Cyclic prosaposin peptides and uses thereof

Abstract

To provide cyclic prosaposin peptides and compositions and uses thereof.SOLUTION: Exemplary uses include use in the treatment of cancer or in the treatment of inflammatory diseases or disorders.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application No. 61 / 970,853, filed March 26, 2014, which is incorporated herein by reference in its entirety.

[0002] government support This invention was made with Government support under R01CA135417 awarded by the National Institutes of Health. The Government has certain rights in the invention. [Background technology]

[0003] 2. Background of the Invention Cancer is a major public health problem, with an estimated 7.6 million cancer deaths occurring in 2008. Many cancers remain incurable, or treatments are suboptimal, only partially effective, or have undesirable side effects such as toxicity. Inflammatory diseases and disorders are also major public health problems, with similar treatment challenges as cancer. Summary of the Invention

[0004] Aspects of the present disclosure are based in part on the discovery that cyclic prosaposin peptides, such as those having the sequence DWLPK (SEQ ID NO: 1), are more stable and more effective in treating animal models of cancer than linear prosaposin peptides. Surprisingly, it was found that the cyclic DWLPK (SEQ ID NO: 1) peptide is more effective in stimulating thrombospondin-1 (Tsp-1) in vitro compared to a cyclic peptide having a glycine substitution at position 3 (i.e., DWGPK, SEQ ID NO: 2). This data is unexpected because the opposite results were obtained with linear prosaposin peptides, i.e., linear DWGPK (SEQ ID NO: 2) was found to have better activity than linear DWLPK (SEQ ID NO: 1). Furthermore, it was also found that the cyclic DWLPK (SEQ ID NO: 1) peptide did not cause toxicity to the liver or spleen of mice treated with the peptide. Another aspect of the present disclosure is based, in part, on the discovery that cyclic DWLPK (SEQ ID NO:1) is effective in treating a mouse model of Crohn's disease.

[0005] Accordingly, aspects of the present disclosure relate to cyclic prosaposin peptides, such as DWLPK (SEQ ID NO:1), and to compositions and methods utilizing such peptides. Some aspects of the present disclosure relate to a method for treating a subject with cancer, the method comprises administering to the subject with cancer an effective amount of a cyclic peptide for treating the cancer, wherein the amino acid sequence of the cyclic peptide is DWLPK (SEQ ID NO: 1).In some embodiments, the cancer is ovarian cancer or melanoma. Another aspect relates to a composition for use in treating cancer, the composition comprising a cyclic peptide, wherein the amino acid sequence of the cyclic peptide is DWLPK (SEQ ID NO: 1). In some embodiments, the cancer is ovarian cancer or melanoma. Yet another aspect of the present disclosure relates to a method for treating a subject with inflammatory disease or disorder, comprising administering to the subject with inflammatory disease or disorder an effective amount of a cyclic peptide for treating inflammatory disease or disorder, wherein the amino acid sequence of the cyclic peptide is DWLPK (SEQ ID NO: 1).In some embodiments, the inflammatory disease or disorder is rheumatoid arthritis, age-related macular degeneration (AMD), Crohn's disease, psoriasis or atherosclerosis.In some embodiments, the inflammatory disease or disorder is Crohn's disease.

[0006] Another aspect of the present disclosure relates to a composition for use in treating inflammatory disease or disorder, the composition comprising a cyclic peptide, wherein the amino acid sequence of the cyclic peptide is DWLPK (SEQ ID NO: 1).In some embodiments, the inflammatory disease or disorder is rheumatoid arthritis, age-related macular degeneration (AMD), Crohn's disease, psoriasis, or atherosclerosis.In some embodiments, the inflammatory disease or disorder is Crohn's disease. Another aspect of the present disclosure relates to a method for stimulating expression of thrombospondin-1 (Tsp-1), the method comprising administering to a subject in need thereof an effective amount of a cyclic peptide for stimulating expression of Tsp-1, wherein the amino acid sequence of the cyclic peptide is DWLPK (SEQ ID NO:1).

[0007] Aspects of the present disclosure relate to cyclic Psap peptides. In some embodiments, the cyclic Psap peptide comprises the amino acid sequence DWLPK (SEQ ID NO: 1), dWLPK (SEQ ID NO: 3, lower case d indicates D-amino acid), DWGPK (SEQ ID NO: 2), or dWGPK (SEQ ID NO: 4, lower case d indicates D-amino acid). In some embodiments, the amino acid sequence of the cyclic Psap peptide is DWLPK (SEQ ID NO: 1), dWLPK (SEQ ID NO: 3, lower case d indicates D-amino acid), DWGPK (SEQ ID NO: 2), or dWGPK (SEQ ID NO: 4, lower case d indicates D-amino acid). In some embodiments, the cyclic Psap peptide comprises the amino acid sequence DWLPK (SEQ ID NO: 1). In some embodiments, the amino acid sequence of the cyclic Psap peptide is DWLPK (SEQ ID NO: 1). Another aspect of the present disclosure relates to a composition comprising a cyclic Psap peptide as described herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a cyclic Psap peptide as described herein and a pharma- ceutically acceptable carrier. The details of one or more embodiments of the disclosure are set forth in the following description. Other features and advantages of the disclosure will be apparent from the following drawings and detailed description of certain embodiments, and from the appended claims. [Brief description of the drawings]

[0008] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Figure 1] FIG. 1 is an exemplary schematic diagram of cyclic DWLPK (SEQ ID NO:1). [Diagram 2] FIG. 2 is a photograph of a Western blot showing the level of thrombospondin-1 (Tsp-1) activation in cells in response to different cyclic or linear peptides (SEQ ID NOs: 1, 3, 2, and 4, from top to bottom, respectively). [Diagram 3]FIG. 3 is a graph depicting the ability of cyclic DWLPK (SEQ ID NO: 1) or linear dWlP (SEQ ID NO: 5) to activate Tsp-1 following incubation in human plasma. [Figure 4] FIG. 4 is a graph showing tumor volume in mice treated with cyclic or linear peptides.

[0009] [Diagram 5] FIG. 5 is a graph showing tumor volume in mice treated with cyclic or linear peptides. [Figure 6] FIG. 6 is a graph showing the total luminous flux of luciferase-expressing tumor cells injected into mice after treatment with cyclic peptides or controls. [Figure 7] FIG. 7 is a series of photographs showing that Tsp-1 is expressed in F4 / 80-expressing macrophages. [Figure 8] FIG. 8 is a series of photographs showing the histology of liver and spleen samples in mice injected with control or cyclic DWLPK (SEQ ID NO:1). [Figure 9] FIG. 9 is a graph showing MPO enzyme activity (an indicator of neutrophil infiltration into the colonic mucosa) in a mouse model of dextran sulfate sodium (DSS)-induced Crohn's disease treated with control, untreated, or treated with cyclic DWLPK (SEQ ID NO: 1).

[0010] [Figure 10] FIG. 10 is a graph showing percent body weight in a mouse model of Crohn's disease that was control-treated, untreated, or treated with cyclic DWLPK (SEQ ID NO:1). [Figure 11] FIG. 11 is a series of photographs showing intestinal histology and macrophage localization in a mouse model of Crohn's disease that was control-treated, untreated, or treated with cyclic DWLPK (SEQ ID NO:1). [Figure 12]12A-12E are diagrams showing stimulation of Tsp-1 and its effect on ovarian cancer cell proliferation and survival. Figure 12A is a Western blot of Tsp-1 and β-actin in WI-38 lung fibroblasts untreated (-) or treated with native DWLP (SEQ ID NO:6), L-amino acids, prosaposin peptide (WT), dWlP (SEQ ID NO:5, lowercase d and 1 indicate D-amino acids) prosaposin peptide (d1,3), or DwLp (SEQ ID NO:7, lowercase w and p indicate D-amino acids) prosaposin peptide (d2,4); Figure 12B is a Western blot of Tsp-1 and β-actin in WI-38 lung fibroblasts treated with metastatic prostate cancer cell conditioned medium alone (-) or treated with DWLP (SEQ ID NO:6) prosaposin peptide (WT), or dWlP (SEQ ID NO:5, lowercase d and 1 indicate D-amino acids) prosaposin peptide (d1,3 FIG. 12C is a Western blot of Tsp-1 and β-actin in mouse lung tissue taken from mice treated for 3 days at doses of 10 or 30 mg / kg / day in combination with 12C-peptide; FIG. 12D is a plot of cell number as measured by Wst-1 assay of patient-derived ovarian cancer cell lines treated with 1 μM recombinant human Tsp-1 (rhTsp-1) for 24, 48, and 72 hours; and FIG. 12E is a FACS analysis of Annexin V and PI of patient-derived ovarian cancer cell lines treated with 1 μM recombinant human Tsp-1 (rhTsp-1) for 48 hours.

[0011] [Figure 13]Figures 13A-13E show the effect of d-amino acid prosaposin peptides on PDX models of metastatic ovarian cancer. Figure 13A is a plot of relative luciferase intensity of metastatic PDX ovarian tumors treated with saline (control), cisplatin 4 mg / kg / QOD (cisplatin), and dWlP (SEQ ID NO: 5) prosaposin peptide 4 mg / kg QD; Figure 13B shows luciferase images of two mice treated with control and two mice treated with dWlP (SEQ ID NO: 5) prosaposin peptide at days 17 (day 0 of treatment) and 48 (day 31 of treatment), each image showing the luciferase scale to the right of the image. 13C is a photograph of the liver of a mouse bearing a metastatic PDX ovarian tumor treated with saline (control) or dWlP (SEQ ID NO: 5) prosaposin peptide (peptide); FIG. 13D is a diagram showing H&E staining of the liver of a mouse bearing a metastatic PDX ovarian tumor treated with saline (control) or dWlP (SEQ ID NO: 5) prosaposin peptide (peptide). The right panel is 5x magnification and the left panel is 20x magnification; and FIG. 13E is a diagram showing FACS analysis of GR1+ / Cd11b+ cells in the ascites of mouse control and dWlP (SEQ ID NO: 5) prosaposin peptide-treated metastatic PDX ovarian tumor-bearing mice after 48 days.

[0012] [Figure 14]14A-14F show the effect of cyclic prosaposin peptides on Tsp-1 expression and PDX models of metastatic ovarian cancer. FIG. 14A is a Western blot of Tsp-1 and β-actin in WI-38 lung fibroblasts that were untreated (-), treated with dWlP (SEQ ID NO:5) prosaposin peptide (d1,3, "L"), or treated with cyclic DWLPK (SEQ ID NO:1) prosaposin peptide ("C"); FIG. 14B is an ELISA of Tsp-1 expression in WI-38 lung fibroblasts that were untreated (-), treated with dWlP (SEQ ID NO:5) prosaposin peptide (d1,3), or treated with cyclic DWLPK (SEQ ID NO:1) prosaposin peptide after incubation in human plasma at 37° C. for up to 24 hours; FIG. 14C is a Western blot of Tsp-1 and β-actin in WI-38 lung fibroblasts that were untreated (-), treated with dWlP (SEQ ID NO:5) prosaposin peptide (d1,3), or treated with cyclic DWLPK (SEQ ID NO:1) prosaposin peptide (10 mg / kg QD); FIG. 14D is a plot of the average metastatic lesions in mice treated with saline (control) and with cyclic DWLPK (SEQ ID NO: 1) prosaposin peptide (peptide); FIG. 14E is an immunofluorescence staining of Gr1 and Tsp-1 expression in metastatic lesions in mice treated with saline (control) and with cyclic DWLPK (SEQ ID NO: 1) prosaposin peptide (peptide); and FIG. 14F is an immunohistochemistry of Tsp-1 expression (leftmost panel) and immunofluorescence staining of TUNEL and DAPI of metastatic lesions in control and cyclic DWLPK (SEQ ID NO: 1) prosaposin peptide-treated mice (peptide).

