WO2000015250A2 - Fibroblast inhibitor - Google Patents

Abstract

There is disclosed the use of an inhibitor of factor Xa activity in the production of a medicament for the treatment of a condition which is characterised by the proliferation of fibroblasts and/or the production of procollagen by fibroblasts.

Description

FIBROBLAST INHIBITOR

The present invention relates to the inhibition of fibroblast proliferation and the inhibition of procollagen production by fibroblasts. In particular, it relates to the use of an inhibitor of factor Xa to inhibit factor Xa-associated stimulation of fibroblasts (resulting in the proliferation of fibroblasts and/or the production of procollagen by fibroblasts) for the prevention or treatment of organ damage. Current therapies for fibroproliferative disorders and excessive connective tissue deposition are inadequate, have little or no specificity and only provide limited improvements for the patient.

For example, treatments for lung fibrosis or adult respiratory distress syndrome rely on immunosuppressive drugs such as corticosteroids. These have both a low success rate and undesirable side effects. Patients with renal sclerosis or atherosclerosis are administered ACE inhibitors which are not tolerated by many patients and only address partial aspects of the pathology. Moreover, no effective treatment exists for numerous fibrotic conditions including hepatic fibrosis, liver cirrhosis, or skin conditions such as keloids and hypertrophic scarring. Recently, there has been a growing interest in identifying molecules and cell types that mediate organ fibrosis in the hope of developing drugs to alleviate these conditions.

Fibroblasts are the principal cell type found in connective tissue. Fibroblasts synthesise, deposit and remodel all of the main extracellular matrix elements, including collagen, the principal structural protein of connective tissue. Fibroblasts synthesise procollagen which is then processed to form collagen. Fibroblasts are the vector cell type that mediates tissue repair and regeneration following injury. They also mediate most fibrotic and connective tissue disorders where an accumulation of extracellular matrix disrupts the normal organisation of the tissue and results in the loss of organ function. Fibrotic and connective tissue disorders can be caused by excessive proliferation of fibroblasts and/or excessive production of procollagen by fibroblasts, which is then converted to collagen. Fibroblasts are mesenchymal cells located in connective tissue. They belong to different tissue compartments and execute different functions from smooth muscle cells, endothelial cells and lymphocytes. Previous work has shown that the coagulation cascade can be a source of mitogenic activity for fibroblasts. The coagulation protease thrombin can regulate gene transcription, protein synthesis and proliferation in fibroblasts after being activated during blood coagulation. Thrombin can induce the release of potent growth factors for mesenchymal cells such as platelet-derived growth factor (PDGF) , (Ohba et al . , 1994). These growth factors induce fibroblast replication which contributes to extracellular matrix (ECM) deposition and connective tissue formation. Factor Vila, factor IXa and factor Xa, which are activated during the coagulation cascade, display strong sequence homologies to thrombin and belong to the same family of vitamin K-dependent serine proteases. ^' Factor Xa (fXa) has been shown to induce smooth muscle cell and endothelial cell replication (Herbert et al . , 1998). Binding of fXa to effector cell protease receptor-1 (EPR-1) is necessary to induce smooth muscle cell proliferation (Herbert et al . , 1998) . Binding of factor Xa to EPR-1 on smooth muscle cells induces the release of PDGF which acts in an autocrine fashion to induce mitosis.

It has been shown that the mitogenic activity of fXa for smooth muscle cells can be abolished by inhibitors of the catalytic site of fXa, as well as inhibitors of the interaction between fXa and EPR-1, including antibodies directed against the interacting regions of either fXa or EPR-1 (Herbert et al . , 1998).

The catalytic activity of fXa is essential for its mitogenic effect for smooth muscle cells (Kho et al . , 1996) , although the substrate that is cleaved has not been identified.

Binding of fXa to its receptor EPR-1 on the surface of B- and T-lymphocytes leads to their activation and predisposes them to proliferate under cytokine stimulation.

To date, the mitogenic effect of fXa has only been shown in smooth muscle cells and endothelial cells in the vasculature and leukocytes.

Based on these studies, US Patent No. 5385885 concerns the use of specific inhibitors of fXa in patients with disorders where smooth muscle cell proliferation is a feature and causes the narrowing of the lumen of blood vessels, in particular in diseases associated with vessel wall hyperplasia. Tick anticoagulant peptide (TAP) and Antistasin

(ASN) are selective inhibitors of fXa and are naturally produced by parasitic organisms. They have the advantage of being highly specific for fXa whilst being fully active in complex physiological fluids. Tick anticoagulant peptide is isolated from the tick Ornithodoros moubata and abolishes catalytic activity of fXa. TAP and its recombinant equivalent (rTAP) bind to fXa in a reversible but highly selective fashion (Jordan et al . , 1992). Antistasin is a specific inhibitor of the fXa catalytic site and is isolated from the Mexican leech Haementeria offinalis. Short synthetic peptides mimicking the core inhibitory region of the molecule, including ASN D-Arg³²-Pro³⁸, have been shown to be active and abolish the catalytic activity of fXa (Ohta et al . ,

1994) .

A number of synthetic peptides mimicking the fXa inhibitory regions of TAP and ASN have been shown to have similar properties and no side effects in vivo . These molecules were used as the drugs of choice in several studies of anti-thrombosis and anti-restinosis agents. For examples of uses of TAP- and ASN-derived compounds in vivo, see Wong et al . , 1996; Schwartz et al . , 1996; Ragosta et al . , 1994; Kotze et al . , 1997. The low molecular weight compound Glu-Gly-Arg

Chloromethylketone (GGACK) is a specific synthetic inhibitor of fXa. It binds irreversibly to the catalytic site of fXa to abrogate its activity. Once the complex fXa/GGACK is formed, it can be purified to obtain a form of catalytically inactive fXa termed DEGR-factor Xa (DEGR-fXa) .

