WO2011103583A2 - Methods and compositions related to anti-angiogenic peptides - Google Patents

Abstract

Embodiments are directed to compositions and methods of using compositions comprising anti-angiogenic peptides derived from the SPARC polypeptide. The methods include treating conditions related to pathological or abnormal vascularization by administering anti- angiogenic compositions.

Description

DESCRIPTION

METHODS AND COMPOSITIONS RELATED TO ANTI-ANGIOGENIC PEPTIDES

[0001] This application claims priority to U.S. Provisional Patent Application serial number 61/306,742 filed on February 22, 2010, which is incorporated herein by reference in its entirety.

[0002] This invention was made with government support under NS 049814 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

I. FIELD OF THE INVENTION

[0003] Embodiments of this invention are directed generally to biology and medicine. Certain embodiments are directed to anti-cancer or anti-angiogenic cyclic peptides or mimetics thereof.

II. BACKGROUND

[0004] Angiogenesis is the process by which new blood vessels form by developing from pre-existing vessels. This multi-step process involves signaling to endothelial cells, which results in (1) dissolution of the membrane of the originating vessel, (2) migration and proliferation of the endothelial cells, and (3) formation of a new vascular tube by the migrating cells (Alberts et al, 1994). While this process is employed by the body in beneficial physiological events such as wound healing and myocardial infarction repair, it is also exploited by unwanted cells such as tumor cells, and in undesirable conditions such as atherosclerosis, inflammatory conditions such as dermatitis, psoriasis, and rheumatoid arthritis, as well as eye diseases such as diabetic retinopathy and macular degeneration.

[0005] Angiogenesis is required for the growth and metastasis of solid tumors. Studies have confirmed that in the absence of angiogenesis, tumors rarely have the ability to develop beyond a few millimeters in diameter (Isayeva et al, 2004). Angiogenesis is also necessary for metastasis formation by facilitating the entry of tumor cells into the blood circulation and providing new blood vessels that supply nutrients and oxygen for tumor growth at the metastatic site (Takeda et al, 2002). [0006] Abnormal neovascularization is also seen in various eye diseases, where it results in hemorrhage and functional disorder of the eye, contributing to the loss of vision associated with such diseases as retinopathy of prematurity, diabetic retinopathy, retinal vein occlusion, and age-related macular degeneration (Yoshida et al, 1999). These conditions are the leading causes of blindness among infants, those of working age and the elderly (Aiello, 1997).

[0007] There is a need for additional compositions and methods for modulating angiogenesis and treating those conditions related to aberrant angiogenesis.

SUMMARY OF THE INVENTION

[0008] The Inventors have previously shown that full-length Secreted Protein Acidic and Rich in Cysteine (SPARC) and a SPARC peptide corresponding to the follistatin domain of the protein (FS-E) potently block angiogenesis and inhibit the growth of neuroblastoma tumors in preclinical models. Peptide FS-E is structurally complex and difficult to produce, limiting its potential as a therapeutic in the clinic. Two smaller and structurally more simple SPARC peptides, FSEN and FSEC, were designed based on the amino acid sequence of the N- and C-terminal loops of the peptide FS-E, respectively. Both peptides FSEN and FSEC have anti-angiogenic activity in vitro and in vivo. Histologic examination of the treated and control neuroblastoma tumors demonstrated that both SPARC peptides induced changes in blood vessel architecture that were consistent with blood vessel normalization. In control neuroblastoma xenografts, the characteristic features of tumor angiogenesis were seen with structurally abnormal, tortuous blood vessels. In contrast, the blood vessels observed in SPARC-peptide treated tumors were thin walled and structurally more normal. These SPARC peptides are anti-angiogenic and cause normalization of blood vessels. These properties, together with simple structure and ease of production, render these peptides suitable for clinical use.

[0009] Certain embodiments are directed to compositions that comprise a cyclic molecule having chemical groups similar to those of FSEN or FSEC. Further embodiments are directed methods of treating conditions related to pathological or abnormal vascularization by administering anti-angiogenic compositions described herein. [0010] Certain aspects of the invention are directed to cyclic anti-angiogenic peptides of at st or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids comprising an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:l or SEQ ID NO:2. In certain embodiments, the molecule will be a non-peptide polymer that mimics the peptide backbone and has functional groups similar to or identical to those in the peptides described herein. In certain aspects, the peptide or peptide mimetic comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO:l or SEQ ID NO:2. In further aspects, the peptide or peptide mimetic comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2. In still further aspects, the peptide or peptide mimetic comprises the amino acid sequence of SEQ ID NO.l or the amino acid sequence of SEQ ID NO:2.

