Patent Application
20140056964
The invention relates to compositions, methods, and uses for wound healing.
Wound healing is an intricate process in which an organ, such as the skin, is repaired after injury. In normal skin, the epidermis and dermis form a protective barrier against the external environment. Once this protective barrier is broken, wound healing is set in motion to once again repair the protective 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 pan in weakening or breaking the protective barrier. For example, diseases such as diabetes and psoriasis can disrupt the protective barrier. 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, chemical, heat, or other trauma to the skin (see, for e.g., Sen et al., 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 $25 billion annually, there is a need in the art for the identification of compounds, compositions, and methods to promote wound healing and/or prevent the occurrence or re-occurrence of such wounds.
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, fibromodulin, fibrillin-1, fibrillin-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 not released in the absence of Granzyme B or when Granzyme B was inhibited by DCI. In addition, it has been shown that Granzyme B cleaves the proteoglycan substrates, biglycan and betaglycan at a P1 residue of Asp (biglycan: D91, betaglycan: D558).
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 frailty, 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-KO mice and that Granzyme B deficiency protects against skin thinning due, at least 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 healing by, for example, stimulating collagen 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 limitation, 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 following: Azepino[3,2,1-hi]indole-2-carboxamide, 5-[[(2S,3S)-2-[(2-benzo[b]thien-3-ylacetyl)amino]-3-methyl-1-oxopentyl]amino]-1,2,4,5,6,7-hexahydro-4-oxo-N-(1H-1,2,3-triazol-5-ylmethyl)-, (2S,5S)-(compound 20 from Willoughby et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2197-2200) referred to herein as Willoughby 20 and different batches of Willoughby 20 are referred to herein as JT25102B and JT00025135; Bio-x-IEPDP-(OPh)2; (2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-4-oxo-N-(1H-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-(1H-1,2,3-triazol-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide; (2S,5S)-5-([(2R)-3-methyl-2-pyridin-2-ylbutanoyl]amino)-4-oxo-N-(1H-1,2,3-triazol-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide; (2S,5S)-4-oxo-5-{[N-(phenylacetyl)-L-isoleucyl]amino}-N-(1H-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-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-[[(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, serp-2, ZINC05723764, ZINC05723787, ZINC05316154, ZINC05723499, ZINC05723646, ZINC05398428, ZINC05723503, ZINC05723446, ZINC05317216, ZINC05315460, ZINC05316859, and ZINC05605947. Alternatively, the Granzyme B inhibitor may be selected from one or more of the following: Willoughby 20, NCI 644752, NCI 644777, ZINC05317216, 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 antifungal; an anti-inflammatory; a barrier; a moisturizer; and a sealant. Optionally, the another wound treatment may be selected from one or more of the following: 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; 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. Optionally, the Granzyme B inhibitor may be formulated for topical application in a wound covering, a wound filler, or an implant. Optionally, 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 healing 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. Optionally, 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 small molecules. Optionally, the peptides may be antibodies. Optionally, the antibodies may be monoclonal antibodies.
Optionally, the Granzyme B inhibitor used herein may be selected from one or more of the following: Azepino[3,2,1-hi]indole-2-carboxamide, 5-[[(2S,3S)-2-[(2-benzo[b]thien-3-ylacetyl)amino]-3-methyl-1-oxopentyl]amino]-1,2,4,5,6,7-hexahydro-4-oxo-N-(1H-1,2,3-triazol-5-ylmethyl)-, (2S,5S)-(compound 20 from Willoughby et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2197-2200) referred to herein as Willoughby 20; Bio-x-IEPDP-(OPh)2; (2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-4-oxo-N-(1H-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-(1H-1,2,3-triazol-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide; (2S,5S)-5-{[(2R)-3-methyl-2-pyridin-2-ylbutanoyl]amino}-4-oxo-N-(1H-1,2,3-triazol-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide; (2S,5S)-4-oxo-5-{[N-(phenylacetyl)-L-isoleucyl]amino}-N-(1H-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-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-[[(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, serp-2, ZINC05723764, ZINC05723787, ZINC05316154, ZINC05723499, ZINC05723646, ZINC05398428, ZINC05723503, ZINC05723446, ZINC05317216, ZINC05315460, ZINC05316859, and ZINC05605947. Alternatively, the Granzyme B inhibitor may be selected from one or more of the following: Willoughby 20, NCI 644752, NCI 644777, ZINC05317216, 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. Optionally, 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 antifungal; an anti-inflammatory; a barrier; a moisturizer; and a sealant. Optionally, the another wound treatment is selected from one or more of: a wound covering, a wound filler and an implant. Optionally, the another wound treatment is 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 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. Optionally, 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. Optionally, the Granzyme B inhibitor may be selected from one or more of the following: nucleic acids, peptides, and small molecules. Optionally, the peptides may be antibodies. Optionally, the antibodies may be monoclonal antibodies. Optionally, the Granzyme B inhibitor may be selected from one or more of the following: Azepino[3,2,1-hi]indole-2-carboxamide, 5-[[(2S,3S)-2-[(2-benzo[b]thien-3-ylacetyl)amino]-3-methyl-1-oxopentyl]amino]-1,2,4,5,6,7-hexahydro-4-oxo-N-(1H-1,2,3-triazol-5-ylmethyl)-, (2S,5S)-(compound 28 from Willoughby et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2197-2200) referred to herein as Willoughby 20; Bio-x-IEPDP-(OPh)2; (2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-4-oxo-N-(1H-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-(1H-1,2,3-triazol-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide; (2S,5S)-5-{[(2R)-3-methyl-2-pyridin-2-ylbutanoyl]amino}-4-oxo-N-(1H-1,2,3-triazol-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide; (2S,5S)-4-oxo-5-{[N-(phenylacetyl)-L-isoleucyl]amino}-N-(1H-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-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-[[(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, serp-2, ZINC05723764, ZINC05723787, ZINC05316154, ZINC05723499, ZINC05723646, ZINC05398428, ZINC05723503, ZINC05723446, ZINC05317216, ZINC05315460, ZINC05316859, and ZINC05605947. Alternatively, the Granzyme B inhibitor may be selected from one or more of the following: Willoughby 20, NCI 644752, NCI 644777, ZINC05317216, 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. Optionally, 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. Optionally, 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. Optionally, 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; optionally the subject may be a human.
In another aspect, a method of inhibiting release of a cytokine, such as active transforming growth factor-β (TGF-β), wherein the cytokine, e.g. TGF-, is bound to an extracellular 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, finromodulin, 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: Asp91Thr-Thr-Leu-Leu-Asp; or Asp558Ala-Ser-Leu-Phe-Thr; or Asp31Glu-Ala-Ser-Gly; or Asp69Leu-Gly-Asp-Lys; or Asp82Thr-Thr-Leu-Leu-Asp; or Asp261Asn-Gly-Ser-Leu-Ala.
In another aspect, a model for studying age-related wound healing 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 skin of the mouse. Alternatively or in addition, the high-fat feed diet may be sufficient to result in premature aging in non-xanthamatous 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, collagen 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 otherwise 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 healing 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 to result in premature aging of the skin.
In another aspect, a method of screening 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 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 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 screening compounds involved in inhibiting or reducing skin lesions 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 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, 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 following: nucleic 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[3,2,1-hi]indole-2-carboxamide, 5-[[(2S,3S)-2-[(2-benzo[b]thien-3-ylacetyl)amino]-3-methyl-1-oxopentyl]amino]-1,2,4,5,6,7-hexahydro-4-oxo-N-(1H-1,2,3-triazol-5-ylmethyl)-, (2S,5S)-(compound 20 from Willoughby et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2197-2200) referred to herein as Willoughby 20; Bio-x-IEPDP-(OPh)2; (2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-4-oxo-N-(1H-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-(1H-1,2,3-triazol-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide; (2S,5S)-5-{[(2R)-3-methyl-2-pyridin-2-ylbutanoyl]amino}-4-oxo-N-(1H-1,2,3-triazol-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide; (2S,5S)-4-oxo-5-({[N-(phenylacetyl)-L-isoleucyl]amino}-N-(1H-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-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-[[(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, serp-2, ZINC05723764, ZINC05723787, ZINC05316154, ZINC05723499, ZINC05723646, ZINC05398428, ZINC05723503, ZINC05723446, ZINC05317216, ZINC05315460, ZINC05316859, and ZINC05605947. Alternatively, the Granzyme B inhibitor may be selected from one or more of the following: Willoughby 20, NCI 644752, NCI 644777, ZINC05317216, 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 sufficient to promote would healing, thereby promoting wound healing in the subject.