These compounds and their inhibitory actions have been extensively described and studied both in vitro and in vivo in models of blood coagulation, thrombosis, restinosis and arterial wall remodelling. These studies have demonstrated the possibility to deliver effectively in animals, including the rat, rabbit, dog, pig and baboon. In these experiments, the animals were treated successfully with various TAP- and ASN-derived compounds inhibiting the catalytic activity of fXa.

However, these studies have looked only at the capacity of these molecules to abolish the function of fXa in the coagulation cascade, leukocyte and smooth muscle cell proliferation. In addition, Herbert et al . (1998) have demonstrated the possibility to deliver small molecular weight fXa inhibitors such as DX9065 to relieve thrombosis. In this case, the compound was effective in vivo to inhibit factor Xa after simple dilution in saline solution and subcutaneous injection into rabbits.

The antibody 5224 is a monoclonal antibody that inhibits the enzymatic activity of fXa (Zacharski et al . , 1991) . It has also been shown to be a potent inhibitor of the mitogenic activity of factor Xa on smooth muscle cells (Ko et al . , 1996) .

The antibody JC15 is a monoclonal antibody directed against fXa. JC15 inhibits the activity of fXa by interfering with the interaction between fXa and its receptor EPR-1 (Ambrosini et al . , 1997). The antibody JC15 has also been shown to be a potent inhibitor of the mitogenic activity of fXa on smooth muscle cells both in vi tro and in vivo (Herbert et al . ,

1998) . In this instance, JC15 was delivered locally to the carotide in the form of a gel and prevented smooth muscle cell proliferation after injury to the vasculature. Other antibodies directed against the fXa receptor EPR-1, such as antibody 2E1 , were also shown to be active in vivo . By administering antibody 2E1 to mice, Duchosal et al . (1996) were able successfully to inhibit lymphocyte proliferation and graft versus host disease. This experiment demonstrated the possibility to inhibit fXa activity in vivo by preventing the interaction between fXa and EPR-1 with antibodies.

Another anti-EPR-1 antibody, B6, has been shown to block the binding of fXa to EPR-1 in a competitive manner, by binding to the region of the receptor necessary for the interaction with factor Xa (Nicholson et al . , 1996 ) .

Furthermore, it has been demonstrated that short peptides mimicking the region of fXa which interacts with EPR-1 can inhibit fXa in vitro . An example of such a peptide is eu⁸³-Leu⁸⁸- (Gly) (Ambrosini et al . ,

1997, Herbert et al . , 1998).

Hitherto, the observation of fXa mitogenic activity was considered relevant solely to the modulation of the immune response and diseases of the vasculature . Indeed, one previous study failed to show that fXa affects DNA synthesis in fibroblasts (Gasic et al . , 1992) .

Surprisingly - and contrary to this prior teaching - the inventors have found that coagulation factor Xa stimulates proliferation, DNA synthesis, and procollagen gene promoter activity in fibroblasts . Moreover, they have found that this function can be suppressed with inhibitors of the activity of fXa. This includes inhibitors of the catalytic site of fXa and inhibitors of its interaction its receptor. These findings identify coagulation factor Xa as a novel drug target in organ fibrosis .

According to a first aspect of the present invention, there is provided the use of an inhibitor of factor Xa activity in the production of a medicament for the prevention or treatment of organ damage associated with factor Xa stimulation of fibroblasts resulting in the proliferation of fibroblasts and/or the production of procollagen by fibroblasts. The inhibitor of factor Xa activity may be used in the production of a medicament for the prevention of collagen deposition resulting from factor Xa-associated stimulation of fibroblasts.

The inhibitor of factor Xa activity may also be used in the production of a medicament for use in a ethod of preventing or treating a disease by inhibiting the factor Xa-associated stimulation of fibroblast proliferation and/or the factor Xa- associated stimulation of procollagen production by fibroblasts.

Thus the inhibitor of factor Xa activity can be used generally in the production of a medicaments for the treatment of a conditions which are characterised by the proliferation of fibroblasts and/or the production of procollagen by fibroblasts.

Preferably, the inhibitor inhibits factor Xa activity by blocking the catalytic site thereof. Examples of such inhibitors are tick anticoagulant peptide, antistasin, GGACK, DX9065, an antibody which binds to the catalytic site and derivatives thereof . The tick anticoagulant peptide, antistasin or derivative thereof may be obtained from natural sources or by recombinant techniques. GGACK can be synthesised chemically. In either case, appropriate starting materials and methods are known to those skilled in the art .

"Derivatives" of tick anticoagulant peptide, antistasin, GGACK and such antibodies inhibit the activity of fXa. They may be fragments of tick anticoagulant peptide, antistasin, GGACK or such antibodies, or may be tick anticoagulant peptide, antistasin, GGACK or such antibodies modified by the addition of therapeutic or diagnostic or marker compounds, or modified by the addition or deletion of one or more amino acids. They may be combinations of such fragments and such modifications .

A preferred derivative is ASN D-Arg³²-Pro³⁸" When the inhibitor is an antibody which binds to the catalytic site, it is preferably antibody 5224. It is known that factor Xa acts via a cell surface receptor (Herbert et al . , 1998; Nicholson et al . , 1996) . Accordingly, the inhibitor may inhibit factor Xa activity by blocking the interaction between factor Xa and its receptor. Such an inhibitor may be a short peptide which mimics the receptor binding region of fXa.

The inhibitor may prevent such interaction by binding to the receptor binding site of factor Xa. In this case, the inhibitor is preferably an antibody, such as antibody JC15, or a derivative thereof. Alternatively, the inhibitor may prevent such interaction by binding to the fXa binding site of the receptor. In this case, the inhibitor is preferably an antibody, such as B , 2E1, or a derivative thereof. "Derivatives" of such inhibitors and antibodies inhibit the activity of fXa . They may be fragments thereof, or may be such inhibitors and antibodies modified by the addition of therapeutic or diagnostic or marker compounds, or modified by the addition or deletion of one or more amino acids. They may be combinations of such fragments and such modifications. The invention also provides a method for treating a condition characterised by the proliferation of fibroblasts and/or the production of procollagen by fibroblasts, comprising administering a therapeutically effective amount of an inhibitor of factor Xa activity. The invention also relates to a method of preventing the proliferation of fibroblasts and/or the production of procollagen by fibroblasts, comprising administering an inhibitor of factor Xa activity. According to a second aspect of the invention, there is provided a method for treating or preventing a disease associated with factor Xa stimulation of fibroblasts resulting in the proliferation of fibroblasts and/or the production of procollagen by fibroblasts disease in a mammalian patient, which method comprises the step of administering a pharmaceutically effective amount of an inhibitor of factor Xa activity to the mammalian patient.

