| Therapeutic and diagnostic methods for ulcerative colitis and associated disorders -> Monitor Keywords |
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Therapeutic and diagnostic methods for ulcerative colitis and associated disordersRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai, Cyclopeptides, 25 Or More Peptide Repeating Units In Known Peptide Chain StructureTherapeutic and diagnostic methods for ulcerative colitis and associated disorders description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070032426, Therapeutic and diagnostic methods for ulcerative colitis and associated disorders. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application is a continuation of U.S. Ser. No. 10/359,401 filed Feb. 5, 2003, which is a continuation of U.S. Ser. No. 09/779,689 filed Feb. 8, 2001, and now issued as U.S. Pat. No. 6,800,446, which claims priority to U.S. Provisional Ser. No. 60/181,356, filed Feb. 8, 2000, which are incorporated herein by reference. FIELD OF THE INVENTION [0003] This invention relates to the field of therapy and diagnostic methods for ulcerative colitis. Specifically, the method comprises administering a compound or recombinant protein that inhibits interaction between CEP and human tropomyosin. Also included in the invention are methods to screen for drugs useful in treating ulcerative colitis. BACKGROUND OF THE INVENTION [0004] Various scientific and scholarly articles are referenced throughout the specification. These articles are incorporated by reference herein to describe the state of the art to which this invention pertains. [0005] The Ca.sup.2+ dependence of vertebrate skeletal muscle contraction is due entirely to a set of specialized accessory proteins closely associated with actin filaments. If myosin is mixed with pure actin filaments in a test tube, myosin ATPase is activated whether or not Ca.sup.2+ is present; in a normal myofibril, on the other hand, where the actin filaments are associated with accessory proteins, the activation of the myosin ATPase depends on Ca.sup.2+. [0006] One of these accessory proteins is a rigid rod-shaped molecule, called tropomyosin because of similarities to myosin in its x-ray diffraction pattern. Like the myosin tail, tropomyosin is a dimer of two identical. alpha.-helical chains which wind around each other in a coiled coil. By binding along the length of an actin filament, tropomyosin stabilizes and stiffens the filament. [0007] Tropomyosins are present in all eukaryotic cells. Different isoforms of tropomyosin, generated through alternative splicing, are expressed in a tissue-specific manner (Less-Miller, J P, et al., Bioassays 1991; 13: 429-37). In human fibroblast tissue, at least eight isoforms of TMs have been identified. These isoforms range in molecular weights from 30-40 kDa (Lin J J-C, et al., Int Rev Cytol 1997; 170:1-38). Classically, tropomyosins are known to remain intracellular because they lack the signal sequence required for membrane insertion and translocation (Less-Miller, supra). [0008] Human tropomyosin (hTM) is a cytoskeletal microfilament protein. A significant number of ulcerative colitis patients show a preferential immune response to hTMs, in particular, the hTM5 isoform. Thus, hTM is a candidate autoantigen in ulcerative colitis. Using lamina propria lymphocytes from mucosa of patients with ulcerative colitis and ulcerative colitis sera, an autoantibody response to hTM isoforms has been demonstrated in several independent studies, including that of Das, K M, et al. J Immunol. 1993; 150:2487-93. Such an anti-hTM autoantibody response, however, was not seen in patients with Crohn's disease. Recently, these findings were extended to an animal model of colitis using TCR.sup..alpha.-/- mice (Mizoguchi, A., et al. J. Exp Med. 1996; 183: 847-56). Severity of colitis in these mice is directly correlated with the increased titer of anti-TM autoantibodies and the increased number of appendicular B cells producing anti-TM autoantibodies (Mizoguchi, A., et al. J. Exp Med 1996; 184:707-15). [0009] In colon epithelium, the most predominantly expressed hTM isoform is hTM5 (Geng X, et al., Gastroenterology 1998; 1 14:912-22). It is presently unknown whether hTM5 is accessible to anti-TM autoantibodies, particularly when the target protein is expected to be exclusively intracellular. The possibility of externalization of hTM5 in colon epithelium and likelihood of the passive transport of hTM5 with a secretory protein has been considered. One likely candidate for this chaperone function is a colon epithelial-specific protein recognized by the 7E.sub.12H.sub.12 monoclonal antibody. [0010] The monoclonal antibody 7E.sub.12H.sub.12 was raised using highly enriched colonic tropomyosin (earlier named as 40 kDa protein or p40) (Das K M, et al., J. Immunol 1987; 139:77-84). However, 7E.sub.12H.sub.12 does not react with any of the known hTM isoforms in ELISA or immunotransblot analysis, either from muscle as well as from non-muscle epithelial cells (Das K M, et al., Gastroenterology 1997; 112:A955). However, the 7E.sub.12H.sub.12 monoclonal antibody recognizes a cell membrane associated protein present exclusively in the colon epithelium (Das K M, et al. (1987) supra; Das K M, et al. (1997) supra). By immunotransblot analyses, CEP has been identified as a high molecular weight (>200 kDa) protein present in colon epithelial cells but not in small intestinal enterocytes. Among the colon cancer cell lines, LS-180, and DLD-1 cells express the 7E.sub.12H.sub.12-reactive protein but HT-29 cells do not (Hassan T., et al., Clin Exp Immunol. 