[0013] [Figure 15]Figures 15A-N show the expression of CD36 and prosaposin in TMAs of human ovarian cancer patients. Figures 15A-15C show the expression of CD36 in normal human ovaries (magnification: A=5x, B=10x, C=20x); Figures 15D-15F show the expression of CD36 in primary human ovarian tumors (magnification: A=5x, B=10x, C=20x); Figures 15G-15I show the expression of CD36 in human ovarian cancer metastases (magnification: A=5x, B=10x, C=20x); Figures 15J-15L show the expression of CD36 in human ovarian cancer lymph nodes (magnification: A=5x, B=10x, C=20x). Shows CD36 expression in metastases (magnification: A=5x, B=10x, C=20x); Figure 15M is a plot of CD36 staining index for normal human ovaries, primary human ovarian tumors, human ovarian cancer metastases, and human ovarian cancer lymph node metastases; Figure 15N is a plot of prosaposin (Psap) staining index for normal human ovaries, primary human ovarian tumors, human ovarian cancer metastases, and human ovarian cancer lymph node metastases.

[0014] [Figure 16] FIG. 16 shows that the psap peptide had no effect on body weight. [Figure 17] FIG. 17 shows pancreatic cancer cells expressing the Tsp-1 receptor. [Figure 18] FIG. 18 shows that the cyclic prosaposin peptide DWLPK (SEQ ID NO:1) has approximately two-fold greater activity than the linear peptide. [Figure 19] FIG. 19 shows that the cyclic prosaposin peptide DWLPK (SEQ ID NO: 1) has significant antitumor activity against pancreatic cancer. [Figure 20] FIG. 20 shows that the cyclic prosaposin peptide DWLPK (SEQ ID NO:1) regresses metastatic pancreatic cancer and inhibits metastasis.

[0015] Detailed Description of the Invention Prosaposin peptides (herein referred to as Psap peptides), originally derived from fragments of saposin A, have previously been shown to be effective in treating multiple types of cancer when used in linear form (see, e.g., PCT Publications WO2009002931, WO / 2011 / 084685 and WO / 2013 / 096868, and U.S. Patent Applications 12 / 640,788 and 13 / 516,511, all of which are incorporated by reference in their entireties herein). As described herein, cyclic Psap peptides, particularly cyclic DWLPK (SEQ ID NO: 1) peptides, are unexpectedly more stable and effective than linear Psap peptides. Moreover, cyclic DWLPK (SEQ ID NO: 1) was found to have no toxic effects on the liver or spleen. Moreover, cyclic DWLPK (SEQ ID NO: 1) had better activity than other cyclic versions of amino acid substituted peptides (e.g., DWGPK, SEQ ID NO: 2). This finding was surprising because linear versions of substituted peptides were found to be more active than linear versions of DWLPK (SEQ ID NO: 1).

[0016] As further described herein, cyclic Psap peptide DWLPK (SEQ ID NO: 1) has been found to be effective in a mouse model of Crohn's disease. Without intending to be bound by theory, it is believed that cyclic Psap peptide activates or enhances the expression of Tsp-1, and the expression of Tsp-1 reduces the inflammation associated with Crohn's disease. Tsp-1 has previously been shown to be secreted in response to inflammation, promote the resolution of the inflammatory process, and promote the phagocytosis of damaged cells (see, for example, Lopez-Dee et al. Thrombospondin-1: Multiple Paths to Inflammation. (2011) Mediators of Inflammation; Vol. 2011; Article ID 296069). Thus, because the therapeutic effect of cyclic Psap peptides was observed in a model of Crohn's disease, a typical inflammatory disease, it is believed that cyclic Psap peptides will also be effective for treating other inflammatory diseases, such as rheumatoid arthritis and psoriasis, as well as other diseases in which inflammation is involved, such as atherosclerosis and age-related macular degeneration (AMD). Accordingly, aspects of the present disclosure relate to cyclic Psap peptides and their use in compositions and methods for treating diseases, such as cancer and inflammatory disorders or diseases, as well as other diseases in which inflammation is involved.

[0017] Cyclic Psap peptides Prosaposin (Psap) is a saposin precursor protein consisting of approximately 524-527 amino acids, including a 16 amino acid signal peptide. The full-length precursor polypeptide undergoes cotranslational glycosylation and modification in the endoplasmic reticulum and Golgi system to generate a 70-72 kDa precursor protein. After transport to the lysosome, cathepsin D mediates its proteolytic processing to generate a 35-53 kDa intermediate molecular form, then a 13 kDa glycoprotein, and finally an 8-11 kDa mature partially glycosylated form of the individual saposin molecule (O'Brien JS, and Kishimoto Y, The FASEB J., 5: 301-8, 1991; Kishimoto Y. et al., J. Lipid Res. 33:1255-67, 1992). Prosaposin is processed into four cleavage products, saposin A, B, C, and D. The amino acid sequences of the Psap preproprotein isoforms A, B, and C, and the cleavage product saposin A are as follows:

[0018] Psap preproprotein isoform A (SEQ ID NO:9)

[0019] Psap preproprotein isoform B (SEQ ID NO:10)

[0020] Psap preproprotein isoform C (SEQ ID NO:11)

[0021] Saposin A SLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCES (SEQ ID NO: 12) Aspects of the present disclosure relate to cyclic Psap peptides, compositions comprising cyclic Psap peptides, and uses thereof. In some embodiments, the cyclic Psap peptide comprises the amino acid sequence DWLPK (SEQ ID NO: 1), dWLPK (SEQ ID NO: 3, lower case d indicates D-amino acid), DWGPK (SEQ ID NO: 2), or dWGPK (SEQ ID NO: 4, lower case d indicates D-amino acid). In some embodiments, the amino acid sequence of the cyclic Psap peptide is DWLPK (SEQ ID NO: 1), dWLPK (SEQ ID NO: 3, lower case d indicates D-amino acid), DWGPK (SEQ ID NO: 2), or dWGPK (SEQ ID NO: 4, lower case d indicates D-amino acid). In some embodiments, the cyclic Psap peptide comprises the amino acid sequence DWLPK (SEQ ID NO: 1). In some embodiments, the amino acid sequence of the cyclic Psap peptide is DWLPK (SEQ ID NO: 1).

[0022] In some embodiments, the cyclic peptide is less than 10, less than 9, less than 8, less than 7, less than 6, or less than 5 amino acids in length. In some embodiments, the cyclic peptide is between 5-10, 5-9, 5-8, 5-7, or 5-6 amino acids in length. In some embodiments, the cyclic peptide is 5 amino acids in length. The peptides described herein can be chemically synthesized and isolated by biochemical methods known in the art, such as solid-phase peptide synthesis using t-BOC (tert-butyloxycarbonyl) or FMOC (9-fluorenylmethyloxycarbonyl) protecting groups, as described in "Peptide synthesis and applications" by John Howl, Methods in molecular biology Vol. 298, Ed. and "Chemistry of Peptide Synthesis" by N. Leo Benoiton, 2005, CRC Press, (ISBN-13: 978-1574444544) and "Chemical Approaches to the Synthesis of Peptides and Proteins" by P. Lloyd-Williams, et al., 1997, CRC-Press, (ISBN-13: 978-0849391422). Solid-phase peptide synthesis, developed by RB Merrifield, 1963, J. Am. Chem. Soc. 85 (14): 2149-2154, was a breakthrough that enabled the chemical synthesis of peptides and small proteins.

[0023] Cyclic Psap peptides can be synthesized using any method known in the art. Examples of synthesis methods include, but are not limited to, recombinant synthesis, solution phase synthesis, solid phase synthesis, or chemical ligation (e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001;Current Protocols in Molecular Biology, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York;Schnolzer, MA, P.; Jones, A.; Alewood, D.; Kent, SBH (2007). "In Situ Neutralization in Boc-chemistry Solid Phase Peptide Synthesis". Int. J. Peptide Res. Therap. 13 (1-2): 31-44;Albericio, F. (2000). Solid-Phase Synthesis: A Practical Guide (1 ed.). Boca Raton: CRC Press. p. 848; and Nilsson BL, Soellner MB, Raines RT (2005). "Chemical Synthesis of Proteins". Annu. Rev. Biophys. Biomol. Struct. 34: 91-118; and U.S. Patent Nos. 4,749,742, 4,794,150, 5,552,471, 5,637,719, 6,001,966, 7,038,103, 7,094,943, 7,176,282, and 7,645,858, which are incorporated by reference in their entireties.Psap peptides and methods for making Psap peptides are also known in the art (see, e.g., PCT Publications WO2009002931, WO / 2011 / 084685 and WO / 2013 / 096868, the entireties of which are incorporated herein by reference). Cyclic peptides are polypeptide chains that are linked together by peptide bonds or other covalent bonds to form a ring structure. Methods for designing and synthesizing cyclic peptides are well known in the art (see, e.g., U.S. Patent Nos. 5,596,078, 5,990,273, 7,589,170 and U.S. Patent Publication No. 20080287649) and are commercially available (e.g., cyclic peptide synthesis services are provided by Selleck Chemicals, Abbiotec, Abgent, AnaSpec Global Peptide Services, LLC., INVITROGEN™ and rPeptide, LLC or GenScript). Cyclic peptides can be cyclized, for example, head-to-tail (C-terminus-N-terminus), head-to-side chain, side chain-to-tail, or side chain-to-side chain (see, for example, White et al. Contemporary strategies for peptide macrocyclization. Nature Chemistry (2011); 3:509-524). Cyclization reagents such as PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate), BOP (benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate), DIPEA (N,N-diisopropylethylamine), DMTMM BF4 (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate) or FDPP (pentafluorophenyl diphenylphosphinate), or combinations thereof, can be used during or after peptide synthesis to induce cyclization. An exemplary method involves the use of a photolabile auxiliary, such as HnB (2-hydroxy-6-nitrobenzyl), in a ring-contraction strategy to cyclize a pentapeptide.After introduction of this auxiliary at the N-terminus of the peptide, the peptide is contacted with BOP and DIPEA, which results in an intramolecular O to N acyl transfer, which in turn results in a cyclic pentapeptide. The photodegradable auxiliary can then be removed.

[0024] In some embodiments, the Psap peptides may be modified, such as by oligomerization or polymerization (e.g., dimers, trimers, multimers, etc.), modification of amino acid residues or the peptide backbone, crosslinking, conjugation, pegylation, glycosylation, acetylation, phosphorylation, fusion to additional heterologous amino acid sequences (e.g., antibodies or antibody Fc domains, serum transferrin or portions thereof, albumin, or transthyretin), or other modifications that substantially alter the stability, solubility, or other properties of the peptide while substantially retaining or improving therapeutic activity. Conjugation may be, for example, to a polymer. Suitable polymers include, for example, polyethylene glycol (PEG), polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polyoxyethylated polyols, heparin, heparin fragments, polysaccharides, cellulose and cellulose derivatives including methylcellulose and carboxymethylcellulose, starch and starch derivatives, polyalkylene glycols and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers, and α,β poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixtures thereof. Conjugation may be via a linker, for example, a peptide or chemical linker. Methods of modifying peptides are well known in the art (see, e.g., U.S. Pat. Nos. 5,180,816, 5,596,078, 5,990,273, 5,766,897, 5,856,456, 6,423,685, 6,884,780, 7,610,156, 7,256,258, 7,589,170, and 7,022,673, and PCT Publication WO 2010 / 014616; the contents of which are incorporated herein by reference).

