| F11 receptor (f11r) antagonists as therapeutic agents -> Monitor Keywords |
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  F11 receptor (f11r) antagonists as therapeutic agentsRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Immunoglobulin, Antiserum, Antibody, Or Antibody Fragment, Except Conjugate Or Complex Of The Same With Nonimmunoglobulin MaterialF11 receptor (f11r) antagonists as therapeutic agents description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20050265992, F11 receptor (f11r) antagonists as therapeutic agents. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] The present application is a Continuation-in-Part of PCT/US2003/39890, filed on Dec. 16, 2003, which claims priority of U.S. Provisional Application No. 60/438,669 filed on Jan. 3, 2003. FIELD OF THE INVENTION [0002] The present invention relates to protein and peptide chemistry, as well as crystallography and organic chemistry. The present invention is directed to a cell adhesion molecule (CAM) and fragments thereof, and more particularly to a CAM designated as the F11 receptor (F11R), or a polypeptide fragment thereof. The present invention also relates to F11R-antagonists and methods for the prevention and treatment of excessive bleeding following a wound injury, inflammatory diseases of the nervous system, thrombosis, inflammatory thrombosis, atherothrombosis, angiogenesis, plaque formation, cancer, immunothrombocytopenia (ITP), heart attacks, stroke, disorders of platelet and endothelial cell dysfunctions and other disorders involving thrombus formation. BACKGROUND OF THE INVENTION [0003] The vasculature is recognized as a dynamic metabolic organ that exists under normal physiological conditions in an intact, undisturbed state (Karsan, et al. In: Hematology: Basic Principles and Practice, 3rd Ed. Hoffman, et al. (eds) 2000; pp. 1770-82). Endothelial cells (EC), which line the exposed (luminal) surface of blood vessels, are normally not thrombogenic. That is, healthy EC do not attract nor bind circulating platelets (Cines, et al. Blood 1998, 91: 3527-61; May, et al. Thromb Haemost 1999, 82: 962-70). It is well known that the physiological function of the endothelium is to facilitate blood flow by providing a highly thromboresistant surface to flowing blood that inhibits platelet adhesion and clotting (Cines, et al.). However, under inflammatory conditions, the nonthrombotic surface of EC can be transformed to a prothrombotic surface following exposure to cytokines (May, et al.; Diquelou et al. Thromb Haemost 1995, 74: 778-83), resulting in procoagulant activity and a predisposition to thrombosis (May, et al.; Dardik, et al. Br J Haemarol. 2000, 109:512-8; Andre, et al. Blood 2000, 96:3322-8). Indeed, the adhesion, accumulation and recruitment of non-stimulated platelets on cytokine-stimulated EC have been reported, with studies implicating the Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1; Rosenblum, et al. Stroke 1995, 27:709-11); beta 1 integrin (Bombeli et al. J Exp. Med 1998, 187:329-39), von Willebrand factor (Dardik, et al.; Andre, et al.), and tissue factor (Verheul, et al. Blood 2000, 96:4216-21) in these processes. Thus, under inflammatory conditions, cytokines induce alterations in EC which result in the adhesion of non-stimulated platelets. [0004] Recently, a novel adhesion protein of the immunoglobulin (Ig) superfamily has been described with properties indicating a potential triggering role in the pathogenesis of inflammatory thrombosis, atherosclerosis and other disorders involving thrombosis formation. This protein was identified first on the surface of human platelets and called the F11 receptor (F11R; Komecki, et al. J Biol Chem 1990, 265: 10042-8; Naik, et al. Biochem J 1995, 311: 155-62), and then on the surface of murine endothelial and epithelial cells and called JAM (Martin-Padura, et al. J. Cell Biol. 1998, 142:117-27.). [0005] The human platelet F11 receptor (F11R) is a surface glycoprotein duplex (32 and 35 kD at core protein: 29 kDa) member of the immunoglobulin superfamily. The F11R was first discovered as the target of a potent stimulatory monoclonal antibody, M.Ab.F11, that induces platelet secretion followed by aggregation (Komecki, et al.; Naik, et al.; Komecki, et al. J Lab Clin. Med. 1988, 111:618-26; Wang et al. Biochem. J 1995, 311: 401-6; Kornecki, et al. In: Leukocyte Typing V. Schlossman, et al. (eds.) Oxford University Press 1195: 1241-3; Sobocka, et al. Blood 1997, 90: 10, Supplement 1, Part 2, Nov. 15, 2996a.; Sobocka, Ph.D. Thesis, 1998: SUNY Downstate, Brooklyn, N.Y., Presented Jun. 10, 1998; published Sep. 15, 1998; Sobocka, et al. Blood 2000, 95:2600-9; Babinska, et al. Thromb Haemost 2002, 87: 712-21). Signal transduction mechanisms for platelet secretion and aggregation