1995; 100:457-62). SUMMARY OF THE INVENTION [0011] In accordance with the present invention, it has been found that hTM5 is externalized in colon epithelium but not in small intestinal epithelium, despite the lack of a signal peptide. Furthermore, hTM5 is specifically associated with the colon epithelial-specific protein (CEP), and both are found to be secreted by LS-180 colon cancer cells. [0012] The first aspect of the invention-is due to the new appreciation that hTM is externalized in the colon epithelium and thus can stimulate the immune system and provide its antigenic role. The physical interaction of hTM with CEP is also now appreciated as important for the release of hTM outside the cell. Since an autoantibody response to hTM is associated with ulcerative colitis, the condition can be treated by decreasing the externalization of hTM in the colon. The first aspect of the invention is therefore a prophylactic and therapeutic method for treating or preventing ulcerative colitis and other diseases associated with an autoantigen response to hTM in patients in need of such a treatment. In preferred embodiments, the hTM isoform is hTM5. In a preferred embodiment, this treatment method comprises administering a compound to target cells. The compound inhibits the externalization of hTM and/or interaction between CEP and hTM within target cells. In preferred embodiments, the target cells are colon cells. In a more preferred embodiment, the compound inhibits the interaction between CEP and hTM by physically binding either within the cell. In a particularly preferred embodiment, the compound is a recombinant protein that has a functional hTM binding domain from CEP or CEP-like proteins and competes for hTM binding it vivo. In another preferred embodiment, the compound decreases or causes a decrease in the expression of the CEP protein in target cells. In another preferred embodiment, the compound reduces the release of hTM from colon cells. The compound may also prevent secretion of the CEP-hTM complex from the target cells. In a more preferred embodiment, the compound affects the organization of the cytoskeleton and/or inhibits active secretion. In a most preferred embodiment, the compound is phorbol-12-myristate-13-acetate, monensin or methylamine. [0013] Another aspect of the invention is a prophylactic or therapeutic method to treat ulcerative colitis and other diseases associated with an autoantigen response to hTM that uses the specific binding of CEP to hTM to decrease or remove the autoantigenic nature of hTM. One embodiment of the method entails administering a recombinant protein that comprises a functional hTM binding site from CEP operably linked to a non-antigenic protein. Another embodiment of the method entails tolerization by repeated oral feeding of the hTM and/or CEP. [0014] Another aspect of the invention is method to identify drugs that are useful for treating ulcerative colitis and other diseases associated with an autoantigen response to hTM which targets the disassociation of the CEP-hTM complex. In a preferred embodiment, intracellular association of CEP and hTM is determined by hTM secretion from human colon cells, with a decrease in hTM secretion indicative of a drug with therapeutic properties. In a more preferred embodiment, the LS-180 cells are used. [0015] Another aspect of the invention is a diagnosis method for detecting diseases associated with an autoantigen response to hTM which entails detecting CEP-hTM complexes in affected tissue. Presence of CEP-hTM complexes are indicative of disease. In one embodiment, the complexes, or a part of them, are detected in the extracellular space of the affected tissue or by sensitized lymphocytes form colonic mucosa. In another embodiment, the CEP-hTM complexes are detected in intracellular space of the affected tissue. In a preferred embodiment, the tissue is the colon epithelium. [0016] Other features and advantages of the present invention will be better understood by reference to the drawings, detailed description and example that follow. BRIEF DESCRIPTION OF THE DRAWINGS [0017] FIG. 1: Cell surface expression of hTM5 on colon epithelial cells: [0018] Colon epithelial cells were isolated from freshly obtained surgical specimens (normal segments) and analyzed by flow cytometry using anti hTM mAbs, (A) CG3 (anti-hTM5), C) LC-24 (anti-HTM4), and D) CG1 (anti-hTM1) and with anti-CEP mAb, 7E.sub.12H.sub.12 (B). The reactivities of respective isotype control mAbs are shown by a dotted line. Panel E shows a histogram of percent positive cells in the flow cytometric analysis of epithelial cells from six different colon specimens using CG3 mAb. The isotype control mAb, MOPC-lgM was used for each sample to determine the background staining. [0019] FIG. 2: Flow cytometric analysis of ileal epithelial cells: [0020] Epithelial cells isolated from ileum (A and B) do not show reactivity with either CG3 (A) or with 7E.sub.12H.sub.12 (6) mAbs. The background staining with isotype control mAbs is shown by the dotted lines. The data are representative of two ileal and two jejunal specimens. Panel C shows a Western blot analysis using ceillysates (10 ug proteins in each lane) of three different colon and one ileal specimens with CG3 mAb. hTM5 reactivity is clearly evident at 31 kDa. Panel D shows Western blot analysis using 7E.sub.12H.sub.12 mAb against total cell Iysates (10 ug proteins in each lane) from colon epithelial cells (lane-1), jejunal epithelial cells (lane-2), LS-180 colon cancer cells (lane-3), and the cell free culture medium from the dish where LS-180 cells were grown (lane-4) after 8% SDS-PAGE. CEP is present in freshly prepared colon epithelial cells and LS-180 cells, but not in jejunal epithelial cells. CEP is also seen in the LS-180 culture medium. CEP is the only protein at >200 kDa reactive to 7E.sub.12H.sub.12 mAb. [0021] FIG. 3: Flow cytometric analysis of colon cancer cell lines with anti-hTM5 mAbs: Continue reading about Therapeutic and diagnostic methods for ulcerative colitis and associated disorders... Full patent description for Therapeutic and diagnostic methods for ulcerative colitis and associated disorders Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Therapeutic and diagnostic methods for ulcerative colitis and associated disorders 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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