[0011] Further aspects are directed to an anti-angiogenic composition comprising at least one cyclic peptide of at least, at most, or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:l or SEQ ID NO:2. [0012] Certain aspects are directed to an anti-angiogenic agent comprising a cyclic peptide having the formula: X_! QNHH AKHGKVX₂ (SEQ ID NO:l) or X₁ELDEN TPMX₂ (SEQ ID NO:2); wherein Xj and/or X₂ are chemical moieties and X] is or can be coupled to X₂ forming a cyclic peptide. In certain aspects Xi and X₂ are coupled by a disulfide bond. In a further aspect, Xi and X₂ can be independently selected from mercaptopropionyl (Mpr), mercaptovaleryl (Mvl), cysteine, penicillamine (Pen), p^p-pentamethylene-p- mercaptopropionic acid (Pmp), or amino-puP-pentamethylene-P-mercaptopropionic acid (Pmc). In still a further aspect, X\ and X₂ are cysteine. Any pair of functional groups that are known to be capable of chemical coupling are contemplated for X\ and X₂. In a further aspects, a peptide described herein can be cyclized using a non-amino acid linker or cyclizing moiety.

[0013] In other aspects, an anti-angiogenic agent can comprise a cyclic polymer having the formula: XiQNHHAKHGKVX₂ or X₁ELDENNTPMX₂; wherein Xi and/or X₂ are chemical moieties and

is coupled to X₂ forming a cyclic molecule. In certain aspects Xi and X₂ are coupled by a disulfide bond. In further aspects Xi and X₂ are independently selected from mercaptopropionyl (Mpr), mercaptovaleryl (Mvl), cysteine, penicillamine (Pen), βι,β-^nt^am^pthvlene-P-mercaptopropionic acid (Pmp), or amino-Pi,P-pentamethylene-P- opionic acid (Pmc). In certain aspects X_! and X₂ are cysteine. [0014] Embodiments include methods of inhibiting pathological vascularization in a subject comprising contacting a tissue or tumor having or suspected of having pathological vascularization with an effective amount of cyclic peptide of 5, 6, 7, 8, 9, 10, 11, 12 to 20 or more amino acids comprising an amino acid sequence that is at least 70, 80, or 90% identical to the amino acid sequence of SEQ ID NO:l or SEQ ID NO:2. In certain aspects the pathological vascularization is associated with a tumor or an ophthalmologic disorder. The tumor can be a sarcoma, carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell, carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, ganglioneuroblastoma, neuroblastoma, or retinoblastoma. In certain aspects the tumor is a neuroblastoma. The opthamlologic disorder can be neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasias, uveitis, retinopathy of prematurity, macular degeneration, or corneal graft neovascularization.

[0015] In certain aspects the cyclic peptide is administered locally or systemically. In a further aspect the cyclic peptide is administered orally, intravascularly, topically, intraocularly, or intratumorally. The cyclic peptide can be administered at a dose of 0.001, 0.01, 0.1, 0.5, 1, 5, 10, 15 mg/kg/day to 5, 10, 15, 20, 50, 100 mg/kg/day. In certain aspects 0.001, 0.01, 0.1, 0.5, 1, 5, 10, 15 mg to 5, 10, 15, 20, 50, 100 mg of a composition or a polymer described here can be administered per dose.

[0016] The methods can further comprising a second anti-tumor therapy. In certain aspects the second anti-tumor therapy is a chemotherapy, a radiotherapy, an immunotherapy, an anti- angiogenic therapy, or surgery. In particular aspects the second anti-tumor therapy is py. The chemotherapy can be, but is not limited to paclitaxel, Abraxane, fluorouracil, irinotecan, vitamin D, taxol, doxorubicin, etoposide, Tarceva, Gefitinib, Fluoro- Sorafenib, Sorafenib, or PF-2341066.

[0017] Certain aspects are directed to methods of treating cancer comprising administering to a subject having cancer or at risk for developing cancer or at risk for recurrence of cancer an effective amount of a peptide composition comprising a cyclic peptide having an amino acid sequence of SEQ ID NO:l or SEQ ID NO:2.

[0018] Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. The embodiments in the Example section are understood to be embodiments of the invention that are applicable to all aspects of the invention.

[0019] The terms "inhibiting," "reducing," or "prevention," or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result. [0020] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0021] It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.

[0022] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0023] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." It is also contemplated that anything listed using the term "or" may also be specifically excluded.