The wound may be a chronic wound, such as a chronic skin wound, such as a pressure ulcer.
In one embodiment, cleavage of an extracellular matrix protein is inhibited. In one embodiment, the extracellular matrix protein is selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodulin, fibrillin-1, fibrillin-2, and fibulin-2. In one embodiment, the extracellular matrix protein is decorin.
In one embodiment, release of TGFβ bound to an extracellular 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 sufficient to prevent skin 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 aging.
In one embodiment, cleavage of an extracellular matrix protein is inhibited. In one embodiment, extracellular matrix protein is selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodulin, fibrillin-1, fibrillin-2, and fibulin-2. In one embodiment, the extracellular matrix protein is decorin.
In one embodiment, release of TGFβ bound to an extracellular 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 inhibited. In one embodiment, the extracellular matrix protein is selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodulin, fibrillin-1, fibrillin-2, and fibulin-2. In one embodiment, the extracellular matrix protein is decorin.
In one embodiment, release of TGFβ bound to an extracellular matrix protein is inhibited. In one embodiment, the extracellular matrix protein is decorin.
In another aspect, the present invention provides methods 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.
In one embodiment, cleavage of an extracellular matrix protein is inhibited. In one embodiment, the extracellular matrix protein is selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodulin, fibrillin-1, fibrillin-2, and fibulin-2. In one embodiment, the extracellular matrix protein is decorin
In one embodiment, release of TGFβ bound to an extracellular matrix protein is inhibited. In one embodiment, the extracellular 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 healed skin wound of a subject.
In one embodiment, cleavage of an extracellular matrix protein is inhibited. In one embodiment, the extracellular matrix protein is selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodulin, fibrillin-1, fibrillin-2, and fibulin-2. In one embodiment, the extracellular matrix protein is decorin
In one embodiment, release of TGFβ bound to an extracellular matrix protein is inhibited. In one embodiment, the extracellular matrix protein is decorin.
In one aspect, the present invention provides methods for inhibiting release of TGFβ bound to an extracellular protein, comprising contacting the extracellular proteoglycan with a Granzyme B inhibitor, thereby inhibiting release of TGFβ bound to the extracellular protein.
In one embodiment, the protein is selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibromodulin, fibrillin-1, fibrillin-2, and fibulin-2. In one embodiment, the protein is decorin
In another aspect, the present invention provides methods inhibiting extracellular 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 in any of the foregoing methods is wherein the Granzyme B inhibitor is selected from one or more of the following:
In one embodiment, the Granzyme B inhibitor for use in any of the foregoing methods is formulated for topical administration. 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 collagen; an elastin; a tissue growth promoter; an enzymatic debriding preparation; an antifungal; an anti-inflammatory; a barrier; a moisturizer; and a sealant. In 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 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.
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 healing 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 skin 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
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. In one embodiment, 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.
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 in promoting wound healing in a subject. In one embodiment, the wound is a skin wound. In one embodiment, the 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, 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.
Until recently Granzyme B (Granzyme B) was thought to act within cells to mediate cell destruction. This cytotoxic enzyme effectively kills virally infected 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 herein, 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 essential to find effective and alternative solutions to wound care. Currently most wound care is focused on treating symptoms, but wound repair and closure is challenging if Granzyme B is still destroying the ECM proteins needed to maintain skin integrity.
Granzyme B (Granzyme B, also referred to herein at GZMB) is a pro-apoptotic serine protease found in the granules of cytotoxic lymphocytes (CTL) and natural killer (NK) cells. Granzyme B is released towards target cells, along with the pore-forming protein, perforin, resulting in its perforin-dependent internalization into the cytoplasm and subsequent induction of apoptosis (see, for e.g., Medema et al. 1997). 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, neutrophil, dendritic) or non-immune (keratinocyte, chondrocyte) cells and has been to possess extracellular matrix remodeling activity (Choy et al., 2004 and Buzza et al., 2005).
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, fibrillin-1, fibrillin-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 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 P1 residue of Asp (biglycan: D91, betaglycan: D558).
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 frailty, 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-KO mice and that Granzyme B deficiency protects against skin 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 cleavage in vitro and in vivo and promote wound healing by, for example, stimulating collagen organization, decreasing scarring and increasing the tensile strength of skin.
Accordingly, the present invention provides, among others, methods for promoting wound healing, inhibiting release of TGFβ bound to an extracellular matric proteins, e.g., extracellular proteoglycans, methods of 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. In another aspect, use of a Granzyme B inhibitor in the preparation of a medicament for promoting wound healing 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) repairs itself after injury. In normal skin, the epidermis (outermost layer) and dermis (inner or deeper layer) exists in a steady-state equilibrium, forming a protective barrier against 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 healing is divided into four sequential, yet overlapping, phases: (1) hemostasis, (2) inflammatory, (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 minutes post-injury, platelets (thrombocytes) aggregate at the injury site to form a fibrin clot. This clot acts to control active bleeding (hemostasis).
In the inflammatory phase, bacteria and debris are phagocytosed and removed, and factors are released that cause the migration and division of cells involved in the proliferative phase.
The proliferative phase is characterized by angiogenesis, collagen deposition, granulation tissue formation, epithelialization, and wound contraction. In angiogenesis, new blood vessels are formed by vascular endothelial cells.[5] In fibroplasia and granulation tissue formation, fibroblasts grow and form a new, provisional extracellular matrix (ECM) by excreting collagen and fibronectin. Concurrently, re-epithelialization of the epidermis occurs, in which epithelial cells proliferate and ‘crawl’ atop the wound bed, providing cover for the new tissue.
In contraction, the wound is made smaller by the action of myofibroblasts, which establish a grip on the wound edges and contract themselves using a mechanism similar to that in smooth muscle cells. When the cells' roles are close to complete, unneeded cells undergo apoptosis.[
In the maturation and remodeling phase, collagen is remodeled and realigned along tension lines and cells that are no longer needed are removed by apoptosis.
In one embodiment, the methods include administering a Granzyme 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 inhibitor to the wound 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 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 reparative process to produce anatomic and functional integrity of the injured site. Chronic wounds are those that are detained in one or more of the phases of wound healing. 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 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or about 35 days), or which have not completely healed in about eight weeks (or about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or about 65 days). Chronic wounds, as used herein, also refer to wounds in which inflammation 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 skilled in the art will appreciate that wounds may occur in other epithelial tissue. As a non-limiting example, the term “wound” encompasses, without limitation, skin ulcers, which can include: venous skin ulcers, arterial skin ulcers, pressure ulcers, and diabetic skin ulcers. Wounds can also include, without limitation, lacerations, and burns (e.g. heat, chemical, radioactivity, UV burns) of the epithelial tissue. In one embodiment, a chronic skin 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 desired 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, and weight, and the ability of the inhibitor 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 indicated 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 inhibition of extracellular proteoglycan cleavage, e.g., decorin cleavage, in comparison to the normal level of extracellular proteoglycan cleavage, e.g., decorin cleavage, detected in the absence of the Granzyme B inhibitor. Inhibition of extracellular proteoglycan cleavage, e.g., decorin cleavage, is achieved when the value 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%, 15%, 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 skill 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 inhibited. 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 extracellular 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 administering or applying a Granzyme B inhibitor for a time and in an amount sufficient such that release of TGFβ or other growth factor or cytokine bound to an extracellular matrix protein, e.g., an extracellular proteoglycan, selected from the group consisting of decorin, biglycan, betaglycan, syndecan, brevican, fibrillin-1, fibrillin-2, and fibulin-2 is inhibited. In one embodiment, release of TGFβ bound to decorin is inhibited.