[0025] In some embodiments, the cyclic Psap peptide is functionally modified to enhance stability. In some embodiments, chemical modifications to the Psap peptide include, but are not limited to, alkyl, alkoxy, hydroxyalkyl, alkoxyalkyl, alkoxycarbonyl, alkenyl, alkynyl, cycloalkyl, amino, alkylamino, aminoalkyl, dialkylamino, aminodialkyl, halogen, heteroatom, carbocycle, carbocyclyl, carbocyclo, carbocyclic, aryl, aralkyl, aralkoxy, aryloxyalkyl, heterocycle, heterocyclyl, heterocyclic, heteroaryl, and / or aliphatic groups. In some embodiments, the cyclic peptide is fused / conjugated to at least one therapeutic molecule.

[0026] In some embodiments, amino acid substitution variants of cyclic Psap peptides resulting from the substitution of one or more D-amino acids with the analogous L-amino acids are contemplated herein. In some embodiments, there is one D-amino acid substitution. In some embodiments, there are two or more D-amino acid substitutions. In some embodiments, there are three, four, or five D-amino acid substitutions. In some embodiments, the D-amino acid substitutions are evenly spaced, e.g., every other amino acid. In some embodiments, the D-amino acid substitutions are to aspartic acid (D). The L and D convention for amino acid configurations refers not to the optical activity of the amino acid itself, but to the optical activity of the isomer of glyceraldehyde from which the amino acid could theoretically be synthesized (D-glyceraldehyde is dextrorotatory; L-glyceraldehyde is levorotatory). Exemplary D-amino acid substitution variants include cyclic dWLPK (SEQ ID NO:3, lower case d indicates a D-amino acid) and cyclic dWGPK (SEQ ID NO:4, lower case d indicates a D-amino acid).

[0027] The cyclic peptides described herein can be further modified or derivatized. Modified or derivatized polypeptides will typically substantially retain the activity of the base polypeptide (before modification / derivatization). Examples of modifications and derivatives are pegylation, glycosylation, acetylation, amidation, and phosphorylation. Methods of acetylation (e.g., N-terminal acetylation) and amidation (e.g., C-terminal amidation) are well known to those skilled in the art. Methods of modifying, derivatizing, and derivatizing polypeptides are described in published international application WO 2010 / 014616, the contents of which are incorporated herein by reference. In some embodiments, the cyclic peptides described herein can be conjugated or otherwise covalently linked (e.g., using a chemical linker) to other molecules. One such form of linkage is via a non-amide bond (e.g., a disulfide bond). In some embodiments, the cyclic peptide is linked to a polymer that enhances serum half-life. In some embodiments, the cyclic peptide is covalently linked (e.g., via a linker molecule) to an antibody or a domain thereof (e.g., one or more constant domains in an Fc domain) that is suitable for enhancing the half-life of the molecule. In some embodiments, the polypeptide is linked to an Fc domain (e.g., IgG, IgA, IgM, IgD, or IgE).

[0028] In some embodiments, the cyclic peptides described herein are linked to non-amino acid polymers. Polymers such as polyethylene glycol can be used for the purpose of improving serum half-life. Suitable polymers include, for example, polyethylene glycol (PEG), polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polyoxyethylated polyols, heparin, heparin fragments, polysaccharides, cellulose and cellulose derivatives including methylcellulose and carboxymethylcellulose, starch and starch derivatives, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycol and derivatives thereof, polyvinyl ethyl ether, and α,β poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixtures thereof. Such polymers may or may not have their own biological activity. Polymers can be covalently or non-covalently conjugated to the cyclic peptide. Methods of conjugation to increase serum half-life are known in the art and can be found, for example, in U.S. Pat. Nos. 5,180,816, 6,423,685, 6,884,780, and 7,022,673, which are incorporated by reference herein in their entireties.

[0029] In some embodiments, the cyclic peptide is conjugated to a therapeutic molecule, hi some embodiments, the therapeutic molecule is an anti-angiogenic therapeutic molecule, an anti-VEGF agent, a chemotherapeutic agent, or an anti-inflammatory agent. Various versions of the cyclic peptides described herein, including modified, conjugated, or derivatized cyclic peptides encompassed by this disclosure, are expected to retain a significant amount of the biological activity exhibited by the cyclic peptides (e.g., as reported in the Examples section herein). In some embodiments, about 100% activity is retained in a given assay. In some embodiments, about 90%, 80%, 70%, 60%, or 50% activity is retained. One such activity is the ability to stimulate the expression of Tsp-1. Stimulation of expression is a significant, reproducible amount of increased expression resulting from contact of the cyclic peptides described herein with appropriate target cells, compared to the same or sufficiently similar target cells not contacted with the cyclic peptide (control target cells). Methods for determining the expression-inducing activity of Tsp-1 are described herein and are well known to those skilled in the art.

[0030] The amino acid abbreviations are shown below. Alanine, Ala, A Isoleucine, Ile, I Leucine, Leu, L Valin, Val, V Phenylalanine, Phe, F Tryptophan, Trp, W Tyrosine, Tyr, Y Asparagine, Asn, N Cysteine, Cys, C Glutamine, Gln, Q Methionine, Met, M Serine, Ser, S Threonine, Thr, T Aspartic acid, Asp, D Glutamic acid, Glu, E Arginine, Arg, R Histidine, His, H Lysine, Lys, K Glycine, Gly, G Proline, Pro, P

[0031] Other Cyclic Peptide Modifications It should be understood that modified versions of the cyclic peptides described herein are encompassed in the present disclosure.Modifications to the cyclic peptides described herein can be carried out as described in US Published Application 20080090760 and / or US Published Application 20060286636, each of which is incorporated herein by reference in its entirety.The following provides a non-limiting description of various other peptide modifications that are encompassed within the scope of the present disclosure.

[0032] The present disclosure includes chemical derivatives of the cyclic peptides described herein, so long as they substantially retain the activity of the underivatized cyclic peptides. "Chemical derivatives" is a subset of the peptide derivatives described herein and refers to the subject cyclic peptides having one or more residues chemically derivatized by reaction of a functional side group. In addition to derivatization of the side groups, chemical derivatives can have one or more backbone modifications, including, for example, α-amino substitutions such as N-methyl, N-ethyl, N-propyl, and α-carbonyl substitutions such as thioesters, thioamides, guanidino, and the like. Such derivatized molecules include, for example, molecules in which free amino groups are derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups, or formyl groups. Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters, or other types of esters or hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-im-benzyl. Further included as chemical derivatives are peptides that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. Further included as chemical derivatives are peptides that contain one or more non-natural amino acids, including but not limited to those available for peptide synthesis from commercial suppliers (e.g., Bachem Catalog, 2004 pp. 1-276).For example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; ornithine may be substituted for lysine; β-alanine may be substituted for alanine; norleucine may be substituted for leucine; phenylglycine may be substituted for phenylalanine; and L-1,2,3,4-tetrahydronorharman-3-carboxylic acid or H-β-(3-benzothienyl)-Ala-OH may be substituted for tryptophan.

[0033] In certain embodiments, chemical modifications to peptides include, but are not limited to, the inclusion of alkyl, alkoxy, hydroxyalkyl, alkoxyalkyl, alkoxycarbonyl, alkenyl, alkynyl, cycloalkyl, amino, alkylamino, aminoalkyl, dialkylamino, aminodialkyl, halogen, heteroatom, carbocycle, carbocyclyl, carbocyclo, carbocyclic, aryl, aralkyl, aralkoxy, aryloxyalkyl, heterocycle, heterocyclyl, heterocyclic, heteroaryl, and / or aliphatic groups. The terms "alkyl", "alkoxy", "hydroxyalkyl", "alkoxyalkyl", and "alkoxycarbonyl", when used alone or as part of a larger moiety, are intended to include both straight and branched chains containing 1 to 12 carbon atoms. The terms "alkenyl" and "alkynyl", when used alone or as part of a larger moiety, are intended to include both straight and branched chains containing 2 to 12 carbon atoms. The term "cycloalkyl", when used alone or as part of a larger moiety, is intended to include cyclic C3-C12 hydrocarbons that are fully saturated or contain one or more units of unsaturation, but are not aromatic. Lower alkyl refers to alkyl groups containing 1 to 6 carbons.

[0034] The term "amino" refers to the NH2 group. The term "alkylamino" or "aminoalkyl" refers to an amino group in which one of the hydrogen atoms is replaced by an alkyl group. The term "dialkylamino" or "aminodialkyl" refers to an amino group in which a hydrogen atom is replaced by an alkyl group, where the alkyl groups may be the same or different. The term "halogen" means F, Cl, Br, or I. The term "heteroatom" means nitrogen, oxygen, or sulfur having a carbocyclic ring structure, including any oxidized form of nitrogen and sulfur, and the quaternized form of any basic nitrogen. The term "nitrogen" also includes a substitutable nitrogen of a heterocycle. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur, or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR+ (as in N-substituted pyrrolidinyl). As used herein, the term "carbocycle", "carbocyclyl", "carbocyclo", or "carbocyclic" refers to an aliphatic ring system having 3 to 14 members. The terms "carbocycle", "carbocyclyl", "carbocyclo", or "carbocyclic" whether saturated or partially unsaturated also refer to rings that are optionally substituted. The terms "carbocycle", "carbocyclyl", "carbocyclo", or "carbocyclic" also include an aliphatic ring that is fused to one or more aromatic or non-aromatic rings, such as in decahydronaphthyl or tetrahydronaphthyl, where the radical or point of attachment is on the aliphatic ring.

[0035] The term "aryl", when used alone or as part of a larger moiety, such as in "aralkyl", "aralkoxy", or "aryloxyalkyl", refers to an aromatic ring group having 6 to 14 members, such as phenyl, benzyl, phenethyl, 1-naphthyl, 2-naphthyl, 1-anthracyl, and 2-anthracyl. The term "aryl" also refers to an optionally substituted ring. The term "aryl" may be used interchangeably with the term "aryl ring". "Aryl" also includes fused polycyclic aromatic ring systems in which an aromatic ring is fused to one or more rings. Examples include 1-naphthyl, 2-naphthyl, 1-anthracyl, and 2-anthracyl. Further included within the scope of the term "aryl", as used herein, are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phenanthridinyl, or tetrahydronaphthyl, where the radical or point of attachment is on the aromatic ring.

[0036] The terms "heterocycle", "heterocyclyl" or "heterocyclic" as used herein include non-aromatic ring systems having 4 to 14 members, preferably 5 to 10 members, in which one or more, preferably one to four, ring carbons are each replaced by a heteroatom. Examples of heterocycles include 3-1H-benzimidazol-2-one, (1-substituted)-2-oxo-benzimidazol-3-yl, 2-tetrahydro-furanyl, 3-tetrahydrofuranyl, 2-tetrahydropyranyl, 3-tetrahydropyranyl, 4-tetrahydropyranyl, [1,3]-dioxalanyl, [1,3]-dithiolanyl, [1,3]-dioxanyl, 2-tetrahydrothiophenyl, 3-tetrahydrothiophenyl, 2-morpholinyl, 3-morpholinyl, 4 ... Examples of aryl include aryl, 2-thiomorpholinyl, 3-thiomorpholinyl, 4-thiomorpholinyl, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, 1-piperazinyl, 2-piperazinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 4-thiazolidinyl, diozolonyl, N-substituted diozolonyl, 1-phthalimidinyl, benzoxanyl, benzopyrrolidinyl, benzopiperidinyl, benzoxolanyl, benzothiolanyl, and benzothianyl. As used herein, further included within the scope of the term "heterocyclyl" or "heterocyclic" are groups in which a non-aromatic heteroatom-containing ring is fused to one or more aromatic or non-aromatic rings, such as in indolinyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the non-aromatic heteroatom-containing ring. The terms "heterocycle", "heterocyclyl", or "heterocyclic" also refer to rings that are optionally substituted, whether saturated or partially unsaturated.