[0024] As used in this specification and claim(s), the words "comprising" (and any form of such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0025] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. DESCRIPTION OF THE DRAWINGS

[0026] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [0027] FIG. 1. SPARC peptides FSEN and FSEC. SPARC peptides FSEN and FSEC were designed to correspond to the N-terminal and C-terminal regions of peptide FS-E, respectively. The native cysteine linkage was preserved in peptide FSEN; the unpaired cysteine was replaced with alanine. To maintain the conformation of peptide FSEC, cysteine 4 was linked with cysteine 3 instead of cysteine 2. [0028] FIG. 2. SPARC peptides FSEN and FSEC inhibit endothelial cell migration. A

- Peptide FSEN inhibited basic fibroblast growth factor (bFGF)-stimulated endothelial migration with an EC₅₀ of ~2 nM. B - Peptide FSEC displayed a strong dose-dependent inhibition of bFGF-stimulated endothelial migration with an EC₅₀ ~1 pM. Dark circles represent bFGF-stimulated migration; light circles represent basal migration in the absence of an activator.

[0029] FIG. 3. Inhibition of neovascularization by peptides FSEN and FSEC in the Matrigel plug assay. A - Gross appearance of Matrigel plugs containing bFGF alone (positive control), phosphate-buffered saline (PBS, negative control), and bFGF with 10 uM SPARC peptides FSEC, FSEN, or scrambled control peptides scFSEN and scFSEC. B - For ' ^' analysis of angiogenesis and blood vessel architecture endothelial cells were visualized with green CD31 immunofluorescence, and pericytes were detected with red anti- a-smooth muscle actin (SMA) antibody. Representative photographs at x400 magnification are shown. C - The relative quantity of endothelial cells and pericytes was estimated by calculating the area occupied by green and red fluorescence (in pixels). There were statistically significant decreases in the blood vessel area and quantity of pericytes in the Matrigel plugs containing SPARC peptides compared to the positive control with bFGF alone (single asterisk) and from the negative control (double asterisk).

[0030] FIG. 4. Inhibition of neuroblastoma tumor progression by the SPARC peptides FSEN and FSEC in the preclinical model of neuroblastoma. A - Treatment with the SPARC peptide FSEC resulted in a statistically significant (p<0.05) decrease in the average size of xenografted neuroblastoma tumors starting from day 4 until the end of the treatment period. The average tumor weight was reduced to 26% (p=0.01) of average control tumor weight. The average weight of tumors treated with peptide FSEN was reduced to 88%, but the decrease was not statistically significant (p=0.83). Scrambled peptide did not affect tumorigenicity of neuroblastoma xenografts (102%, p=0.97). B - Representative photographs of neuroblastoma tumors treated with the SPARC peptides. C - Normalization of the blood vessels in the SPARC peptide-treated xenografts. H&E staining of areas with large blood vessels at x200 and x400 magnification shows areas of extensive hemorrhage and microvascular proliferation (MVP) in control tumors and tumors treated with the scrambled peptides.

[0031] FIG. 5. Inhibition of tumor-induced angiogenesis by peptides FSEN and FSEC in the animal model. For quantitative analysis of angiogenesis in the mouse xenografts, paraffin sections were stained with green CD31, and red SMA immunofluorescence. A - Angiogenesis was quantified by calculating the area occupied by green CD31 -positive endothelial cells and red SMA-positive pericytes. The quantity of tumor blood vessels was statistically significantly decreased in the SPARC peptide-treated xenografts compared to vehicle treated control (p < 0.001; marked with an asterisk). Treatment with the scrambled peptide did not affect angiogenesis in the xenografted tumors. B - Representative photographs at x 100 magnification. [0032] FIG. 6. Blood vessel architecture in the peptide-treated murine neuroblastoma xenografts. Endothelial cells and pericytes were visualized with green CD31 and red SMA spectively. Aberrant blood vessels architecture was evident at the x400 magnification in the control xenografts treated with the vehicle or scrambled peptide. Peptide-treated tumors had more structurally normal, thin- walled blood vessels.

DETAILED DESCRIPTION OF THE INVENTION

[0033] Neuroblastoma tumors are one model for vascularized tumors. Neuroblastoma tumors exhibit a broad spectrum of clinical behavior, reflective of their biologic heterogeneity (Maris et al, 2007). Although significant progress has been made in the successful treatment of neuroblastoma tumors with favorable biology, more effective therapeutic strategies are still needed for children with high-risk neuroblastoma (Oppedal et al, 1989). A strong correlation between high-risk tumors and angiogenesis has been reported, suggesting that blood vessels may be clinically relevant therapeutic targets. In support of this hypothesis, preclinical studies have demonstrated that neuroblastoma tumor growth can be significantly impaired following treatment with anti-angiogenic agents ((Reiher et al, 2002; Katzenstein et al, 1999; Chesler et al, 2007).