induced by M.Ab.F11 following its initial binding to F11R include: crosslinking of the F11R to the Fc.gamma.RII (Naik, et al.), activation and translocation of specific PKC isozymes (Wang, et al.), phosphorylation of the F11R through activation of PKC (Naik, et al.; Wang, et al.), phosphorylation of the F11R following induction of platelet aggregation by the physiological agonists thrombin and collagen and by M.Ab.F11 itself (Sobocka, et al. 1997; Sobocka; Sobocka, et al. 2000; Babinska, et al.), and phosphorylation of myosin light chain and pleckstrin, leading to shape change and granular secretion respectively (Kornecki, et al. 1990). Following secretion, this signal transduction pathway culminates in the activation of latent fibrinogen receptors and platelet aggregation (Kornecki, et al. 1990). Partial amino acid sequences representing 30% of the length of purified F11R have been reported Kornecki in 1995 (Naik, et al.). Cloning of the full-length cDNA for the platelet F11R has revealed that it is a cell adhesion molecule (CAM), a member of the immunoglobin superfamily (Sobocka, et al. 1997; Sobocka; Sobocka, et al. 2000). As a CAM, the F11R participates in mechanisms underlying adhesion of platelets, endothelial cells, and epithelial cells (Martin-Padura, et al.; Sobocka, et al. 2000). [0006] The conclusion that, in addition to its role as a receptor that triggers signal transduction leading to secretion, the F11R also serves as a CAM involved in platelet adhesion was supported by the high degree of sequence similarity found between the human platelet F11R and an adhesion protein called Junctional Adhesion Molecule (JAM), a protein found in murine endothelial cells (Martin-Padura, et al. 1998). Comparison of the murine JAM sequence to the previously-reported sequences of the human platelet F11R (Naik, et al.) revealed over 70% homology of JAM to the N-terminus (23 amino acids) of F11R and to two enzyme-digested products of F11R. In addition, both the human platelet F11R core protein and the murine JAM protein were found to contain a single transmembrane domain and two pairs of cysteine residues in their extracellular domains that allow formation of intermolecular disulfide bridges forming characteristic Ig-like folds. It is now well established that the protein referred to as JAM (Martin-Padura, et al, 1998; Ozaki, et al. J. Immunol 1999, 163: 553-7; Williams, et al. Mol. Immunol. 1999, 36: 1175-88; Liu, et al. J. Cell Science 2000, 113: 2363-74; Gupta, et al. IUBMB Life 2000, 50: 51-6; Naik, et al. J. Cell Science 2001, 114: 539-47), is the murine ortholog of the human F11R (Kornecki, et al. 1990; Naik, et al 1995; Kornecki, et al. 1988; Wang, et al.; Kornecki, et al 1995; Sobocka, et al. 1997; Sobocka; Sobocka, et al. 2000; Babinska, et al.). JAM was localized at intercellular junctions of mouse endothelial and epithelial cells (Martin-Padura, et al.). Similarly, the platelet antibody M.Ab.F11 was found to recognize F11R molecules present at intercellular junctions of cultured human umbilical vein endothelial cells (Sobocka, et al. XVIII ISTH Congress, July, 2001, Paris, France, Abs# P1902; Babinska et al., manuscript submitted, 2005). A recent study conducted by the inventors (Babinska, et al.2002) has determined that two domains of F11 R are critical for the induction of platelet aggregation by M.Ab.F11 and the adhesion of platelets to M.Ab.F11. Heretofore, the role of F11R in physiological and pathophysiological processes involving the adhesion of platelets to cytokine-inflamed endothelial cells has remained unknown. The inventors have now determined that the N-terminus of F11R and the first Ig fold of F11R contain protein sequences which are critical for the adhesion of platelets to endothelial cells, and that the recombinant soluble F11R protein and F11R-peptides block approximately 60% of the force of adhesion of platelets to cytokine-treated EC, demonstrating the involvement of the F11 R protein in platelet-endothelial cell interactions, which under pathological conditions, result in thrombosis, atherosclerosis and other disorders involving thrombosis formation. SUMMARY OF THE INVENTION [0007] The present invention provides the full length cDNA sequence of the F11 receptor (F11R) (SEQ ID NO: 6) and the encoded F11R amino acid sequence (SEQ ID NO: 7). The present invention also provides F11R-antagonists including peptide antagonists and peptidomimetic drugs that inhibit the biological action of the F11R protein. [0008] The present invention is directed to methods and compositions for treating F11R-mediated disorders such as thrombosis, atherosclerosis, plaque formation, heart attacks, inflammatory diseases of the nervous system, stroke and all other clinical disorders involving thrombus formation, as