WO2009055934A1 - Treatment of fibrillinopathy or elastinopathy using granzyme inhibitors - Google Patents

Abstract

A method of medical treatment or prevention fibrillinopathy or an elastinopathy in a subject, the method including administering a therapeutically effective amount of a granzyme B inhibitor and/or granzyme A inhibitor to the subject is provided. In other aspects uses of granzyme B and/or granzyme A inhibitors for treatment and/or prevention of a fibrillinopathy or an elastinopathy, or for preparation of medicaments for treatment of a fibrillinopathy or an elastinopathy are provided.

Description

TREATMENT OF FIBRILLINOPATHY OR ELASTINOPATHY USING

GRANZYME INHIBITORS

Technical Field

This invention relates to the field of structural protein pathology. More particularly to the treatment of structural protein pathology by granzyme inhibition.

Background

The granzymes are highly conserved group of serine proteases, with five members (A, B, H, K and M) in humans and ten members (A-G, K, M-N) in mice (Sattar R. et al. Biochem Biophys Res Commun 308, 726-35 (2003). Granzyme B (GrB or cytotoxic T-lymphocyte (CTL)-associated gene transcript- 1 - Brunet JF. et al. Nature 322, 268-71 (1986)), has been reported as being involved in anti-viral and anti-tumour functions, and is associated with autoimmunity, transplant rejection, graft- versus-host disease, and thymocyte development (Barry M. & Bleackley RC. Nat Rev Immunol 2, 401-9 (2002)). Granzyme A (GrA) is also involved in immune-mediated killing, and is expressed by both innate and adaptive immune cytotoxic cells.

GrB is reported to have a contribution to CTL-mediated target cell apoptosis. GrB-deficient mice possess a normal phenotype, with the exception of a slightly reduced CTL-mediated target cell apoptosis, anti-viral responses and tumour cell clearance (Revell PA. et al. J Immunol 174, 2124-31 (2005); and Heusel JW. et al. Cell 76, 977-87 (1994)), suggesting a redundancy in immune mediated cells removal. GrB-deficient recipient mice exhibit reduced allograft vasculopathy (Choy JC. et al. Am J Transplant 5, 494-9 (2005)), and its deficiency in mice leads to increased susceptibility to allergen-induced asthma (Devadas, S. et al. Immunity 25, 237-47 (2006)).

Buzza MS. et al. report that plasma GrA levels in normal individuals are between 15-35 pg/ml and plasma GrB levels in normal individuals are up to 15pg/ml (Biol. Chem. 387:827-837 (2006)). Skjelland, et al. teach that plasma levels of granzyme B are increased in patients with lipid rich carotid plaques, but also teach that normal plasma levels of GrB can be up to 1 OOpg/ml, while patients with unstable plaques have plasma levels of GrB over about 100pg/ml (and up to about 650pg/ml) and patients with stable plaques have plasma levels of GrB between about 25pg/ml and about 400pg/ml (Atherosclerosis, 195:el42-el46 (2007)). Choy JC. et al. reported increased levels of GrB in patients with advanced atherosclerosis (Mod Pathol 16, 460-70 (2003)). GrB has also been associated with aortic aneurisms and with atherosclerosis plaque destabilization (Choy et al. Arterioscler. Thromb. Vase. Biol; 24; 2245-2250, (2004)). Kim et al, show that macrophages express granzyme B in the lesion areas of atherosclerosis and rheumatoid arthritis (Immunology Letters, 111, 57-65, (2007)). Increased GrB levels in Chronic Obstructive Pulmonary Disease (COPD) patients were reported in bronchoalveolar lavage (BAL) derived T-cells (Hodge et al. J. of COPD 3:179-187 (2006)). Also, GrB produced protein fragments are reported in Sjogren's Syndrome patients (Huang M. et al. Clin Exp Immun 142:148-154 (2005)). Additionally, GrA and GrB are reported in BAL fluids from patients with inflammatory lung disease (Tremblay et al. J Immunology 165:3966-3969 (2000)). Thewissen et al. compares GrA and GrB levels in rheumatoid arthritis (RA), multiple sclerosis (MS), and between healthy individuals, and reports no change in GrA levels between healthy patients and RA or MS patients for GrA and reports a decrease in GrB levels for MS patients relative to healthy patients (Clinical Immunology 123:209-218 (2007)). GrA released in the brain maybe associated with autoimmune disorders of the nervous system (Suidan et α/.PNAS 91 :8112- 8116 (1994)). Vernooy et al. report increased GrA expression in type II pneumocytes of patients with severe COPD (Am J Respir Crit Care Med 175:464-472 (2007)). GrB has also been reported to cleave vitronectin, fibronectin, and laminin (Buzza MS. et al. JBC vol. 280(25):23549-23558 (2005)). Furthermore, GrB has been associated with acute coronary syndrome (Tsuru R. et al. Heart 94:305-310 (2008) e-published June 25, 2007). GrB has also been reported on in association with rheumatoid arthritis (Goldbach-Mansky et al. Ann Rheum Dis. 64:715-721 (2005); Kraan et al. Ann Rheum Dis 63:483-488 (2004); and Villanueva et al. Arthritis Res Ther 7:R30-R37 (2005)) in inflammatory lung disease (Tremblay et al. J Immunology 165:3966-3969 (2000)), in Chronic Obstructive Pulmonary Disease (Hodge et al. J. of COPD 3:179-187 (2006), and Sjogren's Syndrome (Rosen et al. Crit Rev Oral Biol Med 15(3): 156-164 (2004)). Immunodiagnostic methods for Granzymes A and B are known (for example WO 99/54737). GrB inhibitors are also known (for example WO 03/065987).

Summary

This invention is based, in part, on the discovery of the contribution granzyme A makes to certain diseases, including fibrillinopathies and elastinopathies, and in particular to the extracellular activity of granzyme A whereby granzyme A cleaves extracellular matrix proteins such as fibronectin and fibrillin- 1.

This invention is also based, in part, on the discovery of the contribution granzyme B makes to certain diseases, including fibrillinopathies, elastinopathies and actinic elastosis, and in particular to the extracellular activity of granzyme B whereby granzyme B cleaves extracellular matrix proteins such as elastin, Fibulin-2, Fibrillin- 1, and Fibrillin- 2.

Fibrillinopathies may be selected from one or more of the following: Marfan syndrome; autosomal dominant Weill-Marchesani syndrome; severe neonatal Marfan syndrome; dominant ectopia lentis; familial ascending aortic aneurysm; isolated skeletal features of Marfan syndrome (or Familial Marfanoid habitus); Beals syndrome (or Arachnodactyly, Contractural Beals Type, Beals-Hecht Syndrome, and Congenital Contractural Arachnodactyly (CCA)); Familial mitral valve prolapse syndrome (MVP); mitral valve prolapse, myopia, minimal or no aortic dilation, subtle skeletal changes and skin changes (MASS phenotype); or Shprintzen-Goldberg syndrome.

Elastinopathies may may be selected from one or more of the following: supravalvular aortic stenosis; Williams syndrome; and Cutis laxa.

In one aspect of the present invention, there is provided a method for diagnosis of fibrillinopathies and/or elastinopathies in a subject suspected of having a fibrillinopathy or an elastinopathy, the method including: determining the concentration of GrB and/or GrA in a blood plasma or serum sample from the subject; and comparing the concentrations to the corresponding concentration in a control sample, wherein an elevated concentration of GrB and/or GrA is indicative of a fibrillinopathy or an elastinopathy.

The method may further include determining the concentration of one or more of: elastin; Fibronectin; Fibulin-2; Fibrillin-1; and Fibrillin-2; with reference to the control sample as indicative of fibrillinopathy, elastinopathy, or actinic elastosis as described herein. The concentration of GrB or GrA or elastin or fibrillin or fibulin or fibronectin may be determined by an immunodiagnostic assay. The immunodiagnostic assay may be an enzyme-linked Immunosorbent assay (ELISA), enzyme-linked immunosorbent spot (ELISPOT), dot blot, western blot, or other proteomics assay etc. The elastin or fibrillin or fibulin or fibronectin, may be an elastin degradation product or a fibrillin degradation product or fibulin degradation product or a fibronectin degradation product. The method may further include one or more of: diagnostic imaging; clinical diagnosis and alternative laboratory diagnostics.

In accordance with another aspect of the invention, there may be provided a method of preventing or treating a fibrillinopathy or an elastinopathy in a subject in need thereof, the method including adminmay betering to the subject one or more of: a Granzyme B (GrB) inhibitor and Granzyme A (GrA) inhibitor.

In accordance with another aspect of the invention, there is provided a use of a Granzyme B (GrB) inhibitor or a Granzyme A (GrA) inhibitor in the manufacture of a medicament for the prevention or treatment of a fibrillinopathy or an elastinopathy in a subject in need thereof.

