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Title:
GRANZYME B INHIBITOR COMPOSITIONS, METHODS AND USES FOR PROMOTING WOUND HEALING
Document Type and Number:
WIPO Patent Application WO/2012/076985
Kind Code:
A2
Abstract:
Methods of promoting wound healing in a subject is disclosed. The method include applying a Granzyme B (Granzyme B) inhibitor to the wound. The wound may be a skin wound. The Granzyme B inhibitor may be comprised of nucleic acids, or peptides, including but not limited to antibodies, or small molecules.

Inventors:
HIEBERT PAUL R (CA)
KNIGHT DARRYL A (CA)
GRANVILLE DAVID J (CA)
BOIVIN WENDY A (CA)
COOPER DAWN M (CA)
Application Number:
IB2011/003207
Publication Date:
June 14, 2012
Filing Date:
December 06, 2011
Export Citation:
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Assignee:
UNIV BRITISH COLUMBIA (CA)
HIEBERT PAUL R (CA)
KNIGHT DARRYL A (CA)
GRANVILLE DAVID J (CA)
BOIVIN WENDY A (CA)
COOPER DAWN M (CA)
International Classes:
A61K31/55
Domestic Patent References:
WO2007101354A12007-09-13
WO2003065987A22003-08-14
Foreign References:
Other References:
See references of EP 2648735A4
Attorney, Agent or Firm:
ERRATT, Judy, A. (160 Elgin Street Suite 2600,Ottawa, Ontario, K1P 1C3, CA)
Download PDF:
Claims:
We claim:

1. A method of promoting wound healing in a subject, the method comprising administering a Granzyme B (Granzyme B) inhibitor to the subject for a time and in an amount sufficient to promote would healing, thereby promoting wound healing in the subject.

2. A method of promoting wound healing in a subject, the method comprising applying a Granzyme B (Granzyme B) inhibitor to the wound, for a time and in an amount sufficient to promote would healing, thereby promoting wound healing in the subject.

3. The method of claim 1 or 2, wherein the wound is a chronic wound.

4. The method of claim 3, wherein the chronic wound is a chronic skin wound.

5. The method of claim 4, whereinthe chronic skin would is a pressure ulcer.

6. The method of claiml or 2, wherein cleavage of an extracellular matrix protein is inhibited.

7. The method of claim 6, wherein the extracellular matrix protein is selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodulin, fibrillin- 1, fibrillin-2, and fibulin-2.

8. The method of claim 7, wherein the extracellular matrix protein is decorin.

9. The method foe laim 1 or 2, wherein release of TGF bound to an extracellular matrix protein is inhibited.

10. The method of claim 9, wherein the extracellular matrix protein is decorin.

11. A method of preventing skin tearing of a subject, comprising applying a Granzyme B inhibitor to the skin of the subject for a time and in an amount sufficient to prevent skin tearing, thereby preventing skin tearing in the subject.

12. The method of claim 11, wherein the skin tearing is associated with a chronic wound.

13. The method of claim 11, wherein the skin tearing is associated with aging.

14. The method of claim 11 , wherein cleavage of an extracellular matrix protein is inhibited.

15. The method of claim 14, wherein the extracellular matrix protein is selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodulin, fibrillin- 1, fibrillin-2, and fibulin-2.

16. The method oc claims 14, wherein the extracellular matrix protein is decorin.

17. The method of claim 11 , wherein release of TGF bound to an extracellular matrix protein is inhibited.

18. The method of claim 17, wherein the extracellular matrix protein is decorin. 19. A method for inhibiting hypertrophic scarring of a wound, comprising applying a Granzyme B inhibitor to the skin of the subject for a time and in an amount sufficient to prevent skin hypertrophic scarring of a wound, thereby inhibiting hypertrophic scarring of a wound.

20. The method of claim 19, wherein cleavage of an extracellular matrix protein is inhibited.

21. The method of claim 20, wherein the extracellular matrix protein is selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodulin, fibrillin- 1, fibrillin-2, and fibulin-2.

22. The method of claim 20, wherein the extracellular matrix protein is decorin. 23. The method of claim 19, wherein release of TGF bound to an extracellular matrix protein is inhibited.

24. The method of claim 23, wherein the extracellular matrix protein is decorin.

25. A method for increasing collagen organization in the skin of a subject, comprising applying a Granzyme B inhibitor to the skin of the subject in an amount and for a time sufficient to increase collagen organization in the subject, thereby increasing collagen organization in the skin of the subject.

26. The method of claim 25, wherein cleavage of an extracellular matrix protein is inhibited.

27. The method of claim 26, wherein the extracellular matrix protein is selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodulin, fibrillin- 1, fibrillin-2, and fibulin-2.

28. The method of claim 26, wherein the extracellular matrix protein is decorin 29. The method of claim 25, wherein release of TGF bound to an extracellular matrix protein is inhibited.

30. The method of claim 29, wherein the extracellular matrix protein is decorin.

31. A method for increasing the tensile strength of a healing or healed skin wound of a subject, comprising applying a Granzyme B inhibitor to the skin of the subject in an amount and for a time sufficient to increase increase the tensile strength of the healing or healed skin wound of the subject, thereby increasing the tensile strength of a healing or healed skin wound of a subject.

32. The method of claim 31, wherein cleavage of an extracellular matrix protein is inhibited.

33 The method of claim 32, wherein the extracellular matrix protein is selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodulin, fibrillin- 1, fibrillin-2, and fibulin-2.

34. The method of claim 32, wherein the extracellular matrix protein is decorin

35. The method of claim 31, wherein release of TGF bound to an extracellular matrix protein is inhibited.

36. The method of claim 35, wherein the extracellular matrix protein is decorin.

37. A method for inhibiting release of TGF bound to an extracellular protein , comprising contacting the extracellular proteoglycan with a Granzyme B inhibitor, thereby inhibiting release of TGF bound to the extracellular protein .

38. The method of claim 38, wherein the protein is selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodulin, fibrillin- 1, fibrillin-2, and fibulin-2.

39. The method of claim 38, wherein the protein is decorin

40. A method of inhibiting extracellular decorin cleavage, comprising contacting decorin with a Granzyme B inhibitor, thereby inhibiting extracellular decorin cleavage.

41. The method of any one of claims 1-40, wherein the Granzyme B inhibitor is selected from the group consisting of a nucleic acid molecule, a peptide, an antibody, and a small molecule.

42. The method of claim 41, wherein the antibody is a monoclonal antibody.

43. The method of any one of claims 1-40, wherein the Granzyme B inhibitor is selected from one or more of the following:

2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4- oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-acetamido-3- methylpentanamido)-4-oxo-l, 2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((R)-3-methyl-2-(pyridin-2- yl)butanamido)-4-oxo- 1 , 2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-3-methyl-2-(2- phenylacetamido)pentanamido)-4-oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((lH-l,2,4-triazol-3-yl)methyl)-5-((2S,3S)-2-acetamido-3- methylpentanamido)-4-oxo-l, 2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((lH-pyrazol-3-yl)methyl)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4- oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-pyrazol-4-yl)methyl)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4- oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-imidazol-4-yl)methyl)-5-((2S,3S)-2-acetamido-3-methylpentanamido)- 4-oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(thiazol-5-ylmethyl)- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide; (2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-N-(isoxazol-3-ylmethyl)-4-oxo l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(thiazol-2-ylmethyl)- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-N-(isoxazol-5-ylmethyl)-4-oxo l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(thiazol-4-ylmethyl)- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyrimidin-5- ylmethyl)- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyridazin-4- ylmethyl)- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyridin-2-ylmethyl)- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyridin-3-ylmethyl)- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyridin-4-ylmethyl)- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-N-(imidazo[l,2-a]pyrimidin-2- ylmethyl)-4-oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-N-((3a,7a- dihydrobenzo[d]thiazol-2-yl)methyl)-4-oxo- 1,2,4,5, 6,7-hexahydroazepino[3 ,2,1- hi]indole-2-carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((R)-3-methyl-2-(pyridin-2-yl)butanamido)-4- oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((S)-3-methyl-2-(pyridin-2-yl)butanamido)-4- oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-3-methyl-2-(2- phenylacetamido)pentanamido)-4-oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide; (2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(2,3-difluorophenyl)acetamido)- 3-methylpentanamido)-4-oxo-l, 2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(dimethylamino)acetamido)-3- methylpentanamido)-4-oxo-l, 2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(benzo[b]thiophen-3- yl)acetamido)-3-methylpentanamido)-4-oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l- hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-(2-(dimethylamino)acetamido)-

3- methylpentanamido)-4-oxo-l, 2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-(2-(benzo[b]thiophen-3- yl)acetamido)-3-methylpentanamido)-4-oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l- hi]indole-2-carboxamide;

(R)-N-((2S,5S)-2-((lH-l,2,3-triazol-4-yl)methylcarbamoyl)-4-oxo-l,2,4,5,6,7- hexahydroazepino[3,2,l-hi]indol-5-yl)-3-acetyl-5,5-dimethylthiazolidine-4- carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-3-methyl-2-(2-oxopyrrolidin-l- yl)pentanamido)-4-oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-(2-cyclopentylacetamido)-4-oxo- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((S)-2-acetamido-2-cyclopropylacetamido)-

4- oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((S)-2-acetamido-2-cyclopentylacetamido)- 4-oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

Bio-x-IEPDp-(OPh)2;

azepino[3,2,l-hi]indole-2-carboxamide;

(4S)-4-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-[[(2S)-4-hydroxy-l,4- dioxobutan-2-yl]carbamoyl]pyrrolidin-l-yl]-5-oxopentanoic acid; (4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-5-[[(2S,3S)-3-hydroxy-l- [ [(2S)-4-hydroxy- 1 ,4-dioxobutan-2-yl] amino] - 1 -oxobutan-2-yl] amino] -5 -oxopentanoic acid;

5 -chloro-4-oxo-3 - [2- [2-(phenylmethoxycarbonylamino) propanoylamino]

propanoylamino] pentanoic acid;

5-chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylamino) propanoylamino]

propanoylamino] pentanoic acid;

ZINC05723764;

ZINC05723787;

ZINC05316154;

ZINC05723499;

ZINC05723646;

ZINC05398428;

ZINC05723503;

ZINC05723446;

ZINC05317216;

ZINC05315460;

ZINC05316859;

ZINC05605947;

an isocoumarin;

a peptide chloromethyl ketone;

a peptide phosphonate;

a Granzyme B inhibitory nucleic acid molecule;

an anti-Granzyme B antibody;

an inhibitory Granzyme B peptide;

a SerpB9 polypeptide, or fragment thereof; 5-chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylamino)

propanoylaminojpropanoylamino] pentanoic acid;

Ac-IEPD-CHO;

(4S)-4-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-[[(2S)-4-hydroxy-l,4- dioxobutan-2-yl]carbamoyl]pyrrolidin-l-yl]-5-oxopentanoic acid;

Ac-IETD-CHO;

(4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-5-[[(2S,3S)-3-hydroxy-l- [ [(2S)-4-hydroxy- 1 ,4-dioxobutan-2-yl] amino] - 1 -oxobutan-2-yl] amino] -5 -oxopentanoic acid;

(2S ,5 S)-4-oxo-5 - { [N-(phenylacetyl)-L-isoleucyl] amino } -N- ( 1 H- 1 ,2,3 -triazol-4- ylmethyl)- 1,2,4,5, 6,7-hexahydroazepino[3 ,2,l-hi]indole-2-carboxamide; 5-chloro-4- oxo-3-[2-[2-(phenylmethoxycarbonylamino) propanoylamino] propanoylamino] pentanoic acid;

5-chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylamino) propanoylamino]

propanoylamino] pentanoic acid;

(4S)-4-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-[[(2S)-4-hydroxy-l,4- dioxobutan-2-yl]carbamoyl]pyrrolidin-l-yl]-5-oxopentanoic acid;

(4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-5-[[(2S,3S)-3-hydroxy-l- [ [(2S)-4-hydroxy- 1 ,4-dioxobutan-2-yl] amino] - 1 -oxobutan-2-yl] amino] -5 -oxopentanoic acid,

a Serp2 polypeptide, or fragment thereof;

a CrmA polypeptide or fragment thereof; and

a SerpinA3 polypeptide or fragment thereof. 44. The method of any one of claims 1 -43, wherein the Granzyme B inhibitor is formulated for topical administration.

45. The method of any one of claims 1-44, wherein the Granzyme B inhibitor is formulated for co- administration with another wound treatment.

46. The method of claim 45, wherein another wound treatment is selected from one or more of: a topical antimicrobial; a cleanser; a wound gel; a collagen; an elastin; a tissue growth promoter; an enzymatic debriding preparation; an antifungal; an antiinflammatory; a barrier; a moisturizer; and a sealant.

47. The method of claim 45, wherein the another wound treatment is selected from one or more of: a wound covering, a wound filler, and an implant.

48. The method of claim 45, wherein another wound treatment is selected from one or more of: absorptive dressings; alginate dressings; foam dressings; hydrocolloid dressings; hydrofiber dressings; compression dressing and wraps; composite dressing; contact layer; wound gel impregnated gauzes; wound gel sheets; transparent films; wound fillers; dermal matrix products or tissue scaffolds; and closure devices.

49. The method of any one of claims 1-48, wherein the subject is a mammal.

50. The method of any one of claims 1-49, wherein the subject is a human.

51. Use of a Granzyme B inhibitor to promote wound healing in a subject.

52. Use of a Granzyme B inhibitor in the preparation of a medicament for promoting wound healing in a subject.

53. The use of claim 51 or 52, wherein the wound is a skin wound.

54. The use of claim 53, wherein the skin wound is a chronic skin wound.

55. The use of any one of claims 51-54, wherein the Granzyme B inhibitor is selected from the group consisting of a nucleic acid molecule, a peptide, an antibody, and a small molecule.

56. The use of any one of claims 51-54, wherein the Granzyme B inhibitor is selected from one or more of the following: In one embodiment, a Granzyme B inhibitor is selected from the group consisting of

2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4- oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-acetamido-3- methylpentanamido)-4-oxo-l, 2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((R)-3-methyl-2-(pyridin-2- yl)butanamido)-4-oxo- 1 , 2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide; (2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-3-methyl-2-(2- phenylacetamido)pentanamido)-4-oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((lH-l,2,4-triazol-3-yl)methyl)-5-((2S,3S)-2-acetamido-3- methylpentanamido)-4-oxo-l, 2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((lH-pyrazol-3-yl)methyl)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4 oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-pyrazol-4-yl)methyl)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4 oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-imidazol-4-yl)methyl)-5-((2S,3S)-2-acetamido-3-methylpentanamido)- 4-oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(thiazol-5-ylmethyl)- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-N-(isoxazol-3-ylmethyl)-4-oxo- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(thiazol-2-ylmethyl)- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-N-(isoxazol-5-ylmethyl)-4-oxo- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(thiazol-4-ylmethyl)- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyrimidin-5- ylmethyl)- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyridazin-4- ylmethyl)- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyridin-2-ylmethyl)- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyridin-3-ylmethyl)- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide; (2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyridin-4-ylmethyl)- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-N-(imidazo[l,2-a]pyrimidin-2- ylmethyl)-4-oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-N-((3a,7a- dihydrobenzo[d]thiazol-2-yl)methyl)-4-oxo- 1,2,4,5, 6,7-hexahydroazepino[3 ,2,1- hi]indole-2-carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((R)-3-methyl-2-(pyridin-2-yl)butanamido)-4- oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((S)-3-methyl-2-(pyridin-2-yl)butanamido)-4- oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-3-methyl-2-(2- phenylacetamido)pentanamido)-4-oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(2,3-difluorophenyl)acetamido)- 3-methylpentanamido)-4-oxo-l, 2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(dimethylamino)acetamido)-3- methylpentanamido)-4-oxo-l, 2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(benzo[b]thiophen-3- yl)acetamido)-3-methylpentanamido)-4-oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l- hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-(2-(dimethylamino)acetamido)- 3-methylpentanamido)-4-oxo-l, 2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-(2-(benzo[b]thiophen-3- yl)acetamido)-3-methylpentanamido)-4-oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l- hi]indole-2-carboxamide; (R)-N-((2S,5S)-2-((lH-l,2,3-triazol-4-yl)methylcarbamoyl)-4-oxo-l,2,4,5,6,7- hexahydroazepino[3,2,l-hi]indol-5-yl)-3-acetyl-5,5-dimethylthiazolidine-4- carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-3-methyl-2-(2-oxopyrrolidin-l- yl)pentanamido)-4-oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-(2-cyclopentylacetamido)-4-oxo- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((S)-2-acetamido-2-cyclopropylacetamido)- 4-oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((S)-2-acetamido-2-cyclopentylacetamido)-

4- oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

Bio-x-IEPDp-(OPh)2;

azepino[3,2,l-hi]indole-2-carboxamide;

(4S)-4-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-[[(2S)-4-hydroxy-l,4- dioxobutan-2-yl]carbamoyl]pyrrolidin-l-yl]-5-oxopentanoic acid;

(4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-5-[[(2S,3S)-3-hydroxy-l- [ [(2S)-4-hydroxy- 1 ,4-dioxobutan-2-yl] amino] - 1 -oxobutan-2-yl] amino] -5 -oxopentanoic acid;

5 -chloro-4-oxo-3 - [2- [2-(phenylmethoxycarbonylamino) propanoylamino]

propanoylamino] pentanoic acid;

5- chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylamino) propanoylamino]

propanoylamino] pentanoic acid;

ZINC05723764;

ZINC05723787;

ZINC05316154;

ZINC05723499;

ZINC05723646;

ZINC05398428; ZINC05723503;

ZINC05723446;

ZINC05317216;

ZINC05315460;

ZINC05316859;

ZINC05605947;

an isocoumarin;

a peptide chloromethyl ketone;

a peptide phosphonate;

a Granzyme B inhibitory nucleic acid molecule;

an anti-Granzyme B antibody;

an inhibitory Granzyme B peptide;

a SerpB9 polypeptide, or fragment thereof;

5-chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylamino)

propanoylaminojpropanoylamino] pentanoic acid;

Ac-IEPD-CHO;

(4S)-4-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-[[(2S)-4-hydroxy-l,4- dioxobutan-2-yl]carbamoyl]pyrrolidin-l-yl]-5-oxopentanoic acid;

Ac-IETD-CHO;

(4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-5-[[(2S,3S)-3-hydroxy-l-

[ [(2S)-4-hydroxy- 1 ,4-dioxobutan-2-yl] amino] - 1 -oxobutan-2-yl] amino] -5 -oxopentanoic acid;

(2S ,5 S)-4-oxo-5 - { [N-(phenylacetyl)-L-isoleucyl] amino } -N- ( 1 H- 1 ,2,3 -triazol-4- ylmethyl)- 1,2,4,5, 6,7-hexahydroazepino[3 ,2,l-hi]indole-2-carboxamide; 5-chloro-4- oxo-3-[2-[2-(phenylmethoxycarbonylamino) propanoylamino] propanoylamino] pentanoic acid;

5-chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylamino) propanoylamino]

propanoylamino] pentanoic acid; (4S)-4-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-[[(2S)-4-hydroxy-l,4- dioxobutan-2-yl]carbamoyl]pyrrolidin-l-yl]-5-oxopentanoic acid;

(4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-5-[[(2S,3S)-3-hydroxy-l- [ [(2S)-4-hydroxy- 1 ,4-dioxobutan-2-yl] amino] - 1 -oxobutan-2-yl] amino] -5 -oxopentanoic acid,

a Serp2 polypeptide, or fragment thereof;

a CrmA polypeptide or fragment thereof;

a SerpinA3 polypeptide or fragment thereof.

57. The use of any one of claims 51-56, wherein the Granzyme B inhibitor is formulated for topical administration.

58. The use of any one of claims 51-57, wherein the Granzyme B inhibitor is formulated for co administration with another wound treatment.

59. The use of claim 58, wherein the another wound treatment is selected from one or more of: a topical antimicrobial; a cleanser; a wound gel; a collagen; a elastin; a tissue growth promoter; an enzymatic debriding preparation; an antifungal; an antiinflammatory; a barrier; a moisturizer; and a sealant.

60. The use of any one of claims 51-59, wherein the subject is a mammal.

61. The use of any one of claims 51-59, wherein the subject is a human.

62. A Granzyme B inhibitor for use in promoting wound healing in a subject.

63. The Granzyme B inhibitor of claim 62, wherein the wound is a skin wound.

64. The Granzyme B inhibitor of claim 62 or 63, wherein the Granzyme B inhibitor is selected from the group consisting of a nucleic acid moelcue, a peptide, and antibody, and a small molecule.

65. The Granzyme B inhibitor of any one of claims 62-64, wherein the Granzyme B inhibitor is formulated for topical administration.

66. The Granzyme B inhibitor of any one of claims 62-65, wherein the Granzyme B inhibitor is formulated for co- administration with another wound treatment.

67. The Granzyme B inhibitor of any one of claims 62-66 selected from the group consisting of wherein the Granzyme B inhibitor is selected from one or more of the following:

2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4- oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-acetamido-3- methylpentanamido)-4-oxo-l, 2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((R)-3-methyl-2-(pyridin-2- yl)butanamido)-4-oxo- 1 , 2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-3-methyl-2-(2- phenylacetamido)pentanamido)-4-oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((lH-l,2,4-triazol-3-yl)methyl)-5-((2S,3S)-2-acetamido-3- methylpentanamido)-4-oxo-l, 2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((lH-pyrazol-3-yl)methyl)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4- oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-pyrazol-4-yl)methyl)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4- oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-imidazol-4-yl)methyl)-5-((2S,3S)-2-acetamido-3-methylpentanamido)- 4-oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(thiazol-5-ylmethyl)- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-N-(isoxazol-3-ylmethyl)-4-oxo- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(thiazol-2-ylmethyl)- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-N-(isoxazol-5-ylmethyl)-4-oxo- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(thiazol-4-ylmethyl)- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide; (2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyrimidin-5- ylmethyl)- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyridazin-4- ylmethyl)- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyridin-2-ylmethyl) l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyridin-3-ylmethyl) l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyridin-4-ylmethyl) l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-N-(imidazo[l,2-a]pyrimidin-2- ylmethyl)-4-oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-N-((3a,7a- dihydrobenzo[d]thiazol-2-yl)methyl)-4-oxo- 1,2,4,5, 6,7-hexahydroazepino[3 ,2,1- hi]indole-2-carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((R)-3-methyl-2-(pyridin-2-yl)butanamido)-4- oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((S)-3-methyl-2-(pyridin-2-yl)butanamido)-4- oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-3-methyl-2-(2- phenylacetamido)pentanamido)-4-oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2 carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(2,3-difluorophenyl)acetamido)- 3-methylpentanamido)-4-oxo-l, 2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(dimethylamino)acetamido)-3- methylpentanamido)-4-oxo-l, 2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide; (2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(benzo[b]thiophen-3- yl)acetamido)-3-methylpentanamido)-4-oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l- hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-(2-(dimethylamino)acetamido)-

3- methylpentanamido)-4-oxo-l, 2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-(2-(benzo[b]thiophen-3- yl)acetamido)-3-methylpentanamido)-4-oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l- hi]indole-2-carboxamide;

(R)-N-((2S,5S)-2-((lH-l,2,3-triazol-4-yl)methylcarbamoyl)-4-oxo-l,2,4,5,6,7- hexahydroazepino[3,2,l-hi]indol-5-yl)-3-acetyl-5,5-dimethylthiazolidine-4- carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-3-methyl-2-(2-oxopyrrolidin-l- yl)pentanamido)-4-oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-(2-cyclopentylacetamido)-4-oxo- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((S)-2-acetamido-2-cyclopropylacetamido)-

4- oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((S)-2-acetamido-2-cyclopentylacetamido)- 4-oxo- 1,2,4,5, 6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

Bio-x-IEPDp-(OPh)2;

azepino[3,2,l-hi]indole-2-carboxamide;

(4S)-4-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-[[(2S)-4-hydroxy-l,4- dioxobutan-2-yl]carbamoyl]pyrrolidin-l-yl]-5-oxopentanoic acid;

(4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-5-[[(2S,3S)-3-hydroxy-l- [ [(2S)-4-hydroxy- 1 ,4-dioxobutan-2-yl] amino] - 1 -oxobutan-2-yl] amino] -5 -oxopentanoic acid;

5 -chloro-4-oxo-3 - [2- [2-(phenylmethoxycarbonylamino) propanoylamino]

propanoylamino] pentanoic acid; 5-chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylamino) propanoylamino] propanoylamino] pentanoic acid;

ZINC05723764;

ZINC05723787;

ZINC05316154;

ZINC05723499;

ZINC05723646;

ZINC05398428;

ZINC05723503;

ZINC05723446;

ZINC05317216;

ZINC05315460;

ZINC05316859;

ZINC05605947;

an isocoumarin;

a peptide chloromethyl ketone;

a peptide phosphonate;

a Granzyme B inhibitory nucleic acid molecule;

an anti-Granzyme B antibody;

an inhibitory Granzyme B peptide;

a SerpB9 polypeptide, or fragment thereof;

5-chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylamino)

propanoylaminojpropanoylamino] pentanoic acid;

Ac-IEPD-CHO;

(4S)-4-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-[[(2S)-4-hydroxy-l,4- dioxobutan-2-yl]carbamoyl]pyrrolidin-l-yl]-5-oxopentanoic acid;

Ac-IETD-CHO; (4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-5-[[(2S,3S)-3-hydroxy-l- [ [(2S)-4-hydroxy- 1 ,4-dioxobutan-2-yl] amino] - 1 -oxobutan-2-yl] amino] -5 -oxopentanoic acid;

(2S ,5 S)-4-oxo-5 - { [N-(phenylacetyl)-L-isoleucyl] amino } -N- ( 1 H- 1 ,2,3 -triazol-4- ylmethyl)-l,2,4,5,6,7-hexahydroazepino[3 ,2,l-hi]indole-2-carboxamide; 5-chloro-4- oxo-3-[2-[2-(phenylmethoxycarbonylamino) propanoylamino] propanoylamino] pentanoic acid;

5-chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylamino) propanoylamino]

propanoylamino] pentanoic acid;

(4S)-4-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-[[(2S)-4-hydroxy-l,4- dioxobutan-2-yl]carbamoyl]pyrrolidin-l-yl]-5-oxopentanoic acid;

(4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-5-[[(2S,3S)-3-hydroxy-l- [ [(2S)-4-hydroxy- 1 ,4-dioxobutan-2-yl] amino] - 1 -oxobutan-2-yl] amino] -5 -oxopentanoic acid,

a Serp2 polypeptide, or fragment thereof;

a CrmA polypeptide or fragment thereof; and

a SerpinA3 polypeptide or fragment thereof.

68. The Granzyme B inhibitor of claim 66, wherein the another wound treatment is selected from one or more of: a topical antimicrobial; a cleanser; a wound gel; a collagen; a elastin; a tissue growth promoter; an enzymatic debriding preparation; an antifungal; an anti- inflammatory; a barrier; a moisturizer; and a sealant.

69. A method for identifying a compound useful for promoting chronic wound healing, comprising

providing an indicator composition comprising decorin and Granzyme B;

contacting the indicator composition with each of a plurality of test compounds; and

determining the effect of each of the plurality of test compounds on the cleavage of decorin, and selecting a compound that inhibits the cleavage of decorin in the indicator composition, therby identifying a compound useful for promoting chronic wound healing.

Description:
GRANZYME B INHIBITOR COMPOSITIONS, METHODS AND USES FOR PROMOTING WOUND H EALING

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Appl ication No. 61 /420,230 filed on December 6, 2010 and U.S. Provisional Application No. 61 /493,265 filed on June 3, 201 1 , the entire contents of which arc incorporated herei n by this reference.

FIELD OF THE INVENTION

The invention relates to compositions, methods, and uses for wound heal ing.

BACKGROUND OF THE INVENTION

Wound healing is an intricate process in which an organ, such as the skin, is repaired after injury. In normal skin, the epiderm is and dermis form a protective barrier against the external environment. Once this protective barrier is broken, wound heal ing is set in motion to once again repair the protecti ve barrier.

The protective barrier can be weakened and/or ultimately broken by

environmental factors such as exposure to UV light, chemical, heat or mechanical injury to the skin. Additionally, biologic and genetic factors can play a part in weakening or breaking the protective barrier. For example, diseases such as d iabetes and psoriasis can disrupt the protective barrie . Further, natural conditions such as biological and/or environmentally-induced aging can result in disruption or thinning of the skin's protective barrier. Immobilization or obsesity may also lead to disruption or thinning of the skin's protective barrier. All of these conditions can lead to skin tearing or ulceration caused by pressure, ischemia, friction, chem ical, heat, or other trauma to the skin (see, for e.g., Sen el ai , 2009). In many cases these wounds may not heal completely or properly due to these underlying conditions.

Given the high costs for health care of subjects having chronic wounds, or wounds that fail to close properly or recur, approximately S25 bi ll ion annual ly, there is a need in the art for the identi fication of compounds, compositions, and methods to promote wound healing and/or prevent the occurrence or re-occurrence of such wounds.

SUMMARY

In some embodiments, the present invention is based, at least in part, on the discovery that Granzyme B cleaves the extracellular matrix proteins, decorin, biglycan, betaglycan, syndecan, brevican, fibromodul in, fibril l in- 1 , fibri l li n-2, and fibulin-2 in vitro and cleavage of decorin, biglycan, betaglycan by Granzyme B is concentration- dependent. Cleavage of decorin, biglycan, and betaglycan by Granzyme B releases active TGF-β. The release of TGF-β was specific to cleavage of decorin, biglycan, and betaglycan by Granzyme B as TGF-β was nol released in the absence of Granzyme B or when Granzyme B was inhibited by DC1. In addition, it has been shown that Granzyme B cleaves the proteoglycan substrates, biglycan and betaglycan at a P I residue of Asp (biglycan: D 9 ' , betaglycan: D 558 ).

In some embodiments, the present invention is further based, at least in part, on the discovery that, in vivo, deletion of Granzyme B delays the onset of skin frai lly, hair loss, hair graying and the formation of inflammatory subcutaneous skin lesions or xanthomas in the ApoE knockout mouse. I t has also been shown that Granzyme B is expressed in areas of collagen and decorin dcgradalion and remodelling in the skin of apoE- θ mice and that Granzyme B deficiency protects against skin thinning due, at lest in part, to inhibition of decorin cleavage and/or an increase in dermal thickness.

Furthermore, the present invention demonstrates that inhibitors of Granzyme B downmodulate decorin cleavage in vitro and in vivo and promote wound heal ing by, for example, stimulating col lagen organization, decreasing scarring and increasing the tensile strength of skin.

Accordingly, in one aspect, there is provided a method of promoting wound healing in a subject. The method involves applying a Granzyme B (Granzyme B) inhibitor to the wound. The wound may be, without l imitation, a skin wound.

