Abstract:
The present invention comprises the use of chondroitin sulphate (CS-E) or an active fragment thereof for the treatment of diseases or conditions related to collagen fibril formation. Said compounds can be administrated either by oral, topical, injectable or by any other suitable route.

Description:
TECHNICAL BACKGROUND 
       [0001]    Many proteins of the extracellular matrix (ECM) are modified post-translationally by addition of oligosaccharide chains and are thus known as glycoproteins. The oligosaccharides are linked either O-glycosidically to serine or threonine residues, or N-glycosidically to an asparagine residue. Proteoglycans are glycoproteins that are substituted with a particular class of carbohydrate polymers, known as the glycosaminoglycans (GAGs). Proteoglycans are found in the ECM, at the cell surface and intracellularly in storage granules. In the ECM they contribute to the structure and organisation, and at the cell surface often function as receptors and/or co-receptors. All glycosaminoglycans (with the exception of hyaluronan) are synthesised on a core protein acceptor, and they are thus an integral component of proteoglycans (Wight et al., 1981; Heinegård and Paulsson, 1984, review). 
         [0002]    Glycosaminoglycans (GAGs) are named to indicate that one of the monosaccharides in the repeating sequence of disaccharides is an amino sugar. The other monosaccharide is an uronic acid (glucuronic acid or iduroic acid), with the exception of keratan sulphate where it is a galactose. While other oligosaccharide substituents may be branched, GAG chains are linear (again, with the exception of keratan sulphate). Proteoglycans may be substituted with one (e.g. decorin) and up to some one hundred (e.g. aggrecan) GAG chains. 
         [0003]    There are 4 types of glycosaminoglycans: hyaluronic acid, chondroitin sulphate/dennatan sulphate, heparan sulphate/heparin and keratan sulphate. The disaccharides in all glycosaminoglycan chains except hyaluronan are sulphated, increasing their negative charge and leading to an extended conformation of the chain. The molecule will occupy large solvent domains, observed as a high viscosity of a solution. This property is essential in cartilage and is the basis on which the tissue&#39;s resistance lies. 
         [0004]    The repeating disaccharide sequence in CS is glucuronic acid-N-acetyl-galactosamine (GlcA-GalNAc), see  FIG. 1 . Chondroitin sulphate is found in several forms, named chondroitin-4 sulphate, -6 sulphate and -D and -E respectively. These forms differ in the sulphation of saccharides. CS-E is a highly sulphated species, which is attached to perlecan in the I and V domains. 
         [0005]      FIG. 1 . Basic structure of Chondroitin sulfate. Repeating dimeric ullits of GlcA β1-3 GalNAc. All hydroxy positions may be sulfated or/and epimerised. 
         [0006]    The various positions open for sulfatation are numbered. 
         [0007]    Chondroitin sulphate/dermatan sulphate is found in all extracellular matrices. Cartilage and invertebral disc are the tissues richest in chondroitin sulphate (Wight et al., 1981, review). Chondroitin sulphate is synthesised by specific enzymes located in the Golgi. The polymers are assembled onto a linker tri-saccharide. The hydroxyl group of serine residues followed by a glycine in the protein is substituted with a xylose and two successive galactose residues. Thereafter alternating monosaccharides of glucuronic acid and N-acetylgalactosamine are added successively to form the chain. Some glucuronate residues are converted to iduronate by an epimerase and sulfation is the last event just prior to secretion (Wight et al., 1981, review). In cartilage aggrecan, a member of the family hyalectins, is a chondroitin sulphate proteoglycan and is substituted with some one hundred CS chain and some thirty keratan sulphate chains. Aggrecan molecules are clustered along HA strands bound via their N-terminal globular domain. A protein known as link protein contacts both the HA-binding G1 domain of the aggrecan molecule and HA, and stabilises the complex. In this manner hundreds of aggrecan molecules are joined at one end to the HA. Thus, in cartilage matrix chondroitin sulphate is by far the most abundant GAG. 
         [0008]    Perlecan was first identified as a large heparan sulphate proteoglycan isolated from the Engelbrecht-Holm-Swarm (EHS) murine basement membrane tumour. In basement membranes, it has been shown to bind several different classes of molecules. In each instance the core protein, the heparan sulphate (HS) side chains or both in concert, are involved in mediating the interaction. The proteoglycan binds to extracellular matrix components integral to basement membrane such as collagen IV, nidogen, laminin, and fibronectin (Timpl, R. and Brown, J. C. (1996)  Bioassays  18, 123-132). Perlecan has also been shown to bind extracellular matrix components outside the basement membrane, e.g. PRELP and collagen type I (Bengtsson, E., Mörgelin, M., Sasaki, T., Timpl, R., Heinegård, D., and Aspberg, A. (2002)  J. Biol. Chem ). Perlecan supports cell-attachment both by binding and clustering integrins (Brown, J. C., Sasaki, T., Gohring, W., Yamada, Y., and Timpl, R. (1997)  Eur. J Biochem.  250, 39-46). Binding to growth factors has been shown for both the HS side-chains (FGF-2 (Aviezer, D., Hecht, D., Safran, M., Eisinger, M., David, G., and Yayon, A. (1994)  Cell  79, 1005-1013)) and the core protein (progranulin, (Gonzalez, E. M., Mongiat, M., Slater, S. J., Baffa, R., and Jozzo, R. V. (2003)  J Biol Chem )). Based on its interactions, perlecan is assumed to have a role in basement membrane integrity. 