In accordance with another aspect of the invention, there is provided a use of a Granzyme B (GrB) inhibitor or a Granzyme A (GrA) inhibitor for the prevention or treatment of a fibrillinopathy or an elastinopathy in a subject in need thereof.

In accordance with another aspect of the invention, there is provided a use of a pharmaceutical composition comprising a Granzyme B (GrB) inhibitor or a Granzyme A (GrA) inhibitor for the prevention or treatment of a fibrillinopathy or an elastinopathy in a subject in need thereof.

In accordance with another aspect of the invention, there is provided a kit or a commercial package including: a pharmaceutical composition including: a GrB inhibitor; or a GrA inhibitor; and a pharmaceutically acceptable carrier; and instructions for the use thereof for treating a fibrillinopathy or an elastinopathy.

The fibrillinopathy or the elastinopathy may be selected from one or more of: Marfan syndrome; autosomal dominant Weill-Marchesani syndrome; severe neonatal Marfan syndrome; dominant ectopia lentmay be; may beolated skeletal features of Marfan syndrome; Beals syndrome; familial ascending aortic aneurysm; Familial mitral valve prolapse syndrome (MVP); mitral valve prolapse, myopia, minimal or no aortic dilation, subtle skeletal changes and skin changes (MASS phenotype); Shprintzen-Goldberg syndrome; supravalvular aortic stenosmay be; Williams syndrome; and Cutmay be laxa. The fibrillinopathy may be Marfan syndrome. The fibrillinopathy may be autosomal dominant Weill-Marchesani syndrome. The fibrillinopathy may be severe neonatal Marfan syndrome. The fibrillinopathy may be isolated skeletal features of Marfan syndrome. The fibrillinopathy may be dominant ectopia lentmay be. The fibrillinopathy may be Beals syndrome. The fibrillinopathy may be MVP. The fibrillinopathy may be MASS phenotype. The fibrillinopathy may be Shprintzen-Goldberg syndrome. The elastinopathy may be supravalvular aortic stenosis. The elastinopathy may be Williams syndrome. The elastinopathy may be Cutmay be laxa. The subject may be a human.

The GrA or GrB inhibitor may be formulated for oral administration. The GrA or GrB inhibitor may be formulated for administration by injection. The GrA or GrB inhibitor may be formulated for topical administration. The GrA or GrB inhibitor may be formulated for topical application to a device.

In accordance with another aspect of the invention, there is provided a method of preventing or treating actinic elastosis in a subject in need thereof, the method including administering to the subject a Granzyme B (GrB) protein, peptide, fragment thereof or variant thereof.

In accordance with another aspect of the invention, there is provided a use of a GrB protein, peptide, fragment thereof or variant thereof in the manufacture of a medicament for the prevention or treatment of actinic elastosis in a subject in need thereof.

In accordance with another aspect of the invention, there is provided a use of a GrB protein, peptide, fragment thereof or variant thereof for the prevention or treatment of actinic elastosis in a subject in need thereof.

In accordance with another aspect of the invention, there is provided a use of a pharmaceutical composition including a GrB protein, peptide, fragment thereof or variant thereof for the prevention or treatment of actinic elastosis in a subject in need thereof.

Brief Description of the Drawings

Figure 1 shows the percentage area of the aortic root in ApoE KO (white bar) and ApoE/GrB DKO (black bar) mice fed a Western diet. N=2 for the DKO mice, N=4 for the ApoE KO mice. Values for each section was calculated (sum of the plaque area) / (total aortic root area) * 100%. For each animal, 3-7 sections of aortic roots were analyzed for % lesion area and averaged.

Figure 2 Representative artery sections from mice fed a Western diet for 30 weeks. (A)C57 WT, (B) GrB-/-, (C) ApoE-/-, (D) ApoE/GrB-DKO.

Figure 3 Skin of dehaired mice. C57 BL/6 (wt), GrB -/-, ApoE -/- and GrB/ ApoE double knockout mice (DKO) with hair removed show the varying skin conditions associated with the gene knockout phenotypes. Figure 4 Representative skin samples at 30 weeks. Hair follicles are deeply embedded in both the GrB-KO and GrB/ApoE-DKO. Hair follicle density is also much greater in the GrB/ApoE DKO. Xanthoma formation is visible in the ApoE-KO and absent from GrB/ApoE-DKO. There also appears to be a change in the extracellular matrix composition in the GrB-KO and DKO mice as the white area/processing artifact is always present in these tissues in the dermis and likely due to changes in hydrophobicity in the matrix and fixation procedures.

Figure 5 granzyme B degrades elastin in vitro. Granzyme B was incubated with ³H-elastin for 7 days at room temperature. Elastase was incubated with ³H-elastin for 2 hours. Supernatants containing the soluble elastin cleavage fragments were collected and counted. Data is represented as fold increase in radioactivity over the control (elastin only). (n = 2)

Figure 6A illustrates extracellular proteins visualized by Western blot for biotin. Increased levels of fragmented extracellular matrix proteins were observed in granzyme A-treated plates.

Figure 6B illustrates supernatants treated with granzyme A for the indicated times were probed for fibronectin and several fragments were observed, as indicated by the arrows, by Western blot.

Detailed Description

A sample from the subject and a normal sample from a normal subject may be blood plasma samples, bronchiole lavages or other bodily fluids. A "subject" and a "normal subject" differ, at least in part, in that the normal subject is known to not have, or at least suspected of not having, a fibrillinopathy or an elastinopathy, or actinic elastosis, and is not at risk for having or developing a fibrillinopathy or an elastinopathy or actinic elastosis as described herein. Provided that the sample from the subject and the normal sample from the normal subject are taken from the same tissue type or bodily fluid type, then the sample from the subject may be compared to the normal sample from the normal subject for the purpose of identifying a subject for treatment or prevention of a fibrillinopathy or an elastinopathy or actinic elastosis as described herein.

Granzyme A or B inhibitors include any molecule that inhibits the inhibiting the GrA and/or GrB protein, either directly or indirectly, for example by up regulating endogenous inhibitors (e.g., PI9) and/or shutting down transcription of the GrA and/or GrB gene or translation of the GrA and/or GrB transcript. DNA/RNA can be used to inhibit the protein directly (aptamers) or the transcription/translation of GrA and/or GrB. Granzyme A or Granzyme B inhibitors include, but are not limited to, peptides, antibodies (for example, polyclonal; monoclonal, fragments (F(ab')2 and Fab)), small molecules, scFc, peptidomimetics, siRNA, antisense molecules (such as RNA and other nucleic acid molecules) etc. Furthermore, a GrA and/or GrB inhibitor may not be specific for GrA and/or GrB alone and may be a broad spectrum inhibitor of granzymes (as a familiy) or an inhibitor of serine protease.

Many Granzyme A inhibitors are known to a person of skill in the art and are, for example, 3,4-dichloroisocoumarin and others are described in: Granzyme A Released Upon Stimulation of Cytotoxic T Lymphocytes Activates the Thrombin Receptor on Neuronal Cells and Astrocytes, Suidan, et al., Proceesings of the National Academy of Sciences of the United States of America, VoI 91 , No. 17 pp 8112-8116, ( 1994); Granzyme A Is an Inter leukin 1 β-converting Enzyme, Irmler, et al., J. Exp. Med., Vol. 181, pp 1917-1922, (1995); Granzymes (lymphocyte serine proteases): characterization with natural and synthetic substrates and inhibitors, Kam et al., Biochimica et Biophysica Acta, Vol. 1477, pp307-323, (2000); Nuclear war: the granzyme A-bomb, Lieberman, et al., Current Opinion in Immunology, Vol. 15, pp 553-559, (2003).; and Selective Chemical Functional Probes of Granzymes A and B Reveal Granzyme B Is a Major Effector of Natural Killer Cell-Mediated Lysis of Target Cells, Mahrus, et al., Chemistry & Biology, VoI 12, pp 567-577, (2005). Methods of identifying a granzyme A inhibitor are described elsewhere in this application.

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 CA. et al. BioorgMed Chem Lett. 12:2197-2200 (2002); Hill GE. et al. J Thorac Cardiovasc Surg 110:1658-1662 (1995); Sun J. et al J Biol Chem 271 :27802-27809 (1996); Sun J. et al. J Biol Chem 272:15434-15441 (1997); Bird et al. MoI. Cell. Biol. 18, 6387-6398 (1998); Kam et al Biochim Biophys Acta 1477(1 -2):307:23 (2000); and Bio-x-IEPD^p-(OPh)₂ as described in Mahrus S. and Craik CS. Chemistry & Biology 12:567-577 (2005). 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). Further examples of granzyme B inhibitors are: Z-AAD-CMK (IUPAC name: 5-chloro-4-oxo-2-[2-[2-(phenylmethoxycarbonylamino) propanoylamino]propanoylamino] pentanoic acid) MF: C19H24C1N3O7 CID: 16760474; Ac-IEPD-CHO; Granzyme B Inhibitor IV or Casρase-8 inhibitor III (IUPAC: (4S)-4- [[(2S)-2-acetamido-4-methylpentanoyl]amino]-5-[2-[[(2S)-4-hydroxy-l,4-dioxobutan-2- yl]carbamoyl]pyrrolidin-l-yl]-5-oxopentanoic acid) 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-l-[[(2S)-4- hydroxy-l,4-dioxobutan-2-yl]amino]-l-oxobutan-2-yl]amino]-5-oxopentanoic acid) MF: C21H34N4O10 CID: 16760475. Methods of identifying a granzyme B inhibitor are described elsewhere in this application.