The Granzyme B inhibitor may be selected from one or more of the following: nucleic acids, peptides, and small molecules. Optionally, the peptide may be an antibody. Optionally, the antibody may be a monoclonal antibody. The Granzyme B inhibitor may be selected from one or more of the fol lowing: Azepino[3,2, l -hi]indole-2-carboxamide, 5-[[(2S,3S)-2-[(2-benzo[b]lhicn-3- ylacetyl)amino]-3-methyl- 1 -oxopentyl]amino]- 1 ,2,4,5,6,7-hexahydro-4-oxo-N-( I H- l ,2,3-triazol-5-ylmethyl)-,(2S,5S)- (compound 20 from Willoughby el al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2 197-2200) referred to herein as

Willoughby 20 and different batches of Willoughby 20 are referred to herein as

JT25102B and JT00025 135; Bio-x-IEPD p -(OPh) 2 ; (2S,5S)-5-[(N-acclyl- L- isoleucyl)amino]-4-oxo-N-( l H-tetraazol-5-ylmcthyl)- l ,2,4,5 ,6,7- hexahydroazepino[3,2, 1 -hi]indole-2-carboxamide; ' (2S,5S)-5-[(N-acetyl-L- isoleucyl)amino]-4-oxo-N-( l H- 1 ,2, 3-triazol -4-ylmethyl)- l ,2,4,5,6,7- hexahydroazepino[3,2, l -hi] indole-2-carboxamide; (2S,5S)-5- { [(2 R)-3-methyl-2- pyridin-2-ylbutanoyl]amino } -4-oxo-N-( 1 H- 1 ,2,3-triazol-4-ylmethyl)- 1 ,2,4,5,6,7- hexahydr oazepino[3,2, l -hi]indole-2-carboxamide; (2S,5S)-4-oxo-5- { [N-(phenylacetyl)- L-isoleucyl]amino} -N- ( l H- 1 ,2,3-triazol-4-ylmethyl)- l ,2,4,5 ,6,7-hexahydroazepi no[3 ,2, 1 -hi]indole-2-carboxamide; 5-chloro-4-oxo-3-[2-[2-(phenylmethoxycarbonylamino) propanoylamino] propanoylamino] pentanoic acid; 5-ch loro-4-oxo-2-[2-[2- (phenylmethoxycarbonylamino) propanoylamino] propanoylamino] pentanoic acid; (4S)-4-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-[[(2 S)-4-hydroxy- l ,4- dioxobutan-2-yl]carbamoyl]pyrrol idin- l -yl]-5-oxopentanoic acid; (4S)-4-[[(2S,3S)-2- acetamido-3-methylpentanoyl]amino]-5-[[(2S,3S)-3-hydroxy- 1 -[[(2S)-4-hydroxy- 1 ,4- dioxobutan-2-yl]amino]- l -oxobutan-2-yl]amino]-5-oxopentanoic ac id, protease inhibitor-9 or derivatives thereof, CrmA, scrp-2, ZrNC05723764, ZINC05723787, ZI C053 16154, ZrNC05723499, ZINC05723646, ZI NC05398428, Z1NC05723503 , ZINC05723446, ZINC053 1 72 1 6, ZINC053 1 5460, ZI NC053 1 6859, and ZINC05605947. Alternatively, the Granzyme B inhibitor may be selected from one or more of the following: Willoughby 20, NCI 644752, NCI 644777, ZI NC053 1 72 1 6, and NCI 630295.

Optionally, the Granzyme B inhibitor may be formulated for topical

administration. The Granzyme B inhibitor may be formulated for co-administration with another wound treatment. Another wound treatment may be selected from one or more of the following: a topical antimicrobial; a cleanser; a wound gel; a collagen; an elastin; a tissue growth promoter; an enzymatic debriding preparation; an anti fungal ; an antiinflammatory; a barrier; a moisturizer; and a sealant. Optiona l ly, the another wound treatment may be selected from one or more of the fol lowing: a wound covering, a wound filler, and an implant. Optionally, the another wound treatment may be selected from one or more of the following: absorptive dressings; alginate dressings; foam dressings; hydrocolloid dressings; hydrofibcr dressings; compression dressing and wraps; composite dressing; contact layer; wound gel impregnated gauzes; wound gel sheets; transparent films; wound fil lers; dermal matrix products or tissue scaffolds; and closure devices. Optionally, the Granzyme B inhibitor may be formulated for topical application in a wound covering, a wound filler, or an implant. Optional ly, the

Granzyme B inhibitor may be formulated for impregnation in a wound covering, a wound filler or an implant. The subject may be a mammal ; optionally, the subject may be a human.

In another aspect, use of a Granzyme B inhibitor to promote wound heal ing in a subject is disclosed. In another aspect, use of a Granzyme B inhibitor in the preparation of a medicament for promoting wound healing in a subject is disclosed. Optiona lly, the wound may be a skin wound. Optionally, the Granzyme B inhibitor may be selected from one or more of the following: nucleic acids, peptides and smal l molecules.

Optionally, the peptides may be antibodies. Optional ly, the antibodies may be monoclonal antibodies.

Optionally, the Granzyme B inhibitor used herein may be selected from one or more of the following: Azepino[3,2, l -hi]indolc-2-carboxamide, 5-[[(2S,3S)-2-[(2- benzo[b]thien-3-ylacetyl)amino]-3-methyl- 1 -oxopcntyljamino]- 1 ,2,4 ,5,6,7-hexahydro-4- oxo-N-( l H- l ,2,3-triazol-5-ylmethyl)-,(2S,5S)- (compound 20 from Wil loughby el al. (2002) Bioorganic & Medicinal Chemistry Letters 1 2 :2 1 97-2200) referred to herein as Willoughby 20; Bio-x-IEPD p -(OPh) 2 ; (2S,5S)-5-[(N-acelyl-L-isolcucyl)amino]-4-oxo- N-( l H-tetraazol-5-ylmethyl)- 1 ,2,4, 5,6, 7-hcxahydroazepino[3, 2, 1 -hi] indole-2- carboxamide; (2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-4-oxo-N-( I H- l ,2, 3 -triazol -4- ylmethyl)- l ,2,4,5,6,7-hexahydroazepino[3,2, l -hi] indolc-2-carboxamide; (2S,5S)-5- {[(2R)-3-methyl-2-pyridin-2-ylbutanoyl]amino } -4-oxo-N-( 1 H- 1 ,2,3-triazol-4- ylmethyl)- l ,2,4,5, 6,7-hexahydr oazepino[3,2, l -hi] indolc-2-carboxamide; (2S,5S)-4-oxo- 5- {[N-(phenylacetyl)-L-isoleucyl]amino} -N- ( I H- 1 ,2,3-lriazol-4-y!methyl)- 1 ,2,4,5,6,7- hexahydroazepino[3 ,2, l -hi]indole-2-carboxamide; 5-ch loro-4-oxo-3-[2-[2-

(phenylmethoxycarbonylamino) propanoylamino] propanoylamino] pentanoic acid; 5- chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylamino) propanoylamino]

propanoylamino] pentanoic acid; (4S)-4-[[(2S)-2-acclamido-4-melhylpentanoyl]amino]- 5-[2-[[(2S)-4-hydroxy- l ,4-dioxobutan-2-yl]carbamoyl]pyrrolidin- l -yl]-5-oxopentanoic acid; (4S)-4-[[(2S,3S)-2-acetamido-3-methylpcntanoyl]amino]-5-[[(2 S,3S)-3-hydroxy- 1 - [[(2S)-4-hydroxy- 1 ,4-dioxobutan-2-yl]amino]- 1 -oxobutan-2-yl]amino]-5-oxopentanoic acid, protease inhibitor-9 or derivatives thereof, CrmA, serp-2, ZrNC05723764, ZINC05723787, ZI C053 161 54, ZINC05723499, ZINC05723646, ZINC05398428, ZI C05723503, ZI C05723446, ZINC053 1 72 1 6, Z1NC053 1 5460, ZrNC053 16859, and ZINC05605947. Alternatively, the Granzyme B inhibitor may be selected from one or more of the following: Willoughby 20, NCI 644752, NCI 644777, Z1NC053 1 72 1 6, and NCI 630295.

Optionally, the Granzyme B inhibitor being used is formulated for topical administration. Optionally, the Granzyme B inhibitor is formulated for co-administration with another wound treatment. Optional ly, the wound treatment is selected from one or more of: a topical antimicrobial; a cleanser; a wound gel; a collagen; a elastin; a tissue growth promoter; an enzymatic debriding preparation; an anti fungal ; an anti- inflammatory; a barrier; a moisturizer; and a sealant. Optiona l ly, the another wound treatment is selected from one or more of: a wound covering, a wound fil ler and an implant. Optionally, the another wound treatment is selected from one or more of:

absorptive dressings; alginate dressings; foam dressings; hydrocol loid dressings;

hydrofiber dressings; compression dressing & wraps; composite dressing; contact layer; wound gel impregnated gauzes; wound gel sheets; transparent films; wound fi llers; , dermal matrix products or tissue scaffolds; and closure devices. Optional ly, the

Granzyme B inhibitor is formulated for topical application in a wound covering, a wound filler, or an implant. Optionally, the Granzyme B inhibitor is formulated for impregnation in a wound covering, a wound filler or an implant. Optiona lly, the use involves a subject that may be a mammal; optionally, the use involves a subject that may be a human.

In another aspect, a Granzyme B inhibitor for use in promoting wound healing in a subject is disclosed herein. Optionally, the wound may be a skin wound. Optional ly, the Granzyme B inhibitor may be selected from one or more of the following: nucleic acids, peptides, and small molecules. Optional ly, the peptides may be antibodies.

Optionally, the antibodies may be monoclonal antibodies. Optional ly, the Granzyme B inhibitor may be selected from one or more of the fol lowing: Azcpino[3,2, l -hi] indole-2- carboxamide, 5-[[(2S,3S)-2-[(2-benzo[b]thien-3-ylacctyl)amino]-3-melhyl- 1 - oxopentyl]amino]- 1 ,2,4, 5,6, 7-hexahydro-4-oxo-N-( 1 H- l ,2,3 -iriazol-5-ylmethyl)- ,(2S,5S)- (compound 28 from Willoughby el al. (2002) Bioorganic & Medicinal Chemistry Letters 12 :2197-2200) referred to herein as Willoughby 20; Bio-x-lEPD p - (OPh) 2 ; (2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-4-oxo-N-( 1 H-tetraazol-5-ylmethyl)- l ,2,4,5,6,7-hexahydroazepino[3,2, l -hi]indole-2-carboxamide; (2S,5S)-5-[(N-acetyl-L- isoleucyl)amino]-4-oxo-N-( l H- 1 ,2, 3-triazol-4-ylmethyl)- 1 ,2,4,5,6,7- hexahydroazepino[3,2, l -hi] indole-2-carboxamide; (2S,5S)-5- {[(2 R)-3 -methyl-2- pyridin-2-ylbutanoyl]amino } -4-oxo-N-( 1 H- 1 ,2,3-triazol-4-ylmethyl)- 1 ,2,4,5,6,7- hexahydr oazepino[3,2, l -hi]indole-2-carboxamide; (2S,5S)-4-oxo-5- { [N-(phenylacetyl)- L-isoleucyl]amino} -N- ( l H- l ,2,3-triazol-4-ylmethyl)- l ,2,4,5,6,7-hexahydroazepi no[3 ,2, l -hi]indole-2-carboxamide; 5-chloro-4-oxo-3-[2-[2-(phenyl methoxycarbonylamino) propanoylamino] propanoylamino] pentanoic acid; 5-chloro-4-oxo-2-[2-[2- (phenylmethoxycarbonylamino) propanoylamino] propanoylam ino] pentanoic acid; (4S)-4-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-l;[( 2S)-4-hydroxy- l ,4- dioxobutan-2-yl]carbamoyl]pyrrol idin- l -yl]-5-oxopentano [ c acid; (4S)-4-[[(2S,3 S)-2- acetamido-3-methylpentanoyl]amino]-5-[[(2S,3S)-3-hydroxy- 1 -[[(2S)-4-hydroxy- 1 ,4- dioxobutan-2-yl]amino]- l -oxobutan-2-yl]amino]-5-oxopenlanoic ac id, protease inhibitor-9 or derivatives thereof, CrmA, serp-2, ZrNC05723764, ZINC05723787, ZINC053 16154, ZrNC05723499, ZINC05723646, Z1NC05398428, Z1NC05723503, ZINC05723446, ZINC053 172 16, ZINC053 1 5460, Z1NC053 1 6859, and Z1NC05605947. Alternatively, the Granzyme B inhibitor may be selected from one or more of the following: Willoughby 20, NCI 644752, NCI 644777, ZINC053 1 72 1 6, and NCI 630295.

Optionally, the Granzyme B inhibitor may be formulated for topical

administration. Optionally, the Granzyme B inhibitor may be formulated for co- administration with another wound treatment. Optional ly, the another wound treatment may be selected from one or more of: a topical antimicrobial ; a cleanser; a wound gel ; a collagen; an elastin; a tissue growth promoter; an enzymatic debriding preparation; an antifungal; an anti-inflammatory; a barrier; a moisturizer; and a sealant. Optional ly, the another wound treatment may be selected from one or more of: a wound covering, a wound filler and an implant. Optionally, the another wound treatment may be selected from one or more of: absorptive dressings; alginate dressings; foam dressings;

hydrocolloid dressings; hydrofiber dressings; compression dressing & wraps; composite dressing; contact layer; wound gel impregnated gauzes; wound gel sheets; transparent films; wound fillers; dermal matrix products or tissue scaffolds; and closure devices. Optionally, the Granzyme B inhibitor may be formulated for topical application in a wound covering, a wound filler, or an implant. Optiona lly, the Granzyme B inhibitor may be formulated for impregnation in a wound covering, a wound filler or an implant. Optionally, the subject may be a mammal; optional ly the subject may be a human.

In another aspect, a method of inhibiting release of a cytok ine, such as active transforming growth factor- β (TGF- j3 ), wherein the cytokine, e.g. , TGF-, is bound to an extracelular matrix protein, e.g., an extracellular proteoglycan, is disclosed. The method may involve inhibiting a cleavage site in a proteoglycan. The proteoglycan may be selected from any one of the following: biglycan, decorin, finromodul i n, or betaglycan. However, the aforementioned examples are provided as examples only and are not present as limitations. While the method disclosed details TGF- β bound to a proteoglycan, other cytokines and growth factors bound to other proteoglycans may also be considered as suitable targets. Optionally, the method is carried out in vitro.

Optionally, the method is carried out in a subject in vivo. Optionally, the subject may be a mammal. Optionally, the subject may be a human. Optionally, the cleavage sites occur in any one of the following peptide sequences: Asp 9 l Thr-Thr-Lcu-Leu-Asp; or Asp 558 Ala-Ser-Leu-Phe-Thr; or Asp^' Glu-Ala-Ser-Gly; or Asp 69 Leu-Gly-Asp-Lys; or Asp 82 Thr-Thr-Leu-Leu-Asp; or Asp 261 Asn-G Iy-Ser-Leu-Ala^

In another aspect, a model for studying age-related wound heal ing is disc losed.

The model comprises an apolipoprotein E-knock out mouse mainta ined on a high-fat feed diet, wherein the high-fat feed diet is su fficient to result in xanlhomatotic skin lesions on skin of the mouse. Alternatively or in addition, the high- fat feed diet may be sufficient to result in premature aging in non-xanlhamatous skin. As detailed herein, inhibition of Granzyme B by way of Granzyme B inhibitors or through knock-out technology reduces the age-related loss of skin thickness, collagen density, col lagen disorganization, and loss of tensile strength. It is considered that based on the results herein that a Granzyme B inhibitor could be added to Stage I skin ulcers to restore skin thickness, skin integrity, skin collagenicity, and to inhibit or oihci-wisc reduce progression of the skin ulcer.

In another aspect, a model for studying Granzyme B protein expression in vivo is disclosed. The model comprises an apolipoprotein E-knock out mouse maintained on a high-fat feed diet, wherein the high-fat feed diet is sufficient to result in xanthomatotic skin lesions on the skin of the mouse mouse, and wherein the skin lesions express Granzyme B.

In another aspect, a model for screening compounds involved in repairing wounds is disclosed. The method involves maintaining an apolipoprotein E-knock out mouse on a high-fat feed diet, wherein the high-fat feed diet is sufficient to result in skin lesions on the mouse; administering a compound to the skin lesions on the mouse; and monitoring the skin lesions on the mouse.

In another aspect, a model for studying age-related wound heal ing in skin is disclosed. The model comprises an apolipoprotein E-knock-out mouse maintained on a high-fat feed diet, wherein the high-fat feed diet is su fficient to result in premature aging of the skin.

In another aspect, a method of screeni ng compounds involved in repairing wounds is disclosed. The method may involve maintaining an apolipoprotein E-knock out mouse on a high-fat feed diet, wherein the high-fat feed diet is sufficient to result in skin lesions on the mouse, and wherein the skin lesions express Granzyme B;

administering a compound to the skin lesions on the mouse; and monitoring the skin lesions on the mouse.

In another aspect, a method of screening compounds involved in inhibiting or reducing skin lesions is disclosed. The method may involve ma intaining an

apolipoprotein E-l nock out mouse on a high-fal feed diet, wherein the high-fat feed diet is sufficient to result in skin lesions on the mouse when a compound is not administered to the mouse; administering the compound to the mouse; and monitoring the skin lesions on the mouse.

In another aspect, a method of screeni ng compounds involved in inhibiting or reducing skin lesions is disclosed. The method may involve ma inta ining an

apolipoprotein E-knock out mouse on a high- fat feed diet, wherein the high-fat feed diet is sufficient to result in skin lesions on the mouse when a compound is not administered to the mouse, and wherein the skin lesions express Granzyme B; administering the compound to the skin lesions on the mouse; and monitoring the sk in lesions on the mouse. In another aspect, a method of inhibiting or reducing skin tearing is disclosed. The method may involve applying a Granzyme B inhibitor to the skin. The Granzyme B inhibitor selected may be one or more of the fol lowing: nuc leic acids, peptides, and small molecules. The peptides may be antibodies. The antibodies may be monoclonal antibodies. The Granzyme B inhibitor may be selected from one or more of the following: Azepino[3,2, l -hi]indoIe-2-carboxamide, 5-[[(2S,3S)-2-[(2-benzo[b]thien-3- ylacetyl)amino]-3-methyl- l -oxopentyl]amino]- l ,2,4,5,6,7-hexahydro-4-oxo-N-( 1 H- l ,2,3-triazol-5-ylmethyl)-,(2S,5S)- (compound 20 from Wil loughby el al. (2002) Bioorganic & Medicinal Chemistry Letters 1 2: 2 1 97-2200) referred to herein as

Willoughby 20; Bio-x-IEPD p -(OPh) 2 ; (2S,5S)-5-[(N-acetyl- L-isoleucyl)amino]-4-oxo- N-( l H-tetraazol-5-ylmethyl)- l ,2,4,5,6,7-hexahydroazepino[3,2, l -hi]indole-2- carboxamide; (2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-4-oxo-N-( 1 H- 1 ,2, 3-triazol-4- ylmethyl)- l ,2,4,5,6,7-hexahydroazepino[3 ,2, l -hi] indole-2-carboxamide; (2S,5S)-5- {[(2R)-3-methyl-2-pyridin-2-ylbutanoyl]amino } -4-oxo-N-( 1 H- 1 ,2,3-triazol-4- ylmethyl)- l ,2,4,5,6,7-hexahydr oazepino[3,2, l -hi] indolc-2-carboxamide; (2S,5S)-4-oxo- 5- {[N-(phenylacetyl)-L-isoleucyl]amino} -N- ( 1 H- 1 ,2,3-triazol-4-ylmethyl)- 1 ,2,4,5,6,7- hexahydroazepino[3 ,2, 1 -hi]indole-2-carboxamide; 5-chloro-4-oxo-3-[2-[2- (phenylmethoxycarbonylamino) propanoylamino] propanoylamino] pentanoic acid; 5- chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylamino) propanoylamino]

propanoylamino] pentanoic acid; (4S)-4-[[(2S)-2-acctamido-4-methylpcntanoyl]amino]- 5-[2-[[(2S)-4-hydroxy- l ,4-dioxobutan-2-yl]carbamoyl]pyrrol idin- 1 -yl]-5-oxopentanoic acid; (4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-5-[[(2 S,3S)-3-hydroxy- 1 - [[(2S)-4-hydroxy- l ,4-dioxobutan-2-yl]amino]- 1 -oxobutan-2-yl]amino]-5-oxopentanoic acid, protease inhibitor-9 or derivatives thereof, CrmA, serp-2, ZINC05723764, ZI C05723787, ZINC053 161 54, ZINC05723499, ZI C05723646, ΖΓΝΟ05398428,

ZINC05723503, ΖΓΝΟ05723446, ZINC053 17216, Z1 C053 1 5460, Z1NC053 16859, and ZINC05605947. Alternatively, the Granzyme B inhibitor may be selected from one or more of the following: Willoughby 20, NCI 644752, NCI 644777, ZI ~ NC053 1 72 1 6, and NCI 630295. Further, the Granzyme B inhibitor may be formulated for topical administration.

In another aspect, the present invention provides methods of promoting wound healing in a subject, the method comprising administering a Granzyme B (GrB) inhibitor to the subject for a time and in an amount sufficient to promote would healing, thereby promoting wound healing in the subject.

In another aspect, the present invention provides methods of promoting wound healing in a subject, the method comprising applying a Granzyme B (Granzyme B) inhibitor to the wound, for a time and in an amount suffic ient to promote would healing, thereby promoting wound healing in the subject.

The wound may be a chronic wound, such as a a chronic sk in wound, such as a pressure ulcer.

In one embodiment, cleavage of an extracel lular matrix protein is inhibited. In one embodiment, the extracellular matrix protein is selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodul in, fibri l l in- 1 , fibril l in-2, and fibulin-2. In one embodiment, the extracellular ma trix protei n is decorin.

In one embodiment, release of TGI 7 P bound to a n extracel lu lar matrix protein is inhibited. In one embodiment, the extracellular matrix protein is decorin.

In another aspect, the present invention provides methods of preventing skin tearing of a subject, comprising applying a Granzyme B inhibitor to the skin of the subject for a time and in an amount sufficicnl to prevent sk in tearing, thereby preventing skin tearing in the subject.

In one embodiment, the skin tearing is associated with a chronic wound. In another embodiment, the skin tearing is associated with agi ng.

In one embodiment, cleavage of an extracel lular matrix protein is inhibited. In one embodiment, extracellular matrix protein is selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodul in, fibri ll in- 1 , fibril lin-2, and fibulin-2. In one embodiment, the extracellular matrix protein is decorin.

In one embodiment, release of ΤΰΡβ bound to an extracel lular matrix protein is inhibited. In one embodiment, the extracellular matrix protein is decorin.

In yet another aspect, the present invention provides methods for inhibiting hypertrophic scarring of a wound, comprising applying a Granzyme B inhibitor to the skin of the subject for a time and in an amount sufficient to prevent skin hypertrophic scarring of a wound, thereby inhibiting hypertrophic scarring of a wound. In one embodiment, cleavage of an extracellular matrix protein is i nhibited. In one embodiment, the extracellular matrix protein is selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodulin, fibrillin- 1 , fibril lin-2, and fibulin-2. In one embodiment, the extracellular matrix protein is decorin.

In one embodiment, release of ΤϋΡβ bound to an extracel lular matrix protein is inhibited. In one embodiment, the extracel lular matrix protein is decorin.

In another aspect, the present invention provides methods for increasing col lagen organization in the skin of a subject, comprising applying a Granzyme B inhibitor to the skin of the subject in an amount and for a lime sufficient to i ncrease col lagen organization in the subject, thereby increasing collagen organization in the skin of the subject.

In one embodiment, cleavage of an extracel lular matrix protein is inhibited. In one embodiment, the extracellular matrix protein is selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodul in, fibrill in- 1 , fibril l in-2, and fibulin-2. In one embodiment, the extracellular matrix protein is decorin

In one embodiment, release of TG1 7 bound to an extracel lular matrix protein is inhibited. In one embodiment, the extracel lular matrix protein is decorin.

In another aspect, the present invention provides methods for increasing the tensile strength of a healing or healed skin wound of a subject, comprising applying a Granzyme B inhibitor to the skin of the subject in an amount and for a time sufficient to increase increase the tensile strength of the healing or healed skin wound of the subject, thereby increasing the tensile strength of a healing or hea led skin wound of a subject.

In one embodiment, cleavage of an extracellular matrix protein is inhibited. In one embodiment, the extracel lular matrix protein is selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodul in, fibril l in- 1 , fibri l lin-2, and fibulin-2. In one embodiment, the extracellular matrix protein is decorin

In one embodiment, release of Τΰ Ρβ bound to an extracel lular matrix protein is inhibited. In one embodiment, the extracel lular matrix protein is decorin.

In one aspect, the present invention provides methods for inhibiting release of TGF bound to an extracellular protein, comprising contacting the extracellular proteoglycan with a Granzyme B inhibitor, thereby inhibiting release of TG F bound to the extracellular protein.

In one embodiment, the protein is selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodul in, fibril l in- 1 , fibrill in-2, and fibulin-2. In one embodiment, the protein is decorin

In another aspect, the present invention provides methods inhibiting extracel lular decorin cleavage, comprising contacting decorin with a Granzyme B inhibitor, thereby inhibiting extracellular decorin cleavage.

In one embodiment, the Granzyme B inhibitor for use in any of the foregoing methods is selected from the group consisting of a nucleic acid molecule, a peptide, an antibody, and a small molecule. In one embodiment, the antibody is a monoclonal antibody.

In another embodiment, the Granzyme B inhibitor for use i n any of the foregoing methods is wherein the Granzyme B inhibitor is selected from one or more of the following:

2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-acetamido-3 -methylpentanamido)-4- oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi] indole-2-carboxamide;

(2S,5S)-N-(( 1 H- 1 ,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-acclamido-3- methylpentanamido)-4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3 ,2 , 1 -hi]indole-2- carboxamide;

(2S,5S)-N-(( l H- l ,2,3-triazol-4-yl)methyl)-5-(( )-3-methyl-2-(pyridin-2- yl)butanamido)-4-oxo- l ,2,4,5,6,7-hexahydroazcpino[3,2, l -hi] i ndole-2-carboxamidc;

(2S,5S)-N-(( l H- l ,2,3-triazol-4-yl)methyl)-5-((2S,3S)-3-mclhyl-2-(2- phenylacetamido)pentanamido)-4-oxo- l ,2,4,5 ,6,7-hcxahydroazepino[3,2, l -hi] indole-2- carboxamide;

(2S,5S)-N-(( l H- l ,2,4-triazol-3-yl)mcthyl)-5-((2S,3S)-2-acclamido-3- methylpentanamido)-4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3 ,2, 1 -hi] indolc-2- carboxamide;

(2S,5S)-N-(( I H-pyrazol-3-yl)methyl)-5-((2S,3S)-2-acctamido-3-methylpcntan amido)-4- oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi] indolc-2-carboxamide; (2S,5S)-N-(( l H-pyrazol-4-yl)methyl)-5-((2S,3S)-2-aceUimido-3-mclhylpentan amido)-4- oxo- l ^^S^^-hexahydroazepino ^. l -hiJ indolc^-carboxamidc;

(2S,5S)-N-(( l H-imidazol-4-yl)methyl)-5-((2S,3S)-2-acetamido-3-methy!penta namido)- 4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi] indole-2-carboxamidc;

2S,5S)-5-((2S,3S)-2-acetamido-3-methylpenlanamido)-4-oxo-N-( lhiazol-5-ylmethyl)- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indolc-2-carboxamidc;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpenlanamido)-N-(isoxa zol-3-ylmethyl)-4-oxo- l ,2,4,5,6,7-hexahydroazepino[3,2, l -hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N- (thiazol-2-ylmethyl)- l ,2,4,5,6,7-hexahydroazepino[3 ,2, 1 -hi]indolc-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-mcthylpentanamido)-N-(isoxa zol-5-ylmethyl)-4-oxo- l ,2,4,5,6,7-hexahydroazepino[3,2, ] -hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpcntanamido)-4-oxo-N- (thiazol-4-ylmcthyl)- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indolc-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-mcthylpcnlanamido)-4-oxo-N- (pyrimidin-5- ylmethyl)- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -h i]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpcnlanamido)-4-oxo-N- (pyridazin-4- ylmethyl)- 1 ^^.S^J-hexahydroazepino ^, 1 -hi]indolc-2-carboxamidc;

(2S,5S)-5-((2S,3S)-2-acetamido-3-mcthylpentanamido)-4-oxo-N- (pyrid in-2-ylmcthyl)- l ,2,4,5,6,7-hexahydroazepino[3,2, l -hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-mcthylpentanamido)-4-oxo-N- (pyrid in-3-ylmethyl)- 1 , 2,4,5,6, 7-hexahydroazepino[3, 2, 1 -hi]indolc-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpenianamido)-4-oxo-N- (pyridin-4-ylmethyl)- l ,2,4,5,6,7-hexahydroazepino[3,2, l -hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-melhylpenlanamido)-N-(imida zof 1 ,2-a]pyrimidin-2- ylmethyl)-4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indolc-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-N-((3a,7 a- dihydrobenzo[d]thiazol-2-yl)methyl)-4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3 ,2, 1 - hi]indole-2-carboxamide; (2S,5S)-N-((2H etrazol-5-yl)methyl)-5-((R)-3-methyl-2-(pyridin-2-yl)butanam ido)-4- oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indolc-2-carboxami de;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((S)-3-mclhyl-2-(pyri din-2-yl)butanamido)-4- oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indolc-2-carboxami de;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-3-melhyl- 2-(2- phenylacetamido)pentanamido)-4-oxo-l ,2,4,5,6,7-hcxahydroazepino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S ! 3S)-2-(2-(2,3-dinuorophenyl)acetamido)- 3-methylpentanamido)-4-oxo-l,2,4,5,6,7-hexahydroazcpino[3,2, l -hi]indole-2- carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(dimelh ylamino)acelamido)-3- methylpentanamido)-4-oxo-l ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indolc-2- carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(bcnzo[ b]lhiophen-3- yl)acetamido)-3-methylpentanamido)-4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 - hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-(2-(d imclhylamino)acetamido)- 3-methylpentanamido)-4-oxo-l ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indole-2- carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-(2 -(bcnzo[b]lhiophen-3- yl)acetamido)-3-methylpentanamido)-4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 - hi]indole-2-carboxamide;

(R)-N-((2S,5S)-2-((lH-l,2,3-triazol-4-yl)mcthylcarbamoyl)-4- oxo-l,2,4,5,6,7- hexahydroazepino[3,2,l-hi]indol-5-yl)-3-acetyl-5,5-dimethylt hiazolidine-4- carboxamide;

(2S,5S)-N-(( 1 H- 1 ,2,3-triazol-4-yl)mcthyl)-5-((2S,3S)-3-mclhy l-2-(2-oxopyrrolidin- 1 - yl)pentanamido)-4-oxo- 1 ,2,4,5,6,7-hexahydroazcpino[3,2, 1 -hi]indole-2-carboxamide;

(2S,5S)-N-(( 1 H- 1 ,2,3-triazol-4-yl)mcthyl)-5-(2-cyclopentylacclamido)-4-oxo- l^^^^^-hexahydroazepinotS^ -hilindole^-carboxamide; (2S,5S)-N-(( 1 H- 1 ,2,3-triazol-4-yl)mcthyl)-5-((S)-2-aceiamido-2-cyclopropylac etamido)- 4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indole-2-carboxamide;

(2S,5S)-N-(( l H- l ,2,3-triazol-4-yl)mcthyl)-5-((S)-2-acetamido-2-cyc lopentylacetamido)-

4- oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indole-2-carboxamide;

Bio-x-IEPD p -(OPh) 2 ;

azepino[3,2, l -hi]indole-2-carboxamide;

(4S)-4-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-[[(2 S)-4-hydroxy- l ,4- dioxobutan-2-yl]carbamoyl]pyrrolidin- 1 -yl]-5-oxopentanoic acid;

(4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-5-[[(2 S,3 S)-3-hydroxy- l - [[(2S)-4-hydroxy- l ,4-dioxobutan-2-yl]amino] - l -oxobuian-2-yl]amino]-5-oxopentanoic acid;

5- chloro-4-oxo-3-[2-[2-(phenylmethoxycarbonylamino) propanoylamino]

propanoylamino] pentanoic acid;

5-chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylam ino) propanoylamino]

propanoylamino] pentanoic acid;

ZINC05723764;

ZINC05723787;

ZINC053 16154;

ZI C05723499;

ZINC05723646;

ZINC05398428;

ZINC05723503 ;

ZINC05723446;

ZINC053 17216;

ZINC053 15460;

ZINC053 16859;

ZINC05605947;

an isocoumarin; a peptide chloromethyl ketone;

a peptide phosphonate;

a Granzyme B inhibitory nucleic acid molecule;

an anti-Granzyme B antibody;

an inhibitory Granzyme B peptide;

a SerpB9 polypeptide, or fragment thereof;

5-chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylamino)

propanoyIamino]propanoylamino] pentanoic acid;

Ac-IEPD-CHO;

(4S)-4-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-[ [(2S)-4-hydroxy- 1 ,4- dioxobutan-2-yl]carbamoyl]pyrrol idin- 1 -yl]-5-oxopenianoic acid;

Ac-IETD-CHO;

(4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoy!]amino]-5-[[(2 S,3 S)-3-hydroxy- l - [[(2S)-4-hydroxy- l ,4-dioxobutan-2-yl]amino]- l -oxobutan-2-yl]amino]-5-oxopentanoic acid;

(2S,5S)-4-oxo-5- {[N-(phenylacetyl)-L-isoleucyl]amino } -N- ( 1 H - 1 ,2,3-triazol-4- ylmethyl)- l ,2,4,5,6,7-hexahydroazepino[3 ,2, 1 -hi] indolc-2-ca rboxamide; 5-chloro-4- oxo-3-[2-[2-(phenylmethoxycarbonylamino) propanoylamino] propanoylamino] pentanoic acid;

5-chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylamino) propanoylamino]

propanoylamino] pentanoic acid;

(4S)-4-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-f[(2 S)-4-hydroxy- l ,4- dioxobutan-2-yl]carbamoyl]pyrrol idin- l -yl]-5-oxopcntanoic acid;

(4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-5-[[(2 S,3S)-3-hydroxy- l - [[(2S)-4-hydroxy- l ,4-dioxobutan-2-yl]am ino] - l -oxobutan-2-yl]amino]-5-oxopentanoic acid,

a Serp2 polypeptide, or fragment thereof;

a CrmA polypeptide or fragment thereof; and a SerpinA3 polypeptide or fragment thereof.