[0037] The term "heteroaryl" when used alone or as part of a larger moiety, such as in "heteroaralkyl" or "heteroarylalkoxy", refers to a heteroaromatic ring group having 5 to 14 members. Examples of heteroaryl rings include 2-furanyl, 3-furanyl, 3-furazanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxadiazolyl, 5-oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 1-pyrazolyl, 2-pyrazolyl, 3-pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, and the like. Examples of heteroaryl include aryl, 4-pyrimidyl, 5-pyrimidyl, 3-pyridazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 5-tetrazolyl, 2-triazolyl, 5-triazolyl, 2-thienyl, 3-thienyl, carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, isoquinolinyl, indazolyl, isoindolyl, acridinyl, and benzisoxazolyl. As used herein, further included within the scope of the term "heteroaryl" are groups in which a heteroaromatic ring is fused to one or more aromatic or non-aromatic rings, where the radical or point of attachment is on the heteroaromatic ring. Examples include tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[3,4-d]pyrimidinyl. The term "heteroaryl" also refers to rings that are optionally substituted. The term "heteroaryl" may be used interchangeably with the term "heteroaryl ring" or the term "heteroaromatic".

[0038] An aryl (including aralkyl, aralkoxy, aryloxyalkyl, and the like) or heteroaryl (including heteroaralkyl and heteroarylalkoxy, and the like) group may contain one or more substituents. Examples of suitable substituents on any unsaturated carbon atom of an aryl, heteroaryl, aralkyl, or heteroaralkyl group include halogen, --R0, --OR0, --SR0, 1,2-methylenedioxy, 1,2-ethylenedioxy, protected OH (e.g., acyloxy), phenyl(Ph), substituted Ph, --O(Ph), substituted --O(Ph), --CH2(Ph), substituted --CH2(Ph), CH2CH2(Ph), substituted --CH2CH2(Ph), --NO2, --CN, --N(R0), --NR0C(O)R0, NR0C(O)N(R0), NR0CO2R0, --NR0NR0C(O)R0, --NR0NR0C(O)N(R0), --NR0NR0C2R0, C(O)C(O)R0, C(O) CH2C(O)R0, --CO2R0, --C(O)R0, --C(O)N(R0)2, --OC(O)N(R0)2, S(O)2R0, --SO2N(R0)2, --S(O )R0, --NR0SO2N(R0)2, --NR0SO2R0, --C(=S)N(R0)2, C(=NH)N(R0)2, (CH2)yNHC(O)R0, and --(CH 2) yNHC(O)CH(V--R0)(R0), where each R0 is independently selected from hydrogen, a substituted or unsubstituted aliphatic group, an unsubstituted heteroaryl or heterocycle, phenyl(Ph), substituted Ph, O(Ph), substituted --O(Ph), --CH2(Ph), or substituted --CH2(Ph); y is 0-6; and V is a linker group. Examples of substituents on the aliphatic group or phenyl ring of R0 include amino, alkylamino, dialkylamino, aminocarbonyl, halogen, alkyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy, and haloalkyl.

[0039] The aliphatic group, or the non-aromatic heterocyclic ring, or the fused aryl or heteroaryl ring may contain one or more substituents. Examples of suitable substituents on any saturated carbon of the aliphatic group, or the non-aromatic heterocyclic ring, or the fused aryl or heteroaryl ring include those listed above for the unsaturated carbon of the aryl or heteroaryl group, as well as the following: =O, =S, =NNHR*, =NN(R*)2, =N-, =NNHC(O)R*, =NNHCO2(alkyl), =NNHSO2(alkyl), or =NR*, where each R* is independently selected from hydrogen, an unsubstituted aliphatic group, or a substituted aliphatic group. Examples of substituents on the aliphatic group include amino, alkylamino, dialkylamino, aminocarbonyl, halogen, alkyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy, and haloalkyl.

[0040] Suitable substituents on a nitrogen of a non-aromatic heterocycle include R, --N(R), --C(O)R, --COR, --C(O)C(O)R, --C(O)CHC(O)R, --SOR, --SON(R), C(=S)N(R), --C(=NH)--N(R), and --NRSO; where each R is independently selected from hydrogen, an aliphatic group, a substituted aliphatic group, phenyl (Ph), substituted Ph, --O(Ph), substituted --O(Ph), --CH(Ph), substituted --CH(Ph), or an unsubstituted heteroaryl or heterocycle. Examples of substituents on the aliphatic group or phenyl ring include amino, alkylamino, dialkylamino, aminocarbonyl, halogen, alkyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy, and haloalkyl.

[0041] In certain embodiments, the peptide monomers described herein are dimerized or multimerized by covalent attachment to at least one linker moiety. The linker moiety is preferably, but not necessarily, a C1-12 linking moiety, optionally terminated with one or two --NH-- bonds and optionally substituted with lower alkyl substituents at one or more available carbon atoms. Preferably, the linker comprises --NH--R--NH--, where R is a lower (C1-6) alkylene substituted with a functional group, such as a carboxyl or amino group, that allows for attachment to another molecular moiety (e.g., as may be present on the surface of a solid support during peptide synthesis or in a pharmacokinetic modifier such as PEG). In certain embodiments, the linker is a lysine residue. In certain other embodiments, the linker bridges the C-termini of two peptide monomers by simultaneously attaching to the C-terminal amino acid of each monomer. In other embodiments, the linker bridges the peptides by attaching to the side chain of an amino acid that is not at the C-terminus. When the linker is attached to the side chain of an amino acid other than at the C-terminus of the peptide, the side chain preferably contains an amine, such as found in lysine, and the linker contains two or more carboxy groups capable of forming an amide bond with the peptide.

[0042] The peptide monomers of the present disclosure may be oligomerized using a biotin / streptavidin system. Oligomerization may enhance one or more activities of the peptides described herein. Biotinylated analogs of peptide monomers may be synthesized by standard techniques known to those of skill in the art. For example, peptide monomers may be biotinylated at the C-terminus. These biotinylated monomers are then oligomerized by incubation with streptavidin (e.g., in phosphate-buffered saline (PBS) or HEPES-buffered RPMI medium (Invitrogen), at a molar ratio of 4:1 and at room temperature for 1 hour). In a variation of this method, biotinylated peptide monomers may be oligomerized by incubation with any of a number of commercially available anti-biotin antibodies [e.g., goat anti-biotin IgG from Kirkegaard & Perry Laboratories, Inc. (Washington, DC)].

[0043] In some aspects, the cyclic peptides described herein can be physically linked in series to form a polymer. The cyclic peptides constituting such a polymer can be spaced from one another by peptide linkers. Examples of useful linker peptides include, but are not limited to, glycine polymers ((G)n), including glycine-serine and glycine-alanine polymers (e.g., (Gly4Ser)n repeats, where n=1-8 (SEQ ID NO:8), preferably n=3, 4, 5, or 6). The cyclic peptides described herein can also be joined by the linkage of chemical bonds, such as by disulfide bonds or chemical crosslinking. Polymers of cyclic peptides can be created using molecular biology techniques well known to those of skill in the art. The peptide sequences of the present disclosure can also be linked together using non-peptide crosslinkers (Pierce 2003-2004 Applications Handbook and Catalog, Chapter 6), or other scaffolds, such as HPMA, polydextran, polysaccharides, ethylene glycol, polyethylene glycol, glycerol, sugars, and sugar alcohols (e.g., sorbitol, mannitol).

[0044] In some embodiments, polyethylene glycol (PEG) can function as the linker. In yet another embodiment, the linker moiety can include a molecule that contains two carboxylic acids and is optionally substituted at one or more available atoms with additional functional groups, such as amines, that can be attached to one or more PEG molecules. Such molecules can be represented as --CO--(CH2)n-uX--(CH2)m-CO--, where n is an integer between 0 and 10, m is an integer between 1 and 10, X is selected from O, S, N(CH2)pNR1, NCO(CH2)pNR1, and CHNR1, R1 is selected from H, Boc (tert-butyloxycarbonyl), Cbz, and p is an integer between 1 and 10. In certain embodiments, one amino group of each peptide forms an amide bond with the linker. Optionally, the linker contains one or more reactive amines that can be derivatized with a suitably activated pharmacokinetic (PK) modifier, such as a fatty acid, a homing peptide, a transport agent, a cell permeation agent, an organ targeting agent, or a chelating agent.

[0045] The cyclic peptides described herein may further comprise one or more water-soluble polymer moieties. In some embodiments, these polymers are covalently attached to the cyclic peptides of the present disclosure. In some embodiments, for therapeutic use of the final product preparation, the polymer is pharma- ceutically acceptable. One skilled in the art will be able to select the desired polymer based on considerations such as whether the polymer-peptide conjugate will be used therapeutically, and if so, the desired dose, circulation time, resistance to proteolysis, and other considerations. The water-soluble polymer may be, for example, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, and polyoxyethylated polyols. A preferred water-soluble polymer is PEG.

[0046] The polymer may be of any molecular weight and may be branched or unbranched. Preferred PEGs for use in the present disclosure are linear, unbranched PEGs having a molecular weight of about 5 kilodaltons (kDa) to about 60 kDa (the term "about" indicates that in a formulation of PEG, some molecules will weigh more than the indicated molecular weight and some will weigh less). More preferably, the PEG has a molecular weight of about 10 kDa to about 40 kDa, and even more preferably, the PEG has a molecular weight of 20-30 kDa. Other sizes may be used depending on the desired therapeutic profile (e.g., the desired duration of sustained release; the effect, if any, on biological activity; ease of handling; the degree or lack of antigenicity; and other effects of PEG on the therapeutic peptide known to those of skill in the art).

[0047] The number of attached polymer molecules can vary, for example, 1, 2, 3, or more than 4 water-soluble polymers can be attached to the peptides of the present disclosure. The multiple attached polymers can be the same or different chemical moieties (e.g., PEGs of different molecular weights). Methods for stabilizing peptides known in the art can be used in the methods and compositions described herein.For example, pyrrolinones and glycomimetics can provide stabilization using D-amino acids, reduced amide bonds for peptide backbones, and non-peptide bonds linking side chains, for example but not limited to pyrrolinones and glycomimetics, respectively.The design and synthesis of glycoscaffold peptide mimetics are described by Hirschmann et al. (J. Med. Chem., 1996, 36, 2441-2448, incorporated herein by reference in its entirety).In addition, pyrrolinone-based peptide mimetics present peptide pharmacophores in a stable context with improved bioavailability properties (see, for example, Smith et al., J. Am. Chem. Soc. 2000, 122, 11037-11038), incorporated herein by reference in its entirety.

[0048] Included herein are cyclic conjugates as described herein. These peptides can be conjugated to other polymers in addition to polyethylene glycol (PEG). The polymer may or may not have its own biological activity. Further examples of polymer conjugation include, but are not limited to, polymers such as polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polyoxyethylated polyols, heparin, heparin fragments, polysaccharides, cellulose and cellulose derivatives including methylcellulose and carboxymethylcellulose, starch and starch derivatives, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycol and derivatives thereof, polyvinyl ethyl ether, and α,β-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixtures thereof. Conjugation to polymers can improve serum half-life, among other effects. A variety of chelating agents can be used to conjugate the peptides described herein. These chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminopentaacetic acid (DTPA), ethylene glycol-0,0'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N'-bis(hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED), triethylenetetraminehexaacetic acid (TTHA), 1,4,7,10-tetra-azacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetraacetic acid (TITRA), 1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (TETA), and 1,4,8,11-tetraazacyclotetradecane (TETRA).Methods of conjugation are well known and can be found, for example, in PE Thorpe, et. al, 1978, Nature 271, 752-755; Harokopakis E., et. al., 1995, Journal of Immunological Methods, 185:31-42; SF Atkinson, et. al., 2001, J. Biol. Chem., 276:27930-27935; and U.S. Patent Nos. 5,601,825, 5,180,816, 6,423,685, 6,706,252, 6,884,780, and 7,022,673, which are incorporated by reference herein in their entireties.