Inhibitors of coagulation factor Xa activity can thus be used as antifibrotic agents in a variety of conditions associated with fibroblast proliferation and/or collagen production. Lesions and diseases where organs are damaged at least in part by connective tissue hyperplasia or excessive deposition of extracellular matrix by fibroblasts can be treated or prevented.

The present invention provides methods for inhibiting fibroblast proliferation and/or inhibiting procollagen production by fibroblasts. A compound capable of inhibiting coagulation factor Xa is administered to fibroblasts in an amount effective to inhibit their proliferation and/or to inhibit their production of procollagen with the aim of reducing extracellular matrix deposition by fibroblasts.

Inhibiting the mitogenic effect of factor Xa on fibroblasts offers new therapeutic approaches in a vast array of fibrotic conditions and diseases associated with leakage of plasma proteins, including factors of the coagulation cascade, and excessive connective tissue formation, such as, but not restricted to:

Fibrotic conditions of the lungs caused by

Acute lesions or adult respiratory distress syndrome induced by acid, alkali and inert chemicals, thermal injury, smoke inhalation, sharp or blunt mechanical injury, including blast or crush type injuries, radiation injury, Infections, of parasitic, viral, fungal, bacterial, mycobacterial , schistosomal and protozoal origin, local or systemic, and Helminth infections, Autoimmune diseases, such as rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosis, mixed connective tissue disease and obliterative bronchiolitis , - Idiopathic pulmonary fibrosis,

Granulomatous diseases such as sarcoidosis, Occupational diseases such as Berylliosis, Vasculitic diseases, Thromboembolic diseases,, - Allergic diseases, such as asthma, extrinsic allergic alveolitis, allergic bronchopulmonary aspergillosis ,

Iatrogenic diseases,

Neoplastic diseases, such as lymphangitis carcinomatosis,

Lung transplant diseases, such as obliterative bronchiolitis ,

Haematological diseases, including graft versus host disease and sickle cell disease.

Fibrotic conditions of the skin, hypertrophic scarring and keloid formation caused by

Acute lesions induced by acid, alkali and inert chemicals, thermal injury, sharp or blunt mechanical injury including blast or crush type injuries, radiation injury, Infections, of parasitic, viral, fungal, bacterial, mycobacterial , schistosomal and protozoal origin, local or systemic, and Helminth infections,

Autoimmune diseases, such as systemic sclerosis, systemic lupus erythematosis, mixed connective tissue disease, psoriasis, pemphigus, pemphigoid, Bechets, porphyria cutanea tarda, - Granulomatous diseases, such as sarcoidosis, Occupational diseases, such as thermal burns, Allergic diseases, such as eczema, Neoplastic and metastatic diseases, Transplant diseases including graft versus host disease, - Vascular diseases including venous ulcers, arterial ulcers, diabetic ulcers and neurotrophic ulcers .

Fibrotic conditions of the liver caused by - Acute lesions induced by acid, alkali and inert chemicals, including polyvinylchloride, paraquat and cigarettes, thermal injury, sharp or blunt mechanical injury including blast or crush type injuries, radiation injury, - Infections, of parasitic, viral, fungal, bacterial, mycobacterial, schistosomal and protozoal origin, local or systemic, including those leading to granulomatosis, and Helminth infections, - Autoimmune diseases, such as systemic sclerosis, systemic lupus erythematosis, mixed connective tissue disease,

Vasculitic diseases,

Iatrogenic diseases, including those due to paracetamol,

Neoplastic and metastatic diseases, such as hepatoma,

Liver transplant,

Genetic diseases, such as alphal-antitrypsin deficiency,

Storage diseases, including Gauchers disease and glycogen storage disease, as well as liver cirrhosis due to alcohol consumption.

Fibrotic conditions of the kidney caused by

Acute lesions induced by acid, alkali and inert chemicals, thermal injury, sharp or blunt mechanical injury including blast or crush type injuries, radiation injury, as well as reflux nephropathy and interstitial nephritis, - Infections, of parasitic, viral, fungal, bacterial, mycobacterial , schistosomal and protozoal origin, local or systemic, including those leading to granulomatosis and schistosomiasis, and Helminth infections, - Autoimmune diseases, such as systemic sclerosis, systemic lupus erythematosis, mixed connective tissue disease,

Granulomatous diseases, such as sarcoidosis, Vasculitic diseases, such as Wegener's granulomatosis, polyarteritis nodosa, microscopic polyarteritis and goodpastures syndrome, Iatrogenic diseases, including those due to Aspirin, Transplant diseases, - Storage diseases, including Gauchers disease.

Fibrotic conditions of the genito-urinary tract and strictures caused by

Acute lesions induced by acid, alkali and inert chemicals, thermal injury, sharp or blunt mechanical injury including blast or crush type injuries, including catheterization, radiation injury,

Infections, of parasitic, viral, fungal, bacterial, mycobacterial, schistosomal and protozoal origin, local or systemic, including those leading to granulomatosis, and Helminth infections,

Idiopathic diseases, such as Peyronnies disease, - Granulomatous diseases, such as sarcoidosis. Fibrotic conditions of the gastro-intestinal tract induced by

Acute lesions induced by acid, alkali and inert chemicals, thermal injury, sharp or blunt mechanical injury including blast or crush type injuries, radiation injury, including those leading to post operative adhesions,

Idiopathic diseases,

Granulomatous diseases, . such as sarcoidosis, - Neoplastic and metastatic diseases,

Inflammatory diseases such as Crohns disease.