In another aspect, the present invention provides methods of 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 sufficient 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 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, betaglycan, syndecan, brevican, fibromodulin, fibrillin-1, fibrillin-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 preventing skin tearing in a subject include applying a Granzyme B inhibitor for a time 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, fibrillin-2, and fibulin-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 epidermis 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, 105, or about 110 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 sufficient to prevent skin 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 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, betaglycan, syndecan, brevican, fibromodulin, fibrillin-1, fibrillin-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 time and in an amount sufficient such that release of TGFβ 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 embodiment, release of TGFβ bound to decorin is inhibited.
As used herein, the term “hypertrophic scarring” refers to a cutaneous condition characterized by deposits of excessive amounts of collagen which gives 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 decorin and have elevated levels of TGFβ.
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 time 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 in the skin is a subject have frail skin due to, for example, age, disease, e.g., diabetes, immobilization, 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, 105, or about 110 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 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, betaglycan, syndecan, brevican, fibromodulin, fibrillin-1, fibrillin-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 TGFβ 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 embodiment, release of TGFβ bound to decorin is inhibited.
In other aspect, the present invention provides methods for increasing the tensile 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 healing 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, betaglycan, syndecan, brevican, fibrillin-1, fibrillin-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 increasing the tensile strength of a healing or healed skin wound, e.g. a chronic skin wound include applying a Granzyme B inhibitor for a time and in an amount sufficient such that release of TGFβ 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 embodiment, release of TGFβ bound to decorin is inhibited.
A “healing wound” is a wound in which clotting has occurred, a wound in which temporary replacement of cells and extracellular 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 (TGF-β), bound to an extracellular matrix protein, e.g., an extracellular proteoglycan, e.g., release of active TGF-β. The methods include, contacting the extracellular matrix protein, e.g. an extracellular proteoglycan, with a Granzyme B inhibitor, thereby inhibiting release of the cytokine, e.g., TGFβ, bound to an extracellular 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. Optionally, the cleavage occurs in any one of the following peptide sequences: Asp91Thr-Thr-Leu-Leu-Asp (SEQ ID NO: 1); or Asp558Ala-Ser-Leu-Phe-Thr (SEQ ID NO:2); or Asp31Glu-Ala-Ser-Gly (SEQ ID NO:3); or Asp69Leu-Gly-Asp-Lys (SEQ ID NO:4); or Asp82Thr-Thr-Leu-Leu-Asp (SEQ ID NO:5); or Asp261Asn-Gly-Ser-Leu-Ala (SEQ ID NO:6).
The methods and uses of inhibiting release of a cytokine, e.g. TGFβ, 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, betaglycan, syndecan, brevican, fibromodulin, fibrillin-1, fibrillin-2, and fibulin-2. In 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 extracellular proteoglycan, with a Granzyme B inhibitor, wherein the release of a sequestered cytokine, e.g. TGFβ, is inhibited, thereby inhibiting extracellular matrix protein degradation.
The methods and uses of inhibiting degradation of 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, betaglycan, syndecan, brevican, fibromodulin, fibrillin-1, fibrillin-2, and fibulin-2. In one embodiment, the extracellular 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 extracellular 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 cell, such as a skin 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. Granzyme B inhibitors 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 specific 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. In certain embodiments, a moiety is administered topically, intravenously, intramuscularly, subcutaneously, intradermally, intranasally, orally, transcutaneously, intrathecal, intravitreally, 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 applied 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 inhibitor) 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 cells or tissues of the subject are contacted in vivo.
As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, but not limited to, alleviation or amelioration 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 detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment.
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.
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; Kam 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
In another embodiment, a Granzyme B inhibitor suitable for use in the methods, compositions, and uses of the invention includes, for example, Z-AAD-CMK (IUPAC name: 5-chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylamino) propanoylamino]propanoylamino]pentanoic acid) MF: C19H24ClN3O7 CID: 16760474; Ac-IEPD-CHO; Granzyme B Inhibitor IV or Caspase-8 inhibitor III (IUPAC: (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) MF: C22H34N4O9 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-[[(2S,3S)-3-hydroxy-1-[[(2S)-4-hydroxy-1,4-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[3,2,1-hi]indole-2-carboxamide, 5-[[(2S,3S)-2-[(2-benzo[b]thien-3-ylacetyl)amino]-3-methyl-1-oxopentyl]amino]-1,2,4,5,6,7-hexahydro-4-oxo-N-(1H-1,2,3-triazol-5-ylmethyl)-, (2S,5S)-(compound 20 from Willoughby et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2197-2200) referred to herein as Willoughby 20; Bio-x-IEPDP-(OPh)2; (2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-4-oxo-N-(1H-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-(1H-1,2,3-triazol-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide; (2S,5S)-5-([(2R)-3-methyl-2-pyridin-2-ylbutanoyl]amino)-4-oxo-N-(1H-1,2,3-triazol-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide; (2S,5S)-4-oxo-5-{[N-(phenylacetyl)-L-isoleucyl]amino}-N-(1H-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-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-[[(2S,3S)-3-hydroxy-1-[[(2S)-4-hydroxy-1,4-dioxobutan-2-yl]amino]-1-oxobutan-2-yl]amino]-5-oxopentanoic acid, a protease inhibitor-9 or derivatives thereof, CrmA, serp-2, ZIINC05723764, ZINC05723787, ZINC05316154, ZINC05723499, ZINC05723646, ZINC05398428, ZINC05723503, ZINC05723446, ZINC05317216, ZINC05315460, ZINC05316859, and ZINC05605947.
Alternatively, the Granzyme B inhibitor may be selected from one or more of the following: Willoughby 20, NCI 644752, NCI 644777, ZINC05317216, and NCI 630295. Granzyme B inhibitors may include, but are not limited to, nucleic acids (for example, antisense oligonucleotides, siRNA, RNAi, etc.), peptides and small molecules.
Optionally, the Granzyme B inhibitor used herein may be selected from one of the examples detailed herein, which includes but is not limited to one or more of the following: Azepino[3,2,1-hi]indole-2-carboxamide, 5-[[(2S,3S)-2-[(2-benzo[b]thien-3-ylacetyl)amino]-3-methyl-1-oxopentyl]amino]-1,2,4,5,6,7-hexahydro-4-oxo-N-(1H-1,2,3-triazol-5-ylmethyl)-, (2S,5S)-(compound 20 from Willoughby et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2197-2200) referred to herein as Willoughby 20; Bio-x-IEPDP-(OPh)2; (2S,5S)-5-[(N-acetyl-L-isoleucyl)amino]-4-oxo-N-(1H-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-(1H-1,2,3-triazol-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide; (2S,5S)-5-{[(2R)-3-methyl-2-pyridin-2-ylbutanoyl]amino}-4-oxo-N-(1H-1,2,3-triazol-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide; (2S,5S)-4-oxo-5-{[N-(phenylacetyl)-L-isoleucyl]amino}-N-(1H-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-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-[[(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, serp-2, ZINC05723764, ZINC05723787, ZINC05316154, ZINC05723499, ZINC05723646, ZINC05398428, ZINC05723503, ZINC05723446, ZINC05317216, ZINC05315460, ZINC05316859, and ZINC05605947. Alternatively, the Granzyme B inhibitor may be selected from one or more of the following: Willoughby 20, NCI 644752, NCI 644777, ZINC05317216, 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 deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and any chemical modifications thereof. Such modifications include, but are not limited to backbone modifications, methylations, and unusual base-pairing combinations. As detailed herein, the term “nucleic acid” includes, without limitation, RNAi technologies. For example, RNA compounds used to inhibit Granzyme B may be small interfering RNA (siRNA) 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 nucleic acid molecule” or “interfering nucleic acid” as used herein includes single-stranded RNA (e.g., mature miRNA. ssRNAi oligonucleotides, ssDNAi oligonucleotides), double-stranded RNA (i.e., duplex RNA such as siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, or pre-miRNA), self-delivering RNA (sdRNA; see, e.g. U.S. Patent Publication Nos. 200913120341, 200913120315, and 201113069780, the entire contents of all of which are incorporated herein by reference), a DNA-RNA hybrid (see, e.g., PCT Publication No. WO 2004/078941), or a DNA-DNA hybrid (see, e.g., PCT Publication No. WO 2004/104199) 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 mRNAs which are 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 acids 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 nucleic 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:221625527, 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 motif” or “mismatch region” refers to a portion of an interfering nucleic acid (e.g., siRNA) sequence that does not have 100% 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, 11, 12, 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 strand 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 “small-interfering RNA” or “an siRNA” molecule. In another embodiment, an interfering nucleic acid molecules of the invention is a “self-delivering RNA” or “sdRNA” molecule. In one embodiment, an interfering nucleic acid of the invention mediates RNAi. RNA interference (RNAi) 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, 2431-2432 (2000); Zamore, P. D., et al. Cell 101, 25-33 (2000). Tuschl, T. et al. Genes Dev. 13, 3191-3197 (1999); Cottrell T R, and Doering T L. 2003. Trends Microbiol. 11:37-43; 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., 21- 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 RNAi 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 RNAi.