In one embodiment, the Granzymc B inhibitor for use in any of the foregoing methods is formulated for topical admin istration. In one embodiment, the Granzyme B inhibitor is formulated for co- administration with another wound treatment.

In one embodiment, the another wound treatment is selected from one or more of: a topical antimicrobial ; a cleanser; a wound gel; a col lagen; an clastin; a tissue growth promoter; an enzymatic debriding preparation; an anti ungal; an antiinflammatory; a barrier; a moisturizer; and a sealant. I n another embodiment, the another wound treatment is selected from one or more of: a wound covering, a wound filler, and an implant. In another embodiment, another wound trcaimeni is selected from one or more of: absorptive dressings; alginate dressings; foam dressings; hydrocol loid dressings; hydrofiber dressings; compression dressing and wraps; composite dressing; contact layer; wound gel impregnated gauzes; wound gel sheets; transparent films; wound fillers; dermal matrix products or tissue scaffolds; and closure devices.

In one embodiment, the subject is a mammal . In one embodiment, the subject is a human.

In another aspect, the present invention provides uses of a Granzyme B inhibitor as described herein to promote wound heal ing in a subject.

In yet another aspect, the present invention provides uses of a Granzyme B inhibitor as described herein in the preparation of a medicament for promoting wound healing in a subject.

In one embodiment, the wound is a sk in wound. In one embodiment, the skin wound is a chronic skin wound.

In one embodiment, the Granzyme B inhibitor is selected from the group consisting of a nucleic acid molecule, a peptide, an antibody, and a small molecule. In one embodiment, a Granzyme B inhibitor is selected from the group consisting of

2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-aceiamid o-3-methylpcntanamido)-4- oxo- l ,2,4,5,6,7-hexahydroazepino[3,2, l -hi]indolc-2-carboxamidc; (2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-aceta mido-3- methylpentanamido)-4-oxo-l,2,4,5,6,7-hexahydroazcpino[3,2, 1 -hi] indolc-2- carboxamide;

(2S ) 5S)-N-((lH-l,2,3-triazol-4-yl)mcthyl)-5-((R)-3-methyl- 2-(pyridin-2- yl^utanamidoH-oxo-l^^.S^ -hexahydroazcpinofS^J-hiJindole^-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)mcthyl)-5-((2S,3S)-3-mc lhyl-2-(2- phenylacetamido)pentanamido)-4-oxo- 1,2,4, 5,6, 7-hexahydroazcpino[3, 2,1 -hi] indole-2- carboxamide;

(2S,5S)-N-((lH-l,2,4-triazol-3-yl)mcthyl)-5-((2S,3S)-2-accta mido-3- methylpentanamido)-4-oxo-l ,2,4,5,6,7-hexahydroazcpino[3,2, 1 -hi]indolc-2- carboxamide;

(2S,5S)-N-((lH-pyrazol-3-yl)methyl)-5-((2S,3S)-2-acctamido-3 -melhylpentanamido)-4- oxo-1 ,2,4,5,6,7-hexahydroazepino[3,2,l -hi]indolc-2-carboxamide;

(2S,5S)-N-((lH-pyrazol-4-yl)methyl)-5-((2S,3S)-2-acctamido-3 -mcthylpentanamido)-4- oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l -hi]indolc-2-carboxamide;

(2S,5S)-N-((lH-imidazol-4-yl)methy])-5-((2S,3S)-2-acctamido- 3-mcthylpentanamido)- 4-oxo- l,2,4,5,6,7-hexahydroazepino[3, 2,1 -hi]indole-2-carboxamide;

2S,5S)-5-((2S,3S)-2-acetamido-3-methylpenlanamido)-4-oxo-N-( thiazol-5-ylmethyl)- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamidc;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpcnianamido)-N-(is oxazol-3-ylmethyl)-4-oxo- 1, 2,4,5,6, 7-hexahydroazepino[3, 2, 1 -hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpcntanamido)-4-oxo-N- (thiazol-2-ylmcthyl)- 1,2,4, 5,6, 7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-N-(isoxa zol-5-ylmethyl)-4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpcntanamido)-4-oxo- -(thiazol-4-ylmethyl)- l,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpcntanamido)-4-oxo-N- (pyrimidin-5- ylmethyl)- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indole-2-carboxamidc; (2S,5S)-5-((2S,3S)-2-acetamido-3-methylpenlanamido)-4-oxo-N- (pyridazin-4- ylmethyl)- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indolc-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpcntanamido)-4-oxo-N- (pyridin-2-ylmethyl)- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo - -(pyridin-3-ylmethyl)- l ,2,4,5,6,7-hexahydroazepino[3,2, l -hi]indolc-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N- (pyridin-4-ylmethyl)- l ,2,4,5,6,7-hexahydroazepino[3,2, l -hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-N-(imida zo| 1 ,2-a]pyrimidin-2- ylmethyl)-4-oxo- l ,2,4,5,6,7-hexahydroazcpino[3 ,2, l -hi] i ndole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpcntanamido)-N-((3a,7 a- dihydrobenzo[d]thiazol-2-yl)methyl)-4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 - hi]indole-2-carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-(( )-3-methyl-2-(pyridin-2-yl)butanamido)-4- oxo- 1 , 2,4,5, 6,7-hexahydroazepino[3,2, 1 -hi]indolc-2-carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((S)-3-mcthyl-2-(pyri din-2-yl)butanamido)-4- oxo- 1 ,2,4,5, 6,7-hexahydroazepino[3, 2, 1 -hi] indolc-2-carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-3-methyl-2-( 2- phenylacetamido)pentanamido)-4-oxo- 1 ,2,4, 5,6, 7-hcxahydroazcpino[3, 2, 1 -h i]indole-2- carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(2 ,3 -di nuorophenyl)acetamido)- 3-methylpentanamido)-4-oxo- l ,2,4,5,6,7-hexahydroazepino[3,2 , 1 -hi] indole-2- carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(dimclh ylamino)acelamido)-3- methylpentanamido)-4-oxo- l ,2,4,5,6,7-hexahydroazepino[3 ,2, 1 -hi]indolc-2- carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(bcnzo[ b]thiophcn-3- yl)acetamido)-3-methylpentanamido)-4-oxo- 1 ,2,4, 5,6, 7-hexahydroazepino[3 , 2, 1 - hi]indole-2-carboxamide; (2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-(2-(d imelhylamino)acetamido)-

3- methylpentanamido)-4-oxo-l ,2,4,5, 6,7-hexahydroazepino[3, 2,1 -hi]indole-2- carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-(2-(b cnzo[b]lhiophen-3- yl)acetamido)-3-methylpentanamido)-4-oxo- 1 ,2,4,5,6,7-hcxahydroazcpino[3,2, 1 - hi]indole-2-carboxamide;

(R)-N-((2S,5S)-2-((lH-l,2,3-triazol-4-yl)methylcarbamoyl)-4- oxo-l,2,4,5,6,7- hexahydroazepino[3,2,l -hi]indol-5-yl)-3-acelyl-5,5-dimethylthiazolidine-4- carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-3-mc lhyl-2-(2-oxopyrrolidin-l- yl)pentanamido)-4-oxo-l ,2,4,5,6,7-hexahydroazcpino[3,2, 1 -hi]indole-2-carboxamide;

(2S,5S)-N-(( 1 H- 1 ,2,3-triazol-4-yl)methyl)-5-(2-cyclopcntylacctamido)-4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indolc-2-carboxamide;

(2S,5S)-N-((1H-1 ,2,3-triazol-4-yl)mcthyl)-5-((S)-2-acetamido-2- yclopropylacetamido)- 4-oxo- 1,2,4, 5,6, 7-hexahydroazepino[3, 2,1 -hi]indole-2-carboxamidc;

(2S,5S)-N-(( 1 H- 1 ,2,3-triazol-4-yl)methyl)-5-((S)-2-acctamido-2-cyclopentylac etamido)-

4- oxo-l, 2,4, 5,6,7-hexahydroazepino[3, 2, 1 -hi]indolc-2-carboxamidc;

Bio-x-IEPD p -(OPh) 2 ;

azepino[3,2,l-hi]indole-2-carboxamide;

(4S)-4-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-[ [(2S)-4-hydroxy- 1 ,4- dioxobutan-2-yl]carbamoyl]pyrrolidin- 1 -yl]-5-oxopentanoic acid;

(4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-5-[f(2 S,3S)-3-hydroxy-l- [[(2S)-4-hydroxy-l ,4-dioxobutan-2-yl]amino]-l -oxobutan-2-yl]amino]-5-oxopentanoic acid;

5-chloro-4-oxo-3-[2-[2-(phenylmcthoxycarbonylamino) propanoylamino]

propanoylamino] pentanoic acid;

5- chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylamino) propanoylamino]

propanoylamino] pentanoic acid;

ZINC05723764; ZI C05723787;

ZINC053 16154;

ZI C05723499;

ZINC05723646;

ZINC05398428;

ZINC05723503;

ZINC05723446;

ZINC053 17216;

ZINC053 15460;

ZINC053 16859;

ZINC05605947;

an isocoumarin;

a peptide chloromethyl ketone;

a peptide phosphonate;

a Granzyme B inhibitory nucleic acid molecule;

an anti-Granzyme B antibody;

an inhibitory Granzyme B peptide;

a SerpB9 polypeptide, or fragment thereof;

5-chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylamino)

propanoylamino]propanoylamino] pentanoic acid;

Ac-IEPD-CHO;

(4S)-4-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-[[(2 S)-4-hydroxy- l ,4- dioxobutan-2-yl]carbamoyl]pyrrol idin- 1 -yl]-5-oxopentanoic acid;

Ac-IETD-CHO;

(4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-5-[ [(2S,3S)-3-hydroxy- 1 -

[[(2S)-4-hydroxy- l ,4-dioxobutan-2-yl]amino]- 1 -oxobulan-2-yl]amino]-5-oxopentanoic acid; (2S,5S)-4-oxo-5- {[N-(phenylacetyl)-L-isoleucyl]ami no} -N- ( 1 H- 1 ,2,3-triazol-4- ylmethyl)- 1 ,2,4,5, 6, 7-hexahydroazepino[3 ,2, l -hi]indolc-2-carboxamidc; 5-chloro-4- oxo-3-[2-[2-(phenylmethoxycarbonylamino) propanoylamino] propanoylamino] pentanoic acid;

5-chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylamino) propanoylamino]

propanoylamino] pentanoic acid;

(4S)-4-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-[[(2 S)-4-hydroxy- l ,4- dioxobutan-2-yl]carbamoyl]pyrrol idin- l -yl]-5-oxopcntanoic acid;

(4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]ami no] -5 -[[(2S,3S)-3-hydroxy- l - [[(2S)-4-hydroxy- l ,4-dioxobutan-2-yl]am ino]- 1 -oxobutan-2-yl]amino]-5-oxopentanoic acid,

a Serp2 polypeptide, or fragment thereof;

a CrmA polypeptide or fragment thereof; and

a SerpinA3 polypeptide or fragment thereof.

In one embodiment, the Granzyme B inhibitor is formulated for topical administration. In one embodiment, the Granzyme B inhibitor is formulated for co administration with another wound treatment. I n one embodiment, the another wound treatment is selected from one or more of: a topical anti microbia l; a cleanser; a wound gel; a collagen; a elastin; a tissue growth promoter; an enzymatic debriding preparation; an antifungal; an anti- inflammatory; a barrier; a moisturizer; and a sea lant.

In one embodiment, the subject is a mammal. In one embodiment, the subject is a human.

The present invention further provides a Granzyme B inhibitor for use i n promoting wound healing in a subject. In one embodiment, the wound is a sk in wound. In one embodiment, the wound is a chrionic skin wound.

In one embodiment, the Granzyme B inhibi tor is se lected from the group consisting of a nucleic acid moelcue, a peptide, and antibody, and a small molecule.

In one embodiment, the Granzyme B inhibitor is formulated for topical administration. In one embodiment, the Granzyme B inhibitor is formulated for co- administration with another wound treatment as described herein. BRIEF DESCRIPTION OF TH E DRAWINGS

Figure 1 demonstrates the identification of extracel l lular Granzyme B substrates. Star denotes full length and arrows indicate cleavage fragments.

Figures 2 demonstrates that Granzyme B mediates cleavage of native smooth muscle cell derived decorin and biglycan.

Figures 3A-3C demonstrate dose dependent Granzyme B-mcdiated cleavage of decorin, biglycan and betaglycan.

Figure 4 demonstrates that Granzyme B-mediated cleavage of PGs is inhibited by DCI at 4 h and 24 h and Granzyme B cleavage sites contain aspartic acid at the P I residue.

Figure 5 demonstrates Granzyme B cleavage of decorin, biglycan and betaglycan results in the release of active TG F- j3 .

Figure 6 demonstrates that TGF- β released by Granzyme B is active and induces SMAD-3 and Erk-2 phosphorylation in HCAS Cs.

Figure 7 demonstrates Granzyme B-dcpcndent phosphorylation of SMA D-3 by TGF- β released by Granzyme B cleavage in HCASMCs.

Figure 8 demonstrates an analysis of gross sk in pathology, morbidity and frai lty.

Figure 9 demonstrates skin morphology and xanthoma development.

Figure 10 demonstrates an analysis of skin thickness.

Figure 1 1 demonstrates an analysis of collagen and elastin remodeling in diseased skin.

Figure 12 demonstrates an analysis of Granzyme B expression near areas of decorin and collagen remodeling.

Figure 13 demonstrates loss of dermal collagen density in apoE- O mice rescued by knocking out Granzyme B.

Figure 14 demonstrates Granzyme B cleaves decorin and is present in areas of decorin degradation. Figure 15 demonstrates that inhibition of Granzyme B using a speci fic small molecule inhibitor inhibits betaglycan cleavage.

Figure 16 demonstrates that inhibition of Granzyme B using a speci fic smal l molecule inhibitor inhibits the release of proteoglycan-sequestered TGF- β .

Figure 17 demonstrates that inhibition of Granzyme B using a speci fic smal l molecule inhibitor inhibits decorin cleavage.

Figure 18 demonstrates that inhibition of Granzyme B (Granzyme B) using small molecule inhibitors inhibits ECM cleavage.

Figure 19 demonstrates that inhibition of Granzyme B (Granzyme B) using a small molecule inhibitor inhibits ECM cleavage.

Figure 20 demonstrates that inhibition of Granzyme B (Granzyme B) using NCI644777 inhibits betaglycan cleavage.

Figure 21A demonstrates Granzyme B (Granzyme B) cleavage of fibronccti n (FN) reduces EC adhesion to FN dose dependency a lso shows inhibition of Granzyme B using Willoughby 20.

Figure 21B demonstrates that inhibition of Granzyme B (Granzyme B) using Willoughby 20 inhibits fibroncctin cleavage.

Figure 22 demonstrates that GzmB cleaves plasma fibroncctin (FN) in its soluable form and matrix form.

Figure 23 demonstrates that inhibition of Granzyme B prevents decorin degradation in chronic wounds in vivo.

DETAILED DESCRIPTION

Until recently Granzyme B (Granzyme B) was thought to act within cells to mediate cell destruction. This cytotoxic enzyme effectively ki l ls vi ral ly in fected and malignant cells. However, as described herein, it has shown that Granzyme B when present external to cells wreaks havoc on the extracellular matrix ("ECM") in areas of chronic inflammation and wounds. As also described herei n, once Granzyme B is inhibited, the destructive cascade that is launched in the exterior environment is interrupted and resultant cellular damage is halted. As traumatic injuries are the fifth leading cause of death in North America, it is essentia l to find effective and alternative solutions to wound care. Currently most wound care is focused on treating symptoms, but wound repair and closure is chal lenging i f G ranzyme B is sti ll destroying the ECM proteins needed to maintain skin integrity.

Granzyme B (Granzyme B, also referred to herein at GZM B) is a pro-apoptotic serine protease found in the granules of cytotoxic lymphocytes (CTL) and natural ki ller (N ) cells. Granzyme B is released towards target cel ls, along with the pore-forming protein, perforin, resulting in its perforin-dependent interna lization into the cytoplasm and subsequent induction of apoptosis (see, for e.g. , Medema el al. , 1 97). However, during aging, inflammation and chronic disease, Granzyme B can also be expressed and secreted by other types of immune (e.g. , mast cell , macrophage, neutrophi l, dendritic) or non-immune (keratinocyte, chondrocyte) cells and has been to possess extracel lular matrix remodeling activity (Choy el a/. , 2004 and Buzza el a/. , 2005).

I. Methods of the Invention

In some embodiments, the present invention is based, at least in part, on the discovery that Granzyme B cleaves the extracellular matrix proteins, decorin, biglycan, betaglycan, syndecan, brevican, fibril lin- 1 , fibri llin-2, and fibulin-2 in vitro and cleavage of decorin, biglycan, betaglycan by Granzyme B is concentration-dependent. Cleavage of decorin, biglycan, and betaglycan by Granzyme B re leases acti ve TG F-β. The release of TGF-β is specific to cleavage of decorin, biglycan , and betaglycan by Granzyme B as TGF-β is not released in the absence of Granzyme B or when Granzyme B is inhibited by DCI.

In addition, it has been shown that Granzyme B cleaves the proteoglycan substrates, biglycan and betaglycan at a P I residue of Asp (biglycan: D 9 ' , betaglycan : D 558 ).

In some embodiments, the present invent ion is further based, at least in part, on the discovery that, in vivo, deletion of Granzyme B delays the onset of skin frai lty, hair loss, hair graying and the formation of inflammatory subcutaneous skin lesions or xanthomas in the ApoE knockout mouse. It has also been shown that Granzyme B is expressed in areas of collagen and decorin degradation and remodelling in the skin of apoE- O mice and that Granzyme B de ficiency protects aga inst sk in thinning due in part to an increase in dermal thickness, an increase in collagen density, and/or an increase in collagen organization. Furthermore, the present invention demonstrates that inhibitors of Granzyme B downmodulate decorin and biglycan c leavage in vitro and in vivo and promote wound healing by, for example, stimulating collagen organization, decreasing scarring and increasing the tensi le strength of sk in.

Accordingly, the present invention provides, among others, methods for promoting wound healing, inhibiting release of TGF bound to an extracel lular matric proteins, e.g. , extracellular proteoglycans, methods o f preventing hypertrophic scarring of a wound, and methods of preventing skin tearing.

In one aspect, the present invention provides methods for promoting wound healing in a subject having a wound. The present invention further provides use of a Granzyme B inhibitor to promote wound healing in a subject. I n another aspect, use of a Granzyme B inhibitor in the preparation of a medicament for promoting wound hea ling in a subject is disclosed.

As used herein, the term "wound healing" also known as "cicatrisation", is a process in which the skin (or another organ-tissue) repa irs itsel f a fter inj ury. I n normal skin, the epidermis (outermost layer) and dermis (inner or deeper layer) exists in a steady-state equilibrium, forming a protective barrier aga inst the external environment. Once the protective barrier is broken, the normal (physiologic) process of wound healing is immediately set in motion. The classic model of wound heal ing is divided into four sequential, yet overlapping, phases: ( 1 ) hcmoslasis, (2) in flammatory, (3) proliferative and (4) remodeling. Upon injury to the skin, a set of complex biochemical events takes place in a closely orchestrated cascade to repair the damage. Within m inutes post-injury, platelets (thrombocytes) aggregate at the injury site lo form a fibri n clot. This clot acts to control active bleeding (hemoslasis).

In the inflammatory phase, bacteria and debris are phagocytosed and removed, and factors are released that cause the migration and di vision of cells involved in the proliferative phase.

The proliferative phase is characterized by angiogenesis, collagen deposition, granulation tissue formation, epithelialization, and wound contraction. I n angiogenesis, new blood vessels are formed by vascular endothel ial cel ls. [5] I n fibroplasia and granulation tissue formation, fibroblasts grow and form a new, provisional extracellular matrix (ECM) by excreting collagen and fibronccti n. Concurrently, re-cpithelial ization of the epidermis occurs, in which epithelial cel ls prol i ferate and 'crawl' atop the wound bed, providing cover for the new tissue.

In contraction, the wound is made smal ler by the action of myofibroblasts, which establish a grip on the wound edges and conlraci themsel ves using a mechanism simi lar to that in smooth muscle cells. When the cel ls' roles arc close to complete, unneeded cells undergo apoptosis.[

In the maturation and remodeling phase, collagen is remodeled and real igned along tension lines and cells that are no longer needed are removed by apoptosis.

In one embodiment, the methods include administering a G ranzyme B inhibitor to the subject for a time and in an amount sufficient to promote wound healing, thereby promoting wound healing in the subject having a wound. In one embodiment, the methods include applying a Granzyme B inh ibitor to the wound for a time and in an amount sufficient to promote wound heal ing, thereby promot ing wound heal ing in the subject having a wound.

In one embodiment, the wound is an acute wound.

In one embodiment, the wound is a "chronic wound" or "recurring wound". As used herein, the terms "chronic wound" and "recurring wound" refer to wounds that have failed to proceed through an orderly and timely reparati ve process to produce anatomic and functional integrity of the inj ured site. Chronic wounds are those that are detained in one or more of the phases of wound heal ing. For example, in acute wounds, there is a precise balance between production and degradation of molecules such as collagen; in chronic wounds this balance is lost and degradation plays too large a role. In one embodiment, a "chronic wound" or a "recurring wound" is a wound that has not shown significant healing in about four weeks (or about 1 5, 1 6, 1 7, 1 8, 19, 20, 2 1 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 3 1 , 32, 33 , 34, or about 35 days), or which have not completely healed in about eight weeks (or about 40, 4 1 , 42, 43 , 44, 45, 46, 47, 48, 49, 50, 5 1 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 6 1 , 62, 63, 64 , or about 65 days). Chronic wounds, as used herein, also refer to wounds in which i n flammation has not resolved, wounds that have not been restored to greater than 80% of the injured tissue's original tensile strength, wounds in which decorin is reduced and/or collagen remains disorganized and/or wounds in which there is an absence of collagen thick bundle formation.

Chronic wounds can result from traumatic injury, diabetes, peripheral vascular disease, vein abnormalities, complications following surgery, lymphedema, and many ^ other conditions that compromise circulation. In one embodiment, the chronic wound is a skin wound, however those ski lled in the art wi ll appreciate that wounds may occur in other epithelial tissue. As a non-l imiting example, the term "wound" encompasses, without limitation, skin ulcers, which can include: venous ski n ulcers, arterial skin ulcers, pressure ulcers, and diabetic skin ulcers. Wounds can also i nclude, without limitation, lacerations, and burns (e.g., heat, chemica l , radioactivity, UV burns) of the epithelial tissue. In one embodiment, a chronic sk in wound is a pressure ulcer or bed sore.

Use of an "effective amount" of a Granzyme B inhibitor of the present invention (and therapeutic compositions comprising such agents) is an amount effective, at dosages and for periods of time necessary to achieve the desi red result.

For example, an effective amount of a Granzyme B inhibitor may vary according to factors such as the disease state, age, sex, reproductive state, arid weight, and the ability of the inhibotor to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum response. For example, several divided doses may be provided daily or the dose may be proportionally reduced as i ndicated by the exigencies of the situation.

An "effective amount" or "therapeutically effective amount" of a Granzyme B inhibitor, e.g., which inhibits extracellular proteoglycan cleavage, e.g., decorin cleavage, is an amount sufficient to produce the desired effect, e.g., an i nhibition of extracellular proteoglycan cleavage, e.g., decorin cleavage, in comparison to the normal level of extracellular proteoglycan cleavage, e.g., decorin cleavage, detected i n the absence of the Granzyme B inhibitor. Inhibition of extracellular proteoglycan cleavage, e.g., decorin cleavage, is achieved when the va lue obtained with a Granzyme B inhibitor relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 1 5%, 10%, 5%, or 0%. Suitable assays for measuring and determining extracellular proteoglycan cleavage, e.g., decorin cleavage, are known in the art and described herein and include, e.g., examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays described herein and known to those of ordinary ski l l in the art.

In certain embodiments of the invention, the methods and uses for promoting wound healing in a subject having a chronic wound include administering or applying a Granzyme B inhibitor for a time and in an amount sufficient such that cleavage of an extracellular matrix protein, e.g., an extracellular proteoglycan, is i nhibited. The extracellular matrix protein, e.g., an extracellular proteoglycan, may be selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodulin, fibrillin- 1 , fibrillin-2, and fibulin-2. In one embodiment, the extracel lu lar matrix protein, e.g., an extracellular proteoglycan, is decorin.

In other embodiments, the methods and uses for promoting wound healing in a subject having a chronic wound include admi nistering or applying a Granzyme B inhibitor for a time and in an amount sufficient such that release of ΤΰΡβ or other growth factor or cytokine bound to an extracellular matrix protein, e.g. , an extracel lular proteoglycan, selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibrillin- 1 , fibrillin-2, and fibul in-2 is inhibited. In one embodimenl, release of TGFp 1 bound to decorin is inhibited.

In another aspect, the present invention provides methods o f preventing skin tearing of a subject. Skin tearing may be associated with a wound, such as a chronic wound, such as a chronic skin wound, or aging. The methods include, applying a Granzyme B inhibitor to the skin of the subject for a time and in an amount su fficient to prevent skin tearing, thereby preventing skin tearing in the subject.

In certain embodiments of the invention, the methods and uses for preventing skin tearing in a subject include applying a Granzyme B inhibitor for a time and in an amount sufficient such that cleavage of an extracel lular matrix protein, e.g. , an extracellular proteoglycan, is inhibited. The extracellular matrix protein, e.g. , an extracellular proteoglycan, may be selected from the group consisting o f decorin, biglycan, betaglycan, syndecan, brevican, fibromodul in, fibri llin- 1 , fibril l in-2, and fibulin-2. In one embodiment, the extracel lular matrix protein, e.g. , an extracel lular proteoglycan, is decorin. In other embodiments, the methods and uses for preventing skin tearing in a subject include applying a Granzyme B inhibitor for a lime and in an amount sufficient such that release of TGF bound to an extracellular matrix protein, e.g. , an extracellular proteoglycan, selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodulin, fibrillin- 1 , fibrill in-2, and fibuli n-2 is inhibited. In one embodiment, release of TGF bound to decorin is inhibited.

As used herein, a "skin tear" is a traumatic wound occurring as a result of friction and/or shearing forces which separate the epidcnnis from the dermis, or separate both the epidermis and the dermis from underlying structures. In one embodiment, the skin tear is a wound of an extremity. In one embodiment, the skin tear is a recurring or chronic skin tear, e.g. , a skin tear that had previously occurred in the same area within about 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 05, or about 1 1 0 days prior.

In another aspect, the present invention provides methods of inhibiting hypertrophic scarring of a wound. The methods include, applying a Granzyme B inhibitor to the skin of the subject for a time and in an amount su fficient to prevent ski n hypertrophic scarring of a wound, thereby inhibiting hypertrophic scarring of a wound.

In certain embodiments of the invention, the methods and uses for inhibiting hypertrophic scarring of a wound include applying a Granzyme B inhibitor to the wound for a time and in an amount suffic ient such that cleavage of an extracellular matrix protein, e.g., an extracellular proteoglycan, is inhibited. The extracel lular matrix protein, e.g., an extracellular proteoglycan, may be selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodul in, fi bri llin- 1 , fibri ll in-2, and fibulin-2. In one embodiment, the extracellular matrix protein, e.g.. an extracellular proteoglycan, is decorin.

In other embodiments, the methods and uses for inhibiting hypertrophic scarring of a wound in a subject include applying a Granzyme B inhibitor for a lime and in an amount sufficient such that release of TGFP bound to an extracellular matrix protein, e.g., an extracellular proteoglycan, selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodul in, fi bri llin- 1 , fibri llin-2, and fibulin-2 is inhibited. In one embodiment, release of TGF bound to decorin is inhibited.

As used herein, the term "hypertrophic scarri ng" refers to a cutaneous condition characterized by deposits of excessive amounts of col lagen which gi ves rise to a raised scar, but not to the degree observed with keloids. Like keloids, however, they form most often at the sites of pimples, body piercings, cuts and burns. They often contain nerves and blood vessels. They generally develop after thermal or traumatic injury that involves the deep layers of the dermis. In addition, hypertrophic scars lack dccori n and have elevated levels of TGFp.

In other aspect, the present invention provides methods for increasing collagen organization in the skin of a subject in need thereof. The methods include applying a Granzyme B inhibitor to the skin of the subject in an amount and for a lime sufficient to increase collagen organization in the subject, thereby increasing collagen organization in the skin of the subject.

A subject in need of increasing collagen organization i n the skin is a subject have frail skin due to, for example, age, disease, e.g., diabetes, immobi lization, medication (e.g., long-term corticosteroid use), dehydration, and those having had a previous skin tear within about 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 05, or about 1 10 days prior.

In certain embodiment, the methods and uses for increasing collagen

organization include applying a Granzyme B inhibitor to the skin of the subject in an amount and for a time sufficient such that cleavage of an extracellu lar matrix protein, e.g., an extracellular proteoglycan, is inhibited. The extracel lular matri x protein, e.g. , an extracellular proteoglycan, may be selected from the group consisting o f decorin, biglycan, betaglycan, syndecan, brevican, fibromodul in, fibri ll in- 1 , fibri ll in-2, and fibulin-2. In one embodiment, the extracellular matrix proteoglycan is decorin

In other embodiments, the methods and uses for increasing collagen organization include applying a Granzyme B inhibitor for a time and in an amount sufficient such that release of TGFP bound to an extracellular matrix protein, e.g. , an extracellular proteoglycan, selected from the group consisting o decorin, biglycan, betaglycan, syndecan, brevican, fibromodulin, fibrillin- 1 , fibril l in-2, and fibul i n-2 is inhibited. In one embodiment, release of TGF bound to decorin is inhibited.