[0034] The histologic features of neuroblastoma tumors have also been shown to be prognostic, and an abundance of Schwannian stroma is associated with a more benign tumor phenotype and favorable prognosis (Shimada et al, 1999). Schwann cells are known to secrete factors that induce neuroblastoma differentiation (Ambros et al, 2001; Kwiatkowski et al, 1998). Studies have demonstrated that Schwann cells also influence neuroblastoma tumor growth by secreting inhibitors of angiogenesis (Kwiatkowski et al, 1998), the most potent of which is Secreted Protein Acidic and Rich in Cysteine (SPARC) (Chlenski et al, 2002). SPARC belongs to a group of non-structural components of the extracellular matrix (ECM) that modulate interactions between cells and their environment (Sage et al, 1984; Tai and Tang, 2008). It is highly expressed in a variety of cell types associated with remodeling tissues (Mundlos et al, 1992). Although the mechanism for its anti-angiogenic activity is not well understood, SPARC is capable of interfering with the binding of angiogenic stimulators vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and bFGF to their receptors in endothelial cells, resulting in inhibited proliferation (Hasselaar and Sage et al 1992). SPARC has also been shown to down-regulate VEGF in glioma cells (Yunker et al, 2008). [0035] Previously, the inventors synthesized peptides corresponding to the highly «^^η«Η structural domains of SPARC and tested their ability to inhibit angiogenesis al, 2004). To maintain the structural integrity of the native modules, cysteines within the peptides were linked with disulfide bonds during the synthesis. Minimal to no inhibitory activity was observed with the peptides corresponding to the Kazal module and the a-helix of the EC domain. In contrast, the EGF-like module peptide (FS-E) strongly inhibited endothelial cell migration in vitro and angiogenesis in vivo. Reduction of the two disulfide bonds in the FS-E peptide completely abrogated the angiogenesis inhibitory effects, indicating that structural conformation is critical for this biological activity. The inventors have designed two additional SPARC peptides that structurally correspond to N- and C- terminal loops of the FS-E peptide: FSEN and FSEC, respectively. These peptides are smaller, less structurally complex, and easier to produce than the FS-E peptide. These peptides block angiogenesis and effectively inhibit neuroblastoma tumor growth in a preclinical model.

I. SPARC polypeptide and related peptides

[0036] SPARC, also known as osteonectin, is a matricellular glycoprotein. SPARC has affinity for a wide variety of ligands including cations (e.g., Ca2+, Cu2+, Fe2+), growth factors (e.g., PDGF and VEGF), ECM proteins (e.g., collagen I-V and collagen IX, vitronectin, and thrombospondin-1), endothelial cells, platelets, hydroxyapaptite, and albumin. SPARC expression is developmentally regulated, and is predominantly produced in tissues undergoing remodeling during normal development or in response to injury (see, e.g. , Lane et al, 1994). High levels of SPARC protein are expressed in developing bones and teeth. SPARC is also upregulated in several aggressive cancers, but is absent from the vast majority of normal tissues (Porter et al, 1995). SPARC expression is often induced in a variety of tumors (e.g., bladder, liver, ovary, kidney, gut, and breast).

[0037] The inventors have synthesized peptides corresponding to the highly conserved structural domains of SPARC and tested their ability to inhibit angiogenesis (Chlenski et al, 2004). To maintain the structural integrity of the native modules, cysteines within the peptides were linked with disulfide bonds during the synthesis. Minimal to no inhibitory activity was observed with the peptides corresponding to the Kazal module and the a-helix of the EC domain. In contrast, the EGF-like module peptide (FS-E) strongly inhibited endothelial cell migration in vitro and angiogenesis in vivo. Reduction of the two disulfide bonds in the FS-E peptide completely abrogated the angiogenesis inhibitory effects, indicating that structural conformation is related to its biological activity. Two additional •tides have been designed that structurally correspond to N- and C-terminal loops of the FS-E peptide: FSEN and FSEC, respectively. These peptides are smaller, less structurally complex, and easier to produce than the FS-E peptide. Both peptides block angiogenesis in vitro and in vivo, although FSEC is more potent and effectively inhibits neuroblastoma tumor growth in a preclinical model. [0038] SPARC peptides FSEN (CQNHHAKHGKVC) (SEQ ID NO:3) and FSEC (CELDENNTPMC) (SEQ ID NO:4) were synthesized as cyclic cysteine-linked molecules at Alpha Diagnostics International (San Antonio, TX) using fmoc/tboc chemistry. The purity of synthesis was assessed by high-performance liquid chromatography. The molecular mass of the peptides was checked by mass spectrometry. Control scrambled peptides scFSEN (KCGHKHQCAVHN) (SEQ ID NO:5) and scFSEC (MEPECNLNCTD) (SEQ ID NO:6), which contain the same amino acids as peptides FSEN and FSEC in a random order, were made without special modifications.