         [0009]    Perlecan was originally thought to be substituted with HS exclusively, but later studies revealed that it is also present in a variant partially substituted with chondroitin sulphate (CS) (Couchman, J. R., Kapoor, R., Sthanam, M., and Wu, R. R. (1996)  J Biol Chem  271, 9595-9602). Both the HS- and the HS/CS-substituted variants of perlecan have been found in tissues other than basement membrane, for example cartilage. 
         [0010]    The generation of perlecan null mice revealed two particularly intriguing findings (Arikawa-Hirasawa, E., Watanabe, H., Takani, H., Hassell, J. R., and Yamada, Y. (1999)  Nat Genet.  23, 354-358; Costell, M., Gustafsson, E., Aszódi, A., Morgelin, M., Bloch, W., Hunziker, E., Addicks, K., Timpl, R., and Fässler, R. (1999)  J Cell Biol  147, 1109-1122). First, though mice lacking perlecan did develop grave disorders caused by compromised basement membrane strength or integrity (e.g. rupture of pericardial sac), the initial assembly of basement membranes seemed to be without complication. The second striking finding was the severe skeletal defects exhibited, apparently caused by the lack of perlecan in cartilage. 
         [0011]    Following the publication of these results at least two human hereditary diseases with skeletal deficiencies have been ascribed to an underlying scarcity or complete lack of perlecan, underscoring the relevance of this finding in the mouse model (Nicole, S., Davoine, C. S., Topaloglu, H., Cattolico, L., Barral, D., Beighton, P., Hamida, C. B., Hammouda, H., Cruaud, C., White, P. S., Samson, D., Urtizberea, J. A., Lehmann-Horn, F., Weissenbach, J., Hentati, F., and Fontaine, B. (2000)  Nat Genet.  26, 480-483; Arikawa-Hirasawa, E., Wilcox, W. R., Le, A. H., Silverman, N., Govindraj, P., Hassell, J. R., and Yamada, Y. (2001)  Nat Genet.  27, 431-434). 
         [0012]    In skeletal development, the deposition of a cartilaginous template precedes the formation of bones. The integrity of this template is a prerequisite for proper assembly of the skeleton. Perlecan-null mouse cartilage shows fewer and less organised collagen type II fibrils, and decreased levels of aggrecan, indicating a failure to organise the extracellular matrix (Costell, M., Gustafsson, E., Aszódi, A., Morgelin, M., Bloch, W., Hunziker, E., Addicks, K., Timpl, R., and Fäqssler, R. (1999)  J Cell Biol  147, 1109-1122). 
         [0013]    Mature collagen fibres may contain several different types of bound accessory proteins. They are part in the organisation of these fibres and regulate links to other molecules thereby contributing to the architecture of the fibrillar collagen network. A recent concept is that of modulator molecules, which regulate the early steps in the assembly of collagen monomers to fibres. Our laboratory has found that cartilage oligomeric matrix protein (COMP) accelerates the formation of fibres from monomers (Mörgelin and Heinegård, manuscript). Other molecules have the opposite effect and slow down fibre formation in vitro, e.g. decorin (Vogel, K. G., Paulsson, M., and Heinegård, D. (1984)  Biochem. J.  223, 587-597) and fibromodulin (Hedborn, E. and Heinegård, D. (1989)  J. Biol. Chem.  264, 6898-6905). Gene targeting of these molecules lead to abnormal collagen fibrils and disturbed mechanical properties of the tissues (Danielson, K. G., Baribault, H., Holmes, D. F., Graham, H., Kadler, K. E., and Iozzo, R. V. (1997)  J Cell Biol  136, 729-743; Svensson, L., Aszódi, A., Reinholt, F. P., Fäissler, R., Heinegård, D., and Oldberg, Å. (1999)  J. Biol. Chem.  274, 9636-9647). A picture is emerging where proteins in the vicinity of the cell regulate the early stages of collagen fibre formation. 
         [0014]    Perlecan exists as HS and CS substituted forms and it has been shown that these forms can be used to facilitate collagen fibril formation. To our surprise, the addition of free CS-E was effective in collagen fibril formation, but none of the other CS variants had any significant effect (e.g. CS-D or CS-6). 
         [0015]    A number of publications describing the effect of chondroitin sulphate on wound healing and for treating arthrosis exist (U.S. Pat. No. 5,929,050, JP10120577 and RU2216332). The present invention differs from these significantly as the use of CS-E or active fragments thereof stimulates the formation of collagen based extracellular matrix (ECM) and thus acting as fibrillogenesis agonists or, by modification of CS-E or active fragments thereof, as fibrosis antagonists. 
       DESCRIPTION OF THE INVENTION 
       [0016]    Comprised in the invention is the use of chondroitin sulphate from Perlecan or chondroitin sulphate E (CS-E) or active fragments of CS-E for treatment of various conditions and diseases related to disorders by the facilitation or prevention of collagen fibril formation (CFF) by agonists and antagonists respectively. 
         [0017]    Also comprised is a pharmaceutical acceptable composition containing CS-E or active fragments thereof for treating said conditions and diseases. 
