Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
2002-04-01
2004-09-07
Chan, Christina (Department: 1644)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C530S326000, C514S014800
Reexamination Certificate
active
06787141
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates broadly to compounds which act as modulators or mimics of fibrin fragment E and their use.
Basic Pathology of Chronic Inflammation, Healing and Repair
Fibrinogen is the major circulating plasma protein involved in blood clot formation. Activation of the clotting enzyme cascade, by for example injury or inflammation, results in conversion of prothrombin to thrombin which cleaves two small fibrinopeptides (A and B) from each soluble fibrinogen molecule to give fibrin monomer. Cross linkage of monomers is the final step of the coagulation system that gives solid fibrin. Whole blood includes platelets and forms blood clot in wounds, and is termed thrombus when in abnormal arteries and veins: inflammatory exudate is platelet free and forms fibrin alone.
Fibrin deposition and degradation is a major feature of the pathology of acute and chronic inflammation at any site in the body regardless of the underlying disease aetiology. This process is apparent at the histological level in the healing wound, the organising thrombus, the advanced atherosclerotic plaque, and many other types of pathological lesions including the growing edge of some types of cancer. The fibrin mesh provides a provisional matrix for cell ingrowth, being progressively invaded in wound healing by inflammatory cells (macrophages), new small blood vessels (capillary buds), connective tissue cells (fibroblasts) and the epidermis (squamous epithelium). In the context of the large arteries subject to atherosclerosis, the endothelium of the luminal surface and the smooth muscle cells of the vascular wall invade the fibrin mesh. Secretion of plasminogen activator is the common factor that provides controlled lysis of the fibrin substratum via plasmin degradation, releasing fibrin degradation products. Fibrin degradation products are composed of combinations of two moieties termed fragments D and E. Eventually the fibrin present is replaced by new cells and matrix forming new tissue. These basic features apply to many types of human and animal disease.
Fibrin Degradation Products
Although fibrin may be a factor common to many pathologies involving cell proliferation, it has generally been assumed that its main function was to provide an inert physical matrix to support cell movement. However there has been evidence for some time that fibrinopeptides and fibrin degradation products have biological activity particularly as soluble mediators of chemotaxis, the phenomenon of directional cell movement (1). It has been proposed that fibrin degradation products were a major pathological growth factor common to all sites of chronic inflammation. Using the chick chorioallantoic membrane as an in vivo test model for detection of angiogenic growth factors it has been suggested that fibrin degradation products were angiogenic (2), had stimulatory effects on collagen synthesis (3), and that specifically fibrin fragment E may be the active component (4).
Fibrin deposition and lysis is believed to be relevant to a wide spectrum of human diseases including vascular restenosis, cancer, atherogenesis, rheumatoid arthritis, diabetes and renal diseases.
U.S. Pat. No. 5,981,697 describes generation of antibodies which bind fibrinogen fragments E1, E2 and E3. Bach et al (1998) J Biol Chem 273 pp30719-30728 describes an interaction of the fibrinogen &bgr;15-42 sequence with endothelial cell VE-cadherin. Lee et al (1999), Molecules and Cells 9(1) 7-13 describes the stimulation of production of IL-6 in macrophages by fragment E of fibrin and fibrinogen.
SUMMARY OF THE INVENTION
The production of antibodies to fibrin fragment E has been previously described, the antibodies being derived using an Fd phage combinatorial library of random epitope display to select clones binding and common to both polyclonal rat and rabbit anti E blocking antisera (32). In the present invention, the inventors have individualised a number of compounds which modulate the induction of cell proliferation induced by fibrin degradation products. The present inventors have further demonstrated that fibrin fragment E binds to a cell membrane component of approximately 66 kDa in size using, for example, ligand blotting of SDS-PAGE gels of cell membranes from chick fibroblasts. The presence of a specific binding site for fragment E raises the possibility that agonists and antagonists of its binding could be used to modulate its binding and thus modulate its effects. Such effects include induction of cell proliferation, angiogenesis, fibrogenesis and collagen synthesis. Such agonists and antagonists, such as those disclosed herein, may also have modulatory effects on, for example, cell stimulation per se. Such modulation may be very useful in the control of cell proliferation seen in atherosclerosis, and particularly in post angioplasty restenosis. Using the techniques described below, the inventors have identified a number of compounds which modulate the FDP induced stimulation of cell proliferation. Preferably the variant retains fibrin fragment E activity.
Accordingly, the present invention provides a peptide comprising a sequence selected from the group consisting of:
CRAHSFGSPRPLPVV (SEQ ID NO:1)
SRAHSFGSPRPLPVV (SEQ ID NO:2)
CRAHSFVSPRPLPVV (SEQ ID NO:3)
QPDPHLMMWKLPGFP (SEQ ID NO:4)
or a fragment thereof capable of modulating fibrin fragment E activity.
As described above, fibrin fragment E activity refers to at least one of following activities: induction of cell proliferation, angiogenesis, fibrogenesis and collagen synthesis.
In a further aspect of the invention, there is provided a functional variant of the above peptide, which variant comprises from one to four, preferably from one to three, more preferably one or two, amino acid variations, including substitutions, insertions and deletions. Preferably, the variant retains the capability of modulating fibrin fragment E activity.
In another aspect of the invention there is provided a fusion peptide which comprises a first portion having the amino acid sequence of a peptide according to the invention as defined above and a second portion, attached to the N- or C-terminus of the first portion, which comprises a sequence of amino acid not naturally contiguous to the first portion. Such heterologous peptide fusions are also referred to herein as peptides of the invention.
In a further aspect, the invention provides assay methods for the identification of substances which bind to or modulate the activity of peptides of the invention, either in monomeric or oligomeric form.
In a further aspect of the invention, “analogs” of the peptides of the invention are provided. Analogs are non-peptide compounds which share fibrin fragment E activity, for example the ability to competitively inhibit binding of FDPs for example, fibrin fragment E to the fragment E receptor. Analogs are a further aspect of the invention inasmuch as they are novel. Analogs may be produced by any of the techniques described herein or may be derived using, for example, combinatorial chemical libraries known in the art. Examples of such libraries are reviewed in Newton G R, Exp. Opin. Ther. Patents (1997) 7(10): 1183-1194. Where the term “chemical library” is used herein, those skilled in the art will understand its meaning accordingly.
The invention also provides antibodies and binding fragments thereof capable of selectively binding to peptides or analogs of the invention.
In a further aspect, the invention provides a pharmaceutical composition comprising a peptide, analog or antibody according to the invention together with a pharmaceutically acceptable carrier or diluent.
Peptides, analogs or antibodies and compositions of the invention may be used for a number of purposes. They are also useful as research agents to investigate the receptor for fibrin fragment E and the activation of cell proliferation by fibrin degradation products. They may also be useful in modulating fibrin fragment E activity such as cell proliferation, particularly inhibition of angiogenesis.
Thus in a preferred aspect the pept
Melvin William Thomas
Stirk Christina Maureen
Thompson William Douglas
Chan Christina
Dann Dorfman Herrell & Skillman P.C.
Haddad Maher
The University Court of The University of Aberdeen
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