[0039] The inventors have shown that full length SPARC and a SPARC peptide (FS-E) that corresponds to the highly conserved epidermal growth EGF-like module of the follistatin domain potently inhibit angiogenesis and neuroblastoma tumor growth in preclinical models (Chlenski et al, 2002; Chlenski et al, 2004; Chlenski et al, 2006). The structure of the FS-E peptide is complex, and the inventors have demonstrated that its anti-angiogenic function is conformation-dependent (Chlenski et al, 2004). In this study two structured SPARC peptides were designed and synthesized that correspond to the N- (FSEN) and C-terminal (FSEC) portions of peptide FS-E in order to develop a therapeutic that may be suitable for clinical use. Because proper structural conformation was shown to be imperative to maintain activity for the FS-E peptide, peptides FSEN and FSEC were folded into their native conformation by linking the end cysteines during synthesis with disulfide bonds. Due to the close proximity of the two central cysteines in the peptide FS-E (Hohenester et al, 1997), cysteine 4 was linked with cysteine 3 instead of cysteine 2. This allowed the inventors to produce peptides that correspond to amino acid sequences in the N- and C-terminal loops without disturbing the native folding. It was found that both FSEN and FSEC function as inhibitors of angiogenesis, although the FSEC peptide was more potent. Peptide FSEC also significantly suppressed neuroblastoma tumor growth in a mouse xenograft model. Consistent with its properties as an inhibitor of angiogenesis, significantly reduced numbers of endothelial and perivascular cells were present in the SPARC peptide-treated tumors

1 ' ' controls. [0040] Although the anti-tumor and anti-angiogenic effects of peptide FSEN were less potent, both SPARC peptides had profound effects on the architecture of tumor-induced blood vessels. In contrast to the structurally abnormal blood vessels that were seen in the control tumors, thin walled blood vessels were detected in the peptide-treated tumors, suggesting that treatment with FSEN and FSEC induced blood vessel normalization. Accordingly, hemorrhage was not detected in the peptide-treated tumors, whereas significant hemorrhage was detected in the control tumors.

[0041] It is well established that cancer blood vessels are not structurally normal (Jain, 2005). Multiple layers of hypertrophic endothelial cells alternate with areas in which endothelial cell coverage is lacking. Corresponding abnormalities in the deposition of the basement membrane are also commonly observed. Perivascular smooth muscle cells, which provide both mechanical and physiological support for the endothelial monolayer in normal blood vessels, fail to co-localize with endothelial cells in neoplastic blood vessels. These abnormalities disrupt the integrity of the blood vessels, resulting in a heterogeneous blood supply of the tumor tissue, vessel leakiness, and hemorrhage. Our recent evaluation of blood vessel architecture in a series of neuroblastoma tumors demonstrated that structurally abnormal blood vessels are commonly seen in high-risk tumors, and the presence of MVP was statistically significantly associated with decreased survival (Peddinti et al., 2007).

[0042] Normalization of neoplastic blood vessels has been demonstrated with other anti- angiogenic therapeutics (Jain, 2005), and recently the extent of vascular normalization following treatment with an anti-VEGF therapy has been shown to be predictive of outcome in patients with glioblastoma (Sorensen et al., 2009). Emerging evidence also indicates that by normalizing the abnormal structure and function of tumor vasculature, anti-angiogenic agents can alleviate hypoxia and increase the efficacy of conventional therapies (Jain, 2005). A recently complete phase I dose-escalation study testing an anti-VEGF agent has provided evidence of both vascular normalization and sensitization of rectal tumors to radiation (Willett et al, 2005; Jain et al, 2006).

[0043] As used herein, a "peptide," "protein," or "polypeptide" refers to a molecule comprising at least, at most, or about 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid residues. In some embodiments, a wild-type version of a peptide or polypeptide are employed, however, in many embodiments of the invention, a modified peptide or is employed. The terms described above may be used interchangeably. A "modified peptide" or "modified polypeptide" or a "variant" refers to a peptide or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type or parent peptide or polypeptide.