In other aspect, the present invention provides methods for increasing the tensi le strength of a healing or healed skin wound, e.g., a chronic skin wound, of a subject. The methods include applying a Granzyme B inhibitor to the skin of the subject in an amount and for a time sufficient to increase the tensile strength of the heal ing or healed skin wound of the subject. In certain embodiment, the methods and uses for increasing the tensile strength of a healing or healed skin wound of a subject include applying a Granzyme B inhibitor to the skin of the subject in an amount and for a time sufficient such that cleavage of an extracellular matrix protein, e.g., an extracellular proteoglycan, is inhibited. The extracellular matrix protein, e.g., an extracellular proteoglycan, may be selected from the group consisting of decorin, biglycan, bctaglycan, syndecan, brevican, fibril lin- 1 , fibrillin-2, and fibulin-2. In one embodiment, the extracel lular matrix protein, e.g. , an extracellular proteoglycan, is decorin.

In other embodiments, the methods and uses for increasing the tensi le strength of a healing or healed skin wound, e.g. , a chronic ski n wound i nclude applying a Granzyme B inhibitor for a time and in an amount su fficient suc h that release of TGFP bound to an extracellular matrix protein, e.g. , an extracellular proteoglycan, selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodulin, fibrillin- 1 , fibrillin-2, and fibulin-2 is inhibited. In one embodi ment, release of TGFP bound to decorin is inhibited.

A "healing wound" is a wound in which clotti ng has occurred, a wound in which temporary replacement of cells and extracel lular matrix has occurred, a wound in which resolution of inflammation has occurred, and/or a wound in which synthesis and organization of cells and extracellular matrix in a manner that restores tissue functionality and structure has occurred.

In another aspect, the present invention provides methods for inhibiting release of a cytokine, e.g., transforming growth factor- β (TG F- β ), bound to an extracel lular matrix protein, e.g., an extracellular proteoglycan, e.g. , release of active TG F- j3 . The methods include, contacting the extracel lular matrix protein, e.g. , an ex tracellular proteoglycan, with a Granzyme B inhibitor, thereby inhibiti ng release of the cytokine, e.g., TGFp, bound to an extracel lular matrix protein, e.g.. an extracellular proteoglycan,. The methods may also involve inhibiting a cleavage site in the extracellular matrix protein, e.g., an extracellular proteoglycan. Optional ly, the cleavage occurs in any one of the following peptide sequences: Asp 9 l Thr-Thr-Leu-Leu-Asp (SEQ ID NO: 1 ); or Asp 558 Ala-Ser-Leu-Phe-Thr (SEQ I D NO:2); or Asp 3 l Glu-A la-Scr-Gly (SEQ I D NO: 3); or Asp 69 Leu-Gly-Asp-Lys (SEQ I D NO:4); or Asp 82 Thr-Thr-Leu- Leu-Asp (SEQ I D NO:5); or Asp 261 Asn-Gly-Ser-Leu-Ala (SEQ ID NO:6). The methods and uses of inhibiting release of a cytokine, e.g., TG Fp, bound to an extracellular matrix protein, e.g., an extracellular proteoglycan, may be performed in vitro or in vivo. The extracellular matrix protein, e.g.. an extracellular proteoglycan, may be selected from the group consisting of decorin, biglycan, belaglycan, syndecan, brevican, fibromodulin, fibril 1 in- 1 , fibri llin-2, and fibul in-2. I n one embodiment, the extracellular matrix protein, e.g. , an extracellular proteoglycan, is decorin.

In another aspect, the present invention provides methods for inhibiting extracellular matrix protein degradation. The methods include contacting the extracellular matrix protein, e.g., an extracel lular proteoglycan, with a Granzyme B inhibitor, wherein the release of a sequestered cytokine, e.g. , TG F , is inhibited, thereby inhibiting extracellular matrix protein degradation.

The methods and uses of inhibiting degradation of an extracel lular matrix protein, e.g., an extracellular proteoglycan, may be performed in vitro or in vivo. The extracellular matrix protein, e.g., an extracellula r proteoglycan, may be selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodulin, fibrillin- 1 , fibrillin-2, and fibulin-2. In one embod iment, the extracel lular matrix protein, e.g., an extracellular proteoglycan, is decorin.

In yet another aspect, the present invention provides methods of inhibiting extracellular decorin cleavage. The methods include, contacting the extracellular decorin with a Granzyme B inhibitor, thereby inhibiting extracel lular decorin cleavage.

The methods and uses of inhibiting decorin cleavage may be performed in vitro or in vivo. In certain embodiments, the methods include contacting a cel l , such as a ski n cell, with a Granzyme B inhibitor such that the expression and/or activity of decorin are increased in the epidermal-dermal junction of the skin.

The Granzyme B inhibitor for use in the methods, uses and compositions described herein may be a nucleic acid, a peptide, an antibody, such as a humanized antibody, or a small molecule. Granzymc 13 inhibilors for use in any of the methods, uses, and compositions of the invention are described in detail below.

The term "subject" or "patient" is intended to include mammalian organisms. Examples of subjects or patients include humans and non-human mammals, e.g. , non- human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In speci fic embodiments of the invention, the subject is a human.

The term "administering" includes any method of delivery of a Granzyme B inhibitor or a pharmaceutical composition comprising a Granzyme B inhibitor into a subject's system or to a particular region in or on a subject. I n certa in embodiments, a moiety is administered topically, intravenously, intramuscularly, subcutaneously, intradermally, intranasally, orally, transcutaneously, intrathecal, intravitrcal ly, intracerebral, or mucosally.

In one embodiment, the administration of the Granzyme B inhibitor is a local administration, e.g., administration to the site of a wound, e.g., a chronic skin wound. In one embodiment the administration of the Granzyme B inhibitor is topical

administration to the site of a wound, e.g. , a chronic skin wound.

As used herein, the term "applying" refers to administration of a Granzyme B inhibitor that includes spreading, covering (at least in part), or laying on of the inhibitor. For example, a Granzyme B inhibitor may be appl ied to the skin of a subject or applied to a wound by spreading or covering the skin with an inhibitor. In addition, a Granzyme B inhibitor may be applied to the skin or wound using, for example, a wound covering comprising the inhibitor.

As used herein, the term "contacting" (i .e., contacting a protein, a cell, e.g., a host cell, or a subject with a Granzyme B inh ibitor) includes incubating the Granzyme B ' inhibitor and the, e.g., cell, together in vitro (e.g., adding the moiety to cells in culture) as well as administering the moiety to a subject such that the moiety and cel ls or tissues of the subject are contacted in vivo.

As used herein, the terms "treating" or "treatment" re fer to a benefic ial or desi red result including, but not limited to, alleviation or amel ioration of one or more symptoms, diminishing the extent of a disorder, stabilized (i.e., not worsening) state of a disorder, amelioration or palliation of the disorder, whether delectable or undetectable.

"Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment. II. Granzyme B Inhibitors

A Granzyme B inhibitor for use in any of (he compositions, methods and uses of the present invention may be a nucleic acid molecule, a peptide, an antibody, such as a humanized antibody or a camelid antibody, or a small molecule.

Many Granzyme B inhibitors are known to a person of skill in the art and are, for example, described in international patent application published under WO 03/065987 and United States patent application published under US 2003/0148511 ; Willoughby et al., 2002; Hill et al., 1995; Sun J. et al„ 1996; Sun J. et al., 1997; Bird et al., 1998; am et al., 2000; and Mahrus and Craik, 2005.

A Granzyme B inhibitor for use in any of the compositions, methods and uses of the present invention may be a nucleic acid molecule, a peptide, an antibody, such as a humanized antibody or a camelid antibody, or a small molecule.

In one embodiment, a Granzyme B inhibitor is selected from the group consisting of

2S,5S)-N-((2H-tetrazol-5-yl)mcthyl)-5-((2S,3S)-2-acctamid o-3-mcthylpcnlanamido)-4- oxo- 1 ,2,4,5,6,7-hexahydroazcpino[3,2, 1 -hi]indolc-2-carboxamidc;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-accta mido-3- methylpentanamido)-4-oxo- 1,2,4, 5,6, 7-hexahydroazepino[3, 2,1 -hi] indolc-2- carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((R)-3-mcthyl -2-(pyridin-2- yl)butanamido)-4-oxo- 1 ,2,4,5,6,7-hexahydroazcpino[3,2,l -hi]indolc-2-carboxamidc;

(2S,5S)-N-(( 1 H- 1 ) 2,3-triazol-4-yl)methyl)-5-((2S,3S)-3-mclhy l-2-(2- phenylacetamido)pentanamido)-4-oxo-l ,2,4,5,6,7-hcxahydroazcpino[3,2, l-hi]indole-2- carboxamide;

(2S,5S)-N-((lH-l,2,4-triazol-3-yl)mcthyl)-5-((2S,3S)-2-ac ctamido-3- methylpentanamido)-4-oxo-l,2,4,5,6,7-hcxahydroazcpino[3,2, 1 -hi]indole-2- carboxamide;

(2S,5S)-N-((lH-pyrazol-3-yl)methyl)-5-((2S,3S)-2-acetamido-3 -methylpenlanamido)-4- oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indolc-2-carboxamidc; (2S,5S)-N-(( l H^yrazol-4-yl)methyl)-5-((2S,3 S)-2-acciamido-3-mcthylpcnlanamido)-4 oxo- l ,2,4,5,6,7-hexahydroazepino[3,2, l -hi]indolc-2-carboxamide;

(2S,5S)-N-(( l H-imidazol-4-yl)methyl)-5-((2S,3S)-2-acctamido-3-methylpenta namido)- 4-oxo- l ,2,4,5,6,7-hexahydroazepino[3 ,2, l -hi]indole-2-carboxamidc;

2S,5S)-5-((2S,3S)-2-acetamido-3-methy]penlanamido)-4-oxo-N-( lhiazol-5-ylmethyl)- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indole-2-carboxamidc;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpcntanamido)-N-(isoxa zol-3-ylmethyl)-4-oxo 1 ,2 ( 4,5,6,7-hexahydroazepino[3,2, 1 -hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methy]pcntanamido)-4-oxo-N- (thiazol-2-ylmethyl)- l ,2,4,5,6,7-hexahydroazepino[3 ,2, 1 -hi]indole-2-carboxamide,

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpenlanamido)-N-(isoxa zol-5-ylmethyl)-4-oxo l ,2,4,5,6,7-hexahydroazepino[3,2, l -hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpenlanamido)-4-oxo-N- (thiazol-4-ylmethyl)- l ,2,4,5,6,7-hexahydroazepino[3,2, l -hi]indole-2-carboxamidc;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N- (pyrimidin-5- ylmethyl)- l ,2,4,5,6 ) 7-hexahydroazepino[3 ,2, l -hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpcntanamido)-4- xo-N-(pyridazin-4- ylmethyl)- l ,2,4,5,6,7-hexahydroazepino[3 ,2, 1 -hi]indolc-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-mcthylpentanamido)-4-oxo-N- (pyridin-2-ylmethyl)- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indo!e-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-mcthylpcntanamido)-4-oxo-N- (pyrid in-3-ylmcthyl)- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N- (pyridin-4-ylmethyl)- l ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indole-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)- -(imidazo[ 1 ,2-a]pyrimidin-2- ylmethyl)-4-oxo- l ,2,4,5,6,7-hexahydroazepino[3 ,2, 1 -hi] indolc-2-carboxamide;

(2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-N-((3a,7 a- dihydrobenzo[d]thiazol-2-yl)methyl)-4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 - hi]indole-2-carboxamide; (2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((R)-3-methyl-2-(pyri din-2-yl)butanamido)-4- oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indolc-2-carboxami de;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((S)-3-mcthyl-2-(pyri din-2-yl)butanamido)-4- oxo- 1,2,4, 5,6, 7-hexahydroazepino[3, 2,1 -hi] indolc-2-carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-3-melhyl- 2-(2- phenylacetamido)pentanamido)-4-oxo- 1,2,4, 5,6, 7-hcxahydroazcpino[3,2,l-hi]indole-2- carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(2,3-di fluorophenyl)acetamido)- 3-methylpentanamido)-4-oxo-l ,2,4,5,6, 7-hexahydroazcpino[3, 2,1 -hi] indole-2- carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(dimcth ylamino)acetamido)-3- methylpentanamido)-4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indole-2- carboxamide;

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(benzo[ b]lhiophen-3- yl)acetamido)-3-methylpentanamido)-4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 - hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)mcthyl)-5-((2S,3S)-2-(2-(d imethylamino)acetamido)- 3-methylpentanamido)-4-oxo- 1,2,4, 5,6,7-hexahydroazepino[3, 2,1 -hi]indolc-2- carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)mcthyl)-5-((2S,3S)-2-(2 -(bcnzo[b]lhiophen-3- yl)acetamido)-3-methylpentanamido)-4-oxo- 1 ,2,4,5,6,7-hexahydroazcpino[3,2, 1 - hi]indole-2-carboxamide;

(R)-N-((2S,5S)-2-((lH-l,2,3-triazol-4-yl)mclhylcarbamoyl)-4- oxo-l,2,4,5,6,7- hexahydroazepino[3,2,l-hi]indol-5-yl)-3-acetyl-5,5-dimethyll hiazolidine-4- carboxamide;

(2S,5S)-N-(( 1 H- 1 ,2,3-triazol-4-yl)mcthyl)-5-((2S,3S)-3-mcthy l-2-(2-oxopyrrolidin- 1 - yl)pentanamido)-4-oxo-l,2,4,5,6,7-hexahydroazcpino[3,2, 1 -hi]indolc-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-(2-cyclopenty lacetamido)-4-oxo- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide; (2S,5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((S)-2-acctamido -2-cyclopropylacctamido)- 4-oxo-l,2,4,5 ) 6 ) 7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide;

(2S,5S)-N-((lH-l,2,3-triazol-4-y])mcthyl)-5-((S)-2-acciamido -2-cyc!openrylacetamido)-

4- oxo- 1 ,2,4,5,6 1 7-hexahydroazepino[3,2, 1 -hi]indole-2-carboxamidc;

Bio-x-IEPD p -(OPh) 2 ;

azepino[3,2,l-hi]indole-2-carboxamide;

(4S)-4-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-f[(2 S)-4-hydroxy-l,4- dioxobutan-2-yl]carbamoyl]pyrrolidin-l -yl]-5-oxopentanoic acid;

(4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-5-[[(2 S,3S)-3-hydroxy-l- [[(2S)-4-hydroxy-l,4-dioxobutan-2-yl]amino]-l-Dxobulan-2-yl] amino]-5-oxopentanoic acid;

5- chloro-4-oxo-3-[2-[2-(phenylmethoxycarbonylamino) propanoylamino]

propanoylamino] pentaiioic acid;

5-chloro-4-oxo-2-[2-[2-(phenylmcthoxycarbonylamino) propanoylamino]

propanoylamino] pentanoic acid;

ZINC05723764;

ZINC05723787;

ZI C05316154;

ZINC05723499;

ZINC05723646;

ZI C05398428;

ZI C05723503;

ZFNC05723446;

ZINC05317216;

ZINC05315460;

ZINC05316859;

ZINC05605947;

an isocoumarin; a peptide chloromethyl ketone;

a peptide phosphonate;

a Granzyme B inhibitory nucleic acid molecule;

an anti-Granzyme B antibody;

an inhibitory Granzyme B peptide;

a SerpB9 polypeptide, or fragment thereof;

5-chloro-4-oxo-2-[2-[2-(pheny!methoxycarbonylami no)

propanoylamino]propanoylamino] pentanoic acid;

Ac-IEPD-CHO;

(4S)-4-[[(2S)-2-acetamido-4-methylpcntanoyl]amino]-5-[2-[ [(2S)-4-hydroxy- l ,4- dioxobutan-2-yl]carbamoyl]pyrrol idin- l -yl]-5-oxopenlanoic acid;

Ac-IETD-CHO;

(4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-5-[[(2 S,3S)-3-hydroxy- l - [[(2S)-4-hydroxy- l ,4-dioxobutan-2-yl]amino]- l -oxobulan-2-yl]amino] -5-oxopentanoic acid;

(2S,5S)-4-oxo-5- { [N-(phenylacetyl)-L-isolcucyl]amino} - - ( 1 H- l ,2,3-triazol-4- ylmethyl)- 1 ,2,4,5,6,7-hexahydroazepino[3 ,2, 1 -hi] indole-2-carboxamidc; 5-chloro-4- oxo-3-[2-[2-(phenylmethoxycarbonylamino) propanoylamino] propanoylamino] pentanoic acid;

5-chloro-4-oxo-2-[2-[2-(phenylmcthoxycarbonylamino) propanoylamino] propanoylamino] pentanoic acid;

(4S)-4-[[(2S)-2-acetamido-4-methylpcntanoyl]amino]-5-[2-[[(2 S)-4-hydroxy- l ,4- dioxobutan-2-yl]carbamoyl]pyrrolidin- 1 -yl]-5-oxopenlanoic acid;

(4S)-4-[[(2S ) 3S)-2-acetamido-3-mcthylpcntanoyl]amino]-5-[[(2S,3S)-3 -hydroxy- l - [[(2S)-4-hydroxy- 1 ,4-dioxobutan-2-yl]amino]- 1 -oxobutan-2-yl]amino]-5-oxopcntanoic acid,

a Serp2 polypeptide, or fragment thereof;

a CrmA polypeptide or fragment thereof; and a SerpinA3 polypeptide or fragment thereof.

In another embodiment, a Granzyme B inhibitor suitable for use in the methods, compositions, and uses of the invention includes, for example, Z-AAD-C (IUPAC name: 5-chloro-4-oxo-2-[2-[2-(phenylmcthoxycarbonylamino)

propanoylamino]propanoylamino] pcntanoic acid) MP: CI9H24CIN307 CID:

16760474; Ac-IEPD-CHO; Granzyme B Inhibitor IV or Caspasc-8 inhibitor III (IUPAC: (4S)-4-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-[[(2 S)-4-hydroxy-l,4- dioxobutan-2-yl]carbamoyl]pyrrolidin-l-yl]-5-oxopenlanoic acid) MF: C22H34N409 CID: 16760476; and Ac-IETD-CHO; Caspase-8 Inhibitor I or Granzyme B Inhibitor II (IUPAC: (4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-5-[[(2 S,3S)-3- hydroxy-l-[[(2S)-4-hydroxy-l,4-dioxobutan-2-yl]amino]- 1 -oxobutan-2-yl]amino]-5- oxopentanoic acid) MF: C21H34N4O10 CID: and 16760475.

In yet another embodiment, a Granzyme B inhibitor for use in the methods, compositions, and uses of the invention may include any one or more of the following: Granzyme B inhibitor is selected from one or more of the following: Azepino[3,2,l - hi]indole-2-carboxamide, 5-[[(2S,3S)-2-[(2-benzo[b]lhicn-3-ylacetyl)amino]-3-mclhyl- I -oxopentyljamino]- 1 ,2,4,5,6,7-hcxahydro-4-oxo-N-( I I I- 1 ,2,3-lriazol-5-ylmcthyl)- ,(2S,5S)- (compound 20 from Willoughby el al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2197-2200) referred to herein as Willoughby 20 ' ; Bio-x-IEPD p - (OPh) 2 ; (2S,5S)-5-[fN-acetyl-L-isoleucyl)amino]-4-oxo-N-( I H-iciraazol-5-ylmethyl)- l,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indolc-2-carboxamide; (2S,5S)-5-[(N-acetyl-L- isoleucyI)amino]-4-oxo-N-(I H- 1 ,2, 3-triazol-4-ylmethyl)- 1 ,2,4,5,6,7- hexahydroazepino[3,2,l-hi] indolc-2-carboxamide; (2S,5S)-5- {[(2 )-3-methyl-2- pyridin-2-ylbutanoyl]amino }-4-oxo-N-( 1 II- 1 ,2,3 -triazol-4-y Imethyl)- 1 ,2,4,5,6,7- hexahydr oazepino[3,2,l -hi]indole-2-carboxamide; (2S,5S)-4-oxo-5-{[N-(phenylacetyl)- L-isoleucyl]amino}-N- (lH-1 ,2,3-lriazol-4-yl methyl)- 1 ,2,4,5,6,7-hcxahydroazcpino[3 ,2,l-hi]indole-2-carboxamide; 5-chloro-4-oxo-3-[2-[2-(phcnylmcthoxycarbonylamino) propanoylamino] propanoylamino] pcntanoic acid; 5-chloro-4-oxo-2-[2-[2- (phenylmethoxycarbonylamino) propanoylamino] propanoylamino] pcntanoic acid; (4S)-4-[[(2S)-2-acetamido-4-methylpcntanoyl]amino]-5-[2-[[(2 S)-4-hydroxy- 1 ,4- dioxobutan-2-yl]carbamoyl]pyrrolidin-l-yl]-5-oxopentanoic acid; (4S)-4-[[(2S,3S)-2- acetamido-3-methylpentanoyl]amino]-5-[[(2S,3S)-3-hydroxy- 1 -[[(2S)-4-hydroxy-l ,4- dioxobutan-2-yl]amino]-l -oxobutan-2-yl]amino]-5-oxopenlanoic acid, a protease inhibitor-9 or derivatives thereof, CnnA, serp-2, ZFNC05723764, Z1NC05723787, ZINC053 16154, ZINC05723499, ZINC05723646, Z1 NC05398428, Z1NC05723503, ZI C05723446, ZI C053 1 72 16, ZINC053 1 5460, ZI NC053 1 6859, and ZINC05605947.

Alternatively, the Granzyme B inhibitor may be selected from one or more of the following: Willoughby 20, NCI 644752, NCI 644777, ZINC053 1 72 1 6, and NCI 630295. Granzyme B inhibitors may include, but are not limited to, nucleic acids (for example, antisense oligonucleotides, siRNA, RNAi, etc.), peptides and smal l molecules.

Optionally, the Granzyme B inhibitor used herein may be selected from one of the examples detailed herein, which includes but is not li mited to one or more of the following: Azepino[3,2, l -hi] indole-2-carboxamidc, 5 -| [(2S,3S)-2-[(2-bcnzo[b]thicn-3- ylacetyl)amino]-3-methyl- 1 -oxopentyl]amino]- 1 ,2,4,5,6,7-hexahydro-4-oxo-N-( 1 H- l ) 2,3-triazol-5-ylmethyl)-,(2S,5S)- (compound 20 from Wil loughby el al. (2002) Bioorganic & Medicinal Chemistry Letters 1 2:2 197-2200) re ferred to herein as

Willoughby 20; Bio-x-IEPD p -(OPh) 2 ; (2S,5S)-5-[(N-acetyl- L-isolcucyl)amino]-4-oxo- N-( l H-tetraazol-5-ylmethyl)- 1 ,2,4, 5,6, 7-hexahydroazepino[3, 2, 1 -hi] indole-2- carboxamide; (2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-4-oxo-N-( 1 H- 1 ,2, 3 -triazol-4- ylmethyl)- l ,2,4,5,6,7-hexahydroazepino[3 ,2, l -h i] indole-2-carboxamidc; (2S,5S)-5- {[(2R)-3-methyl-2-pyridin-2-ylbutanoyl]amino } -4-oxo-N-( I H- 1 ,2,3-triazol-4- ylmethyl)- 1 ,2,4,5,6,7-hexahydr oazepino[3,2, 1 -hi]indolc-2-carboxamidc; (2S,5S)-4-oxo- 5- {[N-(phenylacetyl)-L-isoleucyl]amino} -N- ( I H- 1 ,2,3-triazol-4-ylmethyl)- l ,2,4,5,6,7- hexahydroazepino[3 ,2, l -hi] indole-2-carboxamide; 5-chloro-4-oxo-3-[2-[2- (phenylmethoxycarbonylamino) propanoylamino] propanoylamino] pentanoic acid; 5- chloro-4-oxo-2-[2-[2-(phenylmcthoxycarbonylamino) propanoylami no]

propanoylamino] pentanoic acid; (4S)-4-[[(2S)-2-acciamido-4 -methylpcntanoyl]amino]- 5-[2-[[(2S)-4-hydroxy- l ,4-dioxobutan-2-yl]carbamoyl]pyrrol idin- 1 -yl]-5-oxopentanoic acid; (4S)-4-[[(2S,3S)-2-acetamido-3-mcihy lpcntanoyl]amino]-5-[[(2S,3S)-3-hydroxy- 1 - [[(2S)-4-hydroxy- 1 ,4-dioxobutan-2-yl]amino]- 1 -oxobutan-2-yl]amino]-5-oxopentanoic acid, protease inhibitor-9 or derivatives thereof, CrmA , scrp-2, ZINC05723764, ZINC05723787, ZrNC053161 54, ZINC05723499, ZI C05723646, ZINC05398428, ZI C05723503, ZI C05723446, ZINC053 1721 6, Zl NC053 1 5460, ZINC05316859, and ZrNC05605947. Alternatively, the Granzyme B inhibitor may be selected from one or more of the following: Willoughby 20, NCI 644752, NCI 644777, ZINC053 1721 6, and ' NCI 630295. In one embodiment, a Granzyme B inhibitor for use in any of the compositions, uses and methods of the invention is a nucleic acid molecule.

As used herein, the term "nucleic acid" refers to a dcoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and any chemical modifications thereof. Such modi fications include, but are not l imited to backbone modifications, methylations, and unusual base-pairing combinations. As detai led herein, the term "nucleic acid" includes, without limitation, NAi technologies. For example, RNA compounds used to inhibit Granzyme B may be smal l interfering NA (si RNA) compounds.

In one embodiment, a Granzyme B inhibitor for use in the compositions, uses and methods of the invention is an interfering nucleic acid molecule.

The term "interfering nuc leic acid molecule" or "interfering nucleic acid" as used herein includes single-stranded RNA (e.g., mature mi RNA , ssRNA i ol igonucleot ides, ssDNAi oligonucleotides), double-stranded RNA (i.e. , duplex RNA such as si RNA , Dicer-substrate dsRNA, shRNA, ai R A, or prc-mi R A), sel f-del ivering RNA (sdRNA; see, e.g. U.S. Patent Publication Nos. 20091 3 1 2034 1 , 2009 1 3 1 203 1 5, and

201 1 13069780, the entire contents of all of which arc incorporated herein by reference), a DNA-PvNA hybrid (see, e.g., PCT Publication No. WO 2004/07894 1 ), or a DNA-DNA hybrid (see, e.g., PCT Publ ication No. WO 2004/ 10 1 9) that is capable of reducing or inhibiting the expression (and, thus, the activity) of a target gene or sequence (e.g., by mediating the degradation or inhibiting the translation of mR As which arc

complementary to the interfering RNA sequence) when the interfering nucleic acid is in the same cell as the target gene or sequence. Interfering nucleic acid thus refers to a single-stranded nucleic acid molecules that are complementary to a target mRNA sequence or to the double-stranded RNA formed by two complementary strands or by a single, self-complementary strand. Interfering nucleic ac ids may have substantial or complete identity to the target gene or sequence, or may comprise a region of mismatch (i.e., a mismatch motif). The sequence of the interfering nuc leic acids can correspond to the full-length target gene, or a subsequence thereof (e.g.. the gene for Granzyme B, the nucleotide and amino acid sequence of which is known and may be found in for example GenBank Accession No, GI : 22 1625527, the entire contents of which are incorporated herein by reference, and SEQ ID NO:8). Preferably, the interfering nucleic acid molecules are chemically synthesized. The disclosures of each of the above patent documents are herein incorporated by reference in their entirety for all purposes.

As used herein, the term "mismatch moti f or "m ismatch region" refers to a portion of an interfering nucleic acid (e.g., si RN A) sequence lhat does not have 1 00% complementarity to its target sequence. An interfering nucleic acid may have at least one, two, three, four, five, six, or more mismatch regions. The mismatch regions may be contiguous or may be separated by 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 1 2, or more nucleotides. The mismatch motifs or regions may comprise a single nucleotide or may comprise two, three, four, five, or more nucleotides.

An interfering nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule, complementary to an mRNA sequence or complementary to the coding slra nd of a gene. Accordingly, an interfering nucleic acid is an antisense nucleic acid and can hydrogen bond to the sense nucleic acid.

In one embodiment, an interfering nucleic acid of the invention is a "smal l- interfering RNA" or "an si R A" molecule. In another embodiment, an interfering nucleic acid molecules of the invention is a "self-deliveri ng RNA" or "sdRNA" molecule. In one embodiment, an interfering nucleic ac id of the invention mediates RNAi. RNA interference (RNA i) is a post-transcriptional, targeted gene-silencing technique that uses double-stranded RNA (dsRNA) to degrade messenger RNA

(mRNA) containing the same sequence as the dsRNA (Sharp, P. A. and Zamore, P. D. 287, 243 1 -2432 (2000); Zamore, P. D., el a/. Cell 10 1 , 25-33 (2000). Tuschl , T. el a/. Genes Dev. 13, 3 191 -3197 ( 1999); Cottrcll T R, and Doering T L. 2003. Trends Microbiol. 1 1 :37-43; Bushman F. 2003. Mol. Therapy. 7 :9- 10; McManus M T and Sharp P A. 2002. Nat Rev Genet. 3 : 737-47). The process occurs when an endogenous ribonuclease cleaves the longer dsRNA into shorter, e.g. , 2 1 - or 22-nucleotide-long RNAs, termed small interfering RNAs or siRNAs. The smaller RNA segments then mediate the degradation of the target mRNA. Kits for synthesis of RNA i are commercially available from, e.g. New England Biolabs or Ambion. In one embodiment one or more of the chemistries described herein for use in antisense RNA can be employed in molecules that mediate RNA i. Interfering nucleic acid includes, e.g. , siRNA and sdRNA, of about 10-60, 10-50, or 10-40 (duplex) nucleotides in length, more typically about 8- 1 5, 10-30, 10-25, or 1 0- 25 (duplex) nucleotides in length, about 10-24, (duplex) nucleotides in length (e.g., each complementary sequence of the double-stranded si RN A is 1 0-60, 10-50, 10-40, 10-30, 10-25, or 10-25 nucleotides in length, about 10-24, 1 1 -22, or I 1 -23 nucleotides in length, and the double-stranded siRNA is about 10-60, 10-50, 10-40, 10-30, 10-25, or 10-25 base pairs in length). siRNA and sdRNA duplexes may comprise 3 '-overhangs of about 1 , 2, 3, 4, 5, or about 6 nucleotides and 5 ' -phosphate termini . Examples of siRNA and sdRNA include, without limitation, a double-stranded polynucleotide molecule assembled from two separate stranded molecules, wherein one strand is the sense strand and the other is the complementary antisense strand; a double-stranded polynucleotide molecule assembled from a single stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; a double- stranded polynucleotide molecule with a hairpin secondary structure having self- complementary sense and antisense regions; and a circular single-stranded

polynucleotide molecule with two or more loop structures and a stem having self- complementary sense and antisense regions, where the circular polynucleotide can be processed in vivo or in vitro to generate an active double-stranded si RNA (or sdRNA) molecule. As used herein, the terms "siRNA" and "sd RNA ' include RNA-RNA duplexes as well as DNA-RNA hybrids (sec, e.g. , PCT Publ ication No. WO

2004/078941 ).

Preferably, siRNA and sdRNA are chemica lly synthesized. siRNA and sdRNA can also be generated by cleavage of longer dsRNA (e.g. , dsRNA about 5 , about 10, about 1 5, about 20, about 25, or greater nucleotides in length) wi th the E. co/i RNase 111 or Dicer. These enzymes process the dsRNA into biologica l ly active si RNA (see, e.g., Yang el al., Proc. Natl. Acad Sci: USA , 99:9942-9947 (2002); Calegari et al. , Proc. Natl. Acad. Sci. USA, 99: 14236 (2002); Byrom et al.. A bion TechNotes, 10( l ):4-6 (2003); Kawasaki et al., Nucleic Acids Res. , 3 1 :9 1 -987 (2003); Knight et al. , Science, 293 :2269-2271 (2001 ); and Robertson el al.. J. Biol. Chem., 243 : 82 ( 1968)). Preferably, dsRNA are at least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotides in length. A dsRNA may be as long as 1000, 1 500, 2000, 5000 nucleotides in length, or longer. The dsRNA can encode for an entire gene transcript or a partial gene transcript. In certain instances, siRNA or sdR A may be encoded by a plasmid (e.g., transcribed as sequences that automatically fold into duplexes with hai rpin loops).