Fibrotic conditions of the cardiac muscle induced by

Acute lesions induced by acid, alkali and inert chemicals, thermal injury, sharp or blunt mechanical injury including blast or crush type injuries, radiation injury,

Infections, of parasitic, viral, fungal, bacterial, mycobacterial, schistosomal and protozoal origin, local or systemic, including those leading to granulomatosis, and Helminth infections,

Autoimmune diseases, such as systemic lupus erythematosis , - Granulomatous diseases, such as sarcoidosis,

Hypertension and ischaemic cardiomyopathy.

Fibrotic conditions of the eyes induced by - Acute injury induced by acid, alkali and inert chemicals, thermal injury, sharp or blunt mechanical injury including blast or crush type injuries, radiation injury, including those leading to posterior synechiae . Infections, of parasitic, viral, fungal, bacterial, mycobacterial, schistosomal and protozoal origin, local or systemic, including those leading to granulomatosis, and Helminth infections .

In accordance with the present invention, inhibitors of fXa activity may be administered to individuals . Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time- course of treatment, will depend on the severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors. An inhibitor of fXa activity may be administered alone or in combination with another inhibitor of fXa activity. The or each inhibitor may be administered alone or in combination with other treatments, either simultaneously or sequentially. In addition to the inhibitor of fXa activity, the medicament for the treatment of conditions characterised by the proliferation of fibroblasts may comprise a pharmaceutically-acceptable excipient, carrier, buffer, stabiliser or other materials well- known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the fXa inhibitor. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or parenteral e.g. by injection (intravenous, cutaneous or subcutaneous) or by administration to the buccal or nasal mucosae, or by inhalation.

Formulations may be such as to provide a sustained release preparation for oral or parenteral use, such as by trans-dermal administration.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycois such as ethylene glycol, propylene glycol or polyethylene glycol may be included. For intravenous, cutaneous or subcutaneous injection, the fXa inhibitor will be in the form of a parenterally acceptable aqueous solution which has suitable pH, isotonicity and stability. Those of skill in the art are able to prepare suitable solutions using, for example, isotonic vehicles such Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included. EXAMPLES

The invention will be described in more detail in the following Examples, which refer to the accompanying drawings, in which:

Figure 1 is a graph illustrating the effect of varying concentrations of fXa on the proliferation of human foetal lung fibroblasts;

Figure 2 is a graph illustrating the effect of varying concentrations of fXa on the synthesis of DNA by human foetal lung fibroblasts (assessed by measurement of ³H-thymidine incorporation) ;

Figure 3 is a graph comparing the effect of thrombin, fXa, fIXa, and recombinant platelet-derived growth factor (rPDGF-AB) on the proliferation of human foetal lung fibroblasts; Figure 4 is a graph comparing the effect of thrombin, fXa, fIXa, and rPDGF-AB on the synthesis of DNA by human foetal lung fibroblasts (assessed by measurement of ³H-thymidine incorporation) ;

Figure 5 is a graph comparing the effect of thrombin, fXa, fIXa, and rPDGF-AB on the number of human foetal lung fibroblasts;

Figure 6 is a graph comparing the effect of two commercial preparations of fXa on the proliferation of human foetal lung fibroblasts;

Figure 7 is a graph illustrating the effect of rTAP on the proliferation of human foetal lung fibroblasts;

Figure 8 is a graph illustrating the effect of ASN D-Arg³²-Pro³⁸ on the proliferation of human foetal lung fibroblasts; Figure 9 is a graph illustrating the effect of

GGACK on the proliferation of human foetal lung fibroblasts;

Figure 10 is a graph comparing the effect of thrombin, fXa, and catalytically-inactive fXa (DEGR- fXa) on the proliferation of human foetal lung fibroblasts;

Figure 11 is a graph illustrating the effect of anti-fXa antibody (5224) on the synthesis of DNA by human foetal lung fibroblasts (assessed by measurement of ³H-thymidine incorporation) ;

Figure 12 is a graph illustrating the effect of anti-fXa antibody (JC15) on the synthesis of DNA by human foetal lung fibroblasts (assessed by measurement of ³H-thymidine incorporation) ; Figure 13 is a graph illustrating the effect of anti-EPR-1 antibody (B6) on the synthesis of DNA by human foetal lung fibroblasts (assessed by measurement of ³H-thymidine incorporation) ; and

Figure 14 is a graph illustrating the effect of fXa on procollagen αl(I) promoter activity and the effect of various inhibitors on this activity. Materials and methods

Coagulation factors, growth factors, and inhibitors

Preparations of purified human factor Xa and factor IXa and Glu-Gly-Arg Chloromethylketone dihydrochloride (GGACK) were purchased from Calbiochem, (La Jolla, CA) . For comparison purposes, a second commercial preparation of purified human factor Xa was obtained from American Diagnostica (Greenwich, CT) . DEGR-factor Xa was prepared, purified and provided by Dr Chris Goodwin, National Heart and Lung Institute (London, UK) . Purified human thrombin was purchased from Sigma (St Louis, MO) . All enzymes and inhibitors were prepared in DMEM cell culture medium, aliquoted and stored at -70°C. Recombinant human PDGF-AB was obtained from R&D Systems (Minneapolis, MO) and was prepared in phosphate buffered saline supplemented with 0.1% bovine serum albumin, aliquoted and stored at - 70°C. Recombinant tick anticoagulant peptide (rTAP) was a kind gift from Dr M. Scully from the National Heart and Lung Institute (London, UK) . Recombinant tick anticoagulant peptide was originally prepared and provided by Dr G.P. Vlasuk from Corvas Inc. (San Diego, CA) . Antistasin core inhibitory peptide was purchased from Bachem Ltd (Staffron Walden, UK) .

Antibodies

A monoclonal antibody against human factor Xa, 5224, was supplied by American Diagnostica Inc. (Greenwich CT) . Antibody 5224 is an monoclonal antibody (IgGl) raised in mouse against human factor Xa that recognises epitopes in the vicinity of the catalytic site and inhibits its activity (Zacharski et al . , 1991; Ko et al . , 1996).