[0044] In certain embodiments the size of a peptide, protein, or polypeptide (parent or modified peptide) may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino molecules or more, including all ranges derivable therein, or derivative of a corresponding amino sequence described or referenced herein. It is contemplated that peptides may be modified by truncation, rendering them shorter than their corresponding wild-type form, but also they might be altered by fusing or conjugating a heterologous protein sequence with a particular function (e.g., for targeting, localization, or purification purposes, etc.).

[0045] As used herein, an "amino molecule" refers to any amino acid, amino acid derivative, or amino acid mimic known in the art. The amino molecules may be in any available optical isomer known in the art. For example, the peptide compositions disclosed herein may comprise one or more D-amino acid. In certain embodiments, the residues of the proteinaceous molecule are sequential, without any non-amino molecule interrupting the sequence of amino molecule residues. In other embodiments, the sequence may comprise one or more non-amino molecule moieties. In particular embodiments, the sequence of residues of the proteinaceous molecule may be interrupted by one or more non-amino molecule moieties.

[0046] Accordingly, the term "proteinaceous composition" encompasses amino molecule sequences comprising at least one of the 20 common amino acids, or at least one modified or unusual amino acid.

[0047] Proteinaceous compositions may be made by any technique known to those of skill in the art, including (i) the expression of proteins, polypeptides, or peptides through standard molecular biological techniques, (ii) the isolation of proteinaceous compounds from natural sources, or (iii) the chemical synthesis of proteinaceous materials.

[0048] Amino acid sequence variants of SPARC peptides can be substitutional, insertional, or deletion variants. A variation in a polypeptide of the invention may affect 1, 2, 3. 4. 5. or more non-contiguous or contiguous amino acids of the peptide, as compared to the de. A variant can comprise an amino acid sequence that is at least, at most, or about 50%, 60%, 70%, 80%, or 90%, including all values and ranges there between, identical to any sequence provided or referenced herein. A variant can include 2, 3, 4, 5, or more substitute amino acids or amino acid mimetic.

[0049] Deletion variants typically lack one or more residues of the native or parent peptide. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein. Insertional mutants typically involve the addition of material at a non-terminal point in the polypeptide. This may include the insertion of one or more residues. Terminal additions, called fusion proteins, may also be generated. These fusion proteins include multimers or concatamers of one or more peptide or polypeptide described or referenced herein.

[0050] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions may be non-conservative such that a function or activity of the peptide is affected. Non-conservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.

[0051] Peptides of the invention may be recombinant, or synthesized in vitro. Alternatively, a non-recombinant or recombinant protein may be isolated from bacteria.

[0052] The term "functionally equivalent codon" is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to encode biologically equivalent amino acids. Codon Table

Amino Acids Codons

Alanine Ala A GCA GCC GCG GCU

Cysteine Cys C UGC UGU

Aspartic acid Asp D GAC GAU

Glutamic acid Glu E GAA GAG

Phenylalanine Phe F UUC UUU

Glycine Gly G GGA GGC GGG GGU

Histidine His H CAC CAU

Isoleucine He I AUA AUC AUU

Lysine Lys K AAA AAG

Leucine Leu L UUA UUG CUA CUC CUG CUU

Methionine Met M AUG

Asparagine Asn N AAC AAU

Proline Pro P CCA CCC CCG CCU

Glutamine Gin Q CAA CAG

Arginine Arg R AGA AGG CGA CGC CGG CGU

Serine Ser S AGC AGU UCA UCC UCG UCU

Threonine Thr T ACA ACC ACG ACU

Valine Val V GUA GUC GUG GUU

Tryptophan Trp w UGG

Tyrosine Tyr Y UAC UAU

[0053] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity (e.g., anti-angiogenic activity) where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.

[0054] It is contemplated that in compositions of the invention, there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and/or protein per ml. The concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or any range derivable therein). Of this, about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, ^ A7 AS *g_f 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% may be a SPARC peptide, and may be used in combination with other peptides or polypeptides.

[0055] The present invention contemplates the administration of SPARC peptides to effect an anti-angiogenic and/or anti-cancer therapy or therapeutic effect against the development of a disease or condition associated with neovascularization or hyperproliferative growth.