detailed above. The invention is also directed to methods for the treatment and prevention of excessive bleeding under physiological procedures, including the prevention of excessive bleeding following wound injury. The present invention provides specific compositions containing at least one F11R-antagonist peptide which inhibits, suppresses or causes the cessation of at least one F11R-mediated biological activity in a mammal, and preferably humans. Another embodiment of the present invention is the preparation of peptidomimetic drugs that have a structure that mimics the active site of F11R and thus inhibit its biological action. An example of the relationship of the structure of such a drug to the structure of the F11R protein is the relationship between the structure of morphine and the protein beta-endorphin. [0009] Nucleic acid molecules coding for any of the above F11R-antagonist proteins, fragments and peptides of the present invention, expression vectors which include any of such nucleic acid molecules, as well as related host cells containing such nucleotide sequences or vectors, are also contemplated by the present invention. [0010] Still another embodiment of the present invention is directed to antibodies raised against the F11R-antagonist proteins, fragments, peptidomimetics and peptides of the present invention. [0011] Preferably, the antibodies of the present invention are raised against those F11R sequences and F11R-antagonist peptides whose sequences coincide with the corresponding sequences of a mammalian F11R or Junctional Adhesion Molecule (JAM) proteins. The antibodies of the present invention can recognize, antagonize or neutralize the activity of F11R. Both polyclonal antibodies and monoclonal antibodies of various chimeric combinations are contemplated by the present invention. Examples of such antibodies include M.Ab.F11. [0012] These and other embodiments of the invention will be readily apparent to those of ordinary skill in view of the disclosure herein. BRIEF DESCRIPTION OF THE FIGURES [0013] FIG. 1 shows the 3-D structure of the external domain of the mature human platelet F11R. The two Ig-like folds of the human recombinant F11R protein (F11R) are shown as a backbone structure based on the template of the mouse JAM which shares about 70% homology to that of human F11R. [0014] FIG. 2 shows that potentiation of ADP-induced platelet aggregation by M.Ab.F11 is blocked by F11R peptides (SEQ ID NO: 1 and SEQ ID NO: 4). FIG. 2A demonstrates strong potentiation of aggregation using subthreshold levels of M.Ab.F11 (0.3 .mu.g/ml) and ADP (0.5 .mu.M). No aggregation with ADP or M.Ab.F11 alone, when used at low, subthreshold concentrations. FIG. 2B demonstrates that a subthreshold concentration of M.Ab.F11 (0.3 .mu.g/ml) does not induce platelet aggregation. However, ADP (0.5 .mu.M) plus M.Ab.F11 causes a strong aggregation. FIG. 2C demonstrates inhibition of the potentiation of aggregation by F11R-peptide (SEQ ID NO: 1). SEQ ID NO: 1 (50 .mu.M) preincubated with platelets for about 30 sec prior to the addition of M.Ab.F11 (0.3 .mu.g/ml) followed by the addition of ADP (0.5 .mu.M) inhibited aggregation. Control: aggregation in the absence of SEQ ID NO: 1. FIG. 2D demonstrates inhibition of the potentiation of aggregation by F11R-peptide (SEQ ID NO: 4). SEQ ID NO: 4 preincubated with platelets for about 42 sec. prior to the addition of M.Ab.F11 (0.3 .mu.g/ml) followed by addition of ADP (0.5 .mu.M) inhibited aggregation. Control: aggregation in the absence of SEQ ID NO: 4. FIG. 2E shows no inhibition in the presence of peptide 2, 3 or 5 (SEQ ID NOS: 2, 3, or 5, respectively). [0015] FIG. 3 shows inhibition of M.Ab.F11-induced platelet aggregation by F11R peptides (FIG. 3A); Inhibition of M.Ab.F11 (2.45 .mu.g/ml)-induced platelet aggregation by 50 .mu.M F11R peptide (SEQ ID NO: 1) and by 50 .mu.M F11R peptide (SEQ ID NO: 4) FIG. 3B Control: FIG. 3C shows lack of inhibition of M.Ab.F11 (2.45 .mu.g/ml)-induced platelet aggregation by F11R (SEQ ID NO: 2) (500 .mu.M). [0016] FIGS. 4A-4D show that potentiation of collagen-inducted platelet aggregation by M.Ab.F11 is inhibited by F11R peptides (SEQ ID NOS: 1 and 4). [0017] FIG. 5 shows inhibition of adhesion by F11R peptides (SEQ ID NOS: 1 and 4). [0018] FIG. 6 provides the F11R cDNA sequence (full length) (SEQ ID NO: 6). [0019] FIG. 7 provides the F11R amino acid sequence (SEQ ID NO: 7). Continue reading about F11 receptor (f11r) antagonists as therapeutic agents... Full patent description for F11 receptor (f11r) antagonists as therapeutic agents Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this F11 receptor (f11r) antagonists as therapeutic agents patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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