Granzyme B (also known as GrB, GZMB, granzyme 2, cytotoxic T-lymphocyte- associated serine esterase 1) proteins, peptides, fragments thereof and variants thereof (referred to collectively herein as GrB) are known in the art (for example, EAW66002.1 GI: 119586406; EAW66003.1 GI: 119586407) and maybe formulated for topical administration to treat actinic elastosis. The GrB as described herein for treatment of actinic elastosis should retain its Fibulin-2 cleavage properties.

The granzyme A and/or granzyme B inhibitor or GrB may be formulated for a variety of different suitable routes of administration, such as inhalation, topical, parenteral, enteral and others, depending on the indication to be treated, prevented or ameliorated. Furthermore, a granzyme A and/or granzyme B inhibitor or GrB may be applied topically to an area in need of treatment, prevention or amelioration. Alternatively, the granzyme A and/or granzyme B inhibitor or GrB may be formulated for application to the surface of a device (for example, as a coating on a stent, clip, catheter, coil, bandage or other covering etc.). Alternatively, GrB may be formulated for use in a UV block or post UV exposure formulation, to prevent, treat or ameliorate actinic elastosis.

In another aspect of the present invention, there is provided use of a granzyme A and/or granzyme B inhibitor for treatment of one or more of: a fibrillinopathy and an elastinopathy. A fibrillinopathy may be selected from one or more of: Marfan syndrome; autosomal dominant Weill-Marchesani syndrome; severe neonatal Marfan syndrome; dominant ectopia lentis; familial ascending aortic aneurysm; isolated skeletal features of Marfan syndrome (or Familial Marfanoid habitus); Beals syndrome (or Arachnodactyly, Contractural Beals Type, Beals-Hecht Syndrome, and Congenital Contractural Arachnodactyly (CCA)); Familial mitral valve prolapse syndrome (MVP); mitral valve prolapse, myopia, minimal or no aortic dilation, subtle skeletal changes and skin changes (MASS phenotype); or Shprintzen-Goldberg syndrome. An elastinopathy may be selected from one or more of: Supravalvular aortic stenosis (or SVAS); Williams syndrome (or WS, Williams-Beuren syndrome, WBS); and Cutis laxa (or CL, which may be autosomal dominant, autosomal recessive, and X-linked recessive).

Many molecules, compounds and compositions described herein are generally water soluble and may be formed as salts. In such cases, pharmaceutical compositions in accordance with this aspect of the invention may comprise a salt of such a compound, preferably a physiologically acceptable salt, which are known in the art. Pharmaceutical preparations will typically comprise one or more carriers acceptable for the mode of administration of the preparation, be it by injection, inhalation, topical administration, lavage, or other modes suitable for the selected treatment. Suitable carriers are those known in the art for use in such modes of administration.

Suitable pharmaceutical compositions may be formulated by means known in the art and their mode of administration and dose determined by the skilled practitioner. For parenteral administration, a 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, the 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, 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 art are described in Remington: the Science & Practice of Pharmacy by Alfonso Gennaro, 20^th ed., Lippencott Williams & Wilkins, (2000). Formulations for parenteral administration 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 parenteral delivery systems for modulatory compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation 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 nasal drops, or as a gel. Formulations of antisense nucleic acid molecules are also known to a person of skill in the art. Isis pharmaceuticals is a company that has developed several antisense formulations, including Vitravene™. Such formulations may be used with antisense nucleic acid molecules that are inhibitors of granzyme A and/or granzyme B as appropriate.

Compounds or pharmaceutical compositions in accordance with aspects of the invention or for use as described herein may be administered by means of a medical device or appliance such as an implant, graft, prosthesis, stent, bandage or other covering etc. Also, devices or appliances may be devised which are intended to contain and release such compounds or compositions. An example would be an implant made of a polymeric material adapted to release the compound over a period of time.

An "effective amount" of a pharmaceutical composition as described herein includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduced extracellular matrix protein cleavage, reduced levels of granzyme A and/or granzyme B activity, improved blood flow, and/or a delay or reduction in the severity of the onset of a fibrillinopathy and/or an elastinopathy as described herein. A therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as reduced extracellular matrix protein cleavage, reduced levels of granzyme A and/or granzyme B activity, improved blood flow, improved tissue elasticity, and maintenance of tissue elastin, fibulin and fibrillin- 1 & -2 content as described herein. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount. It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners. The amount of active compound(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It maybe advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.

In general, compounds as described herein should be used without causing substantial toxicity. Toxicity of the compounds as described herein can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LDlOO (the dose lethal to 100% of the population). In some circumstances however, such as in severe disease conditions, it may be necessary to administer substantial excesses of the compositions.

As used herein, a "subject" or "normal subject" may be a human, non-human primate, or a mammal, or a rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc. The subject may be suspected of having or at risk for having a fibrillinopathy and/or an elastinopathy as described herein. Some diagnostic methods for a fibrillinopathy and/or an elastinopathy as described herein and the clinical delineation of diagnoses of a fibrillinopathy and/or an elastinopathy as described herein are known to those of ordinary skill in the art.

Furthermore, subjects may be tested for GrA and/or GrB levels; fibulin-2; fibrillin- 1, fibrillin-2, elastin levels and degradation products thereof; to determine the subject's risk of a poor outcome from the fibrillinopathy and/or elastinopathy. A poor outcome may be the further reduction in tissue elasticity or reduced blood flow. To evaluate a subject's risk, blood samples (7.5 ml) may be collected from normal subjects having or suspected of having a vasculopathy using a purple top EDTA vacutainer tube (BD™). Following collection, the tube may be inverted 5 times for thorough mixing. The tubes may then be then centrifuged for 11 min at 276 x g (Beckman Coulter™). Following centrifugation, the tubes should be separated into 3 distinct layers: a bottom layer of mostly red blood cells, a thin film layer of white blood cells (buffy coat) and a top layer of plasma. Using a sterile transfer pipette, the top layer of plasma down to about lmm from the red blood cells may be removed, with caution so that you are careful not to aspirate the buffy coat, and the plasma can then be placed into a labeled orange top cryotube. The samples may be stored immediately at -8O⁰C until plasma analysis is performed.

For plasma analysis, human granzyme A and/or granzyme B ELISA kits are available from Bender Medsystems™ (for example, catalog number: BMS2027). The kits comprise enzyme-linked immunosorbent assay for quantitative detection of human granzyme A and/or granzyme B. The reagents may be prepared as per the kit's protocols: a) Wash Buffer; b) Dilution Buffer; c) Biotin-Conjugate; d) Granzyme standards; e) Streptavidin-HRP; and f) Colour-giving reagents: Blue-Dye, Green-Dye, Red-Dye. The assays may be performed as per the kit's protocols and the calculation of the results may also be performed as per the kit's protocols. ELISA's could also be used to detect fibrillin- 1, fibrillin-2, elastin, and degradation products thereof.

In another aspect of the present invention, there is provided a method of medical treatment comprising administering a therapeutically effective amount of a granzyme A and/or granzyme B inhibitor to treat a fibrillinopathy and/or an elastinopathy as described herein or GrB to treat, prevent or ameliorate actinic elastosis as described herein.

Prior to treatment with a granzyme A and/or granzyme B inhibitor or GrB, various methods are known in the art for determining the presence or absence of a fibrillinopathy and/or an elastinopathy or actinic elastosis. For example, a fibrillinopathy and/or an elastinopathy or actinic elastosis may be determined by one or more of the following:

(a) blood tests may be used for genetic testing to determine whether a subject has a genetic mutation that could account for a fibrillinopathy and/or an elastinopathy;

(b) clinical examination;

(c) doppler ultrasound may be used to measure blood pressure at various points along an arm or leg, which may assist in gauging the degree of any heart or vessel malformations, as well as the speed of blood flow through arteries;

(d) angiogram (with dye) allows for a view blood flow through the heart, brain, arms or legs, which can show narrow spots and malformations on the X-ray images; (e) tissue biopsy and histological examination; and

(f) other imaging tests may use ultrasound, a computerized tomography (CT) scanning or a magnetic resonance angiogram (MRA) to image the any heart or vessel malformations, or skeletal or joint malformations (with and without contrast), which may be indicative of the a fibrillinopathy and/or an elastinopathy.

