COLLAGEN MIMETIC PEPTIDES FOR TARGETING COLLAGEN STRANDS FOR IN VITRO AND IN VIVO IMAGING AND THERAPEUTIC USE

• US 20130164220A1
• Filed: 11/16/2012
• Published: 06/27/2013
• Est. Priority Date: 11/22/2011
• Status: Active Grant

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Pomper, Martin G., Yu, Michael S., Li, Yang, Summerfield, Daniel, Wang, Allen Yi-Lan, Foss, Catherine A.
• Granted Patent: US 10,426,850 B2
• Time in Patent Office: Days
• US Class Current: 424/9.6
• 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 38/39 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• CPC Class Codes: A61K 49/0056 Peptides, proteins, polyamino acids
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• CPC Class Codes: G01N 33/6887 from muscle, cartilage or connective tissue
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The present invention provides both a caged collagen mimetic peptide (CCMP) having the formula: L-S-Z_m-[Gly-X-Y]_n-^LGly-X-Y-[Gly-X-Y]_n; wherein L is one or more detectable moieties; S is one or more spacer molecules; Z_m is any amino acid where m is an integer of 1 to 10; X is proline or modified proline; Y is proline or modified proline; Gly is glycine; n is an integer from 1 to 20; and ^LGly is a glycine covalently linked to a cage moiety comprising a labile protecting group, as well as a collagen mimetic peptides lacking the labile protecting group (CMP). The inventions are useful for binding collagen and denatured collagen and/or gelatin both in vitro and in vivo, and are useful for targeting any organ or tissue where collagen is present, and can be used for research and diagnostic imaging (both in vivo and in vitro) and also for in vivo therapeutic applications.

Claims

1. A caged collagen mimetic peptide (CCMP) having the formula:

L-S-[Gly-X-Y]_n-^PLGly-X-Y-[Gly-X-Y]_n;

wherein

L is one or more detectable moieties and/or a biologically active compound;

S is one or more spacer molecules;

X is proline or modified proline;

Y is proline or modified proline;

Gly is glycine;

n is an integer from 1 to 20; and

^PLGly is a glycine covalently linked to a cage moiety comprising a photolabile protecting group.

2. The caged collagen mimetic peptide of claim 1, wherein L is selected from the group consisting of:

fluorescent dyes, radioactive isotopes, magnetic beads, metallic beads, colloidal particles, near-infra red dyes, and electron-dense reagents.

3. The caged collagen mimetic peptide of claim 1, wherein L is carboxyfluorescein or 5(6)-carboxytetramethylrhodamine (TAMRA).

4. The caged collagen mimetic peptide of claim 1, wherein L is IRDye-800CW or IRDye 680RD.

5. The caged collagen mimetic peptide of claim 1, wherein X is proline.

6. The caged collagen mimetic peptide of claim 1, wherein Y is hydroxyproline.

7. The caged collagen mimetic peptide of claim 1, wherein n is 4.

8. The caged collagen mimetic peptide of claim 1, wherein pl is -o-nitrobenzyl.

9. The caged collagen mimetic peptide of claim 1, wherein S is a spacer comprising any amino acid Z_m where m is an integer of 1 to 10 or aminohexanoic acid.

10. The caged collagen mimetic peptide of claim 9, wherein Z is glycine and m=3.

21. A collagen mimetic peptide conjugate comprising:

a) the caged collagen mimetic peptide of claim 1; and

b) a biologically active agent selected from the group consisting of;

antibiotics, cell adhesion molecules, contrast agents, detectable labels, growth factors, components of the extracellular matrix, anti-inflammatory compounds, polymers, PEG, small molecules, growth factor inhibitors, and proteinase inhibitors.

22. A nanoparticle comprising the collagen mimetic peptide of claim 1, fixed to the nanoparticle.

23. The nanoparticle of claim 22, wherein the caged collagen mimetic peptide comprises a conjugate selected from the group consisting of an anti-cancer drug, an anti-angiogenic drug, antibiotic, a cell adhesion molecule, a contrast agent, a detectable label, a growth factor, an anti-inflammatory, a component of the extracellular matrix, a polymer, PEG, a therapeutic compound, and a small molecule.

24. A method for detection of collagen in a subject comprising:

a) administering to the subject an effective amount of the collagen mimetic peptide of claim 1;

b) allowing the collagen mimetic peptide, or conjugate or nanoparticle sufficient time to bind collagen and/or gelatin in the subject; and

c) detecting the collagen mimetic peptide, or conjugate or nanoparticle in the subject.

25. The method of detection of claim 24, wherein the collagen bound in b) is type I collagen.

26. The method of detection of claim 24, wherein the collagen bound in b) is denatured.

27. The method of detection of claim 24, wherein the collagen bound in b) is gelatin.

28. The method of detection of claim 24, wherein the collagen bound in b) is digested by matrix metalloproteases.

29. The method of detection of claim 24, wherein the detection is by the use of fluorescence or near infra-red imaging.

30. A method for detection of collagen remodeling by proteinases in a subject comprising:

a) administering to the subject an effective amount of the collagen mimetic peptide of claim 1;

b) allowing the collagen mimetic peptide, or conjugate or nanoparticle sufficient time to bind collagen and/or gelatin in the subject; and

c) detecting the collagen mimetic peptide, or conjugate or nanoparticle in the subject.