[0056] In certain aspects, combinations of anti-angiogenic peptides and compounds are used in the production of a therapeutic composition that is effective at treating neovacularization, pathologic vascularization or cancer. [0057] In certain embodiments, peptides or peptide mimetics include one or more residues capable of forming a bond and cyclizing the peptide. These residues or chemical moieties can be an amino acid or non-amino acid that may be reacted to form a bond between a first and a second residue or moiety. For example, the first or second moiety can be a naturally occurring amino acid (i.e., Ala, Cys, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, or Tyr) or a residue having a sulfhydryl group such as Mpr (mercaptopropionyl), Mvl (mercaptovaleryl), Cys, Pen (Penicillamine), Pmp (Β^β- pentamethylene-B-mercaptopropionic acid), and Pmc (ammo-fit, B-pentamethylene-B- mercaptopropionic acid). In certain embodiments, one of moieties is not Q, K, or R.

[0058] In certain embodiments, a linear peptide may be cyclized using a disulfide bond, between two cysteine residues or between a cysteine and a Mpr, Pen, or Mvl residue. Further examples of linkages that may be used to generate a cyclic peptide include, but are not limited to a peptide bond formed between the NH₂ of the N-terminal residue and the COOH of the carboxy terminal residue, or an amide bond (i.e., -C(O)-NH-) formed between an amino acid side chain (e.g., of a lysine) and a side chain of another amino acid (e.g., Glu or Asp), or an ester bond (i.e., -C(O)-O-) formed between a side chain hydroxyl (e.g., of a threonine) and a C-terminal COOH or COOH of a side chain of a Glu or Asp.

[0059] Cyclization may also be achieved through a click chemistry reaction (see e.g., Hein et al. 2008). For example, a first moiety can be bound to a second moiety for peptides of the invention through a 1 ,4-disubstituted 1,2,3-triazolyl group:

, where

Z¹ and Z² are independently selected -(CH₂)_n- groups, wherein n is an integer from 1 to 7, and wherein if n is an integer from 3 to 7, then one or two non-adjacent CH₂ groups may be replaced with groups independently selected from the group consisting of: -C(O)-, -NH-, - N(CH₃)-, -C(0)- H-, -S-, -C(0)-0-, and -0-. In certain embodiments one CH₂ group may be replaced with groups independently selected from the group consisting of: -C(O)-, -NH-, - N(CH₃)-, -C(0)-NH-, -S-, -C(0)-0-, and -0-. This cyclization through the 1,4-disubstituted 1,2,3-triazolyl group may be achieved, for example, by the Cu'-catalyzed Huisgen 1,3-dipolar cycloaddition of an azide and a terminal alkyne group (see e.g., Hein et al. (2008)). [0060] e bound to a second moiety through a 1,2,3-

triazol-l,

₉ where Z and Z are independently selected -

(CH₂)_n- groups, wherein n is an integer from 1 to 7, and wherein if n is an integer from 3 to 7, then one or two non-adjacent CH₂ groups may be replaced with groups independently selected from the group consisting of: -C(O)-, -NH-, -N(CH₃)-, -C(0)-NH-, -S-, -C(0)-0-, and -0-. In certain embodiments one CH₂ group may be replaced with groups independently selected from the group consisting of: -C(O)-, -NH-, -N(CH₃)-, -C(0)-NH-, -S-, -C(0)-0-, and -0-. This cyclization through the l,2,3,-triazol-l,4-yl group may be achieved, for example, by the Cu^!-catalyzed Huisgen 1,3-dipolar cycloaddition of an azide and a terminal alkyne group (see e.g., Hein et al. (2008)). [0061] In various embodiments, any of the peptides escribed herein, can bear one or more protecting groups. Thus, for example, the carboxyl terminus can be amidated. In various embodiments certain termini and/or side chains bear one or more blocking groups, the C- terminus, and/or N-terminus, and/or internal residues can be blocked with one or more blocking groups as described herein. [0062] A wide number of protecting groups are suitable for this purpose. Such groups include, but are not limited to acetyl, amide, and alkyl groups with acetyl and alkyl groups ilarly preferred for N-terminal protection and amide groups being preferred for carboxyl terminal protection. In certain embodiments, the protecting groups include, but are not limited to alkyl chains as in fatty acids, propeonyl, formyl, and others. Certain carboxyl protecting groups include, but are not limited to amides, esters, and ether-forming protecting groups. In one embodiment, an acetyl group can be used to protect the amino terminus and an amide group can be used to protect the carboxyl terminus. In certain aspects blocking groups include alkyl groups of various lengths, e.g., groups having the formula: CH₃ (CH₂)_n - CO- where n ranges from about 1 to about 20, preferably from about 1 to about 16 or 18, more preferably from about 3 to about 13, and most preferably from about 3 to about 10.