         [0018]    CS-E or active fragments thereof are highly sulphated and thus may be charged compounds and the invention also comprises pharmaceutical acceptable salts, such as alkali metal salts (sodium, potassium, cesium) and alkaline earth salts (e.g. magnesium, zinc, calcium, strontium) and ammonium, as well as organic salts. 
         [0019]    Also comprised in the invention is a formulation for administration of a therapeutically effective amount of CS-E, or active fragments thereof in production of artificial collagen matrices for treatment of disorders by transplantation of cell containing or recruiting scaffolds. 
         [0020]    Indications comprised in this application are conditions and diseases related to disorders in collagen fibrillogenesis, including but not limited to pulmonary fibrosis, wound healing, in particular chronic wound healing, chronic intestinal disease such as ulcerative colitis and Crohns disease, rheumatoid arthritis (RA), osteroarthritis (OA), reconstructive skeletal formation and skeletal repair. 
       CFF Agonists 
       [0021]    Chronic wound healing can be stimulated by application of a CFF agonist, alone or together with exogenous collagen. This is also useful for treatment of acute open wounds. 
         [0022]    CFF agonists also facilitates healing of internal wounds including but not limited to peptic ulcers, ulcerous colitis, Crohn&#39;s disease or other inflammatory bowel disease. 
         [0023]    CFF agonists can be used to facilitate healing after surgical treatment and transplantation. 
         [0024]    In the progressive degenerative joint disease osteoarthritis the articular cartilage collagen is degraded. CFF agonists counteract or reverse this process and/or facilitates the assembly of newly synthesized collagen molecules into fibers. 
         [0025]    CFF agonists can be used to stimulate repair in damaged ligaments and tendons. 
         [0026]    CFF agonist can be used to facilitate bone repair after severe fractures. 
       CFF Agonists in Tissue Engineering 
       [0027]    Tissue engineering can be used to repair damaged tissue (e.g. skin in burn wound healing or cartilage in OA) via autologous cell replacement therapy or transplantation. CFF agonists can be used to stimulate collagen matrix formation in cell or tissue culture for production of transplantable skin, tendon, cartilage, bone or blood vessels from patient cells or tissue or from various types of stem cells. 
         [0028]    CFF agonists can be used to produce well-formed artificial collagen matrix scaffolds for use in healing of e.g. burn wounds and OA cartilage. After implantation these scaffolds would be populated by cells recruited from the surrounding tissue of the patient. 
         [0029]    CFF agonists are also useful in the production of well-organised collagen fibril matrices for corneal implants. 
       Fibrotic Disorders 
       [0030]    Scarring is a natural response of the body to trauma and injury. In fibrotic conditions the normal wound healing response continues out of control, with excessive production and deposition of collagen. This leads to a loss of function when normal tissue is replaced with scar tissue. Fibrosis can affect virtually all organ systems in the body. 
         [0031]    There are many different causes of fibrosis, e.g. trauma, surgery, infection, environmental pollutants and toxins (including alcohol). Some examples of fibrotic conditions are formation of scar tissue following heart attack, kidney fibrosis as a complication of diabetes, lung fibrosis and surgical scar tissue formation between internal organs. 
         [0032]    Acute fibrosis is a response to various forms of trauma, such as injury, infections, surgery, burns, radiation damage and chemotherapeutic treatments. Many chronic conditions, e.g. diabetes, viral infection and hypertension, induce a progressive fibrosis causing continuous loss of tissue function. The liver, kidney and lung are commonly affected. Systemic fibrotic diseases include diabetic nephropathy, scleroderma, idiopathic pulmonary fibrosis and reactive fibrosis following myocardial infarct. 
       CFF Antagonists 
       [0033]    By using CFF antagonists it will be possible to counteract fibrotic processes by blocking the assembly of the collagen fibrils. In many cases this can be achieved by local administration thus avoiding possible side effects from systemic treatment. However, the CFF antagonists may also be administered systemically when suitable. 
         [0034]    CFF antagonists can be used to prevent fibrotic disorders of the skin, including, but not limited to scar formation in wound healing, hypertrophic scarring and keloid, contracture in connection with hypertrophic scarring after burn injury, surgical adherens or skleroderma. Local application of the CFF antagonists would be easy, except in surgical adherens where administration could be achieved by osmotic pump devices or other suitable administrations. 
         [0035]    CFF antagonists can be used for treatment and prevention of idiopathic lung fibrosis. 
         [0036]    CFF antagonists could be used to treat other deep organ fibrosis such as liver fibrosis/cirrhosis and diabetic kidney fibrosis. 
         [0037]    CFF antagonists could be used to prevent heart muscle scarring after myocardial infarction. 
         [0038]    CFF antagonists could be used to prevent or counteract atherosclerosis and restenosis after angioplasty. To achieve the latter, the antagonist could be delivered locally by implantation of modified stents. 
     