Given the coding strand sequences encoding Granzymc B known in the art and disclosed herein (SEQ ID NO:8), an interfering nucleic acid of the invention can be designed according to the rules of Watson and Crick base pa iring. The i nterfering nucleic acid molecule can be complementary to the enti re coding region of Granzymc B mR A, but more preferably is an ol igonucleotide which is anlisense to only a portion of the coding or noncoding region of Granzyme B mRNA. For example, an interfering oligonucleotide can be complementary to the region surrounding the processing site of ubiquitin and Granzyme B mRNA. An interfering NA ol igonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An interfering nucleic acid of the invention can be constructed using chemica l synthesis and enzymatic ligation reactions using procedures known in the art. For example, an interfering nucleic acid (e.g. , an antisense ol igonucleotide) can be chemical ly synthesized using naturally occurring nucleotides or variously modi fied nuc leotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the anlisense and sense nucleic acids, e.g. , phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the interfering nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil , 5-iodouraci l, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmcthyl) uraci l , 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymeihylaminomethyluracil , dihydrouracil, beta-D-galactosylqueosinc, inosine, N6-isopenlcnyladcnine, 1 - methylguanine, 1 -methylinosine, 2,2-dimethylgiianinc, 2-methyladeninc, 2- methylguanine, 3-methylcytosine, 5-methylcytosinc, N6-adcnine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomelhyl-2-lhiouraci l, beta-D- mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic ac id (v), wybuloxosi nc, pseudouracil, queosinc, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouraci l, 4-ihiouraci l, 5-mcthyluraci l, uracil-5- oxyacetic acid methylester, uracil-5-oxyacctic ac id (v), 5-mcthyl-2-thiouraci l , 3-(3- amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. To inhibit expression in cells, one or more interfering nucleic acid molecules can be used.

Alternatively, an interfering nucleic acid can be produced biologica lly using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid wi l l be of an antisense orientation to a target nucleic acid of interest).

The interfering nucleic acids may i ncl ude any UNA compounds which have sequence homology to the Granzyme B gene and which arc capable of modulating the expression of Granzyme B protein. Examples interfering nucleic acids which are capable of modulating expression of Granzyme B are found in : US 6, 1 59,694; US 6,727,064; US 7,098, 192 ; and US 7,307,069, the entire contents of all of which are incorporated herein by reference.

Antisense oligonucleotides directed aga inst Granzyme B have been designed and manufactured by Biognostik (Euromedcx, Mundolshei, France) and arc described in Hernandez-Pigeon, el al. , J. Biol Che . vol. 28 1 , 1 3525- 1 3532 (2006) and Bruno, el al. , Blood, vol. 96, 1914- 1920 (2000).

In another embodiment, a Granzyme B inhibitor for use in the compositions, methods and uses of the invention is a peptide.

As used herein, "peptide" refers to short polymers of amino acids l inked by peptide bonds. Those persons ski lled in the art wi l l understa nd that a peptide bond, which is also know in the art as an amide bond, is a covalenl chemical bond formed between two molecules when the carboxyl group o f one molecule reacts with the amine group of the other molecule, thereby releasing a molecu le of water (H2O). Peptides may be modified in a variety of conventional ways well known to the ski l led artisan.

Examples of modifications include the following. The terminal amino group and/or carboxyl group of the peptide and/or amino acid side cha ins may be modified by alkylation, amidation, or acylation to provide esters, amides or substi tuted amino groups. Heteroatoms may be included in a liphatic modi fying groups. This is done using conventional chemical synthetic methods. Other modi fications include dcamination of glutamyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively; hydroxylation of prol ine and lysine; phosphorylation of hydroxyl groups of serine or threonine; and methylation of amino groups of lysine, arginine, and histidine side chains (see, for e.g. : T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co. San Francisco, Cal i f , 1983). In another aspect, one or both, usual ly one termi nus of the peptide, may be substituted with a lipophilic group, usually aliphatic or aralkyl group, which may include heteroatoms. Chains may be saturated or unsaturated. Conveniently, commercially available aliphatic fatty acids, alcohols and amines may be used, such as capryl ic acid, capric acid, lauric acid, myristic acid and myristyl alcohol, pal mitic acid, palmitoleic acid, stearic acid and stearyl amine, oleic acid, Iinoleic acid, docosahexaenoic acid, etc. (see, for e.g. : U.S. Pat. No. 6,225,444). Preferred are unbranched, natural ly occurring fatty acids between 14-22 carbon atoms in length. Other lipophi l ic molecules include glyceryl l ipids and sterols, such as cholesterol . The l ipophil ic groups may be reacted with the appropriate functional group on the ol igopeptide in accordance with conventional methods, frequently during the synthesis on a support, depending on the site of attachment of the oligopeptide to the support. Lipid attachment is useful where oligopeptides may be introduced into the lumen of the l iposome, along with other therapeutic agents for administering the peptides and agents into a host.

Depending upon their intended use, particularly for admin istration to mammalian hosts, the subject peptides may also be modified by attachment to other compounds for the purposes of incorporation into carrier molecules, changing peptide bioavai labi lity, extending or shortening half-li fe, controlling dislribulion to various tissues or the blood stream, diminishing or enhancing binding to blood components, and the like. The prior examples serve as examples and are non-limiting.

Peptides may be prepared in a number of ways. Chemical synthesis of peptides is well known in the art. Sol id phase synthesis is commonly used and various commercial synthetic apparatuses are available, for exa mple automaied synthesizers by Applied Biosystems Inc., Foster City, Cali f ; Beckman; etc. Sol ution phase synthetic methods may also be used, particularly for large-scale productions.

Peptides may also be present in the form of a sail, general ly in a salt form which is pharmaceutically acceptable. These include inorganic salts of sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and the like. Various organic salts of the peptide may also be made with, including, but not limited to, acetic acid, propionic acid, pyruvic acid, maleic acid, succinic acid, tartaric acid, citric acid, benozic acid, cinnamic ac id, salicylic acid, etc. Peptides can also be made intracel lular!}' in cel ls by introducing into the cells an expression vector encoding the peptide. Such expression vectors can be made by standard techniques. The peptide can be expressed in intracel lularly as a fusion with another protein or peptide (e.g. , a GST fusion). Synthesized peptides can then be introduced into cells by a variety of means known in the art for iniroducing peptides into cells (e.g., liposome and the like).

In one embodiment, a peptide for use in the methods, compositions, and uses of the invention is a serpin. Serpins are a group of natural ly occurring proteins that inhibit serine proteases. In one embodiment, the serpin binds to Granzyme B and has

Granzyme B inhibitory function.

In one embodiment the Granzyme B inhibitor is a PI9 peptide, or a Granzyme B inhibitory fragment thereof (see, e.g., U.S. Patent Publ ication No. 2003/01485 1 1 , the entire contents of which are incorporated herein by reference). P19, also known as SerpinB9 is a human serpin that inhibits Granzyme B (see, e.g. , review in Bird, 1 999 Immunol. Cell Biol. 77, 47-57). The amino acid and nucleotide sequence of SerpinB9 are known and may be found in, for example, Gcnbank Accession No. G I :223941 859, the entire contents of which are incorporated herein by reference, and SEQ ID NOs:9 and 10. In one embodiment, the peptide is SerpinB9 and comprises part or all of the sequence from SerpinB9 that binds directly to Granzyme B, i. e.

GTEAAASSCFVAECCMESG (SEQ. I D NO: I I ). This sequence contains the "reactive center" or "reactive center loop" of SerpinB9. In another embodiment, the Granzyme B inhibitor, e.g., a SerpinB9 peptide comprises the amino acid sequence selected from the group consisting of VEVNEEGTEAAAASSCFVVA ECC ESGPRFCA DHPFL (SEQ ID NO: 18);VEVNEEGTEAAAASSCFVVADCCMESGPRFCADH PFL (S EQ ID NO: 19); VEVNEEGTEAAAASSCFVVAACCM ESG PRFCADH PFL (SEQ I D NO:20); and VEVNEEGRJEAAAASSCFWA ECC ES.G PRFCA DFrPFL (S EQ I D NO:2 1 )

In another embodiment, the Granzyme B inhibitor is a Scrpina3n peptide, or a Granzyme B inhibitory fragment thereof Serpina3n is also known as SerpinA3. The amino acid and nucleotide sequence of SerpinA3 are known and may be found in, for example, Genbank Accession No. GI :73858562, the entire contents of which are incorporated herein by reference, and SEQ I D NOs: 1 2 and 1 3. In another embodiment, the Granzyme B inhibitor is the cowpox virusprotein , CrmA peptide, or a Granzyme B inhibitor)' fragment thereof (sec, e.g. , Quan, el al. ( 1995) 270, 10377- 10379) (the amino acid and nucleotide sequences of CnnA are set forth in SEQ ID NOs: 14 and 1 5). In one embodiment, a Granzyme B inh ibi tor is a CrmA peptide comprising the amino acid sequence

IDV EEYTEAAAATCALVADCASTVTN EFCA DH PFI (SEQ 1 D 0: 22).

In another embodiment, the Granzyme B inhibi tor is a Serp2 peptide, or a Granzyme B inhibitory fragment thereof. Scrp2 is also known as SerpinA3. The amino acid and nucleotide sequence of SerpinA3 arc known and may be found in, for example, Genbank Accession No. GI .582 1901 1 , the ent ire contents of which arc incorporated herein by reference, and SEQ ID NOs: 1 6 and 1 7.

Other suitable Granzyme B inhibitory peptides for use in any of the methods, compositions, or uses of the invention, include, for example, Z-AA D-C H2CI (Z-ALA- ALA-ASP-chloromethylketone), Ac-IEPD-CHO (Ac-l le-G lu-Pro-Asp-CHO), Ac-I ETD- CHO, Ac-AAVALLPAVLLALLAPIETD-cho, and z-l ETD- fmk.

In yet another embodiment, a Granzyme B inhibitor for use in the compositions, methods and uses of the invention is an antibody, e.g. , an anti-Granzymc B antibody. In one embodiment, the an anti-Granzymc B antibody is a human antibody. In another embodiment, the an anti-Granzyme B antibody is a humanized ant ibody. I n another · embodiment, the an anti-Granzyme B antibody is a camcl id antibody.

As used herein, the term "antibody" refers to a composition comprising a protein that binds specifically to a corresponding antigen and has a common, general structure of immunoglobulins. The term antibody speci fical ly covers polyc lonal antibodies, monoclonal antibodies, dimers, multimers, mul tispcc i fic antibodies (e.g.. bispeci fic antibodies), and antibody fragments, so long as they exhibi t the desired biological activity. The term "antibody" includes, without l imitation, camcl id antibodies.

Antibodies may be murine, human, humanized, chimeric, or deri ved from other species. Typically, an antibody will comprise at least two heavy chains and two l ight chains interconnected by disulfide bonds, wh ich when combined form a binding domain that interacts with an antigen. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CM). The heavy chain constant region is comprised of three domains, C H 1 , C H 2 and C||3, and may be of the mu ( μ ), delta ( δ ), gamma ( y ), alpha ( ) or epsi lon ( £ ) isotype. Si mi larly, the l ight cha in is comprised of a light chain variable region (V L ) and a light chain constant region (CL). The l ight chain constant region is comprised of one domain, C L, which may be of the kappa or lambda isotype. The V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each Vn and V[_ is composed of three CDRs and four FRs, arranged from ami no-terminus to carboxy- terminus in the following order: F 1 , CDR 1 , FR2, CDR2, FR3 , CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. , effector cells) and the first component (Clq) of the classical complement system. The heavy chain constant region mediates bind ing of the immunoglobul in to host tissue or host factors, particularly through cel lular receptors such as the Fc receptors (e.g., Fc y RJ, Fc γ RII, Fc y Ri l l, etc.). As used herein, antibody also includes an antigen binding portion of an immunoglobulin that retains the abil ity to bind antigen. These include, as examples, F(ab), a monovalent fragment of V L C L and Vn C H antibody domains; and F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region. The term antibody also refers to recombinant single chain Fv fragments (sc Fv) and bispeci fic molecules such as, e.g. , diabodies, triabodies, and tetrabodies (see, e.g.. U.S. Patent No. 5,844,094).

Antibodies may be produced and used in many forms, inc luding antibody complexes. As used herein, the term "antibody complex" refers to a complex of one or more antibodies with another antibody or with an antibody fragment or fragments, or a complex of two or more antibody fragments.

As used herein, the term "antigen" is to be construed broadly and refers to any molecule, composition, or particle that can bind speci fical ly to an antibody. An antigen has one or more epitopes that interact with the antibody, a lthough it does not necessari ly induce production of that antibody.

As used herein the term "epitope" refers to a determinant capable of speci fic binding to an antibody. Epitopes are chemical features general ly present on surfaces of molecules and accessible to interaction with an antibody. Typical chemical features are amino acids and sugar moieties, having three-dimensional structural characteristics as well as chemical properties including charge, hydrophi I icily, and I ipophil icity.

Conformational epitopes are distinguished from non-con formational epitopes by loss of reactivity with an antibody following a change in the spatial elements of the molecule without any change in the underlying chemical structure. The term "epitope" is also understood by those persons skilled in the art as an "antigenic determinant". For example, an antibody that is secreted by a B cel l recognizes only a portion of a macromolecule; the recognized portion is an epitope. The foregoing example is provided solely as an example and is not intended not l imit the scope of the term "epitope". Epitopes are recognized by numerous cel l types includi ng B cells and T cells.

As used herein, the term "humanized antibody" refers to an immunoglobu lin molecule containing a minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobul i ns (recipient antibody) in which residues from a complementary determining region (CD ) of the recipient arc replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired speci ficity, affinity and capac ity. I n some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non- human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantial ly a l l of at least one, and typically two, variable domains, in which all or substantia l ly a l l of the CDR regions correspond to those of a non-human immunoglobul in and al l or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence. A humanized antibody will also encompass immunoglobulins comprising at least a portion of an immunoglobulin constant region (Fc), generally that of a human immunoglobulin (Jones el ai, 1986; and Reichmann ei al , 1 988). As used herein the term "antibody fragment" refers to a fragment of an antibody molecule. Antibody fragments can include without limitation: single domains, Fab fragments, and single-chai n Fv fragments. As used herein, the term "monoclonal antibody" refers to monospeci fic antibodies that are the same because they are made by clones of a unique parent cel l. As detai led above, the term "antibody" includes without limitation a "monoclonal antibody".

In one embodiment, a Granzyme B inhibitor is a sma ll molecule. As used herein, the term "small molecule" refers lo a low molecular weight organic compound that binds to a biopolymcr such as a protein, a nucleic acid, or a polysaccharide. The foregoing examples of binding partners of a smal l molecule are non-limiting.

Optionally, the Granzyme B inhibitor used herein may be selected from one of the examples detailed herein, which includes but is not l imited to azepinc compounds of the following formula:

or a pharmaceutically acceptable salt or hydrate thereof, wherein: fi is 0, I, or 2; R and R 2 are each independently selected from the group'consisti ' ng pf: hydrogen, C i ^alkyl, C|_6alkoxy, C3.6cycloalkyl, aryl, HET and -N(R I 0 ) 2> wherei n : (a) said C h alky!, C \ . 6alkoxy and are optionally substituted with 1 -3 substi tuents independently selected from the group consisting of halo and hydroxy; and (b) said aryl and HET arc optionally substituted with 1 -3 substituents independently selected from the group consisting of: halo, hydroxy and C alkyI, optional ly substituted with 1 -3 halo groups; or R 1 and R 2 may be joined together with the carbon atom lo which they are attached to form a five or six membered monocyclic ring, optional ly contain ing 1 -3 heteroatoms selected from the group consisting of: S, O and N(R 10 ), wherein sa id ring is optional ly substituted with 1 -3 R 10 groups, with the proviso that R 1 and R 2 arc both not hydrogen ; each of R 3 and R 7 is independently selected from the group consisting of: hydrogen and C alkyI, optionally substituted with I -3 halo groups: each of R 4 , R 5 , R 6 and R 8 is independently selected from the group consisting of: hydrogen, halo, hydroxy and C \ . 4 alkyl, optionally substituted with 1 -3 halo groups; R is H ET, opt ionally substituted with 1 -3 substituents independently selected from the group consisting of: halo, hydroxy and C alkyI, optionally substituted with 1 -3 halo groups; R i0 is selected from the group consisting of: hydrogen, and -C(0)C i_4alkyl , said -C(0)C alkyl optionally substituted with N(R") 2 , HET and aryl, said aryl optionally substi tuted with 1 -3 halo groups; R is selected from hydrogen and opt ional ly substituted with 1 -3 halo groups; HET is a 5- to 10-membered aromatic, partia lly aromatic or non-aromatic mono- or bicyclic ring, containing 1 -4 heteroatoms selected from O, S and N(R 12 ), and optionally substituted with 1 -2 oxo groups; and R 12 is selected from the group consisting of: hydrogen and optional ly substituted with 1 -3 halo groups.

Optionally, the Granzyme B inhibitor used herein may be selected from one of the examples detailed herein, which includes but is not l imited to one or more of the following:

) also referred to herein as (2S,5S)-N- ((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-acetamido-3-methylpe ntanamido)-4-oxo- 1 ,2,4, 5,6,7-hexahydroazepino[3,2, 1 -hi]indole-2-carboxamide,

also referred to herein as (2S,5S)-N- (( l H- l ,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-acetamido-3-methylpen tanamido)-4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indole-2-carboxamide,

; a | so referred to herein as (2S,5S)-N- ((lH-l,2,3 riazol-4-yl)methyl)-5-((R)-3-melhyl-2-(pyridin-2-yl)butanami do)-4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indole-2-carboxamide,

; also referred to herein as (2S,5S)-N-((lH-l,2,3-triazol-4-yl)mcthyl)-5-((2S,3S)-3-mcthy l-2-(2- phenylacetamido)pentanamido)-4-oxo-l ,2,4,5 ,6,7-hcxahydroazepino[3, 2,1 -hi]indole-2- carboxamide,

i a | so referred to herein as (2S,5S)-N- ((lH-l,2,4-triazol-3-yl)methyl)-5-((2S,3S)-2-acctamido-3-met hylpcnlanamido)-4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indole-2-carboxamide,

j a | so referred to herein as (2S,5S)-N- ((lH-pyrazol-3-yl)methyl)-5-((2S,3S)-2-acetamido-3-methylpen tanamido)-4-oxo- l,2,4,5,6 > 7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide,

) also referred to herein as (2S,5S)-N- ((lH-pyrazol-4-yl)methyl)-5-((2S,3S)-2-acetamido-3-mcthylpcn tanamido)-4-oxo- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide,

) also referred to herein as (2S,5S)-N- ((lH-irnidazol-4-yl)methyl)-5-((2S,3S)-2-acetamido-3-methylp entanamido)-4-oxo- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indolc-2-carboxamide,

, also referred to herein as (2S,5S)-5- ((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(thiazol-5 -y I methyl)- 1 ,2,4,5,6,7- hexahydroazepino[3,2,l -hi]indole-2-carboxamicle,

, also referred to herein as (2S,5S)-5- ((2S,3S)-2-acetamido-3-methylpentanamido)-N-(isoxazol-3-ylmc ihyl)-4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indolc-2-carboxamide,

, also referred to herein as (2S,5S)-5- ((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(thiazol-2 -ylmelhyl)-l,2,4,5,6,7- hexahydroazepino[3,2,l-hi]indole-2-carboxamide,

, also referred to herein as (2S,5S)-5- ((2S,3S)-2-acetamido-3-methylpentanamido)- -(isoxazol-5-ylmcthyl)-4-oxo- 1,2,4, 5,6, 7-hexahydroazepino[3,2, l-hi]indolc-2-carboxamide,

, also referred to herein as (2S,5S)-5

((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(thiazo l-4-ylmcthyl)-l ,2,4,5,6,7- hexahydroazepino[3,2,l-hi]indole-2-carboxamide, , also referred to herein as (2S,5S)-5-

((2S,3S)-2-acetamido-3-methylpcntanamido)-4-oxo-N-(pyrimi di n-5-ylmethyl)- 1 , 2,4,5,6, 7-hexahydroazepino[3 ,2, 1 -hi]indolc-2-carboxamide,

, also referred to herein as (2S,5S)-5- ((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyridazin -4-ylmcthyl)- 1 ,2,4,5,6, 7-hexahydroazepino[3, 2, 1 -hi]indolc-2-carboxamide,

also referred to herein as (2S,5S)-5- ((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo- -(pyridi n-2-ylmelhyl)- 1 ,2,4,5,6,7- hexahydroazepino[3,2, 1 -hi] indole-2-carboxamide,

, also referred lo herein as (2S,5S)-5- ((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyridin-3 -ylmethyl)-l ,2,4,5,6,7- hexahydroazepino[3,2,l-hi]indo!e-2-carboxamide,

, also referred to herein as (2S,5S)-5- ((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyridin-4 -ylmethyl)-l ,2,4,5,6,7- hexahydroazepino[3,2,l-hi]indole-2-carboxamide,

also referred lo herein as (2S,5S)-5- ((2S,3S)-2-acetamido-3-methylpentanamido)- -(imidazo[l,2-a]pyrimidin-2-ylmethyl)- 4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indole-2-carboxamide,

, a lso referred to herein as (2S,5S)-5- ((2S,3S)-2-acetamido-3-methylpentanamido)- -((3a,7a-dihydrobenzo[d]thiazol-2- yl)methyl)-4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indoIe-2-carboxamide,

( a | so referred to herein as (2S,5S)-N- ((2H-tetrazol-5-yl)methyl)-5-((R)-3-methyl-2-(pyridin-2-yl)b utanamido)-4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2, 1 -hi]indolc-2-carboxamide,

also referred to herein as (2S,5S)-N- ((2H-tetrazol-5-yl)methyl)-5-((S)-3-methyl-2-(pyridin-2-yl)b utanamido)-4-oxo- 1 ,2,4,5,6,7-hexahydroazepino[3,2 > I -hi]indolc-2-carboxamide,

, also referred to herein as

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-3-methyl- 2-(2- phenylacetamido)pentanamido)-4-oxo- l ,2,4,5,6,7-hcxahydroazepino[3,2, l -hi]indole-2- carboxamide,

t a | so referred to herein as

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(2,3 -di iluorophenyl)acetamido)- 3-methylpentanamido)-4-oxo- l ,2,4,5,6,7-hexahydroazcpi no[3,2, 1 -hi] indolc-2- carboxamide,

j a | so referred to herein as (2S.5S)- N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(dimethylamino) acctamido)-3- methylpentanamido)-4-oxo- 1 ,2,4, 5,6, 7-hexahydroazepino[3 , 2 , 1 -hi] indolc-2- carboxamide,

, also referred to herein as

(2S,5S)-N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(ben zo[b]thiophcn-3- yl)acetamido)-3-methylpentanamido)-4-oxo- 1 ,2,4,5,6,7-hexahydroazcpino[3,2, 1 - hi]indole-2-carboxamide,

) a | so referred to herein as (2S,5S)- N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S > 3S)-2-(2-(dimethylamino)acetamido)-3- methylpentanamido)-4-oxo-l,2,4,5,6,7-hexahydroazepino[3,2,l -hi]indole-2- carboxamide,

t also referred to herein as (2S ) 5S)-N-((lH-l,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-(2- (bcnzo[b]thiophcn-3- yl)acetamido)-3-methylpentanamido)-4-oxo- 1 ,2,4,5,6,7-hcxahydroazepino[3,2, 1 - hi]indole-2-carboxamide,

, also referred to herein as ( )-N-((2S,5S)-2- ((lH-l,2,3-triazol-4-yl)methylcarbamoyl)-4-oxo-l ,2,4,5,6,7-hexahydroazepino[3,2, 1 - hi]indol-5-yl)-3-acetyl-5,5-dimethylthiazolidine-4-carboxami de,

, also referred to herein as (2S,5S)-N- ((lH-l,2,3-triazol-4-yl)methy])-5-((2S,3S)-3-mcthyl-2-(2-oxo pyrrolidin-l- yl)pentanamido)-4-oxo-l ,2,4,5,6,7-hexahydroazcpino[3,2, 1 -hijindole-2-carboxamidc,

, also referred to herein as (2S,5S)-N- (( 1H- 1,2, 3-triazol-4-yl)methyl)-5-(2-cyclopentylacetamido)-4-oxo- 1,2,4,5,6,7- hexahydroazepino[3,2,l -hi]indole-2-carboxamide,

( a | S0 referred to herein as (2S,5S)-N-((1 H- l,2,3-triazol-4-yl)methyl)-5-((S)-2-acetamido-2-cyclopropyla cetamido)-4-oxo- l,2,4,5,6,7-hexahydroazepino[3,2, l-hi]indole-2-carboxamidc,

t a | S0 referred to herein as (2S,5S)-N-((1 H- l,2,3-triazol-4-yl)methyl)-5-((S)-2-acetamido-2-cyclopcntyla cctamido)-4-oxo- l,2,4,5,6,7-hexahydroazepino[3,2,l-hi]indole-2-carboxamide, or salt or solvate thereof. Optionally, the Granzyme B inhibitor used herein may be selected from one of the examples detailed herein, which includes but is not li mited to one or more of the following:

methylpentanoyl]amino]-5-[2-[[(2S)-4-hydroxy- 1 ,4-dioxobutan-2- yl]carbamoyl]pyrrolidin- l -yl]-5-oxopentanoic acid,

also referred to herein as (4S)-4- [[(2S,3S)-2-acetamido-3-methylpcntanoyl]amino]-5-[[(2S,3S)-3 -hydroxy- l -[[(2S)-4- hydroxy- 1 ,4-dioxobutan-2-yl]amino]- l -oxobulan-2-yl]amino]-5-o.xopentanoic acid,

also referred to herein as 5- chloro-4-oxo-3-[2-[2-(phenylmethoxycarbonylamino) propanoylamino]

ropanoylamino] pentanoic acid,

also referred to her 5-chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylamino) propanoylamino] propanoylamino] pentanoic acid, or a salt or solvate thereof.

Optionally , the Granzyme B inhibitors used herein is selected from the following:

, also referred lo herein as ZINC05723764 and NCI 644752,

also referred to herein as ZINC05723787 and NCI 644777,

, also referred to herein as ZINC05316154 and

NCI 641248,

also referred 10 herein as ZINC05723499 and NCI 641235,

, also referred to herein as ZINC05723646 and

NCI 642017,

, also referred to herein as ZINC0539842S and

NCI 641230,

, also referred ιο herein as ZINC05723503 and

NCI 641236,

also referred io herein as ZINC05723446 and

NCI 640985,

, also referred to herein as ZINC05317216 and NCI 618792,

, also referred to herein as Z1NC053 16859 and NCI 618802, and

, also rcfciTcd to herein as ZINC05605947 and NCI 623744, or a salt or solvate thereof.

Optionally, the Granzyme B inhibitor used herein is: or a sa il or solvate thereof.

Optionally, the Granzyme B inhibitor used herein is:

or a salt or solvate thereof.

Optionally, the Granzyme B inhibitor used herein is:

or a salt or solvate thereof.

A Granzyme B inhibitor for use in the methods, composi tions, and uses of the invention may also be a synthetic inhibitor such as, for example, an isocoumarin, a peptide chloromethyl ketone, or a peptide phosphonate (sec, e.g.. Kam ct al ., 2000).

Optionally, the Granzyme B inhibitor used herein is one or more of:

Isocoumarin derivatives (upper left): 3,4-dichloiOisocoumarin, DCI , X= H, Y= CI ; 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin, X=NI l2, Y=0(CH2)3-SC( =NH + 2 )NH ; 4-chloro-3-ethoxy-7-guanidinoisocoumarin,

X=NHC(=NH + 2 )NH 2 ) Y=OCH 2 CH 3 . FUT- 1 75 analogs (upper right). Bottom line : structures of a peptide substrate, a peptide phosphonatc and a 4-amidinophcnylglycinc phosphonate [(4-AmPhGly) p (OPh)2] derivative. The lailcr is an arginine ana log.

III. Pharmaceutical Compositions

Many Granzyme B inhibitors are water-soluble and may be formed as salts. In such cases, compositions of Granzynie B inhibitors may comprise a physiological ly acceptable salt, which are known to a person of ski ll i n the art. Preparations will typically comprise one or more carriers acceptable for the mode of administration of the preparation, be it by topical administration, lavage, epiderma l admi nistration, subepidermal administration, dermal administration, sub-dermal admi nistration, sub-cutaneous administration, systemic administration, injection, inhalation, oral, or other modes suitable for the selected treatment. Suitable carriers arc those known in the art for use in such modes of administration.

Suitable compositions may be formulated by means known in the art and thei r mode of administration and dose determined by a person of sk ill in the art. For parenteral administration, compound may be dissolved in sterile water or sal ine or a pharmaceutically acceptable vehicle used for administration of non-water soluble compounds such as those used for vitamin . For entera l administration, compound may be administered in a tablet, capsule, or dissolved in l iquid form. The tablet or capsule may be enteric coated, or in a formulation for sustained release. Many suitable formulations are known including, polymeric or protein microparticles encapsulating a compound to be released, ointments, pastes, gels, hydrogcls, foams, creams, powders, lotions, oils, semi-solids, soaps, medicated soaps, shampoos, medicated shampoos, sprays, films, or solutions which can be used topically or loca l ly to administer a compound. A sustained release patch or implant may be employed to provide release over a prolonged period of time. Many techniques known to one of sk i l l in the art are described in Remington: the Science & Practice of Pharmacy by Alfonso Gennaro, 20' h ed., Williams & Wilkins, (2000). Formulations may, for example, contain excipients, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxycthylcne-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially use ful delivery systems for modulatory compounds include ethylenc-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholale, or may be oily solutions for administration in the form of drops, or as a gel.

Compositions containing Granzymc B inhibitors may also inc lude penetrating agents. Penetrating agents may improve the abil ity of the Granzymc B inhibitors to be delivered to deeper layers of the skin. Penetrating agents that may be used are known to a person of skill in the art and include, but are not limited to, hyaluronic acid, insulin, liposome, or the like, as wel l as L-arginine or the arginine-contai ning amino acids.

Compounds or compositions of Granzymc B inhibitors may be admi nistered alone or in conjunction with other wound treatments, such as wound preparations, wound coverings, and closure devices.

Optionally, the Granzymc B inhibitor is formulated for topical administration. For example, the formulations for topical administration of a Granzymc B inhibitor may assume any of a variety of dosage forms, including solutions, suspensions, ointments, and solid inserts. Examples are creams, lotions, gels, ointments, suppositories, sprays, foams, liniments, aerosols, buccal and subl ingual tablets, various passive and active topical devices for absorption through the skin and mucous membranes, including transdermal applications, and the like.

The Granzyme B inhibitor may be formulated for co-administration with another wound treatment. The another wound treatment may be selected from one or more of the following: a topical antimicrobial ; a cleanser; a wound gel; a col lagen; an elastin; a tissue growth promoter; an enzymatic dcbriding preparation; an anti funga l ; an antiinflammatory; a barrier; a moisturizer; and a sealant. Optionally, the another wound treatment may be selected from one or more of the fol lowing: a wound covering, a wound filler, and an implant. Optional ly, the another wound treatment may be selected from one or more of the following: absorptive dressings; a lginate dressings; foam dressings; hydrocolloid dressings; hydrofiber dressings; compression dressing and wraps; composite dressing; contact layer; wound gel impregnated gauzes; wound gel sheets; transparent films; wound fillers; dennal matrix products or tissue sca ffolds; and closure devices. Optionally, the Granzyme B inhibitor is formulated for topical application in a wound covering, a wound filler, or an implant. Optional ly, the

Granzyme B inhibitor is formulated for i mpregnation in a wound covering, a wound filler or an implant. The subject contemplated herein may be a mammal , further, the subject contemplated herein may be a human.