[0049] The natural side chains of the genetically encoded amino acids (or stereoisomeric D amino acids) can be replaced with other side chains, for example with the following groups: alkyl, lower (C1-6) alkyl, cyclic 4-, 5-, 6- to 7-membered alkyl, amide, amide lower alkyl amide, di(lower alkyl), lower alkoxy, hydroxy, carboxy and lower ester derivatives thereof, and 4-, 5-, 6-, to 7-membered heterocycles. In particular, proline analogs can be used in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members. The cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic. The heterocyclic groups preferably contain one or more nitrogen, oxygen and / or sulfur heteroatoms. Examples of such groups include furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g., morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g., 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl groups. These heterocyclic groups can be substituted or unsubstituted. When a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl. The peptides can also be readily modified by phosphorylation or other methods (eg, as described in Hruby, et al. (1990) Biochem J. 268:249-262).

[0050] treatment Aspects of the disclosure relate to methods for treating a subject with cancer. In some embodiments, the method comprises administering to a subject with cancer a cyclic Psap peptide as described herein. In some embodiments, the cyclic peptide comprises the amino acid sequence DWLPK (SEQ ID NO: 1), dWLPK (SEQ ID NO: 3, lower case d indicates D-amino acid), DWGPK (SEQ ID NO: 2), or dWGPK (SEQ ID NO: 4, lower case d indicates D-amino acid). In some embodiments, the amino acid sequence of the cyclic peptide is DWLPK (SEQ ID NO: 1), dWLPK (SEQ ID NO: 3, lower case d indicates D-amino acid), DWGPK (SEQ ID NO: 2), or dWGPK (SEQ ID NO: 4, lower case d indicates D-amino acid). In some embodiments, the cyclic peptide comprises the amino acid sequence DWLPK (SEQ ID NO: 1). In some embodiments, the amino acid sequence of the cyclic peptide is DWLPK (SEQ ID NO: 1).

[0051] Another aspect of the present disclosure relates to the use of the composition in the manufacture of a composition and a medicament for treating a subject with cancer. In some embodiments, the composition comprises a cyclic Psap peptide as described herein, for example, a cyclic peptide having the amino acid sequence DWLPK (SEQ ID NO: 1). As used herein, "treating" or "treatment" of cancer includes, but is not limited to, preventing, reducing, or halting the onset of cancer, alleviating or eliminating symptoms of cancer, suppressing or inhibiting the growth of cancer, preventing or reducing metastasis and / or invasion of existing cancer, promoting or inducing regression of cancer, inhibiting or suppressing the proliferation of cancer cells, reducing angiogenesis, and / or increasing the amount of apoptotic cancer cells.

[0052] Another aspect of the present disclosure relates to a method for treating a subject having an inflammatory disease or disorder. In some embodiments, the inflammatory disease or disorder is inflammatory bowel disease (IBD), such as Crohn's disease. In some embodiments, the method comprises administering a cyclic Psap peptide described herein to a subject having an inflammatory disease or disorder (e.g., Crohn's disease). In some embodiments, the cyclic peptide comprises the amino acid sequence DWLPK (SEQ ID NO: 1), dWLPK (SEQ ID NO: 3, lower case d indicates D-amino acid), DWGPK (SEQ ID NO: 2), or dWGPK (SEQ ID NO: 4, lower case d indicates D-amino acid). In some embodiments, the amino acid sequence of the cyclic peptide is DWLPK (SEQ ID NO: 1), dWLPK (SEQ ID NO: 3, lower case d indicates D-amino acid), DWGPK (SEQ ID NO: 2), or dWGPK (SEQ ID NO: 4, lower case d indicates D-amino acid). In some embodiments, the cyclic peptide comprises the amino acid sequence DWLPK (SEQ ID NO: 1). In some embodiments, the amino acid sequence of the cyclic peptide is DWLPK (SEQ ID NO: 1).

[0053] Other aspects of the disclosure relate to compositions and uses of compositions in the manufacture of medicaments for treating a subject having an inflammatory disease or disorder, such as Crohn's disease. In some embodiments, the compositions comprise a cyclic Psap peptide as described herein, such as a cyclic peptide having the amino acid sequence DWLPK (SEQ ID NO:1). As used herein, "treating" or "treatment" of an inflammatory disease or disorder includes, but is not limited to, preventing, reducing, or arresting the onset of an inflammatory disease or disorder, or alleviating or eliminating the symptoms of an inflammatory disease or disorder.

[0054] An effective amount is that amount of cyclic Psap peptide administered that is sufficient to provide a medically desirable result, such as treatment of cancer or an inflammatory disease or disorder. Effective amounts will vary depending on the particular disease or disorder being treated, the age and health of the subject being treated, the severity of the condition, the duration of treatment, the nature of any concomitant therapy, the particular route of administration, and similar factors within the knowledge and experience of the physician. For administration to subjects, such as humans, dosages from about 0.001, 0.01, 0.1, or 1 mg / kg to 50, 100, 150, or 500 mg / kg or more can generally be used.

[0055] In some embodiments, an effective amount is an amount of cyclic Psap peptide that does not cause toxicity to a subject. In some embodiments, an effective amount is an amount of cyclic Psap peptide that causes reduced toxicity to a subject compared to linear Psap peptide. As used herein, the term "non-toxic" or "reduced toxicity" indicates that the cyclic Psap peptide does not induce the occurrence or reduces the extent of one or more adverse reactions in a subject or in a cell, tissue, or organ of a subject to which it is administered. For example, the cyclic Psap peptides described herein do not cause organ or organ system dysfunction or cell death. For example, the cyclic Psap peptides are not nephrotoxic, splenic toxic, and / or hepatotoxic. Methods for measuring toxicity are known in the art (e.g., liver, spleen, and / or kidney biopsy / histology; alanine transferase, alkaline phosphatase, and bilirubin assays for liver toxicity; and creatinine levels for kidney toxicity).

[0056] Cyclic Psap peptides and compositions thereof can be formulated for various modes of administration, including systemic, topical or localized administration. A variety of routes of administration are available. The particular mode selected will depend on the type of cancer being treated and the dosage required for therapeutic effect. The methods of the present disclosure can generally be practiced using any medically acceptable mode of administration, meaning any mode that produces effective levels of the active compound without causing clinically unacceptable side effects. Such modes of administration include, without limitation, oral, rectal, topical, nasal, intradermal, or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intramuscular, or injection. The pharmaceutical compositions described herein can also be administered by intratumoral, peritumoral, intralesional, or perilesional routes to exert local as well as systemic effects.

[0057] Techniques and formulations can generally be found in Remington: The Science and Practice of Pharmacy, Pharmaceutical Press; 22nd edition and other similar references. When administered, the Psap peptides can be applied in pharma- ceutically acceptable amounts and in pharma- ceutically acceptable compositions. Pharmaceutical compositions and pharma- ceutically acceptable carriers are described herein. Such formulations may routinely contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, salts should be pharma- ceutically acceptable, but pharma- ceutically unacceptable salts may also be advantageously used to prepare pharma- ceutically acceptable salts thereof and are not excluded from the scope of this disclosure. Such pharmacologically and pharma- ceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, and the like. Also, pharma- ceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.

[0058] In some embodiments, treatment with cyclic Psap peptides can be combined with another therapy, such as chemotherapeutic agents, radiation, cytostatic agents, anti-VEGF agents, anti-angiogenic factors, p53 reactivators, and / or anti-inflammatory agents for cancer surgery, inflammatory diseases or disorders. In some embodiments, a cyclic Psap peptide (e.g., DWLPK (SEQ ID NO: 1)) can be used to stimulate expression of TspP-1 in a subject in need thereof. In some embodiments, the method includes administering to the subject an effective amount of the cyclic Psap peptide to stimulate expression of TspP-1.

[0059] Compositions and Pharmaceutically Acceptable Carriers Another aspect of the present disclosure relates to a composition comprising a cyclic Psap peptide as described herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a cyclic Psap peptide as described herein and a pharma- ceutically acceptable carrier. In some embodiments, the composition is for use in treating cancer or an inflammatory disease or disorder. In some embodiments, the composition is for use in stimulating Tsp-1 in a subject in need thereof. In some embodiments, the composition comprises an additional agent, such as a chemotherapeutic agent, a cytostatic agent, an anti-VEGF agent, an anti-angiogenic factor, a p53 reactivator, and / or an anti-inflammatory agent.

[0060] As used herein, the term "pharmaceutical acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents, or encapsulating substances that are suitable for administration to a subject, such as a human. A pharmaceutical acceptable carrier is "acceptable" in the sense of being compatible with other ingredients of the formulation and not deleterious to the subject's tissues (e.g., physiologically compatible, sterile, physiological pH value, etc.). The term "carrier" refers to a natural or synthetic, organic or inorganic component that combines with an active ingredient to facilitate application. The components of the pharmaceutical composition can also be co-mixed with the molecules of the present disclosure, and with each other, in a manner such that there is no interaction that would substantially impair the desired pharmaceutical effectiveness. Some examples of materials which can function as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propyl alcohol, glycerol, sorbitol ... (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances used in pharmaceutical formulations.Wetting agents, coloring agents, releasing agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives and antioxidants can also be present in the formulation.

[0061] Pharmaceutical compositions can be conveniently provided in unit dosage form and can be prepared by any method known in the field of pharmacy.The term "unit dose" used in relation to pharmaceutical compositions of the present disclosure refers to physically separate units suitable as unitary dosages for subjects, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier, or vehicle.

[0062] The formulation of the pharmaceutical composition may depend on the route of administration. Injectables suitable for parenteral administration or intratumoral, peritumoral, intralesional or perilesional administration include, for example, sterile injectable aqueous or oily suspensions, which may be formulated using suitable dispersing or wetting agents and suspending agents according to known techniques. Sterile injectable preparations may also be sterile injectable solutions, suspensions or emulsions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-propanediol or 1,3-butanediol. Acceptable vehicles and solvents that may be used are water, Ringer's solution, USP and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any non-irritating fixed oil may be used, including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0063] For topical administration, pharmaceutical compositions can be formulated into ointments, salves, gels or creams as generally known in the art.For topical administration, transdermal delivery systems known in the art can be used.Examples include skin patches. Compositions suitable for oral administration can be presented as discrete units such as capsules, tablets, lozenges, each containing a predetermined amount of the anti-inflammatory agent. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or emulsion.

[0064] Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administration of anti-inflammatory drugs, increasing the convenience of the subject and the physician. Many types of release delivery systems are available and known to those skilled in the art. They include polymer-based systems, such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the aforementioned polymers containing drugs are described, for example, in U.S. Patent 5,075,109. Delivery systems also include non-polymeric systems, such as: lipids, including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-, di-, and tri-glycerides; hydrogel release systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to, (a) erosion systems in which the anti-inflammatory agent is contained within a matrix, such as those described in U.S. Patent Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660, and (b) diffusion systems in which the active ingredient permeates through a polymer at a controlled rate, such as those described in U.S. Patent Nos. 3,832,253 and 3,854,480. Additionally, pump-based hardware delivery systems can also be used, some of which are adapted for implantation.

[0065] The use of long-term sustained release implants may be particularly suitable for treating chronic conditions.Long-term release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of active ingredient for at least 30 days, preferably 60 days.Long-term sustained release implants are well known to those skilled in the art and include some of the release systems described above. In some embodiments, pharmaceutical compositions used for therapeutic administration must be sterile. Sterility is easily achieved by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Alternatively, preservatives can be used to prevent the growth or action of microorganisms. A variety of preservatives are known, such as phenol and ascorbic acid. If the cyclic Psap peptide and / or pharmaceutical composition is highly stable against heat and oxidative denaturation, it will usually be stored in lyophilized form or as an aqueous solution. The pH of the formulation is typically about 6-8, although higher or lower pH values ​​may be appropriate in certain cases.

