: 1) wherein X₃ is C or Hoc, X₄ is R, H, L or W, X₅ is M, F, I or nor-leucine (J), X₆ is any one of the 20 genetically coded L-amino acids or J, X₇ is W, 1-naphthylalanine (B) or 2-naphthylalanine (U), X₈ is D, E, I, L or V, and X₉ is C or Hoc. Similarly, R² comprises the sequence X′₃X′₄X′₅GPX′₆TX′₇X′₈X′₉ (SEQ IN NO: 2) wherein X′₃ is C or Hoc, X′₄ is R, H, L or W, X′₅ is M, F, I or J, X′₆ is any one of the 20 genetically coded L-amino acids or J, X′₇ is W, B or U, X₈′ is D, E, I, L or V, and X′₉ is C or Hoc. Methods for synthesizing the compounds are provided as well, as are pharmaceutical compositions and methods of use.

Claims

1. A method for synthesizing a peptide dimer, comprising:

(a) providing first and second peptide chains linked to a linking moiety L_K, said chains each possessing multiple amino acid residues capable of disulfide bond formation upon oxidation; and

(b) oxidizing in a single oxidation step said peptide chains in a manner effective to preferentially promote formation of disulfide bonds between residues in the same peptide chain relative to formation of disulfide bonds in different peptide chains, and wherein at least 50% of said peptide dimer comprises a peptide chain having an intrapeptide disulfide bond.

2. The method of claim 1, wherein step (b) comprises treatment with an oxidizing composition containing an oxidizing reagent in an amount effective to minimize reaction products in which a residue of the first peptide chain binds to a residue of the second peptide chain.

3. The method of claim 2, wherein the oxidizing reagent is dimethyl sulfoxide.

4. The method of claim 3, wherein the oxidizing composition comprises approximately 15% to 100% (v/v) dimethyl sulfoxide.

5. The method of claim 4, wherein the oxidizing composition comprises approximately 50% to 100% (v/v) dimethyl sulfoxide.

6. The method of claim 5, wherein the oxidizing composition comprises approximately 80% to 100% (v/v) dimethyl sulfoxide.

7. The method of claim 6, wherein the oxidizing composition comprises approximately 100% (v/v) dimethyl sulfoxide.

8. The method of claim 1, wherein the first peptide chain is approximately 10 to 40 amino acid residues in length, binds to the erythropoietin receptor, and contains a sequence of amino acids X3X4X5GPX6TX7X8X9, wherein each amino acid is indicated by standard one-letter abbreviation, wherein X3 is C or homocysteine (Hoc), X4 is R, H, L or W, X5 is M, F, I or nor-leucine (J), X6 is selected from any one of the 20 conventional amino acids and J, X7 is W, 1-naphthylalanine (B) or 2-naphthylalanine (U), X8 is D, E, I, L, or V, and X9 is C or Hoc;

and the second peptide chain is approximately 10 to 40 amino acid residues in length, binds to the erythropoietin receptor, and contains a sequence of amino acids X′

3X′

4X′

5GPX′

6TX′

7X′

8X′

9, wherein each amino acid is indicated by standard one-letter abbreviation, wherein X′

3 is C or Hoc, X′

4 is R, H, L or W, X′

5 is M, F, I or J, X′

6 is selected from any one of the 20 conventional amino acids and J, X′

7 is W, B or U, X′

8 is D, E, I, L or V, and X′

9 is C or Hoc.

9. The method of claim 8, wherein one or more of said amino acid residues are genetically coded L-amino acids.

10. The method of claim 8, wherein the amino terminus of at least one of said peptide chains is modified.

11. The method of claim 10, wherein at least one of said peptide chains comprises a non-naturally occurring amino acid residue.

12. The method of claim 1, wherein at least one of said peptide chains comprises an amino acid residue, wherein a naturally occurring side chain of said amino acid residue is replaced with a non-naturally occurring side chain.

13. The method of claim 1, wherein said first peptide chain binds to the erythropoietin receptor and wherein said second peptide chain binds to the erythropoietin receptor.