"Fibrillinopathies" or "Microfibrillopathies" are caused by defects in the fibrillin- 1 protein (type 1) and defects in the fϊbrillin-2 protein (type 2). While Marfan syndrome (MIM 154700) is the most common type of fibrillinopathy, not all mutations in the FBNl gene cause this syndrome. FBNl mutations cause a spectrum of connective tissue disorders, with a broad range in severity and age of onset. Some FBNl mutations cause a severe disorder that is fatal to newborns, while other mutations cause adult onset fibrillinopathies with a single abnormality, such as a dislocated lens in the eye or an abnormal aorta. Fibrillin- 1 mutations have also been found in several other related connective tissue disorders, such as severe neonatal Marfan syndrome, dominant ectopia lentis (MIM 129600), familial ascending aortic aneurysm, isolated skeletal features of Marfan syndrome(or Familial Marfanoid habitus), and Shprintzen-Goldberg syndrome (MIM 182212). The gene for fibrillin-2 (FBN2) is highly homologous to FBNl, and mutations in FBN2 have been shown to cause a phenotypically related disorder termed congenital contractural arachnodactyly or familial arachnodactyly (MIM 100700). Since mutations in the fibrillin genes are likely to affect the global function of the microfibrils, the term microfibrillopathy may be the most appropriate to designate the spectrum of disease associated with dysfunction of FBNl and FBN2 (Robinson PN. and Godfrey M. J Med Genet. (2000) 37(l):9-25).

"Marfan syndrome" (or Marian's syndrome) is a genetic disorder of the connective tissue and may be inherited as a dominant trait carried by a gene called FBNl, which encodes a connective protein called fibrillin- 1. Complications include defects of the heart valves, aorta, lungs, eyes, dural sac surrounding the spinal cord, skeleton and hard palate.

"Autosomal Dominant Weill-Marchesani syndrome" (or AD WMS) is a connective tissue disorder characterised by short stature, brachydactyly, joint stiffness, and characteristic eye anomalies including microspherophakia, ectopia of the lenses, severe myopia, and glaucoma. Both autosomal recessive (AR) and autosomal dominant (AD) modes of inheritance have been described and a gene for AR WMS has been mapped to chromosome 19pl3.3-pl3.2. However, AD and AR WMS are genetically heterogeneous entities. A 24 nt in frame deletion within a latent transforming growth factor-betal binding protein (LTBP) motif of the fibrillin- 1 gene was found in a AD WMS family (exon 41, 5074_5097del). This deletion cosegregated with the disease and was not found in 186 controls, which suggests that AD WMS and Marfan syndrome are allelic conditions at the fibrillin-1 locus (Faivre L. et al. J Med Genet. (2003) 40(l):34-6).

"Beals syndrome" (or Arachnodactyly, Contractural Beals Type, Beals-Hecht Syndrome, Congenital Contractural Arachnodactyly (CCA)(MIM 121050)) is an extremely rare genetic disorder caused by a mutation in fibrillin-2 gene (FBN2) and characterized by the permanent fixation of certain joints (e.g., fingers, elbows, knees, and hips) in a flexed position (contractures); abnormally long, slender fingers and toes (arachnodactyly); permanently flexed fingers (camptodactyly); and/or abnormally shaped ears resulting in a "crumpled" appearance. Affected individuals may also exhibit front-to- back and side-to-side curvature of the spine (kyphoscoliosis); feet that are abnormally positioned (talipes equinovarus or clubfoot); outward displacement of the fingers (ulnar deviation of the fingers); an abnormally short neck; and/or displacement of the lens of the eye (ectopia lentis). In some cases, affected individuals may have a slight deformity of the valve on the left side of the heart (mitral valve prolapse). Beals syndrome is inherited as an autosomal dominant trait.

Familial mitral valve prolapse syndrome (MVP - MIM 157700), is characterized by a positive family history of mitral valve prolapse and thickening of the mitral valve, but no lens luxation nor aortic problems (Loeys BL. et al. Acta Clinica Belgica (2002) 58(1):233-241). Mitral valve prolapse is a common disorder characterized by systolic displacement or billowing of the mitral leaflets into the left atrium, often accompanied by mitral regurgitation. MVP is genetically heterogeneous and is inherited as an autosomal dominant trait that exhibits both sex- and age-dependent penetrance. Grau JB. et al. provide a detailed review of the genetics of mitral valve prolapse (Clin. Genet. 72: 288- 295, 2007). The presence of additional connective tissue features in skin and skeleton suggests the MASS phenotype (MIM 604308), which stands for mitral valve prolapse, myopia, minimal or no aortic dilatation, subtle skeletal changes and skin changes or striae atrophicae (Loeys BL. et al. Acta Clinica Belgica (2002) 58(1):233-241)). MASS is a Marfan-like syndrome that can be caused by mutation in the gene encoding fibrillin-1 (FBNl). Dietz HC. et al. demonstrated that one basis for the MASS phenotype can be a nonsense frameshift mutation in the FBNl gene (Genomics (1993) 17:468-475). People with MASS or familial MVP should be followed to monitor for progressive dilatation or dissection of the aortic root. Furthermore, mitral valve regurgitation and endocarditis prophylaxis is often needed.

"Elastinopathies" as used herein caused by defects in the elastin protein (for example, supravalvular aortic stenosis (or SVAS), Williams syndrome (or WS, Williams- Beuren syndrome, WBS), and Cutis laxa).

"Williams syndrome" (or WS, Williams-Beuren syndrome, WBS) is a rare neurodevelopmental disorder caused by a deletion of about 26 genes from the long arm of chromosome 7 and is characterized by 'elfin' facial features, a low nasal bridge, an unusually cheerful demeanor and ease with strangers, coupled with unpredictably occurring negative outbursts, a predisposition to violent outbursts, mental retardation coupled with unusual (for persons who are diagnosed as mentally retarded) language skills, a love for music, and cardiovascular problems, such as supravalvular aortic stenosis and transient hypercalcaemia. The deleted region typically includes CLIP2, ELN, GTF2I, GTF2IRD1, and LIMKl. Loss of the ELN gene (elastin), is associated with the connective-tissue abnormalities and cardiovascular disease (specifically supravalvular aortic stenosis (SVAS) and supravalvular pulmonary stenosis (SVPS)) found in many people with this syndrome.

"Supravalvular aortic stenosis" (or SVAS) is an inherited vascular disease and a fixed form of congenital left ventricular outflow tract (LVOT) obstruction that occurs as a localized or a diffuse narrowing of the ascending aorta beyond the superior margin of the sinuses of Valsalva. SVAS is caused by mutations in the elastin gene (ELN) (Park S. et al. Int J MoI Med. (2006) 18(2):329-32). SVAS may occur as a manifestation of elastin arteriopathy, sporadically, as an autosomal-dominant condition or as part of the developmental disorder Williams-Beuren syndrome (WBS). SVAS may be caused by heterozygous genetic lesions involving the elastin (ELN) gene locus on chromosome 7ql 1.23. Point mutations, chromosomal deletions, and translocation involving ELN have also been described in individuals with nonsyndromic SVAS (M. and Urban Z Methods MoI Med. (2006) 126:129-56).

"Cutis laxa" (or CL) as used herein is a rare, inherited or sporadic connective tissue disorder where a subject's skin becomes inelastic and hangs loosely in folds. CL can be autosomal dominant, autosomal recessive, or X-linked recessive in its inherited forms. A serine to proline amino acid substitution in the fibulin 5 (FB LN5) gene has been associated with problems in normal elastogenesis, resulting in a recessive form of CL in humans. The X-linked form is currently classified in the group of copper transport diseases. The precise cause is unknown, but it may be due to abnormal elastin metabolism resulting in markedly reduced dermal elastin content.

Furthermore, the extracellular matrix protein fibulin-2 (a 195-kDa protein) is reported to play a structural role in larger extracellular matrix assemblies. Fibulin-2 has also been shown to be significantly increased in actinic elastosis as compared to sun protected skin. Actinic elastosis (also known as solar elastosis) is a degradation of elastic fibers in the skin associated with photo-aged or UV-damaged skin. Hunzelmann N. et al (British Journal of Dermatology (2001) 145: 217-222) report that fibulin-2 and elastin colocalized in microfibrils and that fibulin-2 is significantly higher in actinic elastosis as compared to sun protected skin. Accordingly, Granzyme B, which has fibulin-2 cleavage properties may provide a suitable treatment for actinic elastosis. Granzyme B proteins, peptides, fragments thereof, and variants thereof, that retain fibulin-2 cleavage properties may be formulated for actinic elastosis treatment, prevention or amelioration as described herein. Furthermore, the production of Granzyme B proteins is further described in the antibody production section below and Granzyme B activity may be tested as described herein.