The anti-factor Xa antibody JC15 was a generous gift from Dr Altieri (Yale University, New Haven CT) . JC15 is a polyclonal antibody raised in mouse against a synthetic peptide mimicking the inter-EGF region Leu⁸³- Leu⁸⁸ of human factor Xa (Altieri and Edgington, 1990) . JC15 prevents the interaction between the protease and its receptor EPR-1 in a competitive manner (Ambrosini et al . , 1997) .

The anti-EPR-1 antibody B6 was a generous gift from Dr. Altieri (Yale University, New Haven CT) . B6 is a monoclonal antibody raised in mouse against effector cell protease receptor-1 (EPR-1) (Altieri and Edginton, 1990) . B6 prevents binding of Factor Xa to EPR-1 in a competitive manner, by binding to the region of the receptor necessary to the interaction with Factor Xa (Nicholson et al . , 1996) Human foetal fibroblasts and primary human fibroblasts Human foetal fibroblasts (HFL-1) were obtained from the American Type Culture Collection, catalogue number CCL-153, and were used at passages no higher than 20 without any noticeable modification of the tested parameters. Human primary cultures of fibroblasts were kind gifts from M. Parsons (Breast dermal fibroblasts) , L. Reynolds (Left ventricular heart fibroblasts) and C. Keerthisingam (Lung alveolar fibroblasts) . Primary fibroblasts were grown from 1 mm³ explants dissected from human normal organs. The explants were cultured in fibroblast culture medium additionned with 10% serum. Fresh culture medium was provided to the cells one day after isolation and every three days thereafter for three weeks. Eventually, fibroblasts were collected by trypsinisation and characterised. All cells were characterised morphologically and by differential immunocytochemical staining for a selection of smooth muscle cell, endothelial and fibroblast markers such as α-smooth muscle actin, von Willebrand factor, Vimentin and myosin. Human adult SV40-transformed kidney fibroblasts were a kind gift from Dr Gill Norman (University College London, London) . The various primary cells, including SV40- transformed kidney fibroblasts, were discarded after 5 to 10 passages. Cells and tissue cul ture

Fibroblasts were routinely cultured in 75 cm² flasks in DMEM containing 10% NCS, 200 IU/ml penicillin, 200 IU/ml streptomycin and 4 mM glutamine. For proliferation and DNA synthesis experiments, cells were detached with trypsin/EDTA (0.05/0.02%), seeded in 96 well tissue culture plates (5000 cells/well) , grown for 24 h in 5% NCS and used before confluence was reached.

Proliferation assay

To investigate the effect of coagulation factors on cell growth, cells were plated in 96 well tissue culture plates in DMEM plus 5% NCS (5000 cells/lOOμl/well) . After 24 h in culture and before confluence, the medium was removed and replaced with fresh DMEM supplemented with the test substances. All enzymes were evaluated in 0% serum, unless specified otherwise. After 48 h in culture, cell replication was assayed using a spectrophotometric assay based on the uptake and subsequent elution of the dye methylene blue according to the method described by Oliver et al . ,

1989. Cell proliferation experiments were performed at subconfluence and in the absence of serum. For direct cell count experiments, cells were plated in 24 well tissue culture plates in DMEM plus 5% NCS (60000 cells/2.5 ml/well). After 24 h in culture and before confluence, the medium was removed and replaced with fresh DMEM supplemented with the test substances. All enzymes were evaluated in 0% serum, unless specified otherwise. After 48 h in culture, adherent cells were detached with trypsin/EDTA (0.05- 0.02%) . Cell number was determined by counting in a hemacytometer . Four to eight replicate wells were counted for each determination. DNA synthesis assaγ

Cells were plated in 5% NCS in a similar fashion to the proliferation assay for 24 h, quiesced in 0% NCS for 24 h and used at subconfluence . The medium was removed and replaced with fresh DMEM supplemented with the test substances. All enzymes were evaluated in 0% serum, unless specified otherwise. After 16 h in culture, ³H-thymidine (2 μCi/ml) was added for pulse labelling and the cells were incubated for an additional 4 h. The cells were then frozen and thawed once to facilitate release of the DNA out of the cells before harvest. Subsequently, the DNA was harvested on Titertek filterpaper from ICN (Costa Meas, CA) with a micro96 cell harvester from Skatron (UK) . The filter was washed twice with an excess amount of H₂0, individual pieces corresponding to each well were then cut and shaken in 5 ml of scintillation fluid Ecoscint A from National Diagnostics (Atlanta, GA) in Pico 2000 polyethylene vials from Packard Instrument (Meriden, CT) . The samples were then left to rest in the dark overnight. Finally, the incorporated radioactivity was measured in a Miniaxiβ liquid scintillation counter from Packard Instrument (Meriden, CT) . The background values due to unlabelled cells in culture or the non- specific absorption of ³H-thymidine onto plastic were both subtracted from all experimental values. The final data was measured in disintegrations per minute (dpm) and expressed in percent stimulation above control values. Lucif erase activi ty assaγ A procollagen l(I) promoter linked to a luciferase reporter gene construct was kindly provided by Dr. Rajendra Raghow (Wang and Raghow, 1996) . HFL-1 were grown to 80% confluence in DMEM + 5% newborn calf serum (NCS) in 12 -well tissue culture plates. For each well, the DNA construct was complexed with an α₅β₆ integrin-targeting peptide (Peptide 6; Hart et al 1997) and lipofectin (Gibco BRL) at the following ratio based on the method previously described (Hart et al 1998) : In order; a) 0.75 μl lipofectin in 100 μl Optimem, b) 40 μl Peptide 6, c) 1 μg DNA in 100 μl Optimem (Vf = 240 μl) .