[0066] subject Aspects of the present disclosure relate to subjects, such as humans, with cancer, and methods of treating such subjects.Cancer can be benign or malignant, and may or may not metastasize.Any type of cancer is contemplated herein, including, but not limited to, leukemia, lymphoma, myeloma, carcinoma, metastatic carcinoma, sarcoma, adenoma, cancer of the nervous system, and genitourinary cancer. Exemplary cancer types include, but are not limited to, adult and pediatric acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, anal cancer, cancer of the appendix, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma, fibrous histiocytoma, brain cancer, brain stem glioma, cerebellar astrocytoma, malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, hypothalamic glioma, breast cancer, male breast cancer, bronchial adenoma, Burkitt's lymphoma, carcinoid tumor, causes Unknown cancer, central nervous system lymphoma, cerebellar astrocytoma, malignant glioma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing family tumors, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal stromal tumors, extracranial germ cell tumors, extragonadal germ cell tumors, ovarian germ cell tumors, gestational trophoblastic tumors, glioma, hairy cell leukemia,

[0067] Head and neck cancer, hepatocellular carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hypopharyngeal cancer, glioma of the hypothalamus and visual pathway, intraocular melanoma, pancreatic islet cell tumors, Kaposi's sarcoma, kidney cancer, renal cell carcinoma, laryngeal cancer, lip and oral cavity cancer, small cell lung cancer, non-small cell lung cancer, primary central nervous system lymphoma, Waldenström macroglobulinemia, malignant fibrous histiocytoma, medulloblastoma, melanoma, Merkel cell carcinoma, malignant mesothelioma, squamous cell neck cancer, multiple endocrine neoplasia syndromes, multiple myeloma, mycosis fungoides, myelodysplastic syndromes, myeloproliferative disorders, chronic myeloproliferative disorders, nasal and paranasal sinus cancer, and nasopharyngeal cancer. In some embodiments, the cancer is melanoma or ovarian cancer, transitional cell carcinoma, uterine sarcoma, thyroid cancer, uterine cancer ...

[0068] Subjects with cancer can be identified using any method known in the art (e.g., blood test, histology, CT scan, X-ray, MRI, physical examination, cytogenetic analysis, urine test, or genetic test). Subjects suspected of having cancer may show one or more symptoms of the disease. Signs and symptoms of cancer are known to those skilled in the art. Some exemplary clinical tests include, but are not limited to, cancer biomarker tests, such as cancer antigen (CA) 15-3, carcinoembryonic antigen (CEA) and HER-2 for breast cancer, human papillomavirus (HPV) E6 and E7 oncoproteins for cervical cancer, alpha-fetoprotein (AFP), AFP fraction L3, P4 / 5 and +II bands for hepatocellular carcinoma (HCC), and ultrasound for hepatocellular carcinoma (HCC), prostate specific antigen (PSA) for prostate cancer, and serum CA-125 for ovarian cancer and HCC.

[0069] Other aspects of the present disclosure relate to subjects, such as humans, having an inflammatory disease or disorder and methods of treating such subjects. Exemplary inflammatory diseases or disorders include, but are not limited to, rheumatoid arthritis, macular degeneration (e.g., age-related macular degeneration, AMD), inflammatory bowel disease (IBD, e.g., Crohn's disease or ulcerative colitis), psoriasis, atherosclerosis, systemic lupus erythematosus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac disease-dermatitis, chronic fatigue syndrome, immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold aggregation disease, CREST syndrome, Degos disease, dermatomyositis, skin ulcers ... Dermatomyositis - Juvenile, Discoid Lupus, Essential Mixed Cryoglobulinemia, Fibromyalgia - Fibromyositis, Graves' Disease, Guillain-Barre, Hashimoto's Thyroiditis, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenic Purpura (ITP), IgA Nephropathy, Insulin-Dependent Diabetes Mellitus (Type I), Juvenile Arthritis, Meniere's Disease, Mixed Connective Tissue Disease, Multiple Sclerosis, Myasthenia Gravis, Pemphigus Vulgaris, Pernicious Anemia, Nodular Multifocal Inflammatory Syndrome Arteritis, chondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, Takayasu's arteritis, temporal arteritis / giant cell arteritis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis. In some embodiments, the inflammatory disease or disorder is rheumatoid arthritis, macular degeneration (e.g., age-related macular degeneration, AMD), inflammatory bowel disease (IBD, e.g., Crohn's disease or ulcerative colitis), psoriasis, or atherosclerosis.

[0070] The subject with inflammatory disease or disorder can be identified by any method known in the art (e.g., blood test, physical examination, CT scan, or MRI).The subject suspected of having inflammatory disease or disorder may show one or more symptoms of the disease or disorder.The signs and symptoms of inflammatory disease or disorder are well known to those skilled in the art. In some embodiments, the inflammatory disease or disorder is inflammatory bowel disease (IBD), such as Crohn's disease.Subjects with IBD, such as Crohn's disease, can be identified using any method known in the art (e.g., blood test, physical examination, fecal occult blood test, colonoscopy, flexible sigmoidoscopy, CT scan, MRI, capsule endoscopy, double balloon endoscopy, small intestine imaging, or barium enema).Subjects suspected of having IBD may show one or more symptoms of disease.The signs and symptoms of IBD are well known to those skilled in the art. EXAMPLES

[0071] Example 1 Cyclic versions of the peptides DWLPK (SEQ ID NO:1), dWLPK (SEQ ID NO:3), DWGPK (SEQ ID NO:2), and dWGPK (SEQ ID NO:4) were synthesized according to methods known in the art (lower case d indicates a D-amino acid) and compared with the linear peptide dWlP (SEQ ID NO:5, lower case d and l indicate a D-amino acid). Cells were cultured and exposed to 4 μg / ml or 10 μg / ml of each peptide, and the level of thrombospondin 1 (Tsp-1) activation was measured by Western blot. Tsp-1 has previously been shown to be activated by saposin A protein. Figure 2 shows that the cyclic peptide DWLPK (SEQ ID NO: 1) was the most effective in increasing the level of Tsp-1, followed by dWGPK (SEQ ID NO: 4), and dWLPK (SEQ ID NO: 3) and DWGPK (SEQ ID NO: 2) were the least effective. This is surprising, since similar tests with linear versions of these peptides showed that linear DWGPK (SEQ ID NO: 2) was more effective than linear DWLPK (SEQ ID NO: 1).

[0072] The cyclic peptide DWLPK (SEQ ID NO: 1) was then tested for stability in human plasma in comparison with the linear peptide dWlP (SEQ ID NO: 5). Each peptide was incubated in human plasma at 37° C. for 2, 4, 8, or 24 hours. The cyclic peptide was found to retain the ability to activate Tsp-1 after 24 hours of incubation in plasma ( FIG. 3 ). The linear peptide, despite being stabilized against degradation by D-amino acid substitutions, had significantly less activity after 24 hours. The cyclic peptide dWGPK (SEQ ID NO: 4) was tested in a mouse model of melanoma. 5 of B16-BL6 melanoma cells were injected into syngeneic C57BL6 mice. Mice were then treated with either the cyclic peptide or linear dWlP (SEQ ID NO: 5) or a control. In the first experiment, treatment with peptide was started at approximately day 12 and tumor volumes were measured over time until about 20-25 days after cell injection (Figure 4). In the second experiment, treatment with peptide was started at day 15 and tumor volumes were measured over time until about 27 days after cell injection (Figure 5). The cyclic peptide was found to be 4 times more effective than the linear peptide, as a 10 mg / kg dose of the cyclic peptide was as effective as a 40 mg / kg dose of the linear peptide (Figure 4). This was confirmed in the second experiment (Figure 5).

[0073] Next, the cyclic peptide DWLPK (SEQ ID NO: 1) was tested in a mouse model of ovarian cancer. In the mouse model of ovarian cancer, one million patient-derived ovarian cancer cells expressing luciferase were injected intraperitoneally into mice. Tumor burden was measured in vivo using luciferase. Treatment with the cyclic peptide began after 40 days and was administered at 10 mg / kg for 10 days. After 10 days of treatment, mice were euthanized and tumors were analyzed histologically. As shown in Figure 6, luciferase output in mice treated with the cyclic peptide decreased over the 10 days of treatment, indicating tumor regression. All cyclic peptide-treated tumors expressed Tsp-1 and were <25% of the size of untreated tumors by histological observation. The cyclic peptide (SEQ ID NO:1) was also shown to stimulate Tsp-1 in mouse macrophages (FIG. 7), indicating that the cyclic peptide can also induce expression of Tsp-1 in the interstitium.

[0074] Finally, administration of the cyclic peptide DWLPK did not result in any toxicity to the liver or spleen, as measured using histology (Figure 8). Taken together, these data indicate that cyclic Psap peptides were more stable and effective than linear Psap peptides. Furthermore, the cyclic peptide DWLPK (SEQ ID NO: 1) appeared to have the greatest activity in vitro and did not induce toxicity.

[0075] Example 2 A model of Crohn's disease was used to test the efficacy of the cyclic peptide DWLPK (SEQ ID NO:1) in treating Crohn's disease. The cyclic peptide was tested in a dextran sulfate sodium (DSS)-induced model of Crohn's disease, where DSS was given to mice in drinking water (3.5% weight:volume) for 7 days. One group of mice was treated with the cyclic peptide and DSS simultaneously, while the other group was treated with DSS only. The cyclic peptide significantly reduced inflammation in this model (Figure 9). Mice treated with the cyclic peptide also lost less weight than mice treated with DSS only (Figure 10). Finally, the cyclic peptide was shown to stimulate Tsp-1 and inhibit macrophage infiltration in these mice (Figure 11). These data suggest that the cyclic Psap peptides, particularly cyclic DWLPK (SEQ ID NO:1), are effective in treating Crohn's disease.

[0076] Example 3 (i) AMD model is used to test the efficacy of the cyclic Psap peptide described herein in treating AMD. Lesions are created on the retina of mice using a laser. Mice are then treated with the cyclic Psap peptide described herein, for example 10 or 40 mg / kg cyclic DWLPK (SEQ ID NO: 1), or with a scrambled peptide control. Treatment is either systemic (e.g., intravenous or intraperitoneal injection) or by intravitreal injection. Lesion healing rate is measured over time. Lesions are expected to heal faster in mice treated with cyclic Psap peptide than in mice treated with control.

[0077] (ii) A rodent model of collagen-induced arthritis (CIA), an autoimmune model similar to rheumatoid arthritis, is used to test the efficacy of the cyclic Psap peptides described herein for treating rheumatoid arthritis. CIA can be induced by priming inbred DBA / 1 male mice intradermally with heterologous or allogeneic collagen II (about 50 μg) in Freund's complete adjuvant and boosting 2 weeks later with the same amount of collagen II in Freund's incomplete adjuvant. Arthritis develops about 3 weeks after the priming dose and reaches a maximum within 8 weeks after priming. Mice have high levels of collagen II-specific antibodies, collagen II-specific T cells, and signs of systemic inflammation (e.g., production of IL-6, TNF, etc.). Locally in the joints, both an overwhelming inflammatory infiltrate (consisting of T cells, macrophages, neutrophils, and fibroblasts) and severe destruction of cartilage and subchondral bone are observed. These characteristics closely mimic the process seen in human rheumatoid arthritis (Myers et al., Life Sciences 61, p 1861-1878, 1997). Mice are then treated with a cyclic Psap peptide as described herein, for example cyclic DWLPK (SEQ ID NO: 1) at 10 or 40 mg / kg, or with a scrambled peptide control. Treatment is either systemic (e.g., intravenous or intraperitoneal injection) or by local administration. Several parameters of disease are measured (e.g., clinical signs and symptoms, symptom onset, progression, severity, and remission). It is expected that one or more of these parameters will be improved (e.g., reduced clinical signs and symptoms, delayed onset, delayed progression, reduced severity, and / or remission) in mice treated with cyclic Psap peptide compared to mice treated with control.