Antibody Production

One methodology is to detect the presence of GrA or GrB or elastin or fibronectin or fibrillin or fibulin specific peptides or proteins. GrA or GrB or elastin or fibronectin or fibrillin or fibulin specific peptides or proteins may also include degradation products thereof. These peptides or proteins may be detected by isolating proteinaceous material from a biological sample and determining the sequence of peptides or proteins so isolated and comparing to the known sequence of GrA or GrB or elastin or fibronectin or fibrillin or fibulin proteins. Preferably, such detecting will make use of an intermediate agent such as an antibody specific for the GrA or GrB or elastin or fibronectin or fibrillin or fibulin peptide or protein as known in the art.

Antibodies to GrA or GrB or elastin or fibronectin or fibrillin or fibulin peptides or proteins may be prepared by a variety of known methods. Such antibodies may be polyclonal, monoclonal, or may be fragments of antibodies.

For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others, may be immunized by injection with a GrA or GrB or elastin or fibronectin or fibrillin or fibulin peptide or protein fragment which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active X substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that GrA or GrB or elastin or fibronectin or fibrillin or fibrillin or fibulin peptides, fragments, or oligopeptides used to induce antibodies have an amino acid sequence consisting of at least five amino acids, and more preferably at least 10 amino acids. It is also preferable that they are identical to a portion of the amino acid sequence of the natural protein, and they may contain the entire amino acid sequence of the GrA or GrB or elastin or fibronectin or fibrillin or fibulin peptide or protein. Short stretches of GrA or GrB or elastin or fibronectin or fibrillin or fibulin amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule.

Peptides corresponding to a GrA or GrB or elastin or fibronectin or fibrillin or fibulin amino acid sequence may be synthesized using methods known in the art, including the recombinant techniques disclosed in the examples below. Such peptides may also be made to incorporate a N-terminal cysteine to facilitate conjugation to other molecules (e.g. to enhance immunogenicity) with such conjugation being mediated by an agent such as m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS). Antibodies that specifically react with the peptide may be purified from the antisera by affinity chromatography, for example by using Cellulofine (Seikagaku Corporation) conjugated with the peptide. The resulting antibodies may be tested by immunoblotting.

Monoclonal antibodies to GrA or GrB or elastin or fibronectin or fibrillin or fibulin peptides or proteins or anti-idiotypic monoclonal antibodies may be prepared using any technique, which provide for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) MoI. Cell Biol. 62:109-120). One process for obtaining the hybridomas as described herein involves starting from spleen cells of an animal, e.g. mouse or rat, previously immunized in vivo or from spleen cells of such animals previously immunized in vitro with an antigen and fusing the immunized cells with myeloma cells under hybridoma-forming conditions; and selecting those hybridomas which secrete the monoclonal antibodies which are capable of specifically recognizing the GrA or GrB or elastin or fibronectin or fibrillin or fibulin peptide or protein.

Selected hybridomas are cultured in appropriate culture medium; and then the secreted monoclonal antibodies are recovered; or alternatively the selected hybridoma is implanted into the peritoneum of a mouse and, when ascites has been produced in the animal; the monoclonal antibodies formed from the ascites are recovered. Monoclonal antibodies as described herein may be prepared by conventional in vitro techniques such as the culturing of immobilized cells using e.g. hollow fibers or microcapsules or such as the culturing of cells in homogeneous suspension using e.g. airlift reactors or stirred bioreactors.

In addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci. 81 :6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce GrA or GrB or elastin or fibronectin or fibrillin or fibulin- specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3). Such single chain antibodies may also be used for production of anti-idiotypic antibodies for use as described herein.

Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites specific for GrA or GrB or elastin or fibronectin or fibrillin or fibulin peptides or proteins or for anti-GrA or GrB or elastin or fibronectin or fibrillin or fibulin antibodies may also be generated. For example, such fragments include, but are not limited to, the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W. D. et al (1989) Science 254:1275-1281). Such fragments when specific for anti-GrA or GrB or elastin or fibronectin or fibrillin or fibulin antibodies may be used for production of anti-idiotypic antibodies or fragments thereof.

Monoclonal antibodies as described herein may be "chimeric", an example of which is an animal antigen-binding variable domain coupled to a human constant domain (Cabilly et al, U.S. Pat. No. 4,816,567; Morrison, S. L. et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Boulianne, G. L. et al., Nature 312:643-646 (1984); Neuberger, M. S. et al, Nature 314:268-270 (1985)). The term "chimeric" antibody describes a polypeptide comprising at least the antigen binding portion of an antibody molecule linked to at least part of another protein such as an immunoglobulin constant domain. However, antibodies as described herein may be conjugated to a variety of moieties including labeling moieties.

Various immunoassays may be used for screening to identify antibodies having a desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between a GrA or GrB or elastin or fibronectin or fibrillin or fibulin antigen and its specific antibody. Monoclonal-based immunoassays utilizing monoclonal antibodies reactive to at least two non-interfering epitopes are preferred, but competitive binding assays may also be employed (Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).

Antibody Assay Methods

One utility of the antibodies and proteins/peptides as described herein is for diagnostic purposes, in particular in assays to detect of quantify the presence of GrA or GrB or elastin or fibronectin or fibrillin or fibulin antibodies or antigen (GrA or GrB or elastin or fibronectin or fibrillin or fibulin protein or peptide) in a sample. In the following, such assays, in particular ELISAS (enzyme-linked immunosorbent assays) and Western blots can be used to detect GrA or GrB or elastin or fibronectin or fibrillin or fibulin proteins or peptides in samples. Numerous immunoassays are known in the art (Methods in Cell Biology, Vol. 37: Antibodies in Cell Biology, Asai, ed., Academic Press, Inc., New York (1993); and Basic and Clinical Immunology, 7^th ed., Stites & Terr, eds., (1991)).

A preferred method for detecting GrA or GrB or elastin or fibronectin or fibrillin or fibulin proteins is the ELISA, in which an antibody typically is bound to an enzyme, such as peroxidase or phosphatase, which can produce colored reaction products from an appropriate buffer. Thus, it utilizes a tagged antigen molecule of known quantity to determine an unlabelled antigen of unknown quantity. Preferably, a GrA or GrB or elastin or fibronectin or fibrillin or fibulin protein according to aspects of invention, or a suitable functional fragment thereof, is used coupled to a conventional tag, such as His6.

Thus, in an ELISA format according to aspects of invention, polypeptides or proteins specific for GrA or GrB or elastin or fibronectin or fibrillin or fibulin are detected and/or quantified, preferably in a biological sample. The sample may be any sample of biological tissue or fluid, such as blood. The sample is pretreated as necessary by dilution in a suitable buffer solution or concentrated, if desired. Any number of standard aqueous buffer solutions may be used, such as TRIS or the like, at physiological pH. Samples are incubated with an excess of the protein according to aspects of the invention as antigen. After rinsing to remove any unbound antigen, the amount of bound antigen is quantitated by adding a solution of enzyme-conjugated antibody that binds to constant domains of antibodies in the sample. Excess conjugated antibody is rinsed away and the activity of the bound enzyme is determined by adding the substrate to the reaction and measuring the formation of products. As the products of the reactions used in ELISA procedures are colored, the amount of product formed can readily be determined by the intensity of the colour that has developed using a spectrophotometer. The activity of the bound enzyme is proportional to the amount of antigen-binding antibody in the sample; therefore, the original concentration of such antibodies can be estimated from a series of control assays employing known concentrations of specific antigens. Similarly, antibodies to GrA or GrB or elastin or fibronectin or fibrillin or fibulin can be detected in a biological sample using bound antigen (GrA or GrB or elastin or fibronectin or fibrillin or fibulin protein or peptide). As an alternative method for detecting GrA or GrB or elastin or fibronectin or fibrillin or fibulin proteins western blots can be utilized taking advantage of the GrA or GrB or elastin or fibronectin or fibrillin or fibulin specific antibodies described above. Biologic samples containing proteins can be assayed by fractionation on polyacrylamide gels under denaturing conditions. Alternatively, tris tricine polyacrylamide gel electrophoresis can be used for improved separation of small peptides in the range from 1 to 100 kDa (Schagger H. and von Jagow G. ((1987) Analytical Biochemistry 166: 368-379 and Klafki H.-W. et al. (1996) Analytical Biochemistry 237, 24-29.). The proteins separated in the gels can then be transferred to a membrane using a variety of methods known in the art. Membranes can then be probed using GrA or GrB or elastin or fibronectin or fibrillin or fibulin specific antibodies in a western blot to identify the proteins of interest in the biological sample preparations. Numerous Western blotting methods are known in the art (ECL western blotting protocol - Amersham; Hsu SM. Methods Enzymol (1990) 184:357-63; Leong MM. and Fox GR. Methods Enzymol (1990) 184:442-51). Immunodiagnostic method for granzymes A and B are also described in WO 99/54737.