The complex was allowed to form at room temperature for lh and finally diluted with 160 μl Optimem (Vf = 400 μl)

Just prior to the addition of the transfection medium, the cells were washed once with DMEM (without serum or antibiotics) . The transfection medium (400 μl) was then added to the cells for 6h. Subsequently, the cells were incubated overnight in DMEM + 10% NCS (16h) . The next morning, the transfected cells were incubated in fresh DMEM (without serum) until treatment with DMEM supplemented with selected growth factors for 24h.

Before harvest, the fibroblast monolayer was washed once with cold PBS and lOOμl passive buffer (Promega) was added to each well and left for 15 min at room temperature . The cells were then harvested by scraping. The extracts were spun for lOmin at 15000rpm at 4°C to remove cell debris and lOμl of the supernatant was assayed immediately with a luciferase assay kit (Promega) according to the manufacturer's instructions. The luciferase activity was measured with a lu inometer (Turner Designs-20/20) . The data was expressed in relative light units per well. Pro tease inhibition protocol

Factor Xa was incubated with rTAP or ASN for 2h at 37°c , with regular shaking, prior to addition to the cells in culture. The same protocol was followed to inhibit factor Xa with GGACK. However, an alternative protocol was adopted for the preparation of purified catalytically inactive fXa. Catalytically inactive fXa was prepared by Chris Goodwin (National Heart and Lung Institute, London) by incubating factor Xa with GGACK (both from Calbiochem, La Jolla, CA) . Factor Xa was diluted in buffer (0.5 mM MgCl₂, 10 M Hepes, 0.15 M NaCl, 4 mM KC1, 11 mM glucose) and the ratio of inhibitor to enzyme was 20:1. The remaining mitogenic activity was not significant after 2 min of incubation with the inhibitor, as measured by chromogenic assay with a specific synthetic substrate for factor Xa (S- 2765 from Chromogenix, Epsom, UK) . The excess of soluble GGACK was then removed by extensive dialysis. The purified complex of GGACK bound to fXa was then termed DEGR-fXa.

Optimisation of inhibitor and antibody concentrations for inhibition experiments

Prior to inhibition experiments, all protease inhibitors and antibodies were evaluated individually over a range of dilutions in the relevant assay. The maximal workable concentrations were determined to obtain optimal neutralisation at doses that do not interfere with basal cell functions. It has generally been found that incubation of fibroblasts with antibodies (antibody 5224) for 48 h affected mildly the basal absorbance level in the methylene blue assay, possibly by enhancing fibroblast replication in a nonspecific fashion. Thus it was felt that experiments with blocking antibodies should be performed in the DNA synthesis assay which allows the use of high concentrations of antibody without affecting basal ³H- thymidine incorporation. This effect' as not observed in the case of protease inhibitors which could be assayed in the proliferation assay. Second, the neutralisation of the activity of fXa with antibodies in vi tro was more effective in the DNA synthesis assay. The shorter incubation time in this assay may favour the kinetics of the inhibition with compounds such as antibodies, which do not bind covalently to the enzyme, but compete for binding sites with the natural ligands. Statistical analysis

Statistical evaluation was performed using an unpaired student t test. A P value < 0.05 was considered significant. Data showing no statistical difference (P>0.05) was denoted NS . All data are expressed as the mean ± standard error of the mean (sem) , along with the number of repeat experiments and the number of replicate for each experimental value (n) .

When the data is expressed as a percentage of stimulation above medium control, the control value (Cells in medium with no addition) has been subtracted from each experimental value . Thus the data represented is relative to the control value which is arbitrarily considered a basal level of 100%. Example 1

Fibroblast replication induced by factor Xa was measured after 48 h incubation in methylene blue colorimetric assays. The results are shown in Figure

1, expressed as % increase above control ± sem, mean of 4 independent experiments with 6 replicates for each value .

It can be seen that factor Xa was a potent inducer of fibroblast proliferation. It induced a maximal increase in replication of up to 173.3+9.6% above medium control at lOOnM. Because the concentration of fXa in human plasma approximates 200nM, it is likely that its conversion will generate levels of active enzyme approaching the maximal stimulatory dose for fibroblast proliferation in vi tro.

Example 2

Fibroblast DNA synthesis induced by factor Xa was measured after 16h incubation in the DNA synthesis assay (³H-thymidine intake assay) . The results are shown in Figure 2 , expressed as % increase above control ± sem, representative experiment with 6 replicates for each value.

Factor Xa was a potent inducer of fibroblast DNA synthesis, stimulating a maximal increase in ³H- thymidine intake of up to 245±40% above medium control at lOOnM.

Example 3

The proliferative effect of factor Xa was compared to that of the potent fibroblast mitogens thrombin and platelet-derived growth factor-AB (PDGF-AB) and the structurally-related protease factor IXa . The results are shown in Figures 3 (methylene blue colorimetric assay; expressed in % increase above control ±sem, mean of 3 independent experiments with 6 replicates for each value) , 4 (³H-thymidine intake assay; expressed in % increase above control ±sem, representative experiment with 6 replicates for each value) , and 5 (direct cell counts, expressed in cells/well ± sem, (24 well plate) , representative experiment with 4-8 replicates for each value) .

The activity of fXa was compared with factor IXa, thrombin and PDGF-AB in the proliferation assay and the DNA synthesis assay (Figures 3 and 4) . These observations were confirmed by a third method of assessing cell proliferation, direct cell counts (Figure 5) . The three assays were performed in identical experimental conditions.

The enzymes were used at 25nM, a stimulatory concentration for both thrombin, factor IXa and factor Xa. Further, it is likely that such concentrations of active coagulation proteases are generated at sites of activation of the coagulation cascade when the concentrations of the corresponding zymogens in the blood approximate 90 nM, 140 nM and 1.4 mM for factor IX, X and prothrombin respectively.

Factor Xa and thrombin at 25nM consistently induced an increase of the same amplitude in both DNA synthesis and proliferation assays. In comparison, factor IXa did not affect fibroblast proliferation or DNA synthesis significantly.

The stimulation by factor Xa was equal or greater than that by an optimal concentration of the potent growth factor PDGF-AB (0.4pM - lOng/ml) and equal to that of thrombin at the same concentration.

