Modifications of cupredoxin derived peptides and methods of use thereof

• US 10,005,821 B2
• Filed: 09/11/2007
• Issued: 06/26/2018
• Est. Priority Date: 10/07/2004
• Status: Active Grant

• Current Assignee: Board of Trustees of The University of Illinois
• Original Assignee: Board of Trustees of The University of Illinois
• Inventors: Das Gupta, Tapas, Chakrabarty, Ananda
• Primary Examiner(s): KINSEY WHITE, NICOLE ERIN
• Application Number:
  US11/853,497
• Publication Number:
  US 20100267608A1
• Time in Patent Office: 3,941 Days
• Field of Search: None
• 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: A61K 38/04 Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof enzyme inhibitors A61K38/005; gastrins A61K38/2207, somatostatins A61K38/31, melanotropins A61K38/34 ; protease inhibitors A61K38/55
• CPC Class Codes: A61K 38/08 Peptides having 5 to 11 amino acids A61K38/043 - A61K38/046 take precedence
• CPC Class Codes: A61K 38/10 Peptides having 12 to 20 amino acids A61K38/043 - A61K38/046 take precedence
• CPC Class Codes: A61K 38/164 from bacteria
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 33/06 Antimalarials
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/21 from Pseudomonadaceae (F)
• CPC Class Codes: C07K 7/00 Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention provides modified cupredoxin derived peptides with pharmacologic activity that have improved pharmacokinetic properties, and methods to use them to treat mammals suffering from various conditions related to the pharmacologic activities. Modifications of the cupredoxin derived peptides include amino acid sequence variants and structural derivations that increase the plasma half-life of the peptide, increase the specific activity of the pharmacologic activity, decrease immunogenicity, and decrease the biotransformation of the peptides. The modified cupredoxin derived peptides can be used in methods to treat mammals for cancer, conditions related to inappropriate angiogenesis, viral and bacterial infections, and specifically HIV and malaria, conditions related to ephrin signaling, and to deliver cargo compounds, including diagnostic compounds, to cancer cells.

Claims

1. A peptide consisting of:

a modified amino acid sequence;

wherein the amino acid sequence is selected from the group consisting of;

SEQ ID NOs;

16-32 and 35-47; and

wherein the modification is one or more selected from the group consisting of;

(1) replacing or substituting one or more amino acid residues with another amino acid residue(s);

(2) acetylating the N-terminus of the amino acid sequence;

(3) amidating the C-terminus of the amino acid sequence; and

(4) covalently bonding one or more PEG molecules to the amino acid sequence.

2. The peptide of claim 1, whereas said peptide comprises at least one improved pharmacokinetic property selected from the group consisting of (1) less susceptible to biotransformation in the patient, (2) excreted from the body of the patient at a slower rate (3) increased stability of its tertiary structure and (4) longer plasma half-life.

3. The peptide of claim 1, wherein said peptide has at least one pharmacologic activity of a wild-type cupredoxin.

4. The peptide of claim 1, wherein said peptide has at least one pharmacologic activity selected from the group consisting of (1) entering a mammalian cancer cell, (2) not entering non-cancerous mammalian cells, (3) entering pre-malignant mammalian cells, (4) killing mammalian cancer cells, (5) killing pre-malignant mammalian cells, (6) inhibiting the growth of a mammalian cancer cell, (7) inhibit HIV-I infection, (8) inhibiting parasitemia of malaria-infected red blood cells, (9) interfering with ephrin signaling system and (10) inhibiting angiogenesis.

5. The peptide of claim 1, which is less susceptible to hydrolysis.

6. The peptide of claim 1, wherein one or more asparagine or serine residues in the sequence of the peptide are replaced with another amino acid residue.

7. The peptide of claim 6, wherein the asparagine or serine residue is replaced with glutamic acid or threonine residues.

8. The peptide of claim 1, which is less susceptible to deamidation.

9. The peptide of claim 1, wherein one or more glycine residues of the peptide are replaced with another amino acid residue.

10. The peptide of claim 9, wherein one or more glycine residues are replaced with a threonine or alanine residue.

11. The peptide of claim 10, wherein one or more glycine residues in the peptide that are equivalent to residues 58 or 63 of Pseudomonas aeruginosa azurin (SEQ ID NO:

1) is replaced.

12. The peptide of claim 1, which consists of SEQ ID NO:

30.

13. The peptide of claim 1, which is less susceptible to oxidation.

14. The peptide of claim 1, wherein one or more methionine or cysteine residues of the peptide are replaced with another amino acid residue.

15. The peptide of claim 14, further comprising replacing one or more glycine residues with a leucine or valine residue.

16. The peptide of claim 15, wherein one or more methionine residues of the peptide that are equivalent to residues 56 or 64 of Pseudomonas aeruginosa azurin (SEQ ID NO:

1) is replaced.