Western Blotting

As an alternative method for detecting GrA or GrB or elastin or fibronectin or fibrillin or fibulin proteins or peptides western blots can be utilized taking advantage of the GrA or GrB or elastin or fibronectin or fibrillin or fibulin specific antibodies described above. Biologic samples containing proteins can be assayed by fractionation on polyacrylamide gels under denaturing conditions. Alternatively, tris tricine polyacrylamide gel electrophoresis can be used for improved separation of small peptides in the range from 1 to 100 kDa (Schagger H. and von Jagow G. ((1987) Analytical Biochemistry 166: 368-379 and Klafki H.-W. et al. (1996) Analytical Biochemistry 237, 24-29.). The proteins seperated in the gels can then be transferred to a membrane using a variety of methods known in the art. Membranes can then be probed using GrA or GrB or elastin or fibronectin or fibrillin or fibulin specific antibodies in a western blot to identify the proteins or peptides or degradation products thereof of interest in the biological sample preparations. Numerous Western blotting methods are known in the art (ECL western blotting protocol - Amersham; Hsu SM. Methods Enzymol (1990) 184:357-63; Leong MM. and Fox GR. Methods Enzymol (1990) 184:442-51).

Alternatively, GrA or GrB enzyme-linked immunosorbent spot (ELISPOT - Czerkinsky C. et al. (1983) J Immunol Methods 65 (1-2): 109-21), dot blots or other proteomic approaches known in the art.

Various alternative embodiments and examples are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.

Examples

Methods and Materials

Mice

All animal protocols were approved by the University of British Columbia (UBC) Animal Care Committee. C57B1/6 mice, C57Bl/6-ApoE-/- and C57Bl/6-GrB-/- mice were obtained from Jackson Laboratories™ (Bar Harbor, ME) {PIEDRAHITA et al 1992. Proc Natl. Acad Sci 89: 4471-4475; HEUSEL et al 1994 Cell 76:977-987). The C57B1/6- ApoE-/- x GrB-/- double knockout (ApoE/GrB-DKO) mice were generated by crossing the C57Bl/6-ApoE-/- and C57Bl/6-GrB-/- mouse strains. Genotyping of the mice was performed using primers and PCR reactions designed for genotyping these lines from Jackson laboratories™ (GrB primers: 5'-TGAAG ATCCT CCTGC TACTG C-3' and 5'- TCCTG AGAAA GACCT CTGCC-3'; ApoE primers: 5'-GCCTA GCCGA GGGAG AGCCG-3¹ and 5'-TGTGA CTTGG GAGCT CTGCA GC-3'). The pups were weaned at 3 weeks of age and then maintained on a 12-hour day and night cycle with food and water provided ad libitum. At 6 - 8 weeks of age, mice were maintained on either regular chow or a Western high fat diet (Harlan Teklad™) for 30 weeks and were sacrificed to collect blood and tissues.

Tissue and blood collection

Animals were overdosed with 2.5% avertin™ (Sigma™) and perfusion fixed with four mL of 4% formalin (Sigma™) at a flow rate of 2 mL/min. The hearts were then rapidly removed, and aortic root sections were OCT-embedded. Skin samples taken from the back were either OCT-embedded (Tissue-Tek™) or immersion-fixed in 10% formalin for 24h before being embedded in paraffin. Blood extracted by cardiac puncture was collected in EDTA microvette tubes (Sarstedt™), spun at 10,000 x g for 7 minutes at 4°C, and the separated serum stored at -80⁰C until required for analysis.

Hair removal

The animal was deeply anesthetized with 2.5% avertin (Sigma) and fur as removed with 10 ml of chemical hair remover (N AIR™, Church and D wight Co.) distributed evenly with a swab. After 15 minutes of incubating, the N AIR™ and fur was removed with warm water and dried with paper tissues.

Immunofluorescence

Immunofluorescence was performed on OCT-embedded frozen sections. Briefly, sections were fixed with acetone for 10 min. Background staining was blocked by incubation of sections with Dako™ protein block (Dako Cytomation™) for 20 minutes then incubation in 10% donkey serum for 1 hour. Sections were incubated in goat anti- granzyme B (Santa Cruz™, 1 :50) and rat anti-mouse macrophage/monocyte (Chemicon, 1 :50) at 4°C overnight, followed by incubation in donkey anti-goat IgG (Alexa Fluor™ 594, 1 :500) and donkey anti-rat IgG (Alexa Fluor™ 488,1 :500) for 30 min at room temperature in the dark. Slides were mounted with VECTASHIELD™ Hard-set mounting medium with DAPI (Vector Laboratories™, Burlingame, CA). Confocal microscopy was performed using a Leica AOBS™ SP2 confocal microscope.

Histological Assessment and Quantitation

Serial 10 μm sections of the aortic roots isolated as described stained with hematoxylin & eosin, Movat's pentachrome, elastic van Gieson or Oil Red O. ImageProPlus™ (MediaCybernetics™, Silver Spring, MD) was used to quantify the lesion area per cross section in ten to twenty sections per mouse which were then averaged to provide mean lesion area per mouse. Statistics

An ANOVA test was performed to determine statistical differences between multiple groups. Statistical differences between two groups were determined using a Student's t-test. For both tests, a p value (alpha error) of 0.05 or less was considered significant.

Example 1

ApoE/Granzyme B Double Knock-out Mice

Four groups of mice consisting of (1) C57B1/6 wild-type, (2) C57/ApoE -/- (ApoE- KO), (3) C57/GrB -/- (GrB-KO), and (4) C57 GrB/ApoE-DKO were fed a normal chow or high fat 'Western' diet (21% fat, 0.2% cholesterol) for 30 weeks. No obvious phenotypic differences were observed in these mice during the first 3 months. Mice were sacrificed and tissues harvested at 30 weeks of age (ApoE KO mice on the Western diet are sacrificed around this age for humane reasons). As reported in the literature, the ApoE- KO mice had developed severe skin xanthomatosis, hair loss, hair discoloration and numerous atherosclerotic lesions. Surprisingly, the GrB/ApoE-DKO mice demonstrated a significant reduction in both frequency and size of atherosclerotic lesions (Figure 1). Atherosclerotic lesions in the ApoE/GrB DKO mice decreased in size to less than 15% of the aortic root area, from more than 40% in the ApoE KO mice fed a Western diet.

Interestingly, this difference in atherosclerotic lesions is not due to a change in blood cholesterol or lipoprotein levels, as there is no difference between the ApoE KO and the ApoE/GrB DKO mice (not shown), whereby both total cholesterol and LDL-C plasma concentrations are the same. No significant differences in HDL, LDL and triglycerides are observed between ApoE KO and ApoE/GrB DKO mice fed a Western diet (not shown). Also the removal of granzyme B activity alone does not have a significant effect on the blood lipid profiles compared to the C57/BL6 (not shown).

At 30 weeks, the DKO mice had no visible xanthomas (Table 1). The DKO mice have smooth and unwrinkled skin. The difference in the incidence of xanthomatosis was surprising, as no previous link between granzyme B and xanthomatosis had been previously identified.

Table 1 : Cutaneous xanthomatosis is abolished in the absence of granzyme B activity.

The fur in the ApoE mice is patchy, discoloured (graying) and held weakly in the skin (easily removed by depilatory), while surprisingly, the ApoE/GrB DKO mice retain their dark fur and does not discolour, and is held firmly in the skin - even more so than the granzyme B KO mice. The hair follicles in the GrB KO and the ApoE/GrB DKO mice are more abundant and embedded deeper in the fatty layer of the skin, compared to the wild- type or the ApoE KO mice (Table 2). A standard Nair-mediated hair removal procedure takes more than 45 minutes in the GrB KO and ApoE/GrB DKO mice, compared to 5 minutes in the wild-type or ApoE KO mice.

Table 2: Hair follicle density and distribution - Values indicate number of follicles

2 per ~8.9mm . N=2 for each strain

ApoE KO mice exhibit signs of premature aging, necessitating sacrifice by about 30 weeks (6-7 months) of age. However, the ApoE/GrB DKO mice remain healthy and vigorous beyond 12 months of age, with no visible signs of aging or illness. This was surprising, as no support or indication of a role for GrB in longevity has been previously reported.

Example 2

Elastin and Granzyme B Distribution in Aortic Sections

Colocalization of granzyme B and macrophages in the lesions of the aortic roots were performed and imaged by confocal microscopy. The lesion of the ApoE-KO mice showed both granzyme B and macrophage staining. However, colocalization of both occurred at specific regions of the plaque: the fibrotic cap and the shoulder regions. Granzyme B staining was localized to the elastic lamellae.