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 8-15, 10-30, 10-25, or 10-25 (duplex) nucleotides in length, about 10-24, (duplex) nucleotides in length (e.g., each complementary sequence of the double-stranded siRNA is 10-60, 10-50, 10-40, 10-30, 10-25, or 10-25 nucleotides in length, about 10-24, 11-22, or 11-23 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 siRNA (or sdRNA) molecule. As used herein, the terms “siRNA” and “sdRNA’ include RNA-RNA duplexes as well as DNA-RNA hybrids (see, e.g., PCT Publication No. WO 2004/078941).
Preferably, siRNA and sdRNA are chemically synthesized. siRNA and sdRNA can also be generated by cleavage of longer dsRNA (e.g., dsRNA about 5, about 10, about 15, about 20, about 25, or greater nucleotides in length) with the E. coli RNase III or Dicer. These enzymes process the dsRNA into biologically active siRNA (see, e.g., Yang et al., Proc. Natl. Acad. Sci: USA, 99:9942-9947 (2002); Calegari et al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002); Byrom et al. Ambion TechNotes, 10(1):4-6 (2003); Kawasaki et al., Nucleic Acids Res., 31:981-987 (2003); Knight et al., Science, 293:2269-2271 (2001); and Robertson et 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, 1500, 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 sdRNA may be encoded by a plasmid (e.g., transcribed as sequences that automatically fold into duplexes with hairpin loops).
Given the coding strand sequences encoding Granzyme 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 pairing. The interfering nucleic acid molecule can be complementary to the entire coding region of Granzyme B mRNA, but more preferably is an oligonucleotide which is antisense 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 RNA oligonucleotide 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 chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an interfering nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense 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-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 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 biologically 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 will be of an antisense orientation to a target nucleic acid of interest).
The interfering nucleic acids may include any RNA compounds which have sequence homology to the Granzyme B gene and which are 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: U.S. Pat. No. 6,159,694; U.S. Pat. No. 6,727,064; U.S. Pat. No. 7,098,192; and U.S. Pat. No. 7,307,069, the entire contents of all of which are incorporated herein by reference.
Antisense oligonucleotides directed against Granzyme B have been designed and manufactured by Biognostik (Euromedex, Mundolshei, France) and are described in Hernandez-Pigeon, et al., J. Biol Chem. vol. 281, 13525-13532 (2006) and Bruno, et 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 linked by peptide bonds. Those persons skilled in the art will understand that a peptide bond, which is also know in the art as an amide bond, is a covalent chemical bond formed between two molecules when the carboxyl group of one molecule reacts with the amine group of the other molecule, thereby releasing a molecule of water (H2O). Peptides may be modified in a variety of conventional ways well known to the skilled artisan. Examples of modifications include the following. The terminal amino group and/or carboxyl group of the peptide and/or amino acid side chains may be modified by alkylation, amidation, or acylation to provide esters, amides or substituted amino groups. Heteroatoms may be included in aliphatic modifying groups. This is done using conventional chemical synthetic methods. Other modifications include deamination of glutamyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively; hydroxylation of proline 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, Calif., 1983).
In another aspect, one or both, usually one terminus 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 caprylic acid, capric acid, lauric acid, myristic acid and myristyl alcohol, palmitic acid, palmitoleic acid, stearic acid and stearyl amine, oleic acid, linoleic acid, docosahexaenoic acid, etc. (see, for e.g.: U.S. Pat. No. 6,225,444). Preferred are unbranched, naturally occurring fatty acids between 14-22 carbon atoms in length. Other lipophilic molecules include glyceryl lipids and sterols, such as cholesterol. The lipophilic groups may be reacted with the appropriate functional group on the oligopeptide 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 liposome, along with other therapeutic agents for administering the peptides and agents into a host.
Depending upon their intended use, particularly for administration 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 bioavailability, extending or shortening half-life, controlling distribution 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. Solid phase synthesis is commonly used and various commercial synthetic apparatuses are available, for example automated synthesizers by Applied Biosystems Inc., Foster City, Calif.; Beckman; etc. Solution phase synthetic methods may also be used, particularly for large-scale productions.
Peptides may also be present in the form of a salt, generally 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 acid, salicylic acid, etc.
Peptides can also be made intracellularly in cells 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 intracellularly 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 introducing 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 naturally 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 P19 peptide, or a Granzyme B inhibitory fragment thereof (see, e.g., U.S. Patent Publication No. 2003/0148511, 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, 1999 Immunol. Cell Biol. 77, 47-57). The amino acid and nucleotide sequence of SerpinB9 are known and may be found in, for example, Genbank Accession No. GI:223941859, 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 pan or all of the sequence from SerpinB9 that binds directly to Granzyme B, i.e. GTEAAASSCFVAECCMESG (SEQ. ID NO: 11). 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 VEVNEEGTEAAAASSCFVVAECCMESGPRFCADHPFL (SEQ ID NO: 18); VEVNEEGTEAAAASSCFVVADCCMESGPRFCADHPFL (SEQ ID NO:19); VEVNEEGTEAAAASSCFVVAACCMESGPRFCADHPFL (SEQ ID NO:20); and VEVNEEGREAAAASSCFVVAECCMESGPRFCADHPFL (SEQ ID NO:21)
In another embodiment, the Granzyme B inhibitor is a Serpina3n 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 ID NOs: 12 and 13.
In another embodiment, the Granzyme B inhibitor is the cowpox virusprotein, CrmA peptide, or a Granzyme B inhibitory fragment thereof (see, e.g. Quan, et al. (1995) 270, 10377-10379) (the amino acid and nucleotide sequences of CrmA are set forth in SEQ ID NOs: 14 and 15). In one embodiment, a Granzyme B inhibitor is a CrmA peptide comprising the amino acid sequence IDVNEEYTEAAAATCALVADCASTVTNEFCADHPFI (SEQ ID NO:22).
In another embodiment, the Granzyme B inhibitor is a Serp2 peptide, or a Granzyme B inhibitory fragment thereof. Serp2 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:58219011, the entire contents of which are incorporated herein by reference, and SEQ ID NOs: 16 and 17.