Optionally, the Granzyme B inhibitor may be formulated for topical administration. Optionally, the Granzyme B inhibitor may be formulated for coadministration with another wound treatment. Optional ly, the wound treatment may be selected from one or more of: a topical ant im icrobial; a cleanser; a wound gel ; a collagen; a elastin; a tissue growth promoter; an enzymatic dcbriding preparation; an antifungal; an anti-inflammatory; a barrier; a moisturizer; and a sealant. Optionally, another wound treatment may be selected from one or more of: a wound covering, a wound filler and an implant. Optional ly, the another wound treatment may be selected from one or more of: absorptive dressings; alginate dressings; foam dressings;

hydrocolloid dressings; hydrofiber dressings; compression dressing and wraps;

composite dressing; contact layer; wound gel impregnated gauzes; wound gel sheets; transparent films; wound fillers; dennal matrix products or t issue scaffolds; and closure devices. Optionally, the Granzyme B inhibilor may be formulated for topical application in a wound covering, a wound filler, or an implant. Opt ionally, the Granzyme B inhibitor may be formulated for impregnation in a wound covering, a wound fil ler or an implant. Optionally, the use may involve a subject that is a mammal; optional ly, the use may involve a subject that is a human.

IV. Animal Models and Screening Methods

In another aspect, a model for studying age-related wound repair is disclosed. The model comprises an apol ipoprotein E-knock out mouse maintained on a high-fat feed diet, wherein the high-fat feed diet is suffic ient to result in xanlhomatolic skin lesions on the mouse, and wherein the high-fat feed diet is su fficient to result in premature aging of non-xanthomatous regions of the sk in. I n skin areas that do not contain xanthomas, these mice also develop evidence of sk in aging in the form of reduced skin thickness, reduced collagen, and reduced elasticity when fed a high-fat diet.

In another aspect, a model for studying Granzyme B protein expression in vivo is disclosed. The model comprises an apolipoprotein E-knock out mouse mainta ined on a high-fat feed diet, wherein the high-fat feed diet is su fficient to result in xanthomatotic skin lesions on the mouse, and wherein the skin lesions express G ranzyme B. Granzyme B is abundant in the epidermal-dermal junction, an area that is prone to damage and separation as skin ages and during skin ulcer formation. This area also contains a large amount of the Granzyme B substrate decorin.

In another aspect, a model for studying premature aging in skin is disclosed. The model comprises an apolipoprotein E-knock out mouse maintained on a high- fat feed diet, wherein the high-fat feed diet is sufficient lo resul t in premature aging of the skin.

In another aspect, a model for screening compounds involved in repairing wounds is disclosed. The method involves maintaining an apol ipoprotein E-knock out mouse on a high-fat feed diet, wherein the high-fat feed diet is suffic ient lo result in accelerated age-related changes in the skin, thinning, and/or skin lesions on the mouse; administering a compound to the skin lesions on the mouse; and monitoring the skin lesions on the mouse. The monitoring contemplated herein i ncludes any biological sign of repair of a skin lesion. Examples of modes by which repa ir can be monitored include, but are not limited to the following: monitoring the presence or absence of newly formed tissue, and monitoring the width and/or size of the lesion, hair loss and/or restoration on the lesion. Other methods that can be employed include, but are not limited to, the following: monitoring the skin surface temperature, measuring transcpidcrmal water loss, monitoring the presence or absence of ECM abnormal ities, elastosis, col lagen morphology, collagen density, the presence of dccorin, and restoration of proper skin thickness. Additionally, skin-stress studies could be employed. Further, and serving as an example, decorin is reduced in areas of wound healing and fibrosis.

In another aspect, a method of screening compounds involved in repairing wounds is disclosed. The method involves maintaining an apol i poprotein E-knock out mouse on a high-fat feed diet, wherein the high-fat feed diet is su ffic ient to result in skin lesions on the mouse, and wherein the skin lesions express Granzyme B; administering a compound to the skin lesions on the mouse; and monitoring the skin lesions on the mouse.

In another aspect, a method of screening compounds involved in inhibiting or reducing skin lesions is disclosed. The method involves ma i ntaining an apol ipoprotein E-knock out mouse on a high-fat feed diet, wherein the high-fat feed diet is sufficient to result in skin lesions on the mouse when a compound is not administered to the mouse; administering the compound to the mouse; and monitori ng the skin lesions on the mouse.

In another aspect, a method of screening compounds involved in inhibiting or reducing skin lesions is disclosed. The method involves mainta ining an apol ipoprotein E-knock out mouse on a high-fat feed diet, wherein the high- fat feed diet is sufficient to result in skin lesions on the mouse when a compound is not administered to the mouse, and wherein the skin lesions express Granzyme B; administering the compound to the skin lesions on the mouse; and monitoring the skin lesions on the mouse.

In another aspect, the present invention provides methods for identi fying a compound useful for promoting chronic wound healing. The methods include providing an indicator composition comprising decorin and Granzyme B; contacting the indicator composition with each of a plurality of test compounds; and determining the effect of each of the plurality of test compounds on the cleavage of dccorin, and selecting a compound that inhibits the cleavage of decorin in the indicator composition, therby identifying a compound useful for promoting chronic wound hea l ing. The methods may further comprise determining the effect of the compound of collagen density and organization, the release of sequestered cytokine, e.g. , TGF-β, the cleavage of an extracellular matrix protein, e.g., an extracel lular proteoglycan, such as biglycan, and/or the tensile strength of skin.

Examples of agents, candidate compounds or test compounds include, but are not limited to, nucleic acids (e.g. , DNA and RNA), carbohydrates, l ipids, proteins, peptides, peptidomimetics, small molecules and other drugs. Agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase l ibraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affi nity chromatography selection. The biological library approach is l imited to peptide libraries, whi le the other four approaches are applicable to peptide, non-peptide oligomer or sma l l molecule l ibraries of compounds (Lam ( 1997) Anticancer Drug Des. 1 2 : 1 45; U.S. Patent No. 5,738,996; and U.S. Patent No. 5,807,683, each of which is incorporated herein in its entirety by reference).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt el al. ( 1993) Proc. Natl. Acad Sci. USA 90:6909; Erb et al. ( 1994) Proc. Natl. Acad. Sci. USA 91 : 1 1422; Zuckermann el al. ( 1 94) J. Med. Chem. 37:2678; Cho el al. ( 1993) Science 261 : 1 303; Carrel l el al. ( 1 994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. ( 1994) Angew. Chem. Int. Ed. Engl. 33 :2061 ; and Gallop et al. ( 1994) J Med. Chem. 37: 1 233 , each of which is incorporated herein in its entirety by reference.

Libraries of compounds may be presented, e.g. , presented in solution {e.g. ,

Houghten ( 1992) Bio/Techniques 1 3 :41 2-42 1 ), or on beads (Lam ( 1 991 ) Nature 354:82- 84), chips (Fodor ( 1993) Nature 364:555-556), bacteria (U.S. Patent No. 5,223,409), spores (Patent Nos. 5,571 ,698; 5,403,484; and 5,223,409), plasm ids (Cul l et al. ( 1992) Proc. Natl. Acad. Sci. USA 89: 1 865- 1 869) or phage (Scott and Smith ( 1 9900 Science 249:386-390; Devlin ( 1990) Science 249:404-406; Cwiria et al. ( 1 990) Proc. Natl. Acad. Sci. USA 87:6378-6382; and Fel ici ( 1991 ) 7. Mol. Biol. 222 : 301 -3 10), each of which is incorporated herein in its entirety by reference.

The indicator composition can be a cel l that expresses the Granzymc b and/or decorin protein, for example, a cell that naturally expresses or has been engineered to express the protein(s) by introducing into the cell an expression vector encoding the protein(s).

Alternatively, the indicator composition can be a cell-free composition that includes the protein(s) (e.g. , a cell extract or a composition that inc ludes e.g. , cither purified natural or recombinant protein).

For example, an indicator cell can be transfectcd with an expression vector, incubated in the presence and in the absence of a test compound, and the effect of the compound on the expression of the molecule or on a biological response can be determined.

A variety of cell types are suitable for use as an i ndicator cel l in the screening assay. Cells for use in the subject assays include eukaryol ic cells. For example, in one embodiment, a cell is a vertebrate cell , e.g. , an avian cel l or a mammalian cell (e.g., a murine cell, or a human cell).

Recombinant expression vectors that can be used for expression of , e.g. , decorin, are known in the art. For example, the cDNA is first iniroduccd into a recombinant expression vector using standard molecular biology techniques. A cDN A can be obtained, for example, by amplification using the polymerase chain reaction ( PCR) or by screening an appropriate cDNA library. The nucleotide sequences of c DNAs for or a molecule in a signal transduction pathway involving (e.g. , human, murine and bacterial) are known in the art and can be used for the design of PCR primers that a llow for amplification of a cDNA by standard PCR methods or for the design of a hybridization probe that can be used to screen a cDNA l ibrary using standard hybridization methods.

In another embodiment, the indicator composition is a cell free composition. Protein expressed by recombinant methods in a host cel ls or culture med ium can be isolated from the host cells, or cell culture medium usi ng standard methods for protein purification. For example, ion-exchange chromatography, gel fi ltration chromatography, ultrafiltration, electrophoresis, and immunoa ffin ity puri fication with antibodies can be used to produce a purified or semi-puri fied protein that can be used in a cel l free composition. Alternatively, a lysate or an extract of cells expressing the protein of interest can be prepared for use as cel l-free composition.

Once a test compound is identi fied that directly or indirectly modulates, e.g., decorin cleavage by one of the variety of methods described hereinbefore, the selected test compound (or "compound of interest") can then be further evaluated for its effect on cells, for example by contacting the compound of interest with cells either in vivo (e.g. , by administering the compound of interest to an organism) or ex vivo (e.g. , by isolating cells from an organism and contacting the isolated ce lls with the compound of interest or, alternatively, by contacting the compound of interest with a cel l l ine) and determining the effect of the compound of interest on the cells, as compared to an appropriate control (such as untreated cells or cells treated with a control compound, or carrier, that does not modulate the biological response).

In another aspect, the invention pertains to a combi nation of two or more of the assays described herein.

Moreover, a compound identified as described herein (e.g., an antisense nucleic acid molecule, or a specific antibody, or a small molecule) can be used in an animal model to determine the efficacy, toxicity, or side, effects of treatment with such a modulator. Alternatively, a modulator identi fied as described herein can be used in an animal model to determine the mechanism of action of such a modulator.

The instant invention also pertains to compounds identi fied in the subject screening assays.

EXAMPLES:

Abbreviations used herein: CTL, cytotoxic lymphocytes; DC I, 3 ,4- dichloroisocoumarin; DMSO, dimethyl sul foxide; ECM , extracel lular matrix; Erk, extracellular signal-regulated kinase; GAG, glycosaminoglycan; Granzyme B,

Granzyme B; HCASMC, human coronary artery smooth muscle cel ls; N K, natural kil ler cell; LAP, latency associated peptide; LLC, large latent TG F- β complex; LTB P, latent TGF- β binding protein; MMP, matrix metal loprotemasc; MT-M M P 1 , membrane lype- matrix metalloproteinase 1 ; SLC, small latent TG F- β complex; TG F- j3 , transforming growth factor beta.

EXAMPLE 1. G ranzyme B cleaves extracellula r matrix protei ns.

Methods. For in vitro extracel lular matrix c leavage assays, cells were grown to confluency and lysed, leaving the intact ECM on the plate. ECM was then biotinylated. Plates were then washed with PBS and incubated at 37°C with Granzyme B and/or with the Granzyme B inhibitor, 3,4-dichloroisocoumarin (DCI), for 4 and 24 hours at room temperature. Supernatant was then collected and assessed for cleavage fragments. Fragments were determined by Western blotti ng or -terminal sequencing.

Confirmation of cleavage was performed subsequently with puri fied substrate.

Results: In order to identify extracel lular Granzyme B substrates, recombinant decorin, biglycan, betaglycan, syndecan, and brevican were incubated with puri fied Granzyme B for 24hours. Reactions were stopped with SDS-PAG E loading bu ffer, run on an SDS-PAGE gel and imaged by Ponceau staining of a nitrocel lulose membrane. As shown in Figure 1 A, Granzyme B cleaves recombinant decorin, biglycan, betaglycan, syndecan, and brevican.

In order to determine if Granzyme B also cleaves smooth muscle cell- (S C-) derived ECM, following 5-7 days of serum starvat ion for ECM synthesis, human coronary artery smooth muscle cells (HCASMCs) were removed from 6 well plates using ammonium hydroxide. Granzyme B was incubated on ECM for 24 hours and supernatants were western blotted for fibri ll in- 1 , fibri l l i n-2 or fibul in-2 (Figure 1 B).

Figure 2 shows that Granzyme B also cleaves smooth musc le cell-derived decorin and biglycan. HCASMCs were incubated at confluency for adequate ECM synthesis. Cells were removed, Granzyme B was incubated with the ECM , and decorin and biglycan cleavage fragments were detected by western immunoblotting.

EXAMPLE 2. Granzyme B cleaves proteoglycans a nd releases sequestered TG F- β from extracellular matrix.

Methods: For ECM cleavage assays, Granzyme B and/or the inhibitor 3 ,4- dichloroisocoumarin (DC1), were incubated for 4 and 24 hours at room temperature, with decorin, biglycan or soluble betaglycan and visual ized by Ponceau staining.

Cleavage fragments were subjected to Edman degradation for cleavage site

identification.

As TGF- β is sequestered by the a forementioned proteoglycans, Granzyme B was incubated with TGF- β bound proteoglycans to determine i f Granzyme B cleavage resulted in the release of sequestered TG F- β . Cytokine release was assessed in supernatants using Western blotting.

To determine if the TGF- β released by Granzyme B was active, supernatants from the above release assay were incubated on human coronary artery smooth muscle cells (HCASMC) and SMAD Erk activation was examined by Western blotting. Results: Granzyme B cleaved decorin, biglycan and bctaglycan, with proteolysis evident at Granzyme B concentrations as low as 25 iiM. Proteolysis was inhibited by DC1 but not the solvent control D SO. Edman degradation analysis determined Granzyme B cleavage sites in the PGs with P I residues of aspartic acid, consistent with Granzyme B cleavage specificity.

In cytokine release assays, TGF- β was l iberated G ranzyme B-dependently from decorin, biglycan, and betaglycan, after 24 h of incubation. TGF- β was not released in the absence of Granzyme B or when Granzyme B was inh ibited by DCI , indicating release from decorin, biglycan and bctaglycan was speci fic. I n addition, the TG F- β liberated by Granzyme B remained active and induced S A D-3 and Erk-2

phosphorylation in HCAS C, after 16 h of incubation (see below).

EXAMPLE 3. Granzyme B cleaves decori n, biglyca n and soluble bctaglycan and releases active transforming growth factor- 0

Methods: Proteoglycan cleavage assays. The recombinant human PGs, decorin (0^g, Abnova, Walnut, CA), biglycan and betaglycan ( 1 .5-5ug, R& D Systems, Minneapolis, MN) were incubated at room temperature for 24 h with 25-500 nM purified human Granzyme B (Axxora, San Diego, CA), in 50 inM Tris buffer, pH 7.4. For inhibitor studies, Granzyme B was incubated in the presence or absence of 200 μ Μ of the serine protease inhibitor 3,4-dichloroisocoumari n (DCI ; Santa Cruz

Biotechnology Inc, Santa Cruz, CA) or inhibitor solvent control , dimethyl sul foxide (DMSO; Sigma-Aldrich, St Louis, MO) for 4h or 24h. A fter incubation, proteins were denatured, separated on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Ponceau stain (Fisher Scienti fic, Waltham, MA) was used to detect cleavage fragments.

N-terminal sequencing. For Edman degradation, 2-5 μ g/lane of biglycan and betaglycan were incubated with 100-500 nM Granzyme B for 24h. Once run on a gel and transferred to a PVDF membrane, cleavage fragments were identi fied by Ponceau staining. The stain was removed by washes with distil led water, the membrane was dried and analyzed at the Advanced Protein Technology Center at the Hospital for Sick K.ids (Toronto, ON). TGF- β release assays. TGF- β release assays were carried out using a method similar to that previously described for the MMPs (Imai el ai, 1997). Briefly, decorin, biglycan and betaglycan (15 pg/mL) were coated onto 48 well tissue culture plastic plates and allowed to incubate overnight at 4°C in PBS, pH 7.4. After blocking with 1% bovine serum albumin, 20 ng of active TGF- β 1 per well (Pcprotcch lnc, Rocky Hill, NJ) was added in DPBS containing calcium and magnesium (#14040, Invitrogen, Carlsbad, CA) for 5 h at RT. Granzyme B, with or without DC1, was then added to the wells. After 24 h, supematants were removed, denatured, and run on a 15% SDS-PAGE gel. Once transferred to a nitrocellulose membrane and blocked with 10% skim milk, the membrane was probed using a rat anti-human TGF- )31 antibody (1 :200, BD

Biosciences, Franklin Lakes, NJ) and IRDyc® 800 conjugated affinity purified anti-Rat IgG (1:3000, Rockland lnc, Gilbertsville, PA). Bands were imaged using the Odyssey Infrared Imaging System (LI-COR Biotechnology, Lincoln, NF).

Human Coronary Artery Smooth Muscle Cell TGF- β bioavailability assays. For bioavailability assays, HCASMCs (Clonctics/Lonza, Walkcrsvillc, MD) at passage 3-5 were seeded in 6 well plates in smooth muscle cell growth media (SmGM, Clonetics) +5% fetal bovine serum (FBS, Invitrogen) and grown to confluence. Al this time, cells were quiesced by serum removal for 24 h, after which time I 50 μ I of release assay supematants (as described above) or a 10 ng TGF- β positive control were added to the cells for 16 h. Cell lysates were assessed by SDS-PAGE/Wesiern blotting for phosphorylated-Erk 1/2 (p-Erkl/2; 1:1000, Cell Signaling Technology, Danvers, MA), total Erk 1/2 (t-Erkl/2; 1:1000, Cell Signaling Technology), phosphorylated-SMAD3 (p- SMAD3; 1:2000, Epitomics, Burlingame, CA), total SMAD3 (1-SMAD3; 1 :500, BD Biosciences) and the loading controls β -actin (1 :5000, Sigma-Aldrich) or β -tubulin (1:3000, Millipore, Billerica, MA). Secondary IRDye® 800 conjugated antibodies (1:3000, Rockland lnc) were utilized and imaged with the Odyssey Infrared Imaging System (LI-COR Biotechnology). Densitomctric analysis was conducted on the Odyssey Infrared Imaging System and displayed graphically by p-SMAD-3/ 0 -tubulin and p-Erk/ β -actin ratios.

Results: Granzyme B cleaves decorin, biglycan and betaglycan. Incubation of decorin, biglycan and betaglycan with Granzyme B resulted in the concentration- dependent generation of multiple cleavage fragments (Fig 3a-c). Full length decorin (-65 kDa) and 4 decorin cleavage fragments at -50 kDa and -30 k Da, were evident following Granzyme B incubation. Biglycan was identi fied at -40 kDa, with cleavage fragments evident at -25 kDa and 1 5 kDa, whi le incubation of recombinant soluble betaglycan (- 100 kDa) with Granzyme B resulted in multiple cleavage fragments at -60 kDa and 40 kDa. As all of these substrates arc PGs and contain glycosaminoglycan (GAG) chains, the apparent W of the full-length proteins and fragments may not be accurate, as glycosylation can alter movement through the gel . As such, several of the proteins and protein fragments are observed as a smear as opposed to a condensed band.

Referring to Figure 3, dose dependent Granzyme B-mcd iatcd cleavage of decorin, biglycan and betaglycan is demonstrated therei n. I ncreasing concentrations of Granzyme B (25, 50, 100 and 200nM) were incubated with decorin (a), biglycan (b), and betaglycan (c) for 24h at RT. As used in Figure 3, the mark * denotes ful l-length protein, arrows indicate cleavage fragments and Λ indicates Granzyme B.

To confirm that decorin, biglycan and betaglycan proteolysis was mediated by Granzyme B, DCI was included in reactions for 4 h or 24 h (Fig 4a-c). Higher concentrations of PG substrates and Granzyme B were uti lized in this assay for opti ma l detection of cleavage fragments. DCI effectively inhibi ted decorin, biglycan and betaglycan cleavage at both time points while the veh ic le control ( DM SO) had no effect (Fig 4a-c).

Granzyme B cleavage site identification. Granzyme B cleavage sites were characterized in biglycan and betaglycan by Edman degradation (Figure 4b-c). N- terminal sequence results for decorin were unable to be obtained due to low fragment yields, despite multiple trials. In biglycan, the cleavage site was identi fied at Asp 9l Thr- Thr-Leu-Leu-Asp, with a P I residue of Asp (Fig 4b). Interestingly, despite sequencing the 6 unique bands for betaglycan, on ly one unique cleavage site was characterized,

Asp 558 Ala-Ser-Leu-Phe-Thr, near the c-tcrminus o f the protein (Fig 2c). The n-terminal sequence of betaglycan fragments labeled by 1 corresponded to the n-tenninus of the protein and the n-terminal sequence of fragments labeled with 2 corresponded to the cleavage site (Fig 4c). The difference in apparent sizes in the SDS- PAGE gel is most likely due to differences in glycosylation. This heterogeneity in glycosylation is evident in the full length protein as it runs as a smear at the top of the gel (denoted by * in Fig 4c). Referring to Figure 4, it is demonstrated therein that Granzyme B-mediated cleavage of PGs is inhibited by DCI at 4 h and 24 h and Granzyme B cleavage sites contain aspartic acid at the P I residue. More speci fically, Granzyme B was incubated with decorin (a), biglycan (b) and bctaglycan (c), +/- DCI and the sol vent control DMSO, for 4h and 24h. Cleavage sites in biglycan and bctaglycan were identi fied by N- terminal Edman degradation. As utilized therein, the mark * denotes full length protein, arrows indicate cleavage fragments, and cleavage sites are displayed on the right.

Granzyme B-dependent cleavage of biglycan, decorin and betaglycan resiills in the release of active TGF- β ' I. As decorin, biglycan and bctaglycan sequester active TGF- β 1 , a TGF- β release assay was uti l ized to determi ne i f Granzyme B-mediatcd cleavage of these proteins resulted in active TGF- β release (Fig. 5). Following 24h of incubation, minimal TGF- β had dissociated from the plate in the absence of Granzyme B, suggesting that the PG/TGF- β complexes were stable throughout the incubation time. After 24h of Granzyme B treatment, TGF- j3 was released into the supernatants, from all three PG 's. This release was inhibited by DCI , suggesting the process was dependent on active Granzyme B. Bctaglycan consistently released more TG F- j3 than decorin and biglycan.

Referring to Figure 5, Granzyme B cleavage of decorin, biglycan and belaglycan is demonstrated to result in the release of active TGF- β . More specifical ly, 48 well plates coated with TGF- β 1 bound decorin, biglycan and bctaglycan were treated with Granzyme B, DCI, and/or the inhibitor solvent control for 24h. Supernatants (containing released TGF- β ) were collected and released TG F- β was detected by Western blotting. This is a representative western blot from 2-3 repeals for each PG.

TGF- β released by Granzyme B remains active and induces SM D and Erk signaling in smooth muscle cells. To determine that the TG F- β released by Granzyme B remained active and was not bound to an inhibitory fragment, supernatants from the betaglycan release assay were incubated on human coronary artery smooth muscle cells for 16h (Fig. 6). TGF- β signaling was examined through the phoshoporylation and activation of SM AD-3 and Erk 1/2. HCASMCs responded wel l to the 1 0 ng TGF- j3 positive control group, with increased SM A D-3 and Erk 1 /2 phosphorylation at 1 6 h. The TGF- β released from betaglycan by Granzyme B induced SMA D and Erk signaling, confirming that the TGF- β released by Granzymc B remained active. As expected, there was limited TGF- β signaling in the absence of Granzymc B or in the presence of DCI. Total Erk and total SMAD levels did not change with TGF- β treatment. Referring to Figure 6, TGF- β which is released by Granzyme B is active and induces S AD-3 and Erk-2 phosphorylation in HCASMCs. More specifically, Granzymc B +/-DCI was incubated on betaglycan/TGF- β complexes for 24h. Supcrnatants (containing released TGF- β ) were added to HCSMC for 16 h and phosphorylaicd Erk as well as phosphorylated SMAD-3 levels were examined. A similar trend in Granzyme B- dependent phosphorylation was observed in two additional experiments (Figure 7).

In the foregoing Examples, three novel extracellular substrates of Granzyme B were identified: decorin, biglycan and betaglycan. Furthermore, it was demonstrated that upon cleavage of these PGs by Granzyme B, active TGF- β is released. Reports have indicated that approximately one third of Granzyme B may be released non-specifically during immune cell engagement/degranulalion and cytotoxic lymphocytes constitutively release Granzyme B in the absence of target cell engagement (sec, for e.g. , Prakash el ai, 2008). Based, in part, on the results obtained herein, Granzymc B plays an extracellular role in pathogenesis. In this study the Granzyme B cleavage sites for biglycan and betaglycan were identi ied. In this study, it was demonstrated that Granzyme B cleaved these PG substrates at a P I residue of Asp (biglycan: D 91 , betaglycan: D 55S ). Further, in the studies described herein TG F- β 1 was released from all three substrates. There was no release evident in the negaiivc control lacking protease or when Granzyme B was co-incubalcd with the irreversible inhibitor DCI. In addition, the TGF- β released by Granzyme B induced SMAD-3 and Erk-2 phosphorylation, thereby confirming that Granzyme B releases active TGF- β and does not alter TGF- β activity.

Betaglycan cleavage consistently released more TGF- β than biglycan and decorin, which may be due to betaglycan having several binding sites for TGF- β and Granzyme B potentially releasing the cytokine from more than one binding site.

In summary, the current Example demonstrates the identification of three novel factors for Granzyme B, and demonstrates how an accumulation of Granzyme B in the extracellular milieu negatively impacts growth factor sequeslralion by the ECM. EXAMPLE 4. A role for Granzymc B in matrix remodelli ng and aging of the skin in apolipoprotein E knockout mice.

Abbreviations used herein: apolipoprotein 1Z (apoE); knockout ( O); double knockout (D O); extracellular matrix (ECM ); Granzymc B (Granzymc B); ultraviolet (UV); high fat diet (HFD); second harmonic generation (S I IG).

Materials and methods: Mice. Al l animal procedures were performed in accordance with the guidelines for animal experimentation approved by the Animal Care Committee of the University of British Columbia. Male C57BL/6 and apoE- O mice were purchased from The Jackson Laboratory (Bar Harbor, E) and housed at The Genetic Engineered Models (G EM) facility (James Hogg Research Centre, UBC/St. Paul 's Hospital, Vancouver, BC). ApoE/Granzymc B double knockout mice were generated on site and also housed at the G EM facil ity. A l l mice were fed ad l ibitum on either a high fat (21 .2% fat, TD.881 37, Harlan Teklad; Madison, WI) or regular chow (equal parts PicoLab Mouse Diet 20: 5058 and PicoLab Rodent Diet 20: 5053, LabDiet; Richmond, IN) diet beginning at 6-8 weeks of age for cither 0, 5, 1 5 or 30 weeks. At their respective time points, mice were weighed, and euthanized by carbon diox ide inhalation. Life span was measured using only mice designated for the 30 week time point and mortality the result of required euthanasia due to severe i llness in the form of open skin lesions and xanthomatous lesions. The degree of disease severity requiring euthanasia was determined in a blinded manner by an independent animal care technician within the GEM facil ity. Briefly, animals were considered for euthanasia i f they appeared to be in distress or pain that could not be al leviated. Because the animals cannot receive pain medication, mice deemed to be suffering because of open skin lesions or severe xanthomas required euthanasia.

Tissue collection and processing. Following euthanasia, mouse back hair was shaved and dorsal skin was removed from the mid lo lower back . Ha l f of the sk in sample was Fixed in 10% phosphate buffered formal in. Fixed sk in sections were processed, embedded in paraffin and cut to 5 μιη cross-sections for histology and

immunohistochemistry. The other hal f of the dorsal skin sample was treated with a hair removing cream to completely remove al l hai r from the surface of the skin. These skin samples were then flash frozen in liquid 2 and stored at -80°C unti l further use for multi-photon microscopy. Histology and immunohistochemistry. Paraffin embedded skin cross-sections were stained with hematoxylin and eosin (H&E) for evaluation of morphology and with picrosirius red to examine collagen content. Luna's clastin was used to examine elastic fibres. Measurement of skin thickness was completed usi ng a 40X objective lens and a calibrated ocular micrometer scale. Measurements were taken across the entire cross- sectional surface of the skin at multiple sites and averaged for each mouse. Col lagen was observed in picrosirius red stained sections using 100% polarized l ight and pictures were taken at a fixed exposure. Granzyme B immunohistochemistry was performed by boi ling deparaffinised slides in citrate buffer (pH 6.0) for 1 5 mi n. Background staining was blocked by incubating sl ides with 10% goat serum. The primary antibody used was a rabbit anti-mouse Granzyme B antibody al a 1 : 1 00 di l ution (Abeam, Cambridge, MA) and was incubated at 4°C overnight. Slides were then incubated with biotinylated goat anti-rabbit secondary antibody at a 1 :350 dilution (Vector Laboratories, Burlingame, CA) followed by ABC reagent (Vector Laboratories). Sta ining was visual ized with DAB peroxidise substrate (Vector Laboratories). Decorin immunohistochemistry was performed by immersing deparaffinised slides in citrate bu ffer (pl l 6.0) at 80°C for 10 min. Slides were blocked with 10% rabbit scrum and a goal anli-mouse dccorin antibody ( 1 μg/ml)(R&D Systems, M inneapolis, M N) was used whi le sl ides incubated at 4°C overnight. Biotinylated rabbit anti-goat secondary aniibody was used ( 1 :350) (Vector Laboratories) along with ABC reagent (Vector Laboratories) and DAB substrate (Vector Laboratories) as described above.

Multi-photon microscopy. Frozen sk in samples with the hai r completely removed were thawed at room temperature and immobil ized on a fiat surface inside a small dish. Skin samples were washed severa l times and immersed in phosphate buffed saline. Second harmonic generation (SHG) signals were emitted by the collagen in the skin samples and quantified as a measure of col lagen density. Methods used were sim ilar to those described previously (Abraham et al., 2009). Briefly, the laser used was a mode- locked femto-second Ti:Sapphire Tsunami (Spectra-Physics, Mountain View, CA) and was focused on the specimen through a 20X/0.5 NA HCX APO L water dipping objective. An excitation wavelength of 880 nm was used and backscaltered SHG emissions from the sample were collected through the objective lens. Leica Con focal Software TCS SP2 was used for the image acquisilion . I mages (8 bit) acquired were frame-averaged 10 times to minimize the random noise. For each sample, about 200-250 Z-section images with a thickness of about 0.63 μπι were acquired at decreasing tissue depths for a total thickness measurement of approximately 1 30- 160 μιτι per sample. These measurements were taken completely within the dermis of each sample as the thinnest dermal layer observed was 250 μιτι, therefore any decrease in signal is due to a decrease in density rather than a lack of dernial collagen material. Z-section images were compiled and finally the 3D image restoration was performed using Volocity software (Improvisions, Inc., Waltham, MA). A noise-remova l filter whose kernel size of 3X3 was applied to these 3D images and SHG signals thai fel l within a set threshold were quantified for the entire 3D image using Velocity soft ware (I mprovisions Inc.).

Statistical analysis. Survival data were analyzed for signi ficance using the Mantel-Cox test with P < 0.05 considered significant. One- or two-way ANOVA with Bonferroni post test was used where appropriate for group comparison analyses with P < 0.05 considered significant.

Results: Morbidity and skin pathology. A l l cases that requi red euthanasia prior to 30 weeks were attributed to severe open or xanthomatoticskin lesions. Consistent with previous reports, apoE- O mice in this study exhibited a marked decline in health compared to wild type controls resulting in increased morbidity and frequency of required euthanasia over a 30 week span (Fig. 8A). Whi le placing wi ld type mice on a HFD did not alter survival over the 30 week span, the necessity for euthanasia was significantly increased when the apoE- KO m ice were led a H FD with only about 69% surviving to the 30 week time point (Fig. 8A).