    
     EXAMPLES 
       [0039]    In vitro fibrillogenesis assay. 
         [0040]    Bovine pepsin-extracted collagen type I was purchased from Vitrogen. Collagen II was pepsin extracted from bovine tracheal cartilage, as previously described (Vogel, K. G., Paulsson, M., and Heinegård, D. (1984)  Biochem. J.  223, 587-597). The fibrillogenesis assay has been previously described (Hedborn, E. and Heinegård, D. (1989)  J. Biol. Chem.  264, 6898-6905). 
         [0041]    Briefly, a solution of collagen monomers (330 nM) was brought to neutral pH by addition of an appropriate volume of 0.012M NaOH, and buffered by 20 mM HEPES, 150 mM NaCl at pH 7.4. Perlecan fragment was added at concentrations equimolar to that of collagen or at one tenth the molar concentration of collagen. The sample was mixed vigorously and briefly, and transferred to a cuvette. The sample was incubated at 37° C. (collagen type I) or 35° C. (collagen type II) in water-jacketed cuvettes in the spectrophotometer, and the absorbance due to light scattering at 400 nm (collagen type I) or 313 nm (collagen type II) was monitored over a duration of 5-18 hrs. The increased absorbance/turbidity depends on increasing fibre formation. 
       CS-E Fibrillogenesis 
       [0042]    Fibrillogenesis was performed as described above. CS-E from squid was purchased from Calbiochem. 0.13 μg/ml corresponding to the molar concentration of GAG chains in previous experiments using recombinant perlecan domain I variants with HS/CS (PG IB) (33 nM). 
         [0043]    The results of the fibrillogenesis experiment is shown in  FIG. 2 . As can be seen from this figure, CS-E but none of the other tested chondroitin sulphate variants (CS-D and CS-6) gives a positive result on collagen fibril formation (CFF). 
         [0044]      FIG. 2  CS-E accelerated collagen fibril formation. Different types of purified CS-chains were tested for effect in the collagen fibrillogenesis assay (panel A). The highly sulphated CS-E had dramatic effects on fibril formation but neither CS-6 nor CS-D had any effect. The stimulatory effect of CS-E was dose-dependent, reaching saturation at concentration of 30 μg/ml (panel B). 
       In Vivo Model 
     Example of In Vivo Model for Studying Wound Healing. 
       [0045]    Groups of 5 ICR male mice weighing 22±2 gms are used. Under hexobarbital (90 mg/kg, i.p.) anesthesia, the shoulder and back region of each animal is shaved. A sharp punch (ID 12 mm) is used to remove the skin including panniculus carnosus and adherent tissues. The wound area, traced onto clear plastic sheets on days  3 ,  5 ,  7 ,  9  and  11 , are quantitated by use of an Image Analyzer (Life Science Resources VISTA, Version 3.0). Test compound and/or vehicle (20 μl, 0.5% carboxymethylcellulose in PBS pH 7.4) is applied topically immediately following injury and once daily thereafter for a total of 10 consecutive days. The wound half closure time (CT 50 ) is determined and unpaired Student&#39;s t-test is applied for comparison between treated and vehicle group at each measurement time point. Differences are considered statistical significance at P&lt;0.05. (Montesinos, M. C., Gadangi, P., Longaker, M., Sung, J., Levine, J., Nilsen, D., Reibman, J., Li, M., Jiang, C. K., Hirschom, R., Recht, P. A., Ostad, E., Levin, R. I. and Crostein, B. N. Wound healing is accelerated by agonists of Adenosine A 2  (Gα s -linked) receptors. J. Exp. Med. 186: 1615-1620, 1997.) 
       Formulation 
       [0046]    Example of a Preparation Comprising a Capsule 
         [0000]    
       