Other suitable Granzyme B inhibitory peptides for use in any of the methods, compositions, or uses of the invention, include, for example, Z-AAD-CH2Cl (Z-ALA-ALA-ASP-chloromethylketone), Ac-IEPD-CHO (Ac-Ile-Glu-Pro-Asp-CHO), Ac-IETD-CHO, Ac-AAVALLPAVLLALLAPIETD-cho, and z-IETD-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-Granzyme B antibody. In one embodiment, the an anti-Granzyme B antibody is a human antibody. In another embodiment, the an anti-Granzyme B antibody is a humanized antibody. In another embodiment, the an anti-Granzyme B antibody is a camelid 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 specifically covers polyclonal antibodies, monoclonal antibodies, dimers, multimers, multispecific antibodies (e.g. bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. The term “antibody” includes, without limitation, camelid antibodies. Antibodies may be murine, human, humanized, chimeric, or derived from other species. Typically, an antibody will comprise at least two heavy chains and two light chains interconnected by disulfide bonds, which 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 (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3, and may be of the mu (μ), delta (δ), gamma (γ), alpha (α) or epsilon (ε) isotype. Similarly, the light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL, which may be of the kappa or lambda isotype. The VH and VL 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 VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, 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 binding of the immunoglobulin to host tissue or host factors, particularly through cellular receptors such as the Fc receptors (e.g., FcγRI, FcγRII, FcγRIII, etc.). As used herein, antibody also includes an antigen binding portion of an immunoglobulin that retains the ability to bind antigen. These include, as examples, F(ab), a monovalent fragment of VL CL and VH CH 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 (scFv) and bispecific molecules such as, e.g., diabodies, triabodies, and tetrabodies (see, e.g. U.S. Pat. No. 5,844,094).
Antibodies may be produced and used in many forms, including 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 specifically to an antibody. An antigen has one or more epitopes that interact with the antibody, although it does not necessarily induce production of that antibody.
As used herein the term “epitope” refers to a determinant capable of specific binding to an antibody. Epitopes are chemical features generally 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, hydrophilicity, and lipophilicity. Conformational epitopes are distinguished from non-conformational 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 an as an “antigenic determinant”. For example, an antibody that is secreted by a B cell 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 limit the scope of the term “epitope”. Epitopes are recognized by numerous cell types including B cells and T cells.
As used herein, the term “humanized antibody” refers to an immunoglobulin molecule containing a minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In 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 substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all 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 et al., 1986; and Reichmann et al. 1988). 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-chain Fv fragments. As used herein, the term “monoclonal antibody” refers to monospecific antibodies that are the same because they are made by clones of a unique parent cell. As detailed above, the term “antibody” includes without limitation a “monoclonal antibody”.
In one embodiment, a Granzyme B inhibitor is a small molecule.
As used herein, the term “small molecule” refers to a low molecular weight organic compound that binds to a biopolymer such as a protein, a nucleic acid, or a polysaccharide. The foregoing examples of binding partners of a small 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 limited to azepine compounds of the following formula:
or a pharmaceutically acceptable salt or hydrate thereof, wherein: n is 0, 1, or 2; R1 and R2 are each independently selected from the group consisting of: hydrogen, C1-6alkyl, C1-6alkoxy, C3-6cycloalkyl, aryl, HET and —N(R10)2, wherein: (a) said C1-6alkyl, C1-6alkoxy and C3-6cycloalkyl are optionally substituted with 1-3 substituents independently selected from the group consisting of halo and hydroxy; and (b) said aryl and HET are optionally substituted with 1-3 substituents independently selected from the group consisting of: halo, hydroxy and C1-4alkyl, optionally substituted with 1-3 halo groups; or R1 and R2 may be joined together with the carbon atom to which they are attached to form a five or six membered monocyclic ring, optionally containing 1-3 heteroatoms selected from the group consisting of: S, O and N(R10), wherein said ring is optionally substituted with 1-3 R10 groups, with the proviso that R1 and R2 are both not hydrogen; each of R3 and R7 is independently selected from the group consisting of: hydrogen and C1-4alkyl, optionally substituted with 1-3 halo groups; each of R4, R5, R6 and R8 is independently selected from the group consisting of: hydrogen, halo, hydroxy and C1-4alkyl, optionally substituted with 1-3 halo groups; R9 is HET, optionally substituted with 1-3 substituents independently selected from the group consisting of: halo, hydroxy and C1-4alkyl, optionally substituted with 1-3 halo groups; R10 is selected from the group consisting of: hydrogen, C1-4alkyl and —C(O)C1-4alkyl, said —C(O)C1-4alkyl optionally substituted with N(R11)2, HET and aryl, said aryl optionally substituted with 1-3 halo groups; R11 is selected from hydrogen and C1-4alkyl, optionally substituted with 1-3 halo groups; HET is a 5- to 10-membered aromatic, partially aromatic or non-aromatic mono- or bicyclic ring, containing 1-4 heteroatoms selected from O, S and N(R12), and optionally substituted with 1-2 oxo groups; and R12 is selected from the group consisting of: hydrogen and C1-4alkyl, optionally 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 limited 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-methylpentanamido)-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)—N-((1H-1,2,3-triazol-4-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,
also referred to herein as (2S,5S)—N-((1H-1,2,3-triazol-4-yl)methyl)-5-((R)-3-methyl-2-(pyridin-2-yl)butanamido)-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)—N-((1H-1,2,3-triazol-4-yl)methyl)-5-((2S,3S)-3-methyl-2-(2-phenylacetamido)pentanamido)-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)—N-((1H-1,2,4-triazol-3-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,
also referred to herein as (2S,5S)—N-((1H-pyrazol-3-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,
also referred to herein as (2S,5S)—N-((1H-pyrazol-4-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,
also referred to herein as (2S,5S)—N-((1H-imidazol-4-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,
also referred to herein as (2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(thiazol-5-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-N-(isoxazol-3-ylmethyl)-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(thiazol-2-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-N-(isoxazol-5-ylmethyl)-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(thiazol-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyrimidin-5-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyridazin-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyridin-2-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyridin-3-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-4-oxo-N-(pyridin-4-ylmethyl)-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)-5-((2S,3S)-2-acetamido-3-methylpentanamido)-N-(imidazo[1,2-a]pyrimidin-2-ylmethyl)-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (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,
also referred to herein as (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[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (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[3,2,1-hi]indole-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-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)—N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(2,3-difluorophenyl)acetamido)-3-methylpentanamido)-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)—N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-(dimethylamino)acetamido)-3-methylpentanamido)-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)—N-((2H-tetrazol-5-yl)methyl)-5-((2S,3S)-2-(2-benzo[b]thiophen-3-yl)acetamido)-3-methylpentanamido)-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)—N-((1H-1,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-(2-(dimethylamino)acetamido)-3-methylpentanamido)-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)—N-((1H-1,2,3-triazol-4-yl)methyl)-5-((2S,3S)-2-(2-(benzo[b]thiophen-3-yl)acetamido)-3-methylpentanamido)-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (R)—N-((2S,5S)-2-((1H-1,2,3-triazol-4-yl)methylcarbamoyl)-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indol-5-yl)-3-acetyl-5,5-dimethylthiazolidine-4-carboxamide,
also referred to herein as (2S,5S)—N-((1H-1,2,3-triazol-4-yl)methyl)-5-((2S,3S)-3-methyl-2-(2-oxopyrrolidin-1-yl)pentanamido)-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
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[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)—N-((1H-1,2,3-triazol-4-yl)methyl)-5-((S)-2-acetamido-2-cyclopropylacetamido)-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (2S,5S)—N-((1H-1,2,3-triazol-4-yl)methyl)-5-((S)-2-acetamido-2-cyclopentylacetamido)-4-oxo-1,2,4,5,6,7-hexahydroazepino[3,2,1-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 limited to one or more of the following:
also referred to herein as Bio-x-IEPDP-(OPh)2,
also referred to herein as azepino[3,2,1-hi]indole-2-carboxamide,
also referred to herein as (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,
also referred to herein as (4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-5-[[(2S,3S)-3-hydroxy-1-[[(2S)-4-hydroxy-1,4-dioxobutan-2-yl]amino]-1-oxobutan-2-yl]amino]-5-oxopentanoic acid,
also referred to herein as 5-chloro-4-oxo-3-[2-[2-(phenylmethoxycarbonylamino) propanoylamino]propanoylamino]pentanoic acid,
also referred to herein as 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 to 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 to herein as ZINC05723499 and NCI 641235,
also referred to herein as ZINC05723646 and NCI 642017,
also referred to herein as ZINC05398428 and NCI 641230,
also referred to herein as ZINC5723503 and NCI 641236,
also referred to herein as ZINC05723446 and and NCI 640985,
also referred to herein as ZINC05317216 and NCI 618792,
also referred to herein as ZINC05315460 and NCI 630295,
also referred to herein as ZINC05316859 and NCI 618802, and
also referred to herein as ZINC05605947 and NCI 623744, or a salt 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.