The results obtained correlated well with each other and were consistent across the three assays.

Together with Examples 1 and 2, these results show that coagulation protease fXa is a potent inducer of both DNA synthesis and cell division in fibroblasts in a fashion reminiscent of thrombin.

Example 4

The proliferative effect on human fibroblasts of commercial preparations of factor Xa by Calbiochem (La Jolla, CA) and American Diagnostica (Greenwich, CT) were compared .

The results are shown in Figure 6 (methylene blue colorimetric assay; expressed in % increase above control ±sem, P values are calculated between the effects of the two preparations at each concentration, representative experiment with 6 replicates for each value) .

Both preparations tested induced very similar increases in fibroblast proliferation, with no marked difference in potency. Indeed, there is no statistical difference between the measured stimulations at most concentrations (P>0.05).

This example rules out the possibility that the mitogenic effect of factor Xa is unique to the

Calbiochem preparation. The proliferative effect observed seems to be representative of the various preparations available commercially.

Example 5 The effect of factor Xa was tested on primary human fibroblasts isolated from normal adult skin, lung and heart .

As seen from Table 1 below, factor Xa induced an increase in replication in all human fibroblasts tested, including primary culture isolated from four normal adult tissues. Methylene blue colorimetric assay; expressed in % increase above control ±sem, representative experiments with 6 replicates for each value . This observation contradicts the previous report that fXa does not induce fibroblast DNA synthesis (Gasic et al . , 1992) and verifies that the response observed in foetal lung fibroblasts in the previous examples is representative of a variety of tissues.

Factor Xa (25nM)

Foetal lung fibroblasts 92.3 ± 4.6 %

Primary adult lung fibroblasts 45.8 ± 3.0 %

Primary adult skin fibroblasts 35.7 ± 1.3 % Primary adult heart fibroblasts 39.9 ± 1.2 %

(SV40-transf^ed) adult kidney fibroblasts 55.7 ± 6.5 %

Table 1

Example 6

Factor Xa was incubated with rTAP for 2h at 37°C prior to addition to the cells in culture. The remaining mitogenic activity was measured after 48h in 0% NCS in a methylene blue colorimetric assay.

The results are shown in Figure 7 (methylene blue colorimetric assay; expressed in % increase above control ±sem, P values are calculated in comparison with control, mean of 2 independent experiments with 6 replicates for each value) .

Recombinant tick anticoagulant peptide inhibited fXa mitogenic activity entirely in fibroblasts, confirming that rTAP can be used to abrogate fXa mitogenic activity. This observation also demonstrates that fXa mitogenic activity for fibroblasts is mediated by its active catalytic site. Example 7

Factor Xa was incubated with antistasin-related peptide, ASN D-Arg³²-Pro³⁸, in the same manner as Example 6.

The results are shown in Figure 8 (methylene blue colorimetric assay; expressed in % increase above control ±sem, P values are calculated in comparison with control, representative experiment with 6 replicates for each value) .

ASN D-Arg³²-Pro³⁸ inhibited the activity of fXa in a dose-dependent fashion, and produced complete inhibition of factor Xa (25 nM) at a concentration of 1 μM. These results illustrate that natural and synthetic peptides comprising ASN core inhibitory sequence are capable of abolishing the mitogenic effect of fXa and confirm that the proteolytic activity of fXa is necessary to fXa mitogenic effect. Example 8

Factor Xa was incubated with GGACK in the same manner as Example 6.

The results are shown in Figure 9 (methylene blue colorimetric assay; expressed in % increase above control ±sem, P values are calculated in comparison with control, representative experiment with 6 replicates for each value) .

GGACK inhibited the activity of fXa in a dose- dependent fashion, and produced complete inhibition of factor Xa (25 nM) at a concentration of 200 nM.

This confirms that the proteolytic activity of fXa is necessary for its mitogenic effect. In addition, it demonstrates that small synthetic inhibitors of factor Xa are useful to downregulate fibroblast proliferation induced by factor Xa.

Example 9

Factor Xa was incubated with DEGR-factor Xa . The results are shown in Figure 10 (methylene blue colorimetric assay; expressed in % increase above control ±sem, P values are calculated in comparison with control, representative experiment with 6 replicates for each value) .

DEGR-factor Xa did not display the mitogenic effect of fXa at a similar concentration. This confirms that the proteolytic activity of fXa is critical to its mitogenic effect and that other domains of the active enzyme are not sufficient to induce the same effect .

Example 10 Factor Xa was incubated with an antibody directed against factor Xa (5224) .

The results are shown in Figure 11 (³H-thymidine intake assay; expressed in % increase above control ± sem, P values are calculated in comparison with control, representative experiment with 6 replicates for each value) .

The antibody directed against factor Xa (5224) inhibited fXa mitogenic activity in fibroblasts. Previously, the antibody 5224 has been shown to neutralise the catalytic activity of factor Xa

(Zacharski et al . , 1991; Ko et al . , 1996). This confirms that the proteolytic activity of fXa is necessary for its mitogenic effect. This also suggests that the inhibitory effect of small molecular weight inhibitors of the catalytic site of factor Xa can be mimicked efficiently by neutralising antibodies.

Because of its high specificity for fXa, the antibody 5224 only inhibits the mitogenic activity of fXa. After incubation with antibody 5224, the preparation of fXa displays no remaining mitogenic activity. Thus, this observation confirms that the totality of the mitogenic activity of the preparation is due solely to fXa, ruling out the participation of eventual contaminating growth factors. Example 11

Factor Xa was incubated with an antibody (JC15) directed against the region of fXa necessary for its interaction with the receptor EPR-1.

The results are shown in Figure 12 (³H-thymidine intake assay; expressed in % increase above control ± sem, P values are calculated in comparison with control, representative experiment with 6 replicates for each value) .