As shown in Fig. 8B, apoE-KO mice exhibited signs of frai lly, hair loss, hair graying and the formation of subcutaneous lesions or xanthomas on thei r backs and shoulders at 30 weeks. These phenotypes were more severe and occurred much earl ier when apoE-KO mice were fed a HFD (Fig. 8B). Of a ll apo E- KO m ice on a regular chow diet in the 30 week group, 9/3 1 (29%) demonstrated evidence of xanthoma/skin pathologies with the earliest case at 1 8 weeks and the majority of the cases (7/9) appearing when examined at 30 weeks. When fed a H FD, 1 3/32 (4 1 %) apoE-KO mice showed evidence of xanthomas/skin pathology wi th 1 0/ 1 3 occurring prior to the 30 week time point. These data demonstrate that a HFD accelerates the frequency and onset of these lesions. Interestingly, the appearance of severe xanthomas was delayed in the HFD-fed D O mice as the first case requiring euthanasia appeared at 1 9.9 weeks with only 3/14 (21 %) total incidence of observed skin pathologies (Fig. 8A). By comparison, at 19.9 weeks, 8 mice from the HFD-fed apoE-KO group (25%) already required euthanasia, with the first occurring as early as 7 weeks (Table 1 ). D O mice fed a regular chow diet appeared to develop xanthomas in some cases (2/ 1 1 or 1 8%) but were never severe enough to require euthanasia prior to 30 weeks (Fig. 8A). These resu lts demonstrate that Granzyme B contributes to lesion severity and that reduced Granzyme B delays the onset of these skin pathologies. Table 1 summarizes the incidence and severity of the skin lesions in al l groups.

Table 1. Summary of xanthoma/skin pathology incidence. CC: C57BL/6 Chow; CH : C57BL/6 High Fat; AC: apoE-KO Chow; AH : apoE- KO H igh Fat ; G DC: DKO Chow; GDH: DKO High Fat.

Weight gain. While a HFD resulted in signi ficant weight gain in C57B L76 control mice at the 30 week time point, apoE-KO mice on a H FD showed no significant increase in weight compared to the chow fed apoE- KO mice and weighed significantly less than the HFD-fed C57BL/6 mice (Fig. 8F) at 30 weeks. When weight gain was examined at the 0, 5, 1 5 and 30 week time points, C57 B L/6 mice on a H FD showed a significant increase in weight as early as 5 weeks on the diet compared to the chow fed controls which remained higher throughout the course of the study (Fig. 8C). ApoE deficiency alone resulted in no difference in weight gain compared to the chow-fed controls until 30 weeks when the chow-fed apoE-KO group stopped gaining weight (Fig.

8D) and adopted a frail phenotype (Fig. 8B). When apoE-KO mice were fed a HFD, they appeared to gain weight at a similar rate to the control group and possibly to a greater extent at 1 5 weeks. However, by 30 weeks they appeared to have actual ly lost weight (Fig. 8E) and often displayed frai l and diseased skin (Fig. SB). To examine the role of Granzyme B in this process, DKO mice on cither a regular chow or a H FD were maintained until 30 weeks. Both groups of DKO mice showed no signi ficant difference in weight at 30 weeks compared to either the C57B L/6 chow- fed control group or the apoE-KO groups (Fig. 8F).

Referring to Figure 8, C57BL/6 chow (CC), C57B L/6 high fat (C H), apoE-KO chow (AC), apoE-KO high fat (AH), DKO chow (G DC) and DKO high fat (GDH). (A) All C57BL/6 wild type mice survived to the 30 week time point on cither a high fat

(n= 18) or regular chow (n= 19) diet whi le 94% of chow- fed apoE-KO mice (n=3 1 ) were kept alive for 30 weeks. A HFD signi ficantly (P<0.0 \ ) reduced survival in apoE-KO mice (n=32) compared to the control CC group with only 69% remaining healthy enough to survive for the 30 week span. 100% of chow-fed DKO mice (n= l 1 ) survived to the 30 week time point while morbidity in the DKO mice fed a HFD (n= 14) appeared to be delayed compared to the AH group. (B) Representative images of mice at the 30 week time point. (C-E) Weight gain over 0, 5, 1 5 and 30 weeks for the CH, AC and AH groups compared to CC. (F) Average weights of the all groups of m ice at the 30 week time point (Error bars represent the mean ± SEM ). (D-F). */ J <0.05 , * * */><0.001 .

More specifically with respect to Figure 8B, the photographs therein depict as follows: C57BL/6 Chow - control mouse, typical healthy size and weight. Normal looking black hair. C57BL/6 High Fat - appears obese compared to the control mouse, hair and skin look otherwise normal. ApoE-KO Chow - this mouse appears frail compared to the control mouse, shows evidence of hair grayi ng and some areas of hair thinning/loss. ApoE-KO High Fat - this mouse also appears more fra il compared to the control mouse and fails to gain weight from the high fat d iet as the C57BL/6 mouse does. This mouse also displays evidence of hair graying, hair loss and inflammatory ski n lesions (xanthomas) that appear on their backs. DKO Chow - These mice appeared to be relatively normal in terms of weight gain compared to the control mice and reduced incidence and severity of the hair loss, graying and sk in lesion formation compared to the apoE-KO Chow group. DKO High Fat - this group also genera l ly appeared healthier than the apoE-KO High Fat group with reduced incidence and severity of hair loss, graying and skin lesions. Skin istopathology. As shown in Fig. 9, the skin of apoE-KO mice is heterogeneous; exhibiting normal "regular" looking skin (Fig. 9A) and other areas featuring xanthomatous lesions (Fig. 9B). These lesions often develop on the backs of the mice and occur with increased severity and frequency with age and when fed a HFD. None of the C57BL/6 wild type mice exhibited xanthomatosis at any time point over the 30 week span regardless of diet. Histological examination of the xanthomatous lesions i n apoE-KO mice revealed skin thickening including that of the epidermis, considerable immune infiltration, loss of normal adipose tissue, ECM alterations and the presence of cholesterol crystals (Fig. 9B). Although xanthoma development was common in apoE- KO mice, not all mice displayed this phenotype and in some instances mice had skin that appeared relatively normal (Fig. 9A).

Referring to Figure 9, ApoE-KO mouse skin is heterogeneous with certain areas of the skin appearing "regular" while other areas contai n xanthomatous lesions (H& E stain). (A) "Regular" looking skin from C57BL/6 chow (CC), C57 BL/6 high fat (CH), apoE-KO chow (ACR), apoE-KO high fat (A HR), DKO chow (G DCR) and DKO high fat (GDHR) at the 30 week time point. (B) Xanthoma from a HFD- fed apoE-KO mouse (AHX) that survived to the 30 week time point demonstrati ng cholesterol crystals (arrows) and (C) from a HFD-fed apoE-KO mouse thai required euthanasia at 14.9 weeks due to the severity of the lesion. E: epidermis; D: dermis; A : adipose tissue. (A scale bar = 200 μ m, B and C scale bar = 100 μ m).

Skin thickness. Skin thinning and atrophy is a characteristic feature that occurs with age both in humans and mice (see, for e.g., Bhaliacharyya and Thomas, 2004). To determine whether apoE-KO mice exhibit this trait, we analyzed formal in fixed skin sections from the mid to lower back of the chow or H D-fed C57BL/6 and apoE-KO mice using H&E staining at 0, 5, 1 5 and 30 weeks and measured total skin thickness including the epidermis, dermis, adipose and skeletal muscle layers (Fig. 1 OA). Due to the heterogeneous nature of the skin in apoE-KO mice and the fact that xanthoma development has a dramatic effect on skin thickness (Fig. 9), skin samples from apoE- KO mice were separated into two groups depending on the histological presence of xanthoma: "regular" skin and xanthoma skin. When fed a HFD, wild type C57B L/6 mice had significantly thicker skin than the control group at 5, 1 5 and 30 week time points (Fig. 10A). ApoE deficiency alone resulted in no'signi ficant di fference over time compared to controls as shown in Fig. 10B. Interestingly, skin from the HFD-fed apoE- KO group, while significantly thinner than the control group at 30 weeks, appeared to be slightly thicker than the control group at 1 5 weeks when thickness was measured over time (Fig. I OC). This demonstrates that these changes arc not simply a result of developmental differences but rather a change that occurs at an accelerated rate over time compared to the wild type controls. To determine whether Granzyme B deficiency protects against skin thinning and frailty, skin from DKO mice on a regular chow or HFD was examined at 30 weeks and compared to the other groups. As mentioned above, the HFD caused a significant increase in skin thickness in the C57B L/6 group while a significant decrease in thickness in the apoE-KO group (Fig. 1 0G). HFD-fed DKO mice displayed a significant increase in total skin thickness compared to the H FD-fed apoE- KO group at 30 weeks (Fig. 10G), demonstrating that G ranzyme B contributes to age- related skin thinning and frailty in apoE-KO mice.

Closer analysis of the individual layers of the sk in revealed that changes in total skin thickness in the "regular" skin samples were due primarily lo changes in the dermal and/or adipose tissue layers. Whi le no signi ficant di fferences were observed in epidermal thickness at the 30 week time point for any of the groups (Fig. I 0D), dermal thickness was significantly thicker in the chow-fed DKO group compared to the chow- fed control group (Fig. 10E). Interestingly, when fed a H FD, the DKO group had a significantly thicker dermis than the HFD-fed apoE-KO group (Fig. 1 0E). Changes in the thickness of the adipose tissue layer varied the most with the H FD-fed C57B L/6 group demonstrating a signi ficant increase and both apoE-KO groups and the chow-fed DKO group having significantly thinner adipose tissue layer than the chow-fed controls (Fig. 10F). HFD-fed DKO mice showed no signi ficant di fference in adipose tissue thickness compared to either the chow-fed controls or the apoE-KO groups (Fig. 10F). These results demonstrate that apoE deficiency results in a decrease in total skin thickness and that this is due in part to a decrease in adipose tissue whi le Granzyme B deficiency protects against skin thinning due in part to an increase in dermal thickness.

Referring to Figure 10, (A-C) skin thickness of C57B L/6 chow (CC), C57BL/6 high fat (CH), apoE-KO chow (ACR) and apoE-KO high fat (A H R) was measured at 0, 5, 15 and 30 weeks using non-diseased "regular" skin sections. Individual skin layers were measured for CC, CH, ACR, AHR, DKO chow (G DCR) and DKO high fat (GDHR) at 30 weeks including the (D) epidermis, (E) derm is, (F) adipose and (G) total skin thickness including skeletal muscle. Error bars represent the mean ± SE . * F<0.05 vs CC; ** <0.01 vs CC; ***P<0.00 l vs CC;†^<0.05 vs A H ;††/><0.01 vs AHR;††† <0.001 vs AHR.

Collagen and elastin abnormalities in the skin ofapoE-KO mice. To investigate the collagen changes occurring in the diseased, xanthoma skin lesions of apoE-KO mice, skin sections were stained with picrosirius red and visual ized using polarized light. As shown in Figures 1 1 C and 1 2K, skin lesions display clear alterations in collagen organization and structure compared to regular skin from control mice (Fig. 1 1 A).

Collagen fibres were often arranged in a more parallel orientation with thinner col lagen bundles in the diseased skin (Fig. 1 1 C), which explains the increased stiffness and skin frailty that was observed in these lesions. Some areas of the dermis d isplayed a near complete loss/degradation of normal collagen and evidence of damage to the dermal- epidermal barrier (Fig. 121).

To examine elastin content in the diseased skin, Luna 's elasti n stain was used. Wild type control mice demonstrated diffuse elastin distribution with thin elastic fibres and minimal large elastin bundles (Fig. I 1 E). In the diseased skin, wc observed increased elastin deposition in the papi llary dermis as well as abnormal elastin bundle deposits in the dermis (Fig. I 1 F and G).

Referring to Figure 1 1 , skin sections from chow- fed C57B L/6 mice display thick dense collagen fibres while apoE-K.0 mice on a HFD frequently display areas of altered collagen morphology with reduced density compared to controls. (A) Images of skin collagen from a C57BL/6 mouse on a chow diet for 30 weeks (E: epidermis; D: dermis; A: adipose tissue). (B) Skin collagen from a "regular" skin sample from an apoE-KO mouse on a HFD for 30 weeks. (C) HFD-fcd apoE-KO mouse skin collagen from a diseased area containing xanthoma. (D) Example of skin col lagen from "regular" skin of a HFD-fed DKO mouse at 30 weeks. (E) Elastin from C57B L/6 mouse on a chow diet for 30 weeks (Elastin stains dark purple - arrows). (F and G) Examples of abnormal elastin deposits (arrows) from 30 week HFD-fcd apoE- KO mice with diseased skin. Picrosirius red stain viewed under polarized l ight (A-D) and Luna's elastin stain (E-G). (A-D and F-G scale bars = 100 u m, E scale bars = 1 0 μ m).

In Figure 1 1 , collagen is monitored. In Figure 1 1 A, col lagen is densely packed in this slide from a normal (non-knock-out mouse). Figure I I B is a sl ide from an apoE-ko mouse. The collagen appears to be packed less densely. I n Figure I 1 C, this slide shows diseased skin from an apoE-ko mouse. The col lagen appears to be l inear and is less elastic. In Figure 1 I D, this slide shows col lagen in a Granzyme B-/- ApoE-ko mouse. The collagen appears to be packed more densely compared with the single knockout apoE-ko mouse tissue. In Figure 1 I E and 1 I F, elastin is mon itored. Figure I I E is data from a normal mouse. The right panel shows elastin fibers (see arrows).

Decorin remodelling and Granzyme IS expression in the skin apoE-KO mice. Collagen disorganization was readi ly observed in the diseased sk in of apoE-KO mice (Fig. 121). While minimal differences in some areas of the skin were observed (Fig. 12B), staining the diseased skin sections with anti-decorin antibody revealed a distinct loss of decorin in other areas of the diseased skin in apoE-KO mice (Fig. 1 2C and D). Areas of decorin degradation corresponded with areas o f collagen loss and remodelling (Fig. 121 and J). DKO mice exhibited increased decorin content in the non-diseased skin sections particularly near the derma l epiderma l junction (Fig. 1 2 E and F). Additionally, areas of decorin loss were not observed in the diseased sk in sections from DKO mice to the same extent as apoE-KO mice (Fig. 1 2G and H). When xanthoma skin sections from apoE-KO mice were stained with anli-Granzymc B ant ibody, Granzyme B was observed in the lesion in specific areas and was often localized to the papi llary dermis near the dermal-epidermal junction (Fig. 12K). Interestingly, these areas of localized Granzyme B expression in the lesions directly corresponded with areas of collagen/decorin loss and remodelling as evidenced by staining seria l sections ( Fig. 1 21 - K.). These results demonstrate that Granzyme B plays a role in collagen remodel l ing in the skin through the cleavage of decorin.

Referring to Figure 1 2, decorin sta ini ng in the sk in from (A) chow-fed C57B L/6 (B-D) HFD-fed apoE-KO, (E-F) HFD fed DKO "regular" skin showing darker decorin staining at the papillary dermis near the dermal epidermal j unction (arrows) and (G-H) DKO diseased skin. Serial sections of HFD-fed apoE-KO sk in stained for (1) col lagen, (J) decorin and, (K and L) Granzyme B. (A-H and L sca le bars = 50 μ m, 1-K scale bars = 200 m)

* In Figure 12A decorin staining is shown in a normal mouse. Thee staining is more intense towards the epidermal-dermal junction. Figure 1 2B shows decorin staining in an apoE-ko mouse. The staining is more di ffuse. Figures I 2C and 1 2 D show nearly absent decorin staining in diseased portions of skin from apoE-KO mouse tissue. Figures 12E and 12F show regular skin from Granzyme B-/-apoE-/- mouse tissue. There is intense decorin staining. Figures 12G and 1 2H show diseased skin from Granzyme B- /-apoE-/- mouse tissue. In Figure 1 2H decorin is shown in the epidermis. Figures 1 21, 12J, and 12 are serial sections monitoring collagen, decorin, and Granzyme B respectively, all from apoE-ko mouse tissue. Figure I 2 L is a zoom-in photo from Figure 12 . There is increased Granzyme B staining in Figure 1 2K.

Collagen density and organization. To determine i apoE- O mice exhibit differences in collagen content in the regular, non-xanlhoma skin, picrosirius red staining was used on formalin fixed skin sect ions and analyzed for changes in col lagen content and structure. Dermal collagen from the chow- led control group exh ibited typical red/orange staining of thick, dense col lagen fibres at the 30 week time point (Fig. 1 1 A). In contrast, HFD-fed apoE-KO mice often displayed derma l collagen that was loosely packed, and less structured that the control group ( Fig. 1 1 B). This was not readily observed in the other groups including the H FD- fed D KO group (Fig. 1 1 D) suggesting a HFD combined with apoE deficiency affects col lagen structure and density in apoE-KO mouse skin, and that Granzyme B deficiency prevents the loss of thick fibre formation and collagen density.

Although analysis of fixed, thin sl iced sections can provide useful information regarding collagen content and structure, important three di mensional and organizational properties may be missed or altered during processing. Wc took advantage of the bifringent properties of col lagen to visual ize col lagen structure and organ ization in unfixed, unstained thick skin samples in three dimensional space using multi-photon microscopy. Highly ordered fibri l-forming col lagens (Type 1 , 11, 111 , etc.) produce second harmonic generation (SHG) signals without the need for any exogenous label (see, for e.g., Zipfel et al., 2003). These SHG signa ls correlate with the density and organization of the collagen matrix rather than total col lagen content. Representative flattened three dimensional SHG images originating from the collagen matrix (grey) are shown in Fig. 13A for al l groups at the 30 week time poi nt. Only non-diseased skin was used for these experiments to ensure any ECM changes observed were not the result of xanthoma formation but rather the result of a more intrinsic aging process. When collagen density was monitored over time, the 0 week time point appeared similar for the C57BL/6 and apoE-KO groups (Fig. 1 3 B- D). The chow-fed control group appeared to show a slight decrease in collagen density over ti me whi le the apoE-KO mice fed a HFD exhibited a reduced SHG signal as a function of age beginning at 5 weeks and at a greater rate than the wild type controls demonstrating an increased rale of collagen modification and disorganization resulting in a signi ficant loss of collagcn density by 30 weeks (Fig. 1 3D). To confirm that the skin used in these experiments was regular, non- xanthoma skin, skin samples were fixed in 1 0% buffered formali n fol lowing these experiments and examined histologically using H&E sta ining. Upon histological examination the HFD-fed apoE- O skin chosen for these experiments did not show evidence of xanthomatosis, confirming the observed changes in col lagen density are an intrinsic property, rather than brought on by xanthoma formation (data not shown). These results demonstrate that age-related EC changes are occurri ng in the skin of HFD-fed apoE-KO mice even in skin sect ions without xanthoma formation at an increased rate compared to wild type mice leading to premature skin aging.

Referring to Figure 1 3, (A) representative 3 D merged plane images of fresh ex- vivo unfixed, unstained mouse "regular" ski n tissues obtained from chow fed C57BL/6 (CC), apoE-KO mice (ACR) and DKO mice (G DCR) and H FD- fed C57BL/6

(CH),apoE-KO mice (AHR) and DKO mice (GDHR) at 30 weeks. Gray colors represent the collagen matrix. (B-D) collagen density as a function of time expressed as the intensity of the SHG signal for the CC, CH, ACR and A H R groups (0, 5, 1 5 and 30 weeks on their respective diets). (C) Col lagen density at the 30 week time point for all six groups. Knocking out Granzyme B restores the SHG signal intensity that is lost in the HFD-fed apoE-KO mice. Error bars represent the mean ± SEM . *P<0.05 vs CC; **P<0.0 \ vs CC;†P<0.05 vs AHR;†† 3 <0.0 1 vs AH R . Scale bars = 80 μ m.

To further examine the role of Granzyme B in the observed loss of collagcn density, DKO mice skin was also examined using SHG a fter being fed a chow or HFD for 30 weeks as this was the time point where the most extreme di fferences were observed. As mentioned, at the 30 week ti me point only the H FD-fed apoE-KO mice exhibited significantly decreased collagen density in the sk in compared to the chow-fed control group as shown by the decreased SHG signal (Fig. 1 3 E) suggesting apoE deficiency combined with a HFD result in a loss of ski n collagen density. Both groups of DKO mice on either diet demonstrated a signi ficant increase i n skin col lagen density when compared to the HFD-fed apoE-KO group, deonsl rat ing that G ranzyme B plays a role in the intrinsic aging process in the skin by facil itating the age-dependant disorganization of dermal collagen (Fig. 1 3 E). Grari2yme B cleaves decorin and is prcscnl in areas of decorin degradation. Referring to Figure 14, the addition of Granzymc B results in degradation and loss of full-length glycosylated decorin by 24 h. This is prevented when the potent Granzyme B inhibitor, compound 20, is included. (Figure 14A; Asterisk = ful l length protein).

Decorin immunostaining (dark gray) in the non-diseased skin from wild type chow

(WT), apoE-KO high fat (ApoE-fat) and D O high fat (D O-fat) groups. Arrows point to increased decorin near the dermal-epidermal junction (scale bars = 50 μηι) (Figure 14B). Decorin immunostaining (dark gray) in diseased skin from ApoE-fat and DKO-fat groups (scale bars = 50 μιη) (Figure 14C). Granzyme B immunostaining (dark gray) in diseased skin from an ApoE-fat mouse. E denotes "epidermis" and D denotes "dermis". Arrow points to the area of the dermal-epidermal junction (scale ba rs = 1 00 μιη) (Figure 14D). Diseased skin section from ApoE- fat mice dual stained for mast cel ls and Granzyme B. Arrows point to Granzyme B (l ight gray) inside masl cells (dark gray) (scale bars = 25 μτη) (Figure 14E).

Discussion of results in the foregoing Example: I n the present study, it was demonstrated that a HFD has a considerable e ffect on skin aging in apoE- KO mice. Not only does it affect the frequency of inflammatory ski n d isease as the mice age, but also results in a frail, thinned skin state along with signi ficant age-related alterations in the structural organization of the ECM . We also demonstrate that the serine protease, Granzyme , B, plays an important role in aging and disease of the sk i n through remodelling of key ECM proteins and proteoglycans. When apoE- KO mice were fed a HFD for 30 weeks, they demonstrated frailty and increased morbidi ty compared to the wild type controls (Fig. 8). This was also observed histologically in the form of increased skin lesions and skin thinning along with a loss of subcutaneous adipose tissue (Fig. 9 and 10). Although xanthoma development occurred regardless of diet in apoE- KO mice, a HFD was required to observe certain intrinsic aging phenotypes such as skin thinning and loss of collagen density (Fig. 10 and 1 3). S imi lar to the onset and severity of skin lesions in apoE-KO mice, a HFD accelerates these aging characteristics and chow-fed apoE-KO mice also display these phenotypes upon examination at a later time point beyond 30 weeks.

In this study, apoE-KO mice fed a regular chow or HFD showed a decrease in adipose layer thickness at 30 weeks while overal l skin thickness in the H FD-fed apoE- KO mouse skin decreased in from the 1 5 to 30 week time point (Fig. 10). This demonstrates that Granzyme B deficiency helps to rescue this skin thinning phenotype in part by preserving dermal thickness (Fig. 10). Analysis of collagen and elastin in the xanthoma skin samples demonstrated considerable remodell ing of collagen along with abnormal elastin deposits resembling solar clastosis often seen in photoaged skin. The presence of age-related changes in the "regular" skin of apoE-KO mice, together with the thickened, remodelled, pro-inflammatory state of the xanthoma skin demonstrate that apoE-KO mice demonstrate features of both intrinsic/chronological skin aging and extrinsic aging similar to photoaging with both resulting in ECM changes that mimic these forms of skin aging in humans.

In this study evidence is provided that the serine protease Granzyme B is expressed in areas of col lagen and decorin degradation and remodell ing in the skin of apoE- .0 mice (Fig. 12). Our results also demonstrate that Granzyme B contributes to the frail/thin skin and lack of dense collagen observed i n aged apoE-KO mice.

Further, the lichenoid expression of Granzyme B observed in the diseased skin samples presents a novel mechanism of lesion formation and ECM degradation.

Lichenoid inflammation is a characteristic feature of several inflammatory skin diseases. The presence of Granzyme B in this area also shows that G ranzyme B is disrupting ECM close to or at the dermal epidermal junction. I ndeed, D O mice demonstrated an apparent increase in decorin staining in the skin near the dermal epidennal junction (see, for e.g., Fig. 12E and F).

In addition to the diseased skin, apoE-KO mice fed a H FD, but not a regular chow diet, demonstrated a signi ficant loss in col lagen density in the dermis as shown by SHG and multi-photon microscopy over a 30 week span in "regular" skin samples (Fig. 13). These experiments were done on fresh thick tissue sections providing a unique look at the collagen content of the skin in 3 D prior to any fixation or processing methods. Subsequent fixation and histological analysis of these skin tissues confirmed that the decrease in collagen density in HFD-fed apoE- KO mice occurred in "regular" non- xanthoma skin and that the decreased SHG signal was not simply due to the presence of xanthoma (data not shown). Staining fixed skin sections with picrosi rius red also demonstrated a more diffuse col lagen pattern in the sk in of apoE-KO mice compared to controls (Fig. 1 I B). Interestingly, we show using DKO mice, that this decrease in collagen density and organization signal may be rescued by blocking Granzyme B activity (see, for e.g. , Fig. 1 1 and 13). In summary, the findings demonstrate that apoE-dcficiency results in an increased pro-inflammatory state in the skin, contributing to ECM remodelling and other age-related changes seen and that a HFD exacerbates these changes through a Granzymc B-mediated mechanism. These findings also demonstrate a novel role for Granzyme B in the skin involving the cleavage of decorin and the remodelling of dermal collagen, a process that has major implications in ECM structure, skin fragility in aging and disease, and wound repair.

EXAMPLE 5. Inhibition of Granzyme B (Granzyme B) using a specific small molecule inhibitor (Willoughby20) inhibits bctaglycan cleavage.

In this Example, inhibition of Granzymc B (Granzyme B) is demonstrated using a specific small molecule inhibitor (Willoughby20) inhibits bctaglycan cleavage. As shown in Figure 15, incubations were performed at room temperature for 24 hours in a total reaction volume of 30 μ I. Samples were run on a 10% gel, imaged with Ponceau stain and scanned. As shown in Figure 15, the asterisk depicts a full length protein; the arrow depicts cleavage fragments.

EXAMPLE 6. Inhibition of Granzymc B (Granzyme B) using a specific small molecule inhibitor (Willoughby20) inhibits the release of proteoglycan-sequestcrcd TGF- β .

In this Example, 20ug/ml bctaglycan was coaled onto 48 well plates and incubated with l Ong of TGF- β . Excess TGF- β was washed off the plate and betaglycan/TGF- β complexes were incubated with Granzyme B +/- inhibitors for 24h at RT. Supernatants (containing released TGF- β ) were col lected and Western blotted for TGF- β . There is little non-specific dissociation of TGF- β into supernatants in the absence of Granzyme B (2; see Figure 16). TGF- β is released by Granzyme B after 24h of incubation (4; see Figure 16). Release is inhibited by Willoughby20 (specific Granzyme B inhibitor) (5; see Figure 16) and partially inhibited by DCI (non-specific serine protease inhibitor) (6; sec Figure 16).

EXAMPLE 7. Inhibition of Granzymc B (Granzymc B) using a specific small molecule inhibitor (Willoughby20) inhibits decorin cleavage. In this Example, incubations were performed al T for 24h in a total reaction volume of 30ul. Samples were run on a 10% gel and imaged by Coomassie Blue stain. With reference to Figure 17, the asterisk = full length protein.

Table A.

EXAMPLE 8. Identification of small molecule inhibitors of G ra nzyme B

(Granzyme B).

Small molecule libraries (ZINC - Irwin JJ and Shoichct BK, 2005. J. Chem Inf ooW 4591 : 177- 1 82; NCI - Voigt. J . . ei al. J. Chem. Inf. Compui. Sci. 2001 , 41 , 702-712) were screened in silico for candidate Granzyme B inhibitory compounds.

Several candidate small molecule inhibitors were identi fied and subjected to an in vitro Granzyme B inhibition assay. More speci fical ly, a continuous colormetric assay for Granzyme B activity was carried out with the substrate Ac- l EPD-pNA. Briefly, 8ug/ml Granzyme B, 20 μ M substrate, and increasing concentrations of the inhibitor of interest was incubated in a final reaction volume o 50ul.The reaction bu ffer consisted of 50mM HEPES pH 7.5, 10% sucrose, 0. 1 % CHAPS and 5mM DTT and reactions were carried out at 37°C. pNA release and Granzyme B inhibition was monitored at 405nm on a Tecan Safire microplate reader. Granzyme B was used in the assay at a concentration of 4 μg/ml (0. 145 μΜ), estimated to be about 80,000 fold h igher than what would be observed in a subject; our findings have indicated that pathological levels of Gr B are above 50 pg ml, to about 150 pg/m. The results are shown in Tabic B.

Table B. Summary of Granzymc H I nhibitor Data.

IC50 [inhibition] - 195 μ M [exhibited low inhibition]

Structure

Target Human Granzyme B

Identifier ZINC05723499; NCI 6 1 235

IC50 [inhibition] ~224 μ M [exhibited low inhibition]

Structure

Target Human Granzyme B

Identifier ZINC05723646; NCI 64201 7

IC50 [inhibition] -250 μ M [exhibited low inhi bition]

Structure

Target Human Granzyme B

Identifier ZINC05398428; NCI 641230

IC50 [inhibition] ~255 μ [exhibited low inhibition]

Structure

Target Human Granzyme B

Identifier ZINC05723503 ; NCI 64 1 236

IC50 [inhibition] -270 μ [exhibited low inhibition]

Structure

Target Human Granzyme B Identifier ZINC05723446; NCI 640985

IC50 [inhibition] -270 μ [exhibited low inhibition]

Structure

Target Human Granzyme B

Identifier ZINC053 1 72 1 6; NCI 61 8792

IC50 [inhibition] ~50 μ M [exhibited high inhibilion]

Structure

Target Human Granzyme B

Identifier ZINC053 1 5460; NCI 630295

IC50 [inhibition] ~ 100 μ M [exhibited high inh ibition]

Structure

Target Human Granzyme B

Identifier ZINC053 I 6859; NCI 61 8802

IC5 0 [inhibition] ~250 μ M [exhibited low inhibil ion]

Structure

Target Human Granzyme B

Identifier ZINC05605947; NCI 623744

IC5 0 [inhibition] -320 μ M [exhibited low inhibition]

As detailed herein and as detailed in Tables A and 13 herein, candidate inhibitors, and IC50 concentration obtained from the i nhibition assay arc set out below. Compounds NCI 644752, NCI 644777, ZINC053 1 72 1 6, NCI 630295 demonstrated an IC 50 of about 100 μΜ or less ("High inhibition"); compounds NCI 64 1 248, NCI 64 1 235, NCI 64201 7, NCI 641230, NCI 641236, NCI 640985, NCI 6 1 8802, NCI 623744 demonstrated an 1C 50 of about 320 μΜ or less ("Low inhibition").

EXAMPLE 9. Small molecule inhibitors in hibit G ranzyme cleavage of decorin.

Details of this Example are shown representatively in Figure 1 8. More specifically, Figure 18 demonstrates that inhibit ion of G ranzyme B (Granzyme B) using small molecule inhibitors inhibits ECM cleavage. As detai led therei n, the asterisk marks the full length protein. The arrow demonstrates cleavage fragments and the star denotes the full length protein.