         
               
               
             
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Per capsule 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Active ingredient, as salt 
                 5 
                 mg 
               
               
                   
                 Lactose 
                 250 
                 mg 
               
               
                   
                 Starch 
                 120 
                 mg 
               
               
                   
                 Magnesium stearate 
                 5 
                 mg 
               
               
                   
                 Total up to 
                 385 
                 mg 
               
               
                   
                   
               
             
          
         
       
     
         [0047]    In case higher amounts of active ingredient are required, the amount of lactose used may be reduced. 
         [0048]    Example of a suitable tablet formulation. 
         [0000]    
       
         
               
               
             
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Per tablet 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Active ingredient, as salt 
                 5 
                 mg 
               
               
                   
                 Potato starch 
                 90 
                 mg 
               
               
                   
                 Colloidal silica 
                 10 
                 mg 
               
               
                   
                 Talc 
                 20 
                 mg 
               
               
                   
                 Magnesium stearate 
                 2 
                 mg 
               
               
                   
                 5% aqueous solution of gelatine 
                 25 
                 mg 
               
               
                   
                 Total up to 
                 385 
                 mg 
               
               
                   
                   
               
             
          
         
       
     
         [0049]    A solution for parenteral administration by injection can be prepared in aqueous solution of a water-soluble pharmaceutically acceptable acid addition salt of the active substance preferably in a concentration of 0.1% to about 10% by weight. 
         [0050]    These solutions may also contain stabilising agents, buffering agents and/or gelating agents such as but not limited to hyaluronan, PEG, HPMC, EHEC, to obtain a controlled release and/or elimination. 
       Example of a Topical Formulation 
       [0051]    A gel for topical administration can be prepared an active substance in a concentration of 0.1% to 10% by weight, optionally containing stabilising agents, buffering agents and/or additional gelating agents such as but not limited to hyaluronan, PEG, HMPC, EHEC to obtain controlled release and/or elimination