31. A method for detection of pathologic tissues having high proteinase activity in a subject comprising:

a) administering to the subject an effective amount of the collagen mimetic peptide of claim 1;

b) allowing the collagen mimetic peptide, or conjugate or nanoparticle sufficient time to bind collagen and/or gelatin in the subject; and

c) detecting the collagen mimetic peptide, or conjugate or nanoparticle in the subject.

32. A method for detection of collagen remodeling in bones and cartilage in a subject comprising:

a) administering to the subject an effective amount of the collagen mimetic peptide of claim 1;

b) allowing the collagen mimetic peptide, or conjugate or nanoparticle sufficient time to bind collagen and/or gelatin in the subject; and

c) detecting the collagen mimetic peptide, or conjugate or nanoparticle in the subject.

33. A method for detection of musculoskeletal disease in a subject comprising:

a) administering to the subject an effective amount of the collagen mimetic peptide of claim 1;

b) allowing the collagen mimetic peptide, or conjugate or nanoparticle sufficient time to bind collagen and/or gelatin in the subject; and

c) detecting the collagen mimetic peptide, or conjugate or nanoparticle in the subject.

34. The method of claim 33, wherein the musculoskeletal disease is Marfan'"'"'s Syndrome.

38. The caged collagen mimetic peptide of claim 1, wherein the CCMP is selected from the group consisting of:

45. A method for detection of collagen or a protein having a collagen like domain in a substrate comprising:

a) contacting the substrate with an effective amount of a solution comprising the caged collagen mimetic peptide of claim 1;

b) exposing the substrate in the solution of a) to UV light for sufficient time to caged collagen mimetic peptide or conjugate to be activated;

c) allowing the activated collagen mimetic peptide, or conjugate sufficient time to bind collagen and/or gelatin in the substrate; and

c) detecting the collagen mimetic peptide, or conjugate in the substrate.

46. The method of claim 45, wherein the substrate is a protein separation gel.

47. The method of claim 46, wherein the substrate is a SDS-PAGE gel.

48. The method of claim 45, wherein the substrate is a fixed tissue section.

49. The method of claim 48, wherein detection of the collagen mimetic peptide, or conjugate in the substrate is through the use of fluorescent imaging.

50. The method of claim 45, wherein the collagen detected is collagen type I, II, III, or IV or gelatin.

51. The method of claim 45, wherein the collagen like domain detected is C1q protein.

11. A collagen mimetic peptide (CMP) having the formula:

L-S-[Gly-X-Y]_n-Gly-X-Y-[Gly-X-Y]_n;

wherein

L is one or more detectable moieties and/or a biologically active compound;

S is one or more spacer molecules;

X is proline or modified proline;

Y is proline or modified proline;

Gly is glycine; and

n is an integer from 1 to 20.

12. The collagen mimetic peptide of claim 11, wherein L is selected from the group consisting of:

fluorescent dyes, radioactive isotopes, magnetic beads, metallic beads, colloidal particles, near-infra red dyes, and electron-dense reagents.

13. The collagen mimetic peptide of claim 11, wherein L is carboxyfluorescein or 5(6)-carboxytetramethylrhodamine (TAMRA).

14. The caged collagen mimetic peptide of claim 11, wherein Z is glycine and m=3.

15. The collagen mimetic peptide of claim 11, wherein X is proline.

16. The collagen mimetic peptide of claim 11, wherein Y is hydroxyproline.

17. The collagen mimetic peptide of claim 11, wherein n is 4.

18. The collagen mimetic peptide of claim 11, wherein pl is -o-nitrobenzyl.

19. The collagen mimetic peptide of claim 11, wherein S is a spacer comprising any amino acid Z_m where m is an integer of 1 to 10, or aminohexanoic acid.

20. The collagen mimetic peptide of claim 19, wherein L is IRDye-800CW or IRDye 680RD.

39. A method for detection of collagen or a protein having a collagen like domain in a substrate comprising:

a) contacting the substrate with an effective amount of a solution comprising the collagen mimetic peptide of claim 11, wherein the solution is pre-heated to a temperature above the melting point of the collagen mimetic peptide;

b) allowing the collagen mimetic peptide, or conjugate sufficient time to bind collagen and/or gelatin in the substrate at a temperature below the melting point of the collagen mimetic peptide; and

c) detecting the collagen mimetic peptide, or conjugate in the substrate.

40. The method of claim 39, wherein the substrate is a protein separation gel.

41. The method of claim 40, wherein the substrate is a SDS-PAGE gel.

42. The method of claim 41, wherein detection of the collagen mimetic peptide, or conjugate in the substrate is through the use of fluorescent imaging.

43. The method of claim 42, wherein the collagen detected is collagen type I, II, III, or IV or gelatin.

44. The method of claim 42, wherein the collagen like domain detected is C1q protein.

35. A method for treatment of a disease associated with collagen denaturation or remodeling in a subject comprising administering to the subject an effective amount of a collagen mimetic peptide conjugate, or a nanoparticle to treat the disease in the subject.

36. A caged collagen mimetic peptide (CCMP) having the formula:

L-S-[Gly-X-Y]_n-^LGly-X-Y-[Gly-X-Y]_n;

wherein

L is one or more detectable moieties and/or a biologically active compound;

S is one or more spacer molecules;

X is proline or modified proline;

Y is proline or modified proline;

Gly is glycine;

n is an integer from 1 to 20; and

^LGly is a glycine covalently linked to a cage moiety comprising a labile protecting group.

37. The caged collagen mimetic peptide of claim 36, wherein the labile protecting group is selected from the group consisting of chemically labile groups, enzymatically labile groups, and photolabile groups.