More specifically, as it relates to Figure 1 8, to synthesize endogenous ECM, human coronary artery smooth muscle cel ls were seeded in 6 well plates and incubated at confluency for 7 days in serum starvation media (SMGM + 0.2% FBS). Cells were removed from the plates by incubation with 0.25M Ammon ium Hydroxide for 20min. This treatment removes cells while leaving the surrounding extracellular matrix intact. Subsequently, Ι ΟΟηΜ Granzyme B and the inhibitor of interest were incubated on the endogenous ECM for 24h. Supernatants from the plates (containing cleavage fragments) were run on an SDS-PAGE gel and Western blotted for fibroneclin or decorin. DMSO solvent control was not included due to redundancy. Asterisk = ful l length protein; Arrow = cleavage fragments, star denotes ful l length. EXAMPLE 10. Inhibition of Granzyme B (G ranzyme B) usi ng a small molecule inhibitor inhibits ECM cleavage.

As shown in Figure 19 herein, to synthesize endogenous ECM , human coronary artery smooth muscle cells were seeded in 6 well plaics and incubated at confluency for 7 days in serum starvation media (SMGM + 0.2% FBS). Cel ls were removed from the plates by incubation with 0.25M Ammonium Hydroxide for 20min. This treatment removes cells while leaving the surrounding extracellular matrix intact. Subsequently, Ι ΟΟηΜ Granzyme B and the inhibitor of interest were incubated on the endogenous ECM for 24h. Supernatants from the plates (containing cleavage fragments) were run on an SDS-PAGE gel and Western blotted for fibroneciin or decorin. DMSO solvent control was not included due to redundancy. Asterisk = fu l l length protein ; A rrow = cleavage fragments, star denotes full length.

EXAMPLE 11. Inhibition of Granzyme B (G ranzyme B) usi ng NC I644777 i nhibits betaglycan cleavage.

As shown in Figure 20 herein, incubations were performed at RT for h in a total reaction volume of 30μ1. Samples were run on a 10% gel, imaged with Ponceau stain and scanned. Compound NCI644752 docs not inhibit G ranzyme B cleavage of betaglycan at Ι ΟΟμΜ (but was previously shown to be effective at 1 50μ Μ agai nst decorin cleavage). Compound NCI644777 partially inhibits Granzyme B at 50μ Μ and completely at Ι ΟΟμΜ. DMSO vehicle control was not included due to redundancy. Asterisk = full length protein; Arrow = cleavage fragments. Obstruction in Granzyme B Betaglycan lane affecting first 2 cleavage fragments.

EXAMPLE 12. Inhibition of Granzyme B (G ranzyme B) using Willoughby 20 inhibits fibronectin cleavage.

As shown in Figure 2 1 A and B herein, incubations as desc ribed herein were performed with fibronectin (FN) and Granzyme B, both in the absence of inhibitor Willoughby 20 and in the presence of inhibitor Wil loughby 20. Compound Willoughby 20 inhibits Granzyme B cleavage of FN at 3. 1 2 nM . Furthermore, Figure 2 1 A shows the results of HMVEC addition (Human M icrovascular Endothelial Cel ls) and subsequent cell count and shows that Granzyme B cleavage of fibronectin (FN) reduces EC adhesion to FN dose dependently. Figure 2 1 B shows Granzyme B speci fical ly and dose dependently cleaves fibronectin resulting in the release of fibroneciin fragments. EXAMPLE 13: Inhibition of G ranzyme B prevents dccorin degradation in chronic wounds in vivo.

As depicted in Figure 23, decorin distribution is impacted by serpina3n in closed wound tissue. Seven week old wild type C57BL/6 m ice were given a 1 cm diameter ful l thickness wound on their backs. Mice were then given either saline (upper panels) or the Granzyme B inhibitor, serpina3n (lower panels), by applying 60 μ Ι of the appropriate solution directly onto the wound immediately following the wounding procedure.

Wounds were allowed to heal for 16 days, at which point mice were sacri ficed and the closed wound tissue harvested. Inimunohislochcmisiry for dccorin revealed differences in the pattern of decorin distribution in the newly formed dermis between the two groups. Saline-treated mice demonstrated gradual increase in decorin close to the dermal-epidermal junction. Serpina3n-trcatcd mice demonstrated intense decorin staining near the dermal-epidermal junction but also demonstrated intense dccorin staining deeper into the dermis, demonstrating that Granzyme B inhibition prevents excessive decorin degradation in acute or chronic wounds. Increased decorin throughout the newly formed dermis thus provides more organized col lagen, less scarring and increased tensile strength.

While specific embodiments of the invention have been described and i llustrated, such embodiments should be considered il lustrative of the invention only and not as limiting the invention as construed in accordance with the accompanying claims. Other features and advantages of the invention wil l be apparent from the fol lowing description of the drawings and the invention, and from the claims.

INFORMAL SEQUENCE LISTINGS

Asp 51 Thr-Thr-Leu-Leu-Asp <biglycan cleavage sequences (SEQ ID NO:l)

Asp 558 Ala-Ser- Leu- Phe-Thr <betaglycan cleavage sequences (SEQ ID NO : 2 )

Asp J1 Glu-Ala-Ser-Gly <decorin cleavage sequences (SEQ ID NO : 3 )

Asp 6s Leu-Gly- Asp- lys <decorin cleavage sequences (SEQ ID NO : 1 )

Asp 82 Thr-Thr-Leu-Leu- Asp cdecorin cleavage sequence >( SEQ ID NO: 5)

Asp 261 Asn-Gly-Ser-Leu-Ala <decorin cleavage sequences (SEQ ID NO:6) Human Granzyme B amino acid sequence;

MQPI LLLLAFLLLPRADAGEI IGGHEAKPHSRPYNIAYLMIWDQ SLKRCGGFLIRDDFVLTAAHCWGSSI VTLGAH I EQEPTQQFI PV RPI PHPAYNP FSNDIMLLQLERKAKRTRAVQPLRLPS AOVKPGOTCSVAGWGQTAPLG HSH TLQEV MTVQEDRKCESDLRHYYDSTI ELCVGDPEI KKTSF GDSGGPLVCN VAQGI VSYGRNNGMPPRACTKVSSFV HWIKKTM RY (SEQ ID NO : 7 )

Human Granzyme B nucleotide sequence:

CCAAGAGCTAAAAGAGAGCAAGGAGGAAACAACAGCAGCTCCAACCAGGGCAGCCTTCCT GAGAAGATGCAACCAATCC TGCTTCTGCTGGCCTTCCTCCTGCTGCCCAGGGCAGATGCAGGGGAGATCATCGGGGGAC ATGAGGCCAAGCCCCACTC CCGCCCCTACATGGCTTATCTTATGATCTGGGATCAGAAGTCTCTGAAGAGGTGCGGTGG CTTCCTGATACGAGACGAC TTCGTGCTGACAGCTGCTCACTGTTGGGGAAGCTCCATAAATGTCACCTTGGGGGCCCAC AATATCAAAGAACAGGAGC CGACCCAGCAGTTTATCCCTGTGAAAAGACCCATCCCCCATCCAGCCTATAATCCTAAGA ACTTCTCCAACGACATCAT GCTACTGCAGCTGGAGAGAAAGGCCAAGCGGACCAGAGCTGTGCAGCCCCTCAGGCTACC TAGCAACAAGGCCCAGGTG AAGCCAGGGCAGACATGCAGTGTGGCCGGCTGGGGGCAGACGGCCCCCCTGGGAAAACAC TCACACACACTACAAGAGG TGAAGATGACAGTGCAGGAAGATCGAAAGTGCGAATCTGACTTACGCCATTATTACGACA GTACCATTGAGTTGTGCGT GGGGGACCCAGAGATTAAAAAGACTTCCTTTAAGGGGGACTCTGGAGGCCCTCTTGTGTG TAACAAGGTGGCCCAGGGC ATTGTCTCCTATGGACGAAACAATGGCATGCCTCCACGAGCCTGCACCAAAGTCTCAAGC TTTGTACACTGGATAAAGA AAACCATGAAACGCTACTAACTACAGGAAGCAAACTAAGCCCCCGCTGTAATGAAACACC TTCTCTGGAGCCAAGTCCA GATTTACACTGGGAGAGGTGCCAGCAACTGAATAAATACCTCTTAGCTGAGTGGAAAAAA AAAAAAAAAAAA (SEQ ID NO: 8)

Huamn Serpin B9

METLSNASGTFAI RLL I LCQD PSHNVFCSPVSISSALAMVLLGA GNTATQ AQALSLNTEEDI HRAFQSLLTEVNK AGTQYLLRTANRLFGEKTCQFLSTF ESCLQFYHAELKELSFIRAAEESRKLHINTWVSK TEG IEELLPGSSIDAETR LVLVNAIYF G WNEPFDETYTREMPFKINQEEORPVQMMYQEATF LAHVGEVRAQLLELPYARKELSLLVLLPDDGV ELSTVE SLTFE LTAWTKPDCM STEVEVLLP FKLQEDYDMESVL HLGIVDAFQQGKADLSAMSAERDLCLSKFVH KS VEVNEEGTEAAAASSCFWAECCMESGPRFGADHPFLFFI RHNRANS I L CG FSSP (SEQ ID NO: 9) Huamn Serpin B9

GCGGGAGTCCGCGGCGAGCGCAGCAGCAGGGCCGGGTCCTGCGCCTCGGGGGTCGGCGTC CAGGCTCGGA GCGCGGCACGGAGACGGCGGCAGCGCTGGACTAGGTGGCAGGCCCTGCATCATGGAAACT CTTTCTAATG CAAGTGGTACTTTTGCCATACGCCTTTTAAAGATACTGTGTCAAGATAACCCTTCGCACA ACGTGTTCTG TTCTCCTGTGAGCATCTCCTCTGCCCTGGCCATGGTTCTCCTAGGGGCAAAGGGAAACAC CGCAACCCAG ATGGCCCAGGCACTGTCTTTAAACACAGAGGAAGACATTCATCGGGCTTTCCAGTCGCTT CTCACTGAAG TGAACAAGGCTGGCACACAGTACCTGCTGAGAACGGCCAACAGGCTCTTTGGAGAGAAAA CTTGTCAGTT CCTCTCAACGTTTAAGGAATCCTGTCTTCAATTCTACCATGCTGAGCTGAAGGAGCTTTC CTTTATCAGA GCTGCAGAAGAGTCCAGGAAACACATCAACACCTGGGTCTCAAAAAAGACCGAAGGTAAA ATTGAAGAGT TGTTGCCGGGTAGCTCAATTGATGCAGAAACCAGGCTGGTTCTTGTCAATGCCATCTACT TCAAAGGAAA GTGGAATGAACCGTTTGACGAAACATACACAAGGGAAATGCCCTTTAAAATAAACCAGGA GGAGCAAAGG CCAGTGCAGATGATGTATCAGGAGGCCACGTTTAAGCTCGCCCACGTGGGCGAGGTGCGC GCGCAGCTGC GGAGCTGCCCTACGCCAGGAAGGAGCTGAGCCTGCTGGTGCTGCTGCCTGACGACGGCGT GGAGCTCAG CACGGTGGAAAAAAGTCTCACTTTTGAGAAACTCACAGCCTGGACCAAGCCAGACTGTAT GAAGAGTACT GAGGTTGAAGTTCTCCTTCCAAAATTTAAACTACAAGAGGATTATGACATGGAATCTGTG CTTCGGCATT TGGGAATTGTTGATGCCTTCCAACAGGGCAAGGCTGACTTGTCGGCAATCTCAGCGGAGA GAGACCTGTG TCTGTCCAAGTTCGTGCACAAGAGTTTTGTGGAGGTGAATGAAGAAGGCACCGAGGCAGC GGCAGCGTCG AGCTGCTTTGTAGTTGCAGAGTGCTGCATGGAATCTGGCCCCAGGTTCTGTGCTGACCAC CCTTTCCTTT TCTTCATCAGGCACAACAGAGCCAACAGCATTCTGTTCTGTGGCAGGTTCTCATCGCCAT AAAGGGTGCA CTTACCGTGCACTCGGCCATTTCCCTCTTCCTGTGTCCCCAGATCCCCACTACAGCTCCA AGAGGATGGG CCTAGAAAGCCAAGTGCAAAGATGAGGGCAGATTCTTTACCTGTCTGCCCTCATGATTTG CCAGCATGAA TTCATGATGCTCCACACTCGCTTATGCTACTTAATCAGAATCTTGAGAAAATAGACCATA ATGATTCCCT GTTGTATTAAAATTGCAGTCCAAATCCCATAGGATGGCAAGCAAAGTTCTTCTAGAATTC CACATGCAAT TCACTCTGGCGACCCTGTGCTTTCCTGACACTGCGAATACATTCCTTAACCCGCTGCCTC AGTGGTAATA AATGGTGCTAGATATTGCTACTATTTTATAGATTTCCTGGTGCTTAGCCTTATAAAAAAG GTTGTAAAAT GTACATTTATATTTTATCTTTTTTTTTTTTTTTTTTCTGAGACGCAGTCTGGCTCTCTGT CGCCCAGGCT GGAGTGCAGTGGCTCGATCTCGGCTCACTGCAAGCTCCGCCTCCCGGGTTCACGCCATTC TCCTGCCTCA GCCTCCCGAGTAGCTGGGACTACAGGCGCCCGCCACCACGCCCGGCTAATTTTTTGTATT TTTAGTAGAG ACGGGGTTTCACCGTGTTAGCCAGGATGGTGTCGATCTCCTGACCTCGTGATCCACCCGC CTCGGCCTCC CAAAGTGCTGGGATTACAGGCTTGAGCCACCGCGCCCGGCTATATTTTATCTTTTATCTT TTTCTTTGAC ATTTACCAATCACCAAGCATGCACCAAACACTGCTTTAGGCACTGGGGACACAAAGGGGA CAGAGCCATC CTCCTTTGACACCTGGTCTTCAGTTCTGTGCCCAACGTATATAGTTTTGACAATGACCAG GTTGGACTGT TTAATGTCTTTCAACTTACCACGTAATCCTCTTGTAGGGATCACATCTT'I'CTTTATGA TATTGTATTTCT

CTACCTCTAACAGTAAAAATTCCATTCAACCCTTAAAGCTCACTTCAAATTCTTCTT TGAGAAGTTTTTC CTTTCTCCGCAACCAGATGTACATATTTGAACTCTCTTTGTACTTGGAGGGCACTTCTTT CGTGGTAGTT CTTTTATTTTTATTAATCTCTGTATCCTTAGATAGTCCTCCAACAACCAAAGGTTGGGAC TCTGTCTTAC ATATCTGGGTGCCCCTCATAGTGCAGTAATAAGTAAGTTGATTATATACGAGCTATGTAA CTTATATTTT TTAATGGTTGGATATCACTGAGTTTTTTTTTTTAAGAATTTTTTTATTGAGGTAAACTTC ACATAACATA AAATTAACTATTTTAAAGTGAGAAGTTCAGTGCCACTTAGTATTGTTAACAATGTTGCAT AACCACCACC TTTATTTAAAGTTCCAAAAAAAATGTTCTCCTCTAAAAGGAAACCCCATCCCATTAAGCA GATACTCTCC

ATTCCTTCCTTCCTCCAGCCCCCAGCAACCACCAATCTGCTTTCTGTCTCTATGGAT TTATCTATTCTTG CTATTTTATATAAATTGAAT GTATGAGACCTTTTGTGTCTGGCTTCTTTCACTTAGTACAAGTTTTTGA GATTTATTTACATAGTAGCATGTATCAACACTTCATTTTTATGGCCAAATAAAATTGTAT TATGTGTTTA TAGCACAATTTATTTATCCACTCATTCATTGATGGACTTTGGGTTGTTTCTGACTTTTGG CTATTGGGAA TAGTGCTGCTATGAATGTTTGTGTACCTGTATTTGTTTGAATGCCTATTTTGCATTCTCT TGGGTATATA TCTAGGAGTGGAACTGCTGGGTCATATGTTAATTCTATGTTTAGCTTTTTGAGGAACAGA CAAACTGTTT TCCACAGCAGTTGAACCATTCCACATTCCCACCAGCAATGTATGAGAATTCCAATTTCTG TCCACTTCCT CACCAACACTTATTATTTTCCTTTTCCTTTTTTTAAAAAAAATAAGTTATGGCCATCTTA GTGGGTGTGA AGTGGTATCTCATTGTGTTTTTTATTTGCATTTCCTATGTAATGAGCTAGAAACTAAAGT ACAAACTAGA TGGGACATCCAGTCCCTTTGATAGATAATGCTGAGTAAAAAATGAGATGAAAGACATTTG TTTGTTTTTA GAACACGAGTGACAGTTTGTTAAAAAGCTTTAGAGGAGGAATGAAAACAAAGTGAAGTAC ACTTAGAAAA GGGCCAAGTGGACATCTTGGATGTCAAGTGCCTAGTTCAGTATCTTTTTTTTTTTTTTTT TTTTTTTTGA GACAGTGCCTCACTCTGTCACCCAGGCTGGAGTGTAGTGGCATGATCTGGGCTCACTGCA ACCTCCTCCT CCTGGATTCAAGCAATTCTCTTGCTTCAGCCTCCCAAGTAGCTGAGACTACAAGCACCCA CCATCACACC CAGCTAATTTTGTATT TTCAGTAGAGACGGGGTTTCGCCACATTGGCCGTGTTGGTCTTGAACTCCTGG CCTCAAGCGATCCGCCTACCTCAGCCTCCCAAAGTGCTAGGATTACAGGCATAAGCCACT GAGCCCAGCC CTAGTTCAGTATCTTTTATGTAAATTACAAACATCTGCAACATTATGTATCATATGCAGA TACTTATTGC ATTTCTTTTATTAGTGGTGAAAGTGTTCTATGCATTTATTGGCTCTTGAATTTCCTCATC TATGAATTGT CATTCATACACCTACTTTTCTGCTTCGTTTTTACATATGTCTTTGCCTATTAAAGATATT ATCCCTCTGT TTTATATTTTCTCTCATTCTTGTATTGCCTTTTAAATTTTGTTATGATGTTTCATTAATA AACAGTGTTT TGT TTCCTCTATAAAAAAAAAAAAAAAA ( SEQ ID NO : 10 )

Fragment f rom Huamn Serpi n B9

GTEAAASSCFVAECCMESG ( SEQ . I D NO : 11 )

Human SerpinA3

ERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTQENQDRGTHVDLGLASA VDFAFSLYKQLVL APD NVI FSPLS I STAXAFLSLGAH TTLTEI LKGLKFNLTETSEAEI HQSFQHLLRTLNQSSDELQLSMGNAMFVKEQLSLLDRFTEDA RLYGSEAFATDFQDSAAA LINDYVKNGTRGKITDLI DLDSQT MVLVNYI FF A WEMPFDPQDTHQSR FYLSKKK WVMVPMMSLHHL I PYFRDEELSCTWELKYTG ASALFI LPDQD MEEVEAMLLPETLKRWRDSLEFREIGELYLP F SI SRDYNLNDI LLQLGI EEAFTSKADLSG ITGARNLAVSQWHKAVLDVFEEGTEASAATAV ITLLSALVETRTI R F NRPFLMI I VPTDTQNI FF SKVTNPKQA ( SEQ I D NO : 12 )

Human SerpinA3

ATTCATGAAAATCCACTACTCCAGACAGACGGCTTTGGAATCCACCAGCTACATCCAGCT CCCTGAGGCA GAGTTGAGAATGGAGAGAATGTTACCTCTCCTGGCTCTGGGGCTCTTGGCGGCTGGGTTC TGCCCTGCTG TCCTCTGCCACCCTAACAGCCCACTTGACGAGGAGAATCTGACCCAGGAGAACCAAGACC GAGGGACACA CGTGGACCTCGGATTAGCCTCCGCCAACGTGGACTTCGCTTTCAGCCTGTACAAGCAGTT AGTCCTGAAG GCCCCTGATAAGAATGTCATCTTCTCCCCACTGAGCATCTCCACCGCCTTGGCCTTCCTG TCTCTGGGGG CCCATAATACCACCCTGACAGAGATTCTCAAAGGCCTCAAGTTCAACCTCACGGAGACTT CTGAGGCAGA AATTCACCAGAGCTTCCAGCACCTCCTGCGCACCCTCAATCAGTCCAGCGATGAGCTGCA GCTGAGTATG GGAAATGCCATGTTTGTCAAAGAGCAACTCAGTCTGCTGGACAGGTTCACGGAGGATGCC AAGAGGCTGT ATGGCTCCGAGGCCTTTGCCACTGACTTTCAGGACTCAGCTGCAGCTAAGAAGCTCATCA ACGACTACGT GAAGAATGGAACTAGGGGGAAAATCACAGATCTGATCAAGGACCTTGACTCGCAGACAAT GATGGTCCTG GTGAATTACATCTTCTTTAAAGCCAAATGGGAGATGCCCTTTGACCCCCAAGATACTCAT CAGTCAAGGT TCTACTTGAGCAAGAAAAAGTGGGTAATGGTGCCCATGATGAGTTTGCATCACCTGACTA TACCTTACTT CCGGGACGAGGAGCTGTCCTGCACCGTGGTGGAGCTGAAGTACACAGGCAATGCCAGCGC ACTCTTCATC CTCCCTGATCAAGACAAGATGGAGGAAGTGGAAGCCATGCTGCTCCCAGAGACCCTGAAG CGGTGGAGAG ACTCTCTGGAGTTCAGAGAGATAGGTGAGCTCTACCTGCCAAAGTTTTCCATCTCGAGGG ACTATAACCT GAACGACATACTTCTCCAGCTGGGCATTGAGGAAGCCTTCACCAGCAAGGCTGACCTGTC AGGGATCACA GGGGCCAGGAACCTAGCAGTCTCCCAGGTGGTCCATAAGGCTGTGCT'I'GA GTATTTGAGGAGGGCACAG

AAGCATCTGCTGCCACAGCAGTCAAAATCACCCTCCTTTCTGCATTAGTGGAGACAA GGACCATTGTGCG TTTCAACAGGCCCTTCCTGATGATCATTGTCCCTACAGACACCCAGAACATCTTCTTCAT GAGCAAAGTC ACCAATCCCAAGCAAGCCTAGAGCTTGCCATCAAGCAGTGGGGCTCTCAGTAAGGAACTT GGAATGCAAG CTGGATGCCTGGGTCTCTGGGCACAGCCTGGCCCCTGTGCACCGAGTGGCCATGGCATGT GTGGCCCTGT CTGCTTATCCTTGGAAGGTGACAGCGATTCCCTGTGTAGCTCTCACATGCACAGGGGCCC ATGGACTCTT CAGTCTGGAGGGTCCTGGGCCTCCTGACAGCAATAAATAATTTCGTTGGAAAAAAAAAAA AAAAAAAAAA AAAAAAAAAAAAAAAAAAA ( SEQ I D NO : 13 )

Cowpox CrmA

MDI FREIASSMKGENVFI SPPS I SSVLTI LYYGANGSTAEQLSKYVEKEAD N DDI SFKSMN VYGRYSAVF DSFLR IGDNFQTVDFTDCRTVDAIN CVDI FTEGKINPLLDE LSPDTCLIoAI SAVY FKA V-JLMPFEKEFTSDYPFYVSPTEM VDVSMMSMYGEAFNHASVKESFG FS I I ELPYVGDTSMWI L DN I DGLESI EQ LTDTNF WCDSMDAMFI D H I PK FKVTGSY LVDALVKLGLTEVFGSTGDYSNMCNSDVSVDAMI H TY I DVNEEYTEAAAATCALVADCASTVTNE FCADH PFI YVIRHVDGKI LFVGRYCS T ( SEQ I D NO : 11 )

Cowpox CrmA TCCATGGAAGAACGAAAGTAGTATAAAAGTAATAAAACAAAAAAAAGAATATAAAAAATT TATAGCCACT TTC TTGAGGACTGTTT CCTGAAGGAAATGAACCTCTGGAATTAGTTAGATATATAGAATTAGTATACA CGCTAGATTATTCTCAAACTCCTAATTATGACAGACTACGTAGACTGTTTATACAAGATT GAAAATATAT TTC TTTTATTGAGTGGTGGTAGTTACGGATATCTAATATTAATATTAGACTATCTCTATCGTC ACACAA CAAAATCGATTGCCATGGATATCTTCAGGGAAATCGCATCTTCTATGAAAGGAGAGAATG TATTCATTTC TCCACCGTCAATCTCGTCAGTATTGACAATACTGTATTATGGAGCTAATGGATCCACTGC TGAACAGCTA TCAAAATATGTAGAAAAGGAGGCGGACAAGAATAAGGATGATATCTCATTCAAGTCCATG AATAAAGTAT ATGGGCGATATTCTGCAGTGTTTAAAGATTCCTTTTTGAGAAAAATTGGAGATAATTTCC AAACTGTTGA CTTCACTGATTGTCGCACTGTAGATGCGATCAACAAGTGTGTTGATATCTTCACTGAGGG GAAAATTAAT CCACTATTGGATGAACCATTGTCTCCAGATACCTGTCTCCTAGCAATTAGTGCCGTATAC TTTAAAGCAA AATGGTTGATGCCATTTGAAAAGGAATTTACCAGTGATTATCCCTTTTACGTATCTCCAA CGGAAATGGT AGATGTAAGTATGATGTCTATGTACGGCGAGGCATTTAATCACGCATCTGTAAAAGAATC ATTCGGCAAC TTTTCAATCATAGAACTGCCATATGTTGGAGATACTAGTATGGTGGTAATTCTTCCAGAC AATATTGATG GACTAGAATCCATAGAACAAAATCTAACAGATACAAATTTTAAGAAATGGTGTGACTCTA TGGATGCTAT GTTTATCGATGTGCACATTCCCAAGTTTAAGGTAACAGGCTCGTATAATCTGGTGGATGC GCTAGTAAAG TTGGGACTGACAGAGGTGTTCGGTTCAACTGGAGATTATAGCAATATGTGTAATTCAGAT GTGAGTGTCG ACGCTATGATCCACAAAACGTATATAGATGTCAATGAAGAGTATACAGAAGCAGCTGCAG CAACTTGTGC GCTGGTGGCAGACTGTGCATCAACAGTTACAAATGAGTTCTGTGCAGATCATCCGTTCAT CTATGTGATT AGGCATGTCGATGGCAAAATTCTTTTCGTTGGTAGATATTGCTCTCCAACAACTAATTAA ATCACATTCT TAATATTAGAATATTAGAATATTATATAGTTAAGATTTTTACTAATTGGTTAACCATTTT TTTAAAAAAA

TAGAAAAAAAACATGTTATATTAGCGAGGGTCGTTATTCTTCCAATTGCAATTGGTA AGATGACGGCC ( SEQ I D NO : 15 )

Human Serp2

MVAKQRI RMANE HSKNITQRGNVA TLR PQEEKYPVG PWLLALFVFWCGSA I FQI I OS I RMGM ( SEQ I D NO : 16 )

Human Serp2

GCCTCTCTCTGGAGTCGGCTAGCCGGGGCTCGGGGAGCGGGGTGCGCAGGGCTCGGGGCC ACGCCTTGCC ACCTGCAGCGCCCGGGTGGGCCGCGGGGGCCTCGGCGGGACGCGCTCGGCCCTGTCGCAG GAGCTAACGC AGGGGGAATCCTTGCAGGTGGGAGCATTTCAGAGCGCACAAGCCATGGTGGCCAAACAGC GGATCCGGAT GGCTAACGAGAAGCACAGCAAAAACATCACCCAGAGGGGGAACGTAGCCAAAACCCTGAG GCCGCAAGAG GAGAAATATCCTGTGGGACCATGGCTGTTGGCACTGTTTGTTTTTGTTGTCTGTGGCTCA GCTATCTTTC AGATCATTCAGAGCATAAGGATGGGCATGTGAGAAAGCCAGGGATTTGACACCACCTCCC TCCCACTGGA GGCGGGAGGACAACGGAAGCGGTCAGCCAGTTTCTGCGGGAAACAAGCAGGCCACACGGA ATAGAAAAAA ACGCTCCCCCACTTGTTCCCTGATCACTTCATCGTGGATGTCAGACCAAATTGCCTTCTC ACAGGACATC TTGGTGCATCCGCGTTCTCAAGCGGAAAGGACATTTTGCTTTTCTGTTGGCAGGATTAGT AGCCACGCGG GTCGTCCGCAGCAGTGCTGTCTTTTTGGTTTTTCCCTTGGTTTCACTAATGCGTGCATGT GGCCCTCTGA ACGATCACTGGTTTACTTTCTATGGATACAATCTCTCCTCCATTGAGAATTGATTTTACA AATAAATGTC TTCGTTCAACCTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAA AA ( SEQ ID NO : 17 )

PI 9 pept ides

VEVNEEGTEAAAASSCFWAECCMESGPRFCADHPFL ( SEQ I D NO : 18 )

VEVNEEGTEAAAASSCFWADCC ESGPR FCADHPFL ( SEQ I D NO : 1 9 )

VEVNEEGTEAAAASSCFWAACCMESGPRFCADHPFL ( S EQ I D NO : : 20 )

VEVNEEGREAAAASSCFWAECC ESGPRFCADHPFL ( SEQ I D NO : : 21 )

CrmA pept ide

IDVNEEYTEAAAATCALVADCASTVTNEFCADHPFI P ( SEQ I D NO : 22 )

REFERENCES

Adams and White. (2004) Eur. J. Pub. Health. 1 (3): 33 1 -34.

Ang e/ o/. (2008) Exp. Gerontol. 43 :615-22.

Bhattacharyya, T. . and Thomas, J. R. (2004) 6:2 1 -5.

Bird el al. ( 1998) Mol. Cell. Biol. 1 8, 6387-6398.

Buzza et al. (2005) J. Biol. Chem. 280:23549-5S. Chamberlain et al. (2010) Am.7. Pathol. 176:1038-1049.

Cho e/a/. (2004) A rterioscler. Thromb. Vase. Biol.24( 12):2245-50.

Creighton, T.E. Proteins: Structure and Molecular Properties, W.H. Freeman & Co. San Francisco, Calif., 1983).

Dallas et al. (1995)7. Cell. Biol. 131:539-549.

Feingold et al. (1995) 7. Invest. Dermatol. 104:246-50.

Fleischmajer et al. (1991) J Struct. Biol. 106:82-90.

Hildebrand et al. (1994) Biochem.7.302(Pt2):527-534.

Hill etal. (1995) J Thorac. Cardiovasc. Surg. 110:1658-1662.

am et al. (2000) Biochi . Biophys. Acta. 1477(l-2):307:23.

ligman, J.H. (1996) Clin. Dermatol. 14:183-95.

Imai et al. (1997) Biochem.7.322(Pt3): 809-814.

Lopez-Casillas el al. (1994). J Cell. Biol. 124:557-568.

Macri et al. (2007) Adv. Drug. Deliv. Rev.59: 1366- 1381.

Mahrus and Craik. (2005) Chemisliy & Biology 12:567-577.

Medema et al. (1997) Eur.7. Immunol.27:3492-3498.

Pan and Clawson (2006) Can: Med. Chem. 13(25): 3083-103.

Patzel (2007) Drug Discov. Today 12(3-4): 139-48.

Peek and Behlke (2007) C rt: Opin. Mol. T e/: 9(2): 110-8.

Prakash et al. (2008) Immunol Cell Biol.87(3):249-54.

Raja etal. (2002) J Biol. Chem.277:49523-49530.

Schiller et al. (2004)7. Dermatol. Sci.35:83-92.

Schonherre/ al. (1995)7 Biol. Chem.270:2776-2783

Sene/a/. (2009) Wound Repair Regen. 17:763-71.

Sune/o/. (1996)7. Biol. Chem.271:27802-27809.

Sun et al. (1997)7. Biol. Chem.272:15434-15441. Taylor AW. (2009) J Leukoc Biol.85:29-33.

Willoughby et al. (2002) Bioorg Med Chem Leu.12:21 7-2200.

Zipfel el al. (2003) Proc. Nail. Acad. Sci. U.S.A. 100:7075-80.