[0078] (iii) A model of psoriasis is used to test the efficacy of the cyclic Psap peptides described herein in treating psoriasis. Imiquimod (IMQ) is applied to the skin of mice to induce psoriasis-like skin inflammation (see, for example, van der Fits et al. Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23 / IL-17 axis. (2009) J. Immunol.;182(9):5836-45). Mice are then treated with the cyclic Psap peptides described herein, for example 10 or 40 mg / kg cyclic DWLPK (SEQ ID NO:1), or with a scrambled peptide control. Treatment is either systemic (e.g., intravenous or intraperitoneal injection) or topical. The healing rate of the skin inflammation is measured over time. Epidermal expression of IL-23, IL-17A and IL-17F is also measured at the end of the experiment. It is expected that mice treated with cyclic Psap peptides will have faster healing of dermatitis and lower levels of IL-23, IL-17A and / or IL-17F than control treated mice.

[0079] (iv) A model of atherosclerosis is used to test the efficacy of the cyclic Psap peptides described herein in treating atherosclerosis. Mice lacking apoE or LDL receptor (LDLR) genes are used as a model of atherosclerosis. Mice are fed a high fat, high cholesterol Western diet or normal chow diet for 8 weeks or more, preferably at least 15 weeks. Mice are treated with the cyclic Psap peptides described herein, for example 10 or 40 mg / kg cyclic DWLPK (SEQ ID NO: 1), or with a scrambled peptide control. Treatment is delivered systemically (e.g., by intravenous or intraperitoneal injection). Lesions developed in the aortic root, brachiocephalic artery (brachiocephalic) and other branches of the aorta, as well as the lungs and carotid arteries are measured in mice after at least 8 weeks. Lesions are expected to heal faster in mice treated with cyclic Psap peptide than in mice treated with control.

[0080] Example 4 summary Nearly 100% of ovarian cancer-related deaths are caused by subsequent organ failure resulting from metastatic seeding of cells from the primary tumor. However, despite an improved understanding of the physiological processes involved in tumor metastasis, there are no clinically approved drugs that have shown significant efficacy in the treatment of advanced metastatic ovarian cancer. Psap has been identified as a potent inhibitor of tumor metastasis via stimulation of p53 and of the anti-oncogene protein thrombospondin-1 (Tsp-1) in bone marrow-derived cells recruited to metastatic sites. It has been demonstrated that ~100% of human serous ovarian tumors express CD36, a receptor that mediates the proapoptotic activity of Tsp-1. We investigated peptides derived from Psap that may be effective in the treatment of this form of ovarian cancer. The activity and stability of the peptides were investigated by developing novel cyclic peptides with drug-like properties derived from Psap. Cyclic Psap peptides were able to significantly regress PDX models of metastatic ovarian cancer.

[0081] Introduction Ovarian cancer is the most lethal gynecologic malignancy and the fourth leading cause of cancer deaths in women. Pathologically, ovarian cancer is classified into multiple subtypes, with epithelial ovarian cancer (EOC) accounting for 90% of cases. Despite an increased understanding of the biology governing EOC progression, the survival rate of patients with stage IV EOC is only 17%. Thus, there is a need for effective therapies that can specifically treat advanced metastatic ovarian cancer. Although many patients respond to platinum agents as first-line therapy, 70% develop resistance (1, 2). Currently, there are no approved therapies for these patients that significantly increase overall survival rates.

[0082] It has been previously reported that small peptides derived from Psap potently inhibit tumor metastasis in multiple tumor models (3, 4). Specifically, Psap and peptides derived from it inhibit the CD11b + / Gr1+ / Lys6C hi It inhibits tumor metastasis by stimulating the broadly acting anti-tumorigenic protein thrombospondin-1 (Tsp-1) in monocytes (3). These monocytes are recruited to the site of future metastatic lesions, called the premetastatic niche, where they persist after colonization and stimulate tumor growth. However, systemic administration of Psap peptide stimulates the production of Tsp-1 in these cells, which renders the site refractory to future metastatic colonization (3). These results demonstrate that stimulation of Tsp-1 in the tumor microenvironment can prevent future metastatic events. Many ovarian cancer patients present with metastatic disease at the time of initial diagnosis. Thus, there is a need for therapeutic agents that can regress or stabilize metastatic lesions. It has been demonstrated that stimulating Tsp-1 in the microenvironment of metastatic and platinum-resistant ovarian cancer PDX models can induce significant regression of established lesions, a remarkable effect achieved by the fact that serous ovarian cancer cells express CD36, the receptor for Tsp-1, which mediates the proapoptotic effects previously observed in endothelial cells (5).

[0083] result Incorporation of d-amino acids enhances the activity of Psap peptides in vivo The identification of both 4- and 5-amino acid peptides derived from the saposin A domain of Psap that, when administered systemically, were able to inhibit both the tail vein model of Lewis lung carcinoma metastasis and the adjuvant model of breast cancer metastasis has been described previously (3). The therapeutic efficacy of peptides is often limited by their stability, or resistance to degradation by proteases. One common method to increase peptide stability in vivo is to incorporate d-amino acids into the sequence, because d-amino acids are not incorporated into naturally occurring proteins and proteases do not recognize them as substrates (6-10). The stability of a 4-amino acid Psap peptide by incorporating d-amino acids at different positions was investigated. Two peptides were synthesized incorporating combinations of d-amino acids at the first (aspartic acid) and third (leucine) or second (tryptophan) and fourth (proline) residues. The activity of these peptides and the natural l-amino acid peptide was tested by measuring their ability to stimulate thrombospondin-1 (Tsp-1) in WI-38 lung fibroblasts in vitro. Western blot analysis found no difference between the three peptides in Tsp-1 stimulation in vitro (Figure 12A).

[0084] The activity of 1,3-d-amino acid Psap peptides and native Psap peptides was tested in vivo. Peptides were administered systemically to C57BL6 / J mice pretreated with conditioned medium (CM) from PC3M-LN4 (LN4) cells, which have previously been shown to mimic the systemic properties of metastatic tumors by suppressing the expression of Tsp-1 in mouse lungs (3,4). After 3 days of treatment with LN4 CM alone or in combination with two different doses (10 mg / kg / day and 30 mg / kg / day) of d- and l-amino acid peptides (independently), protein pools were prepared from the harvested lungs of each treatment group. The level of Tsp-1 expression in the lungs of these mice was assessed by Western blot analysis. It was observed that the 1,3-d-amino acid peptide stimulated Tsp-1 three-fold more than the native peptide (Figure 12B). A 10 mg / kg dose of the native peptide did not significantly stimulate Tsp-1 (Figure 12B). Conversely, a 10 mg / kg dose of the d-amino acid peptide stimulated Tsp-1 to the same extent as a 30 mg / kg dose of the native peptide, which stimulated Tsp-1 approximately three-fold more strongly than the same dose of the native peptide. Based on the observation that the in vitro activities of the two peptides were virtually identical, we concluded that the difference in in vivo activity was due to differences in stability.

[0085] Human serous ovarian cancer cells are sensitive to Tsp-1-mediated killing To test the efficacy of d-amino acid Psap peptides, we determined an appropriate tumor model that would represent a potential clinical application for the peptide. Given that Psap, and peptides derived from it, stimulate Tsp-1 in bone marrow-derived cells recruited to metastatic sites, we sought to identify specific cancer types that expressed CD36, the receptor for Tsp-1 that mediates its proapoptotic activity (5). Serous ovarian epithelial cells and human ovarian cancer cells have been reported to express CD36 (11-13). Fourteen primary human ovarian cancer cell lines derived from ascites of platinum-resistant ovarian cancer patients were surveyed for CD36 expression. All of the patient-derived cells expressed levels of CD36 nearly equivalent to those expressed in human microvascular endothelial cells (Figure 12C). Three of these cell lines were treated with recombinant human Tsp-1 for up to 72 hours to determine its effect on cell number and apoptosis. RhTsp-1 treatment resulted in a reduction in total cell numbers to 50% of the original number of cells plated for all three cell lines (Figure 12D). Furthermore, by FACS analysis, 30%-60% of treated cells were observed to be apoptotic after Tsp-1 treatment, as evidenced by Annexin V positivity (Figure 12E). In contrast, after cisplatin treatment, a much larger proportion of ovarian cancer cells were observed to be necrotic, as evidenced by low Annexin V and high propidium iodide (PI) staining (Figure 12E). These findings suggest that ovarian cancer cells may respond well to treatment with Psap peptides.

[0086] Psap peptide induces regression of established PDX models of ovarian cancer metastasis It has been previously demonstrated that Psap and Psap peptides can inhibit metastasis formation (3,4). 75% of ovarian cancer patients already have disseminated disease at the time of initial diagnosis (E. Lengyel, Ovarian cancer development and metastasis. Am J Pathol 177, 1053-1064 (2010)). For these patients, inhibition of metastasis would be of limited therapeutic benefit. Rather, these patients require therapeutic agents that can regress or at least stabilize existing metastases. We investigated whether the d-amino acid Psap peptide has therapeutic efficacy in a model of established metastatic dissemination. 1 × 10 6 DF14 cells were injected intraperitoneally into SCID mice to mimic the dissemination route of human ovarian cancer. The growth of metastatic colonies in mice in real time was monitored via relative luciferase intensity, with the mean intensity of the luciferase signal ranging from 0.5 to 1 x 10 8 At the time of RLU, treatment with vehicle (saline), d-amino acid peptide (40 mg / kg QD), or cisplatin (4 mg / kg QOD) was initiated. Both peptide and cisplatin were able to regress tumor volume during the first 20 days of treatment, as determined by luciferase intensity (Figures 13A and 13B). However, during these 20 days, half of the mice treated with cisplatin died from side effects of the drug, as evidenced by a 40% decrease in total body weight (Figure 13C). Furthermore, after 20 days, tumors in the group of surviving cisplatin-treated mice began to grow despite continued treatment with cisplatin, and all remaining mice died within 10 days (Figure 13A).

[0087] Conversely, no weight loss was observed in peptide-treated mice (Figure 13C), and tumors continued to shrink until there was no detectable luciferase signal in any of the mice on day 48 (Figures 13A and 13B and Figure 16). These mice were treated for an additional 35 days (83 days total) until the control-treated group showed symptoms associated with pathology. During this treatment period, the luciferase signal did not rise again, and macroscopic examination of the mice did not reveal any metastatic lesions (Figures 13A and 13D). The livers and spleens of Psap peptide-treated mice were examined histologically (H&E) to determine whether there were any micrometastases. No metastatic lesions could be identified in Psap peptide-treated mice (Figure 13E).

[0088] Peritoneal metastasis, as well as pulmonary metastasis, were Cd11b + / Gr1 + We investigated the mobilization of bone marrow-derived cells (3). Ascites was collected from the peritoneal cavity of control-treated mice with DF14 metastases, and fluid was collected from the peritoneal cavity of mice treated with the Psap peptide that showed no signs of metastases. Fluid was sorted by FACS from these mice and Cd11b + / Gr1 + The bone marrow-derived cell population was assessed. FACS analysis revealed that 71-77% of cells in the ascites of control-treated mice were Cd11b + / Gr1 + (FIGS. 13E and 16), whereas only 31.4% of cells in the ascites from peptide-treated mice were Cd11b + / Gr1 + Based on these findings, we conclude that the Psap peptide can dramatically regress established metastases to the point where no detectable lesions can be found.