[0063] Other suitable protecting groups include, but are not limited to Fmoc, t- butoxycarbonyl (t-BOC), 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9- florenecarboxylic group, 9-fluorenone-l-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl- benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4- methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2- pyridinesulphenyl (Npys), l-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6- dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z), 2- bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), cyclohexyloxy (cHxO), t- butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA), and the like.

[0064] Various embodiments this invention also contemplates pegylated forms of the various protected or unprotected peptides of this invention. Pegylation can be used in improve biocompatibility of the peptides and/or to improve serum half-life. Methods of pegylating peptides are well known to those of skill in the art (see, e.g., U.S. Pat. Nos. 7,256,258, 6,552,170, and 6,420,339, and the references cited therein).

A. Polypeptides and Polypeptide Production

[0065] The present invention describes polypeptides, peptides, and proteins and variants thereof for use in various embodiments of the present invention. For example, specific peptides are assayed for or used as anti-angiogenic agents. In specific embodiments, all or part of the peptides of the invention can also be synthesized in solution or on a solid support in anrnrHa ce with conventional techniques. Various automatic synthesizers are y available and can be used in accordance with known protocols. See, for 25745 example, Stewart and Young, (1984); Tam et al, (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference.

[0066] Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence that encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.

B. Pharmaceutical Compositions

[0067] In some embodiments, pharmaceutical compositions are administered to a subject. Different aspects of the present invention involve administering an effective amount of a composition to a subject. Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0068] The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, or human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-cancer agents, can also be incorporated into the composition.

[0069] In addition to the compounds formulated for parenteral administration, such as those for intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; time release capsules; and any other form currently used, including creams, lotions, mouthwashes, inhalants and the like. The compositions can be administered locally or systemically.

[0070] The active compounds of the present invention can be formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes. The preparation of an aqueous composition that contains a compound or compounds of the invention will be known to those of skill in the art in light of the present disclosure.

such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0071] The proteinaceous compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0072] A carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0073] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with the various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0074] Administration of the compositions according to the present invention will typically be via any common route. This includes, but is not limited to oral, nasal, or buccal administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, intraocular, local administration ("locally administered") or intravenous injection. Locally administered refers a localized administration as in injecting or contacting a specific organ or tissue with a composition with minimal (if any) systemic exposure.

[0075] An effective amount of therapeutic composition is determined based on the intended goal - for instance reduction or inhibition of angiogenesis; reduction or inhibition of growth of a tumor; reduction in size of a tumor, or death of some or all cancer cells associated with a tumor; or reduction or amelioration of symptoms related to the presence of a tumor. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment. [0076] Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition.

[0077] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.

C. Lipid Components and Moieties

[0078] In certain embodiments, the present invention concerns compositions comprising ϊ lipids associated with a polypeptide/peptide. A lipid is a substance that is insoluble in water and extractable with an organic solvent. Compounds other than those specifically described herein are understood by one of skill in the art as lipids, and are encompassed by the compositions and methods of the present invention. A lipid component and a non-lipid may be attached to one another, either covalently or non-covalently. [0079] A lipid may be a naturally occurring lipid or a synthetic lipid. However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glucolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. [0080] A polypeptide/peptide, associated with a lipid may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid or otherwise associated with a lipid. A lipid or lipid-peptide composition of the present invention is not limited to any particular structure. For example, they may also simply be interspersed in a solution, possibly forming aggregates which are not uniform in either size or shape. In another example, they may be present in a bilayer structure, as micelles, or with a "collapsed" structure.

[0081] In certain embodiments, a composition may comprise about 1%, about 2%, about 3%, about 4% about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%), about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%), about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or any range therebetween, ar lipid, lipid type, or non-lipid component such as a peptide, polypeptide or other material disclosed herein or as would be known to one of skill in the art. In a non-limiting example, a composition may comprise about 10% to about 20% neutral lipids, and about 33% to about 34% of a cerebroside, and about 1% cholesterol. In another non-limiting example, a liposome may comprise about 4% to about 12% terpenes, wherein about 1% of the micelle is specifically lycopene, leaving about 3% to about 11% of the liposome as comprising other terpenes; and about 10% to about 35% phosphatidyl choline, and about 1% of a non-lipid component. Thus, it is contemplated that compositions of the present invention may comprise any of the lipids, lipid types or other components in any combination or percentage range. D. Combination Therapy

[0082] In order to increase the effectiveness of the peptide compositions described herein, it may be desirable to combine these compositions with other agents or