17. The peptide of claim 16, which is SEQ ID NO:

31 or SEQ ID NO;

32.

18. The peptide of claim 1, which is less susceptible to diketopiperazine and pyroglutamic acid formation.

19. The peptide of claim 1, wherein a glycine residue in positions 1, 2 or 3 from the N-terminus of the peptide is replaced with another amino acid residue.

20. The peptide of claim 1, wherein a proline residue in position 3 from the N-terminus of the peptide is replaced with another amino acid residue.

21. The peptide of claim 1, wherein an asparagine residue at the N-terminus of the peptide is replaced with another amino acid residue.

22. The peptide of claim 1, which is less susceptible to racemization.

23. The peptide of claim 1, wherein one or more amino acid residues of the peptide are replaced with the D-isomer of the amino acid residue.

24. The peptide of claim 23, wherein all of the amino acid residues of the peptide are replaced with the D-isomers of the amino acid residues.

25. The peptide of claim 1, which consists of SEQ ID NO:

45.

26. The peptide of claim 1, which is less susceptible to degradation.

27. The peptide of claim 2, in which the peptide is modified to increase the stability of its tertiary structure.

28. The peptide of claim 27, wherein the peptide is modified to increase the stability of a least one α

-helix.

29. The peptide of claim 1, wherein at least one amino acid residue of the peptide selected from the group consisting of glycine, proline, serine, aspartic acid, alanine, threonine, valine, glutamine, asparagine, cysteine, histidine, lysine, and arginine is replaced with an amino acid residue selected from the group consisting of leucine, isoleucine, phenylalanine, glutamic acid, tyrosine, tryptophan and methionine.

30. The peptide of claim 29, wherein the replaced residue of the peptide is within equivalent residues to residues 53-56, 58-64 and 68-70 of P. aeruginosa azurin (SEQ ID NO. 1).

31. The peptide of claim 29, in which the glutamine at a residue equivalent to residue 57 of P. aeruginosa azurin (SEQ ID NO. 1) is replaced with a tryptophan residue.

32. The peptide of claim 29, in which the threonine at a residue equivalent to residue 52 of P. aeruginosa azurin (SEQ ID NO. 1) is replaced with a tryptophan residue.

33. The peptide of claim 29, in which the threonine at a residue equivalent to residue 61 of P. aeruginosa azurin (SEQ ID NO. 1) is replaced with a tryptophan residue.

34. The peptide of claim 29, in which the glycine at a residue equivalent to residue 63 of P. aeruginosa azurin (SEQ ID NO. 1) is replaced with a tryptophan residue.

35. The peptide of claim 1, wherein one or more PEG molecules are covalently bonded to one or more cysteine residue of the peptide.

36. The peptide of claim 35, wherein one or more PEG molecules are covalently bonded to one or more cysteine residues equivalent to a residue selected from the group consisting of amino acid residues 3, 6, and 112 of Pseudomonas aeruginosa azurin (SEQ ID NO:

1).

37. The peptide of claim 1, wherein a cysteine residue is substituted into the peptide and is covalently bonded to a PEG molecule.

38. The peptide of claim 1, wherein one or more PEG molecules are covalently bonded to the peptide at one or more sites selected from the group consisting of lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, N-terminal amino group, and C-terminal carboxylic acid.

39. The peptide of claim 38, wherein one or more lysine residues or C-terminal carboxylic acids are covalently bonded to a PEG molecule.

40. The peptide of claim 38, wherein one or more residues selected from the group consisting of lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine and tyrosine residue are substituted into the cupredoxin derived peptide and are covalently bonded to a PEG molecule.

41. The peptide of claim 1, wherein one or more amino groups of the peptide are covalently bonded to a PEG molecule.

42. The peptide of claim 1, wherein one or more PEG molecules are randomly covalently bonded to the peptide.

43. The peptide of claim 1, wherein the average molecular weight of the PEG molecules per peptide is about 200 to about 100,000 daltons.

44. The peptide of claim 43, wherein the peptide is covalently bonded to one or more branched PEG molecules.

45. The peptide of claim 44, wherein the branched PEG molecule is about 50 kDa.

46. The peptide of claim 43, wherein the peptide is covalently bonded to one or more linear PEG molecules.

47. The peptide of claim 46, wherein the linear PEG molecule is about 5 kDa.

48. A pharmaceutical composition, comprising the peptide of claim 1 and a pharmaceutically acceptable carrier.

49. A method, comprising administering to the mammal a therapeutically effective amount of the peptide of claim 1.

50. The method of claim 49, wherein said mammal is human.