[0089] Cyclization further stabilizes Psap peptides while enhancing activity Although the results of the peptide treatment of a PDX model of ovarian cancer were very promising, it was hypothesized that the stability and activity of the peptide could be improved. The peptide derived from Psap was located in a region of the protein that contains a 13 amino acid loop between two helices stabilized by a disulfide bond (3). Thus, a 5 amino acid peptide cyclized via backbone (NC) cyclization was synthesized (Figure 14A, Cyclic DWLPK (SEQ ID NO: 1)). The in vitro activity of this peptide was tested based on its ability to stimulate Tsp-1. The cyclic peptide stimulated Tsp-1 three-fold more than the d-amino acid linear peptide (Figure 14B).

[0090] Cyclization also has the effect of increasing the peptide's stability by forcing it into a conformation that is not recognized by most naturally occurring proteases (9, 10, 14-18). The stability of the cyclic peptide was compared against the linear d-amino acid peptide in human plasma. Both peptides in human plasma were incubated at 37 °C for up to 24 h, after which the ability of the plasma / peptide mixtures to stimulate Tsp-1 in WI-38 fibroblasts was tested. The levels of secreted Tsp-1 were measured by ELISA after treatment with the peptide / plasma mixtures, and stimulation of Tsp-1 by the two peptides was found to be nearly equivalent up to 8 h after incubation (Figure 14C). However, after 24 h of incubation in human plasma, the cyclic peptide retained more than 70% of its Tsp-1 stimulating activity, whereas plasma containing the linear peptide was no longer able to stimulate Tsp-1 (Figure 14C). Thus, it was concluded that the cyclic peptide was much more active and stable than the linear d-amino acid peptide.

[0091] Based on these findings, we tested the efficacy of the cyclic peptide using the DF14 model. To further investigate the effect of the peptide on metastatic lesions, mice were treated with 1 × 10 7 Cells were injected and the luciferase signal was 9 × 10 9RLU was reached. Mice were treated with the cyclic peptide for only 10 days to ensure there was enough tumor tissue to analyze. Importantly, after only 10 days of treatment, the average luciferase signal in peptide-treated mice was 4×10 8 (Fig. 14D). Liver, spleen, and omentum were analyzed by H&E and IHC for Tsp-1, Gr1, and TUNEL in both peptide- and control-treated mice. Metastatic lesions in control-treated mice were, on average, 2.3-fold larger than those in peptide-treated mice (Fig. 14E). Consistent with the mechanism of action (MOA) of the Psap peptide, all of the peptide-treated tumors stained positive for Tsp-1 (Fig. 14F) expression. Also consistent with previous observations, all of the Tsp-1-expressing cells were also found to be Gr1 positive (Fig. 14G) (3). Furthermore, all of the lesions in peptide-treated mice contained a significant proportion of TUNEL-positive cells, averaging 40% TUNEL-positive cells / lesion (Figs. 14H and 14I). Conversely, control-treated tumors contained, on average, only 1.4% TUNEL-positive cells / lesion (Figs. 14H and 14I).

[0092] Metastatic serous ovarian tumors express low levels of Psap but have higher CD36 expression than primary tumors The activity of Psap peptides against tumors formed by ovarian cancer cells derived from patients expressing CD36 was previously shown. The prevalence of CD36 expression in human ovarian cancer patients and therefore how widely applicable potential Psap-based therapeutics might be for this disease were investigated. It was hypothesized that prosaposin expression should decrease as tumors progress to the metastatic stage. Therefore, tumor tissue microarrays (TMAs) consisting of 139 patients with metastatic serous ovarian cancer and normal ovarian tissue from 46 patients were used. Tissues were stained for CD36 and Psap expression and the intensity was then scored using the staining index (SI) method (R. Catena et al., Bone marrow-derived Gr1+ cells can generate a metastasis-resistant microenvironment via induced secretion of thrombospondin-1. Cancer Discov 3, 578-589 (2013)). Sixty-one percent of tissues from normal ovaries expressed CD36 with a mean SI of 2.39 (maximum possible score is 9) (Figures 15A-15C, 15M and Table 1). Analysis of 134 primary ovarian tumors revealed that 97% of tumors (130 / 134) stained positive for CD36 with a mean SI of 5.32 (Figures 15D-15F, 15M and Table 1). 121 visceral metastases from 134 patients were examined. 97% (117 / 121) of metastatic lesions stained positive for CD36 with a mean SI of 6.61 (Figures 15G-15I, 15M and Table 1). Finally, 100% (13 / 13) of lymph node metastases stained positive for CD36 with a mean SI of 6.69 (Figures 15J-15L, 15M and Table 1).

[0093] [Table 1]

[0094] The expression of prosaposin in human ovarian cancer patients was investigated, speculating that its levels would decrease with tumor progression based on its mechanism of action. Prosaposin levels in ovarian cancer TMA samples were investigated. Normal ovaries expressed relatively low levels of prosaposin, with a mean SI of 4.29 (Figure 15N and Table 2). Primary ovarian tumors expressed higher levels of prosaposin, with a mean SI of 5.17 (Figure 15N and Table 2). Surprisingly, in visceral and lymph node metastases, prosaposin levels were significantly decreased, with mean SIs of 4.14 and 4.07, respectively (Figure 15N and Table 2). Thus, taken together, CD36 expression is increased in primary ovarian tumors compared to normal ovaries, and CD36 expression in metastatic lesions is further increased compared to primary tumors, while prosaposin expression decreases with tumor progression. These findings suggest that metastatic ovarian tumors suppress prosaposin expression but retain CD36, which could be used to effectively target ovarian cancer with prosaposin-derived therapies. [Table 2]

[0095] Consideration It was previously demonstrated that prosaposin and short five amino acid peptides derived from it can potently inhibit metastasis (3, 4). The development process of cyclic peptides derived from prosaposin has been depicted with significantly higher activity and stability than the native peptide. Incorporation of d-amino acids at the first and third residues of the native linear peptide was demonstrated to increase in vivo activity. The peptide was further modified by cyclizing the five amino acid peptide via backbone NC cyclization to make it more drug-like. The cyclic peptide shows even higher in vivo activity than the d-amino acid linear peptide. Both modified peptides were demonstrated to be more potently able to regress established metastases in a PDX model of ovarian cancer. The cells used in this PDX model were derived from a patient resistant to platinum, the most common first-line treatment for ovarian cancer patients.

[0096] Analysis of human ovarian cancer cell lines derived directly from patient ascites revealed that all tested cell lines expressed CD36, the receptor for Tsp-1, a downstream target of Psap and peptide in bone marrow-derived cells. Recombinant Tsp-1 was demonstrated to induce apoptosis in these serous ovarian cancer cells expressing CD36. The in vitro activity of Tsp-1 against ovarian cancer cells was recapitulated in a PDX model of ovarian cancer, where cyclic Psap peptide upregulated Cd11b mobilized to the peritoneal cavity of metastasis-bearing mice. + / Gr1 + We stimulated the expression of Tsp-1 in bone marrow-derived cells, and induction of Tsp-1 expression in these cells led to a marked induction of apoptosis and marked regression of tumor cells.

[0097] Based on these results, Psap peptides appear to have the potential to inhibit ovarian cancer progression through three distinct mechanisms, all of which are mediated by induction of Tsp-1. The first, demonstrated here, is direct cell killing, mediated by downstream signaling from CD36 triggered by Tsp-1. The second is through the well-established antiangiogenic activity of Tsp-1 (DJ Good et al., A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc Natl Acad Sci USA 87, 6624-6628 (1990)). Third, through Tsp-1 binding to another of its cell surface receptors, CD47, blocking the "don't eat me" signal mediated by CD47 binding to SIRPα on macrophages (P. Burger, P. Hilarius-Stokman, D. de Korte, TK van den Berg, R. van Bruggen, CD47 functions as a molecular switch for erythrocyte phagocytosis. Blood 119, 5512-5521 (2012);A. Saumet, MB Slimane, M. Lanotte, J. Lawler, V. Dubernard, Type 3 repeat / C-terminal domain of thrombospondin-1 triggers caspase-independent cell death through CD47 / alphavbeta3 in promyelocytic leukemia NB4 cells. Blood 106, 658-667 (2005)).

[0098] Finally, an analysis of tumor tissue from 134 patients with serous ovarian cancer revealed that 97% expressed CD36 and that this expression was not only maintained in metastatic lesions, but that the level of CD36 expression actually increased with tumor progression. The most common first-line treatment strategy for ovarian cancer patients is platinum-based chemotherapy (E. Lengyel, Ovarian cancer development and metastasis. Am J Pathol 177, 1053-1064 (2010)). 70% of ovarian cancer patients develop resistance to this treatment (E. Lengyel, Ovarian cancer development and metastasis. Am J Pathol 177, 1053-1064 (2010)). For these patients, there are no effective FDA-approved therapeutics, and therefore the survival rate is ~17% (E. Lengyel, Ovarian cancer development and metastasis. Am J Pathol 177, 1053-1064 (2010)). The findings presented here suggest that Psap-based therapeutics may have important benefits for the majority of ovarian cancer patients, based on their mechanism of action and the widespread CD36 expression on tumor cells in these patients.

[0099] References [Table 3]

[0100] [Table 4]

[0101] Without further elaboration, it is believed that one skilled in the art can utilize the present disclosure to its fullest extent based on the above description. The specific embodiments are therefore to be construed as merely illustrative, and not limiting in any way to the remainder of the disclosure. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein. The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated to the contrary, are understood to mean "at least one." From the above description, those skilled in the art can easily ascertain the essential features of the present disclosure, and can make various changes and modifications to the present disclosure to adapt it to various applications and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are also within the scope of the claims.

Claims

1. 1. A method for treating a subject having cancer, the method comprising: The method comprises administering to a subject having cancer an effective amount of a cyclic peptide to treat the cancer, wherein the amino acid sequence of the cyclic peptide is DWLPK (SEQ ID NO: 1).

2. 2. The method of claim 1, wherein the cancer is ovarian cancer or melanoma.

3. 1. A composition for use in the treatment of cancer, the composition comprising a cyclic peptide, wherein the amino acid sequence of the cyclic peptide is DWLPK (SEQ ID NO:1).

4. The composition of claim 3 , wherein the cancer is ovarian cancer or melanoma.

5. 1. A method for treating a subject having an inflammatory disease or disorder, the method comprising: The method comprises administering to a subject having an inflammatory disease or disorder an effective amount of a cyclic peptide for treating the inflammatory disease or disorder, wherein the amino acid sequence of the cyclic peptide is DWLPK (SEQ ID NO: 1).

6. 6. The method of claim 5, wherein the inflammatory disease or disorder is rheumatoid arthritis, age-related macular degeneration (AMD), Crohn's disease, psoriasis, or atherosclerosis.

7. 7. The method of claim 6, wherein the inflammatory disease or disorder is Crohn's disease.

8. 13. A composition for use in treating an inflammatory disease or disorder, the composition comprising a cyclic peptide, wherein the amino acid sequence of the cyclic peptide is DWLPK (SEQ ID NO:1).

9. 9. The composition of claim 8, wherein the inflammatory disease or disorder is rheumatoid arthritis, age-related macular degeneration (AMD), Crohn's disease, psoriasis, or atherosclerosis.

10. The composition of claim 9, wherein the inflammatory disease or disorder is Crohn's disease.

11. 1. A method for stimulating expression of thrombospondin-1 (Tsp-1), the method comprising: The method comprises administering to a subject in need thereof an effective amount of a cyclic peptide to stimulate expression of Tsp-1, wherein the amino acid sequence of the cyclic peptide is DWLPK (SEQ ID NO:1).

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