Optionally, the Granzyme B inhibitor used herein is:
or a salt or solvate thereof.
A Granzyme B inhibitor for use in the methods, compositions, 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 (see, e.g. Kam et al., 2000).
Optionally, the Granzyme B inhibitor used herein is one or more of:
Isocoumarin derivatives (upper left): 3,4-dichloroisocoumarin, DCI, X=H, Y=Cl; 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin, X=NH2, Y=O(CH2)3—SC(═NH+2)NH2; 4-chloro-3-ethoxy-7-guanidinoisocoumarin, X=NHC(═NH+2)NH2, Y=OCH2CH3. FUT-175 analogs (upper right). Bottom line: structures of a peptide substrate, a peptide phosphonate and a 4-amidinophenylglycine phosphonate [(4-AmPhGly)P(OPh)2] derivative. The latter is an arginine analog.
Many Granzyme B inhibitors are water-soluble and may be formed as salts. In such cases, compositions of Granzyme B inhibitors may comprise a physiologically acceptable salt, which are known to a person of skill in 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, epidermal administration, sub-epidermal administration, dermal administration, sub-dermal administration, sub-cutaneous administration, systemic administration, injection, inhalation, oral, or other modes suitable for the selected treatment. Suitable carriers are those known in the art for use in such modes of administration.
Suitable compositions may be formulated by means known in the art and their mode of administration and dose determined by a person of skill in the art. For parenteral administration, compound may be dissolved in sterile water or saline or a pharmaceutically acceptable vehicle used for administration of non-water soluble compounds such as those used for vitamin K. For enteral administration, compound may be administered in a tablet, capsule, or dissolved in liquid 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, hydrogels, foams, creams, powders, lotions, oils, semi-solids, soaps, medicated soaps, shampoos, medicated shampoos, sprays, films, or solutions which can be used topically or locally 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 skill in the an are described in Remington: the Science & Practice of Pharmacy by Alfonso Gennaro, 20th 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 polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful delivery systems for modulatory compounds include ethylene-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 deoxycholate, or may be oily solutions for administration in the form of drops, or as a gel.
Compositions containing Granzyme B inhibitors may also include penetrating agents. Penetrating agents may improve the ability of the Granzyme 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 well as L-arginine or the arginine-containing amino acids.
Compounds or compositions of Granzyme B inhibitors may be administered alone or in conjunction with other wound treatments, such as wound preparations, wound coverings, and closure devices.
Optionally, the Granzyme B inhibitor is formulated for topical administration. For example, the formulations for topical administration of a Granzyme 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 sublingual 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 collagen; an elastin; a tissue growth promoter; an enzymatic debriding preparation; an antifungal; an anti-inflammatory; a barrier; a moisturizer; and a sealant. Optionally, the another wound treatment may be selected from one or more of the following: 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; 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. Optionally, 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. 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 co-administration with another wound treatment. Optionally, the wound treatment may be 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. Optionally, 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 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. Optionally, the Granzyme B inhibitor may be formulated for topical application in a wound covering, a wound filler, or an implant. Optionally, the Granzyme B inhibitor may be formulated for impregnation in a wound covering, a wound filler or an implant. Optionally, the use may involve a subject that is a mammal; optionally, the use may involve a subject that is a human.
In another aspect, a model for studying age-related wound repair 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 mouse, and wherein the high-fat feed diet is sufficient to result in premature aging of non-xanthomatous regions of the skin. In skin areas that do not contain xanthomas, these mice also develop evidence of skin 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 maintained on a high-fat feed diet, wherein the high-fat feed diet is sufficient to result in xanthomatotic skin lesions on the mouse, and wherein the skin lesions express Granzyme 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 to result 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 apolipoprotein E-knock out mouse on a high-fat feed diet, wherein the high-fat feed diet is sufficient to 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 includes any biological sign of repair of a skin lesion. Examples of modes by which repair 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 transepidermal water loss, monitoring the presence or absence of ECM abnormalities, elastosis, collagen morphology, collagen density, the presence of decorin, 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 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 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 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 screening compounds involved in inhibiting or reducing skin lesions 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 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 identifying 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 decorin, and selecting a compound that inhibits the cleavage of decorin in the indicator composition, thereby identifying a compound useful for promoting chronic wound healing.
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 extracellular 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, lipids, 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 libraries; synthetic library methods requiring deconvolution; the “one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145; U.S. Pat. No. 5,738,996; and U.S. Pat. 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 et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) 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. 37:1233, 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 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al., (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or phage (Scott and Smith (19900 Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol. 222:301-310), each of which is incorporated herein in its entirety by reference.
The indicator composition can be a cell that expresses the Granzyme 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 includes e.g., either purified natural or recombinant protein).
For example, an indicator cell can be transfected 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 indicator cell in the screening assay. Cells for use in the subject assays include eukaryotic cells. For example, in one embodiment, a cell is a vertebrate cell, e.g., an avian cell 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 introduced into a recombinant expression vector using standard molecular biology techniques. A cDNA can be obtained, for example, by amplification using the polymerase chain reaction (PCR) or by screening an appropriate cDNA library. The nucleotide sequences of cDNAs 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 allow 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 library using standard hybridization methods.
In another embodiment, the indicator composition is a cell free composition. Protein expressed by recombinant methods in a host cells or culture medium can be isolated from the host cells, or cell culture medium using standard methods for protein purification. For example, ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies can be used to produce a purified or semi-purified protein that can be used in a cell free composition. Alternatively, a lysate or an extract of cells expressing the protein of interest can be prepared for use as cell-free composition.
Once a test compound is identified 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 cells with the compound of interest or, alternatively, by contacting the compound of interest with a cell line) 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 combination 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 identified 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 identified in the subject screening assays.
CTL, cytotoxic lymphocytes; DCI, 3,4-dichloroisocoumarin; DMSO, dimethyl sulfoxide; ECM, extracellular matrix; Erk, extracellular signal-regulated kinase; GAG, glycosaminoglycan; Granzyme B, Granzyme B; HCASMC, human coronary artery smooth muscle cells; NK, natural killer cell; LAP, latency associated peptide; LLC, large latent TGF-β complex; LTBP, latent TGF-β binding protein; MMP, matrix metalloproteinase; MT-MMP1, membrane type-matrix metalloproteinase 1; SLC, small latent TGF-β complex; TGF-β, transforming growth factor beta.
Methods. For in vitro extracellular matrix cleavage 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 blotting or N-terminal sequencing. Confirmation of cleavage was performed subsequently with purified substrate.
Results: In order to identify extracellular Granzyme B substrates, recombinant decorin, biglycan, betaglycan, syndecan, and brevican were incubated with purified Granzyme B for 24 hours. Reactions were stopped with SDS-PAGE loading buffer, run on an SDS-PAGE gel and imaged by Ponceau staining of a nitrocellulose membrane. As shown in
In order to determine if Granzyme B also cleaves smooth muscle cell- (SMC-)derived ECM, following 5-7 days of serum starvation 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 fibrillin-1, fibrillin-2 or fibulin-2 (
Methods:
For ECM cleavage assays, Granzyme B and/or the inhibitor 3,4-dichloroisocoumarin (DCI), were incubated for 4 and 24 hours at room temperature, with decorin, biglycan or soluble betaglycan and visualized by Ponceau staining. Cleavage fragments were subjected to Edman degradation for cleavage site identification.
As TGF-β is sequestered by the aforementioned proteoglycans, Granzyme B was incubated with TGF-β bound proteoglycans to determine if Granzyme B cleavage resulted in the release of sequestered TGF-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 betaglycan, with proteolysis evident at Granzyme B concentrations as low as 25 nM. Proteolysis was inhibited by DCI but not the solvent control DMSO. Edman degradation analysis determined Granzyme B cleavage sites in the PGs with P1 residues of aspartic acid, consistent with Granzyme B cleavage specificity.