JC15 inhibited the mitogenic activity of fXa from 119.9 23.5% to 39.5 11.7% above medium control in a DNA synthesis assay shown in Figure 12. In contrast, the antibody alone did not affect either fibroblast proliferation or DNA synthesis at the selected concentration . The neutralisation of the activity of fXa with

JC15 suggests that at least two thirds of the proliferative response induced by fXa in fibroblasts is mediated by the interaction between EPR-1 and the residues Leu⁸³-Leu⁸⁸ of fXa. Example 12

Fibroblasts were incubated with an antibody (B6) directed against the region of EPR-1 necessary for its interaction with the receptor fXa .

The results are shown in Figure 13 (³H-thymidine intake assay; expressed in % increase above control ± sem, P values are calculated in comparison with control, representative experiment with 6 replicates for each value) .

B6 inhibited the mitogenic activity of fXa in a DNA synthesis assay shown in Figure 13. In contrast, the antibody alone did not affect DNA synthesis at the selected concentration.

This confirms that the interaction between fXa and its receptor EPR-1 is necessary for its mitogenic effect. This also suggests that this interaction can be efficiently inhibited by neutralising antibodies and provides an alternative way to inhibit the mitogenic activity of fXa for fibroblasts.

Example 13 Procollagen αl(I) gene expression in transfected fibroblasts induced by factor Xa was measured using the luciferase activity assay. The results are shown in Figure 14, expressed as % increase above control. Figure 14 also shows the effect of various inhibitors (rTAP, ASN D-Arg³²-Pro³⁸ or an antibody directed against factor Xa (5224)) on this effect.

Factor Xa was a potent inducer of procollagen αl(I) gene expression, stimulating procollagen αl(I) promoter activity by up to 300% above control) . This activity was inhibited by specific catalytic site inhibitors .

This demonstrates that the proteolytic activity of fXa is necessary for its procollagen l(I) gene expression stimulatory effect. This also demonstrates that inhibitors of factor Xa are useful to downregulate collagen production induced by factor Xa.

It will be appreciated from the foregoing examples that the inventors examined the effect of coagulation factor Xa on fibroblast proliferation with three different methods, evaluating fibroblast DNA synthesis by ³H-thymidine intake assays and cell replication by methylene blue dye colorimetric assays and confirming by direct cell counts. To investigate the effect of coagulation factor Xa on fibroblast procollagen production, the inventors have examined the effect of factor Xa on procollagen promoter activity in fibroblasts using a luciferase assay with fibroblasts transfected with a procollagen αl(I) promoter linked to a luciferase reporter gene. The inventors have demonstrated for the first time and against previous teaching that Factor Xa is a potent stimulator of fibroblast DNA synthesis and proliferation. Factor Xa stimulated replication in all fibroblasts tested, including primary fibroblasts from five different human organs, suggesting that the mitogenic effect of fXa is relevant to fibroblasts independent of tissue origin. The inventors have also demonstrated for the first time that Factor Xa is a potent stimulator of procollagen promoter activity in fibroblasts.

The inventors have shown that compounds related to antistasin, tick anticoagulant peptide, GGACK or antibodies directed against factor Xa inhibit fibroblast proliferation and procollagen promoter activity induced by coagulation fXa . The inhibitory effect of these compounds is mediated by the inhibition of the catalytic activity of fXa, or alternatively by the inhibition of the interaction between fXa and its receptor.

Blocking the proliferation of fibroblasts and/or the procollagen promoter activity of fibroblasts, will reduce the production of collagen by these cells. Hence, the deposition of extracellular matrix, which would otherwise disturb the organisation of the tissue and result in the loss of function of the organ, can be diminished. All inhibitors of fXa and derivatives thereof known to the skilled person fall within the scope of the invention.

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Claims

CLAIMS :

1. The use of an inhibitor of factor Xa activity in the production of a medicament for the prevention or treatment of organ damage associated with factor Xa stimulation of fibroblasts resulting in the proliferation of fibroblasts and/or the production of procollagen by fibroblasts.

2. The use as claimed in claim 1, wherein the inhibitor inhibits factor Xa activity by blocking the catalytic site thereof.

3. The use as claimed in claim 2, wherein the inhibitor is one or more of tick anticoagulant peptide, antistasin, GGACK, DX9065, an antibody which binds to the catalytic site and derivatives thereof .

4. The use as claimed in claim 3 , wherein the tick anticoagulant peptide, antistasin or derivative thereof is obtained from natural sources or by recombinant techniques .

5. The use as claimed in claim 3 or claim 4, wherein the inhibitor is ASN D-Arg³²-Pro³⁸.

6. The use as claimed in claim 3, wherein the antibody is antibody 5224.

7. The use as claimed in claim 1, wherein the inhibitor inhibits factor Xa activity by blocking the interaction between factor Xa and its receptor.

8. The use as claimed in claim 7, wherein the inhibitor binds to the receptor binding site of factor Xa.

9. The use as claimed in claim 8, wherein the inhibitor is an antibody or a derivative thereof.

10. The use as claimed in claim 9, wherein the antibody is antibody JC15.

11. The use as claimed in claim 7, wherein the inhibitor blocks the factor Xa binding site of the receptor.

12. The use as claimed in claim 11, wherein the inhibitor is an antibody or a derivative thereof.

13. The use as claimed in claim 12, wherein the antibody is antibody 2E1.

14. The use as claimed in claim 12, wherein the antibody is antibody B6.

15. The use of an inhibitor of factor Xa activity in the production of a medicament for use in a method of preventing or treating a disease by inhibiting the factor Xa-associated stimulation of fibroblast proliferation and/or the factor Xa-associated stimulation of procollagen production by fibroblasts.

16. The use of an inhibitor of factor Xa activity in the production of a medicament for the prevention or treatment of collagen deposition resulting from factor Xa-associated stimulation of fibroblasts.

17. A method for treating or preventing organ damage caused by factor Xa-associated stimulation of fibroblasts resulting in the proliferation of fibroblasts and/or the production of procollagen by fibroblasts disease in a mammalian patient, which method comprises the step of administering a pharmaceutically effective amount of an inhibitor of factor Xa activity as defined in any of claims 1 to 14 to the mammalian patient.