In order to adhere to the aortic walls, smooth muscle cells require elastin. Aortas of C57 wt, GrB -/-, ApoE -/- and DKO mice were stained with elastic van Gieson (Figures 2 A to 2D). The aortic wall of the ApoE mouse is very thin and elastin staining is markedly reduced compared to the C57 wt. In the DKO mouse, the aorta wall is significantly thicker and elastin staining is correspondingly more intense. GrB also colocalizes with the internal elastic lamina of atherosclerotic plaques and an influx of macrophages in the ApoE -/-.

The increased localization of granzyme B with the internal elastic lamina indicates that it may accumulate on elastin fibers and over time, contribute to degradation of elastin. This in turn would lead to reduced elasticity, production of fragments that enhance inflammation, increased calcification and overall stiffness (hardening) of blood vessels. Reduced elastin in the internal elastic lamina also promotes migration of smooth muscle cells in to the intima (intimal hyperplasia) and the formation of atherosclerotic plaques. Similarly, the degradation of elastin by granzyme B may lead to elastinopathies such as Supravalvular aortic stenosis (or SVAS), Williams syndrome (or WS, Williams-Beuren syndrome, WBS), and Cutis laxa (or CL, which may be autosomal dominant, autosomal recessive, and X-linked recessive).

Example 3

Reduced Cutaneous Inflammation in DKO Mice

The skin of ApoE -/- mice appears much more aged, unhealthy and is very fragile. The skin has markedly reduced elasticity, which is restored in the DKO mice, where granzyme B activity is absent (Figure 3).

In Figure 4, an area of massive immune cell infiltration in the ApoE -/- mice (circled area) is visible, that is not observed in the DKO mice.

This immune cell recruitment and inflammatory response may be a consequence of the chemotactic action of the cleaved extracellular matrix components such as elastin fragments and fibronectin fragments. The presence of granzyme B in the ApoE -/- mice localizes to these sites and may be contributing to the generation of these fragments. This inflammatory effect (and granzyme B localization) is not observed in the DKO mice. Additionally, granzyme B may contribute to matrix degradation and/or remodeling of matrix composition, as areas are 'lost' or left unstained in the fixation process of tissues from GrB -/- or DKO mice. Granzyme B mediated degradation of matrix, in the presence of high lipids (as observed in the ApoE -/- mice) may contribute to the phenotype observed.

Example 4

Granzyme B Binds to the Extracellular Matrix Protein Elastin

An in vitro granzyme B elastin binding assay was conducted in the following manner. Granzyme B at 50, 100 and 300 ng was incubated with 15 μg of human insoluble skin (Sk) and aortic (Ao) elastin (Elastin Products Company Owensville, MO) in PBS for three hours at room temperature. The samples were centrifuged at 1000 x g at room temperature for three minutes and the insoluble elastin collected in the pellet. The supernatants, which contained unbound granzyme B, were denatured with SDS loading buffer and run on a 10% SDS-PAGE gel. Granzyme B was detected by Western blot. Each gel contained three lanes: a first lane related to a sample containing granzyme B in the absence of elastin; a second lane related to the samples containing granzyme B and human insoluble skin elastin; and a third lane related to the sample containing granzyme B and aortic elastin. The lane relating to the sample containing granzyme B in the absence of elastin showed a heavy band in the supernatant and a faint band in the pellet. The lanes relating to the samples containing granzyme B and skin elastin, and granzyme B and aortic elastin both showed heavy bands in the pellet, which bands were much heavier than the faint band seen in the pellet relating to the sample containing granzyme B in the absence of elastin. Furthermore, the band in the supernatant for the sample containing granzyme B and skin elastin was dramatically less pronounced than the supernatant band shown in the sample relating to granzyme B in the absence of elastin. No band appeared in the supernatant sample containing granzyme B and aortic elastin. Hence, there is less granzyme B present in the supernatant, thus indicating that granzyme B was associating with the elastin in the pellet. This phenomenon was dose-dependent and not restricted to the type of elastin used (i.e. skin elastin or aortic elastin). Example 5

Granzyme B Cleaves Extracellular Matrix Proteins

Treatment of human coronary artery smooth muscle cells (SMC) matrix with granzyme B induced a cleavage of a number of extracellular proteins. Extracellular proteins from SMC cultures were biotinylated and incubated with granzyme B. The supernatant was collected at 2, 4 and 24 hours after treatment, and the entire insoluble extracellular protein preparation collected at 24 hours. Extracellular proteins were visualized by Western blot for biotin. Western blot for beta-actin confirmed that the extracellular protein preparation was devoid of intercellular proteins. Western blots for fϊbronectin, phosphorylated FAK (p-FAK), and FAK were also performed on lysates of SMC treated with granzyme B. In the collected insoluble proteins, four protein bands between approximately 50-70 kDa and approximately 236 kDa disappeared 24 hours after treatment with granzyme B and cleavage of fragments approximately 25-39 kDa were evident in the matrix at this same time point. Further, the six proteins and/or cleavage fragments ranging in molecular weight from approximately 29-148 kDa were eluted into the supernatant as early as two hours after granzyme B treatment. To ensure that the SMC extracellular protein preparations used were devoid of intracellular proteins, western blotting for beta-actin was performed on the collected supernatant and extracellular proteins. Beta-actin was apparent in SMC lysates (positive control) but was absent from matrix and supernatant preparations.

To identify extracellular proteins that are cleaved by granzyme B, western blots for fϊbronectin, collagen, and vitronectin on lysates from untreated and granzyme B-treated SMCs were performed. In all SMCs treated with granzyme B for 24 hours, there was a reduction in the total amount of fϊbronectin in lysates collected from SMCs. In the supernatants of granzyme B-treated SMCs at 24hours, a fϊbronectin cleavage product was detected. There was no cleavage of collagen IV or vitronectin was observed. Therefore, granzyme B induces a cleavage of fϊbronectin in SMC extracellular matrixes but does not affect collagen IV or vitronectin.

Also human coronary artery smooth muscle cells were cultured to confluency and serum starved for 48 hours at which time cells were lysed with NH4OH so that the intact extracellular matrix (ECM) remained on the plate. Granzyme B (80nm) was incubated on the ECM for 24 hours at room temperature. Supernatants (containing cleaved ECM) and ECM still attached to the plate were collected and assessed for fibrillin cleavage by Western blot. The results, not shown, may be summarized as follows: Western blots of PBS (negative control), Trypsin (positive control) and GrB supernatants and PBS, Trypsin and GrB ECMs were performed with a fibrillin- 1 antibody, which showed fibrillin- 1 cleavage fragments in the GrB supernatant, GrB ECM, Trypsin supernatant and Trypsin ECM, but not in the PBS supernatant or ECM. Six independent experiments were carried out and 3 representative groups were tested. Results confirm that GrB cleaves fibrillin- 1 in human coronary artery smooth muscle cells. Similarly, the degradation of fibrillin- 1 by granzyme B may lead to fibrillinopathies such as: Marfan syndrome; autosomal dominant Weill-Marchesani syndrome; severe neonatal Marfan syndrome; dominant ectopia lentis; familial ascending aortic aneurysm; isolated skeletal features of Marfan syndrome (or Familial Marfanoid habitus); Beals syndrome (or Arachnodactyly, Contractural Beals Type, Beals-Hecht Syndrome, and Congenital Contractural Arachnodactyly (CCA)); Familial mitral valve prolapse syndrome (MVP); mitral valve prolapse, myopia, minimal or no aortic dilation, subtle skeletal changes and skin changes (MASS phenotype); or Shprintzen-Goldberg syndrome.

Example 6

Granzyme B Binds and Degrades Elastin in vitro

Tritiated elastin was prepared with the modifications as described in Banda, MJ. and Werb, Z. (1981) Biochem J 193: 589-605 and Gordon, S., Werb, Z. and Cohn, Z. A. (1976) in In Vitro Methods in Cell Mediated and Tumor Immunity, eds. Bloom, B.R. and David, J.R. (Academic Press, New York), pages 349-350. 1 mg of skin or aortic elastin was diluted in 1 ml dH₂0 and pHed to 9.2. 1 mCi NaB₃H₄ (PerkinElmer, Waltham MA) and 2 mg of non-radioactive NaB₃H₄ (Sigma, St. Louis, MO) was added. After 2 hours of incubation, the pH was adjusted to 3.0 and the elastin was incubated for an additional 30 minutes. The elastin was centrifuged for 3 minutes at 5000 x g and the pellet was repeatedly washed to remove excess NaB₃H₄. For the cleavage assays, 0.15 mg 3H-elastin was incubated with granzyme B (0.75 μg was added a total of 5 times) at room temperature for 7 days. At day 7 of incubation, 25 μg of elastase (Elastin Products Company, Owensville, MO) was incubated with elastin for 2 hours, as a positive control. After incubations, reactions were centrifuged at 5000 x g for 3 minutes. The radioactivity of the soluble, cleaved elastin fragments in the supernatant was counted in Ready Safe Scintillation Fluid (Beckman-Coulter, Fullerton, CA). The radioactivity of the cleaved, soluble elastin fragments was 4.8 times and 2.7 times higher than background for skin and aortic elastin, respectively (Figure 5). Proteolysis of elastin by elastase yielded a radioactivity increase over background of 14.9 fold for skin elastin and 7.7 fold for aortic elastin. These data show that granzyme B has affinity to elastin and has elastolytic activity.