In cytokine release assays, TGF-β was liberated Granzyme 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 inhibited by DCI, indicating release from decorin, biglycan and betaglycan was specific. In addition, the TGF-β liberated by Granzyme B remained active and induced SMAD-3 and Erk-2 phosphorylation in HCASMC, after 16 h of incubation (see below).
Methods:
Proteoglycan cleavage assays. The recombinant human PGs, decorin (0.5 μg, Abnova, Walnut, Calif.), biglycan and betaglycan (1.5-5 ug, R&D Systems, Minneapolis, Minn.) were incubated at room temperature for 24 h with 25-500 nM purified human Granzyme B (Axxora, San Diego, Calif.), in 50 mM Tris buffer, pH 7.4. For inhibitor studies, Granzyme B was incubated in the presence or absence of 200 μM of the serine protease inhibitor 3,4-dichloroisocoumarin (DCI; Santa Cruz Biotechnology Inc, Santa Cruz, Calif.) or inhibitor solvent control, dimethyl sulfoxide (DMSO; Sigma-Aldrich, St Louis, Mo.) for 4 h or 24 h. After incubation, proteins were denatured, separated on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Ponceau stain (Fisher Scientific, Waltham, Mass.) was used to detect cleavage fragments.
N-Terminal Sequencing.
For Edman degradation. 2-5 μg/lane of biglycan and betaglycan were incubated with 100-500 nM Granzyme B for 24 h. Once run on a gel and transferred to a PVDF membrane, cleavage fragments were identified by Ponceau staining. The stain was removed by washes with distilled water, the membrane was dried and analyzed at the Advanced Protein Technology Center at the Hospital for Sick Kids (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 et al. 1997). Briefly, decorin, biglycan and betaglycan (15 μg/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-β1 per well (Peprotech Inc, Rocky Hill, N.J.) was added in DPBS containing calcium and magnesium (#14040, Invitrogen, Carlsbad, Calif.) for 5 h at RT. Granzyme B, with or without DCI, was then added to the wells. After 24 h, supernatants 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-β1 antibody (1:200, BD Biosciences, Franklin Lakes, N.J.) and IRDye® 800 conjugated affinity purified anti-Rat IgG (1:3000, Rockland Inc, Gilbertsville, Pa.). Bands were imaged using the Odyssey Infrared Imaging System (LI-COR Biotechnology, Lincoln, Nebr.).
Human Coronary Artery Smooth Muscle Cell TGF-β Bioavailability Assays.
For bioavailability assays, HCASMCs (Clonetics/Lonza, Walkersville, 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. At this time, cells were quiesced by serum removal for 24 h, after which time 150 μl of release assay supernatants (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/Western blotting for phosphorylated-Erk 1/2 (p-Erk1/2; 1:1000, Cell Signaling Technology, Danvers, Mass.), total Erk 1/2 (t-Erk1/2; 1:1000, Cell Signaling Technology), phosphorylated-SMAD3 (p-SMAD3; 1:2000, Epitomics, Burlingame, Calif.), total SMAD3 (t-SMAD3; 1:500, BD Biosciences) and the loading controls β-actin (1:5000, Sigma-Aldrich) or β-tubulin (1:3000, Millipore, Billerica, Mass.). Secondary IRDye® 800 conjugated antibodies (1:3000, Rockland Inc) were utilized and imaged with the Odyssey Infrared Imaging System (LI-COR Biotechnology). Densitometric analysis was conducted on the Odyssey Infrared Imaging System and displayed graphically by p-SMAD-3/β-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 (
Referring to
To confirm that decorin, biglycan and betaglycan proteolysis was mediated by Granzyme B, DCI was included in reactions for 4 h or 24 h (
Granzyme B Cleavage Site Identification.
Granzyme B cleavage sites were characterized in biglycan and betaglycan by Edman degradation (
Referring to
Granzyme B-Dependent Cleavage of Biglycan, Decorin and Betaglycan Results in the Release of Active TGF-β1.
As decorin, biglycan and betaglycan sequester active to TGF-β1, a TGF-β release assay was utilized to determine if Granzyme B-mediated cleavage of these proteins resulted in active TGF-β release (
Referring to
TGF-β Released by Granzyme B Remains Active and Induces SMAD and Erk Signaling in Smooth Muscle Cells.
To determine that the TGF-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 16 h (
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/degranulation and cytotoxic lymphocytes constitutively release Granzyme B in the absence of target cell engagement (see, for e.g. Prakash et al., 2008). Based, in part, on the results obtained herein, Granzyme B plays an extracellular role in pathogenesis. In this study the Granzyme B cleavage sites for biglycan and betaglycan were identified. In this study, it was demonstrated that Granzyme B cleaved these PG substrates at a P1 residue of Asp (biglycan: D91, betaglycan: D558). Further, in the studies described herein TGF-β1 was released from all three substrates. There was no release evident in the negative control lacking protease or when Granzyme B was co-incubated 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 sequestration by the ECM.
Abbreviations Used Herein:
apolipoprotein E (apoE); knockout (KO); double knockout (DKO); extracellular matrix (ECM); Granzyme B (Granzyme B); ultraviolet (UV); high fat diet (HFD); second harmonic generation (SHG).
Materials and Methods:
Mice. All 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-KO mice were purchased from The Jackson Laboratory (Bar Harbor, Me.) and housed at The Genetic Engineered Models (GEM) facility (James Hogg Research Centre, UBC/St. Paul's Hospital, Vancouver, BC). ApoE/Granzyme B double knockout mice were generated on site and also housed at the GEM facility. All mice were fed ad libitum on either a high fat (21.2% fat, TD.88137, Harlan Teklad; Madison, Wis.) or regular chow (equal pans PicoLab Mouse Diet 20: 5058 and PicoLab Rodent Diet 20: 5053, LabDiet; Richmond, Ind.) diet beginning at 6-8 weeks of age for either 0, 5, 15 or 30 weeks. At their respective time points, mice were weighed, and euthanized by carbon dioxide 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 illness 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 facility. Briefly, animals were considered for euthanasia if they appeared to be in distress or pain that could not be alleviated. 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 to lower back. Half of the skin sample was fixed in 10% phosphate buffered formalin. Fixed skin sections were processed, embedded in paraffin and cut to 5 μm cross-sections for histology and immunohistochemistry. The other half of the dorsal skin sample was treated with a hair removing cream to completely remove all hair from the surface of the skin. These skin samples were then flash frozen in liquid N2 and stored at −80° C. until 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 elastin was used to examine elastic fibres. Measurement of skin thickness was completed using a 40× 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. Collagen was observed in picrosirius red stained sections using 100% polarized light and pictures were taken at a fixed exposure. Granzyme B immunohistochemistry was performed by boiling deparaffinised slides in citrate buffer (pH 6.0) for 15 min. Background staining was blocked by incubating slides with 10% goat serum. The primary antibody used was a rabbit anti-mouse Granzyme B antibody at a 1:100 dilution (Abcam, Cambridge, Mass.) 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, Calif.) followed by ABC reagent (Vector Laboratories). Staining was visualized with DAB peroxidise substrate (Vector Laboratories). Decorin immunohistochemistry was performed by immersing deparaffinised slides in citrate buffer (pH 6.0) at 80° C. for 10 min. Slides were blocked with 10% rabbit serum and a goat anti-mouse decorin antibody (1 μg/ml) (R&D Systems, Minneapolis, Minn.) was used while slides incubated at 4° C. overnight. Biotinylated rabbit anti-goat secondary antibody was used (1:350) (Vector Laboratories) along with ABC reagent (Vector Laboratories) and DAB substrate (Vector Laboratories) as described above.
Multi-Photon Microscopy.