Example 7

Granzyme A-mediated proteolysis of smooth muscle cell (SMC)-generated extracellular proteins is illustrated in Figures 6A and 6B. SMC were cultured until confluency and then lysed with 0.25 M NH₄OH for 30 min. The remaining extracellular matrix proteins were biotinylated and incubated with granzyme A. Supernatants were collected at 1, 2, 6, 16 and 24 h post-treatment. Figure 6 A illustrates the results whereby extracellular proteins were visualized by Western blot for biotin. Increased levels of fragmented extracellular matrix proteins were observed in granzyme A-treated plates. Figure 6B illustrates the results whereby supernatants treated with granzyme A for the indicated times were probed for fibronectin and several fragments were observed as indicated by the arrows.

Granzyme A cleaves fibrillin- 1 in vitro. Human coronary artery smooth muscle cells were cultured to confluency and serum starved for 48 hours at which time cells were lysed with NH₄OH so that the intact extracellular matrix (ECM) remained on the plate. Granzyme A (10OnM) in PBS was incubated on the ECM for 24 hours at room temperature. Supernatants were collected and assessed for fibrillin presence and size by SDS-PAGE and subsequent fibrillin- 1 Western blot (results not shown). The results, may be summarized as follows: Western blots of PBS (negative control), Trypsin (positive control) and GrA supernatants and PBS, Trypsin and GrA ECMs were performed with a fibrillin- 1 antibody, which showed fibrillin- 1 cleavage fragments in the GrA supernatant, GrA ECM, Trypsin supernatant and Trypsin ECM, but not in the PBS supernatant or ECM. Six independent experiments were carried out and 3 representative groups were tested. Results confirm that GrA cleaves fibrillin- 1 in human coronary artery smooth muscle cells. Furthermore, GrB has also been shown to cleave Fibrillin-2 and Fibulin-2 in human coronary artery smooth muscle cells (HCASMC)-derived ECM (data not shown) and GrB cleavage is attenuated by the granzyme B inhibitor dichloroisocoumarin (DCI). Additionally, the cleavage of fibrillin- 1 and -2 by granzyme B and fibrillin- 1 by granzyme A, may lead to fibrillinopathies such as: Marfan syndrome; autosomal dominant Weill-Marchesani syndrome; severe neonatal Marfan syndrome; dominant ectopia lentis; familial ascending aortic aneurysm; isolated skeletal features of Marfan syndrome (or Familial Marfanoid habitus); Beals syndrome (or Arachnodactyly, Contractural Beals Type, Beals-Hecht Syndrome, and Congenital Contractural Arachnodactyly (CCA)); Familial mitral valve prolapse syndrome (MVP); mitral valve prolapse, myopia, minimal or no aortic dilation, subtle skeletal changes and skin changes (MASS phenotype); or Shprintzen-Goldberg syndrome.

Furthermore, the cleavage of elastin by granzyme B may lead to elastinopathies such as supravalvular aortic stenosis; Williams syndrome(or WS, Williams-Beuren syndrome, WBS); and Cutis laxa.

Furthermore the accumulation of excess fibulin-2 may lead to actinic elastosis.

Claims

WHAT IS CLAIMED IS:

1. A method of preventing or treating a fibrillinopathy or an elastinopathy in a subject in need thereof, the method comprising administering to the subject one or more of: a Granzyme B (GrB) inhibitor and Granzyme A (GrA) inhibitor.

2. The method of claim 1, wherein the fibrillinopathy or the elastinopathy is selected from one or more of: Marfan syndrome; autosomal dominant Weill-Marchesani syndrome; severe neonatal Marfan syndrome; dominant ectopia lentis; isolated skeletal features of Marfan syndrome; Beals syndrome; Familial mitral valve prolapse syndrome (MVP); mitral valve prolapse, myopia, minimal or no aortic dilation, subtle skeletal changes and skin changes (MASS phenotype); Shprintzen-Goldberg syndrome; supravalvular aortic stenosis; Williams syndrome; and Cutis laxa.

3. The method of claim 1 or 2, wherein the fibrillinopathy is Marfan syndrome.

4. The method of claim 1 or 2, wherein the fibrillinopathy is autosomal dominant Weill-Marchesani syndrome.

5. The method of claim 1 or 2, wherein the fibrillinopathy is severe neonatal Marfan syndrome.

6. The method of claim 1 or 2, wherein the fibrillinopathy is isolated skeletal features of Marfan syndrome.

7. The method of claim 1 or 2, wherein the fibrillinopathy is dominant ectopia lentis.

8. The method of claim 1 or 2, wherein the fibrillinopathy is Beals syndrome.

9. The method of claim 1 or 2, wherein the fibrillinopathy is MVP.

10. The method of claim 1 or 2, wherein the fibrillinopathy is MASS phenotype.

11. The method of claim 1 or 2, wherein the fibrillinopathy is Shprintzen-Goldberg syndrome.

12. The method of claim 1 or 2, wherein the elastinopathy is supravalvular aortic stenosis.

13. The method of claim 1 or 2, wherein the elastinopathy is Williams syndrome.

14. The method of claim 1 or 2, wherein the elastinopathy is Cutis laxa.

15. The method of any one of claims 1-14, wherein the GrA or GrB inhibitor is formulated for oral administration.

16. The method of any one of claims 1-14, wherein the GrA or GrB inhibitor is formulated for administration by injection.

17. The method of any one of claims 1-14, wherein the GrA or GrB inhibitor is formulated for topical administration.

18. The method of any one of claims 1-14, wherein the GrA or GrB inhibitor is formulated for topical application to a device.

19. The method of any one of claims 1-18, wherein the subject is a human.

20. Use of a Granzyme B (GrB) inhibitor or a Granzyme A (GrA) inhibitor in the manufacture of a medicament for the prevention or treatment of a fibrillinopathy or an elastinopathy in a subject in need thereof.

21. Use of a Granzyme B (GrB) inhibitor or a Granzyme A (GrA) inhibitor for the prevention or treatment of a fibrillinopathy or an elastinopathy in a subject in need thereof.

22. Use of a pharmaceutical composition comprising a Granzyme B (GrB) inhibitor or a Granzyme A (GrA) inhibitor for the prevention or treatment of a fibrillinopathy or an elastinopathy in a subject in need thereof.

23. The use of any one of claims 20-22, wherein the fibrillinopathy or elastinopathy is selected from one or more of: Marfan syndrome; autosomal dominant Weill-Marchesani syndrome; severe neonatal Marfan syndrome; dominant ectopia lentis; isolated skeletal features of Marfan syndrome; Beals syndrome; Familial mitral valve prolapse syndrome (MVP); mitral valve prolapse, myopia, minimal or no aortic dilation, subtle skeletal changes and skin changes (MASS phenotype); Shprintzen-Goldberg syndrome; supravalvular aortic stenosis; Williams syndrome; and Cutis laxa.

24. The use of any one of claims 20-23, wherein the GrA or GrB inhibitor is formulated for oral administration.

25. The use of any one of claims 20-23, wherein the GrA or GrB inhibitor is formulated for administration by injection.

26. The use of any one of claims 20-23, wherein the GrA or GrB inhibitor is formulated for topical administration.

27. The use of any one of claims 20-23, wherein the GrA or GrB inhibitor is formulated for topical application to a device.

28. The use of any one of claims 20-27, wherein the subject is a human.

29. A commercial package comprising:

(a) a pharmaceutical composition comprising:

(i) a GrB inhibitor; or a GrA inhibitor; and (ii) a pharmaceutically acceptable carrier; and (b) instructions for the use thereof for treating a fibrillinopathy or an elastinopathy.

30. A method of preventing or treating actinic elastosis in a subject in need thereof, the method comprising administering to the subject a Granzyme B (GrB) protein, peptide, fragment thereof or variant thereof.

31. Use of a Granzyme B (GrB) protein, peptide, fragment thereof or variant thereof in the manufacture of a medicament for the prevention or treatment of actinic elastosis in a subject in need thereof.

32. Use of a Granzyme B (GrB) protein, peptide, fragment thereof or variant thereof for the prevention or treatment of actinic elastosis in a subject in need thereof.

33. Use of a pharmaceutical composition comprising a Granzyme B (GrB) protein, peptide, fragment thereof or variant thereof for the prevention or treatment of actinic elastosis in a subject in need thereof.