Frozen skin samples with the hair completely removed were thawed at room temperature and immobilized on a fat surface inside a small dish. Skin samples were washed several 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 collagen density. Methods used were similar 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, Calif.) and was focused on the specimen through a 20×/0.5 NA HCX APO L water dipping objective. An excitation wavelength of 880 nm was used and backscattered SHG emissions from the sample were collected through the objective lens. Leica Confocal Software TCS SP2 was used for the image acquisition. Images (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 μm were acquired at decreasing tissue depths for a total thickness measurement of approximately 130-160 μm per sample. These measurements were taken completely within the dermis of each sample as the thinnest dermal layer observed was 250 μm, therefore any decrease in signal is due to a decrease in density rather than a lack of dermal collagen material. Z-section images were compiled and finally the 3D image restoration was performed using Volocity software (Improvisions, Inc., Waltham, Mass.). A noise-removal filter whose kernel size of 3×3 was applied to these 3D images and SHG signals that fell within a set threshold were quantified for the entire 3D image using Velocity software (Improvisions Inc.).
Statistical Analysis.
Survival data were analyzed for significance 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. All cases that required euthanasia prior to 30 weeks were attributed to severe open or xanthomatotic-skin lesions. Consistent with previous reports, apoE-KO 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 (
As shown in
Weight Gain.
While a HFD resulted in significant weight gain in C57BL/6 control mice at the 30 week time point; apoE-KO mice on a HFD 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 (
Referring to
More specifically with respect to
Skin Histopathology.
As shown in
Referring to
Skin Thickness.
Skin thinning and atrophy is a characteristic feature that occurs with age both in humans and mice (see, for e.g. Bhattacharyya and Thomas, 2004). To determine whether apoE-KO mice exhibit this trait, we analyzed formalin fixed skin sections from the mid to lower back of the chow or HFD-fed C57BL/6 and apoE-KO mice using H&E staining at 0, 5, 15 and 30 weeks and measured total skin thickness including the epidermis, dermis, adipose and skeletal muscle layers (
Closer analysis of the individual layers of the skin revealed that changes in total skin thickness in the “regular” skin samples were due primarily to changes in the dermal and/or adipose tissue layers. While no significant differences were observed in epidermal thickness at the 30 week time point for any of the groups (
Referring to
Collagen and Elastin Abnormalities in the Skin of apoE-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 visualized using polarized light. As shown in
To examine elastin content in the diseased skin, Luna's elastin stain was used. Wild type control mice demonstrated diffuse elastin distribution with thin elastic fibres and minimal large elastin bundles (
Referring to
In
Decorin Remodelling and Granzyme B Expression in the Skin apoE-KO Mice.
Collagen disorganization was readily observed in the diseased skin of apoE-KO mice (
Referring to
In
Collagen Density and Organization.
To determine if apoE-KO mice exhibit differences in collagen content in the regular, non-xanthoma skin, picrosirius red staining was used on formalin fixed skin sections and analyzed for changes in collagen content and structure. Dermal collagen from the chow-fed control group exhibited typical red/orange staining of thick, dense collagen fibres at the 30 week time point (
Although analysis of fixed, thin sliced sections can provide useful information regarding collagen content and structure, important three dimensional and organizational properties may be missed or altered during processing. We took advantage of the bifringent properties of collagen to visualize collagen structure and organization in unfixed, unstained thick skin samples in three dimensional space using multi-photon microscopy. Highly ordered fibril-forming collagens (Type I, II, III, etc.) produce second harmonic generation (SHG) signals without the need for any exogenous label (see, for e.g., Zipfel et al., 2003). These SHG signals correlate with the density and organization of the collagen matrix rather than total collagen content. Representative flattened three dimensional SHG images originating from the collagen matrix (grey) are shown in
Referring to
To further examine the role of Granzyme B in the observed loss of collagen density, DKO mice skin was also examined using SHG after being fed a chow or HFD for 30 weeks as this was the time point where the most extreme differences were observed. As mentioned, at the 30 week time point only the HFD-feed apoE-KO mice exhibited significantly decreased collagen density in the skin compared to the chow-fed control group as shown by the decreased SHG signal (
Granzyme B cleaves decorin and is present in areas of decorin degradation. Referring to
Discussion of Results in the Foregoing Example:
In the present study, it was demonstrated that a HFD has a considerable effect on skin aging in apoE-KO mice. Not only does it affect the frequency of inflammatory skin disease as the mice age, but also results in a frail, thinned skin state along with significant 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 skin through remodelling of key ECM proteins and proteoglycans. When apoE-KO mice were fed a HFD for 30 weeks, they demonstrated frailty and increased morbidity compared to the wild type controls (
In this study, apoE-KO mice fed a regular chow or HFD showed a decrease in adipose layer thickness at 30 weeks while overall skin thickness in the HFD-fed apoE-KO mouse skin decreased in from the 15 to 30 week time point (
In this study evidence is provided that the serine protease Granzyme B is expressed in areas of collagen and decorin degradation and remodelling in the skin of 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 Granzyme B is disrupting ECM close to or at the dermal epidermal junction. Indeed, DKO mice demonstrated an apparent increase in decorin staining in the skin near the dermal epidermal junction (sec. for e.g.,
In addition to the diseased skin, apoE-KO mice fed a HFD, but not a regular chow diet, demonstrated a significant loss in collagen density in the dermis as shown by SHG and multi-photon microscopy over a 30 week span in “regular” skin samples (
In summary, the findings demonstrate that apoE-deficiency 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 Granzyme 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.
In this Example, inhibition of Granzyme B (Granzyme B) is demonstrated using a specific small molecule inhibitor (Willoughby 20) inhibits betaglycan cleavage. As shown in
In this Example, 20 ug/ml betaglycan was coated onto 48 well plates and incubated with 10 ng of TGF-β. Excess TGF-β was washed off the plate and betaglycan/TGF-β complexes were incubated with Granzyme B+/− inhibitors for 24 h at RT. Supernatants (containing released TGF-β) were collected 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
In this Example, incubations were performed at RT for 24 h in a total reaction volume of 30 ul. Samples were run on a 10% gel and imaged by Coomassic Blue stain. With reference to
Small molecule libraries (ZINC—Irwin J J and Shoichet B K, 2005. J. Chem Inf Model 4591:177-182; NCI—Voigt, J. H. et al. J. Chem. Inf Comput. Sci. 2001, 41, 702-712) were screened in silico for candidate Granzyme B inhibitory compounds. Several candidate small molecule inhibitors were identified and subjected to an in vitro Granzyme B inhibition assay. More specifically, a continuous colormetric assay for Granzyme B activity was carried out with the substrate Ac-IEPD-pNA. Briefly, 8 ug/ml Granzyme B, 20 μM substrate, and increasing concentrations of the inhibitor of interest was incubated in a final reaction volume of 50 ul. The reaction buffer consisted of 50 mM HEPES pH 7.5, 10% sucrose, 0.1% CHAPS and 5 mM DTT and reactions were carried out at 37° C. pNA release and Granzyme B inhibition was monitored at 405 nm on a Tecan Safire microplate reader. Granzyme B was used in the assay at a concentration of 4 μg/ml (0.145 μM), estimated to be about 80,000 fold higher 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 Table B.
As detailed herein and as detailed in Tables A and B herein, candidate inhibitors, and IC50 concentration obtained from the inhibition assay are set out below. Compounds NCI 644752, NCI 644777, ZINC05317216, NCI 630295 demonstrated an IC50 of about 100 μM or less (“High inhibition”); compounds NCI 641248, NCI 641235, NCI 642017, NCI 641230, NCI 641236, NCI 640985, NCI 618802, NCI 623744 demonstrated an IC50 of about 320 μM or less (“Low inhibition”).
Details of this Example are shown representatively in
More specifically, as it relates to
As shown in
As shown in
As shown in
As depicted in
While specific embodiments of the invention have been described and illustrated, such embodiments should be considered illustrative 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 will be apparent from the following description of the drawings and the invention, and from the claims.
This application claims priority to U.S. Provisional Application No. 61/420,230 filed on Dec. 6, 2010 and U.S. Provisional Application No. 61/493,265 filed on Jun. 3, 2011, the entire contents of which are incorporated herein by this reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB11/03207 | 12/6/2011 | WO | 00 | 10/28/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61420230 | Dec 2010 | US | |
| 61493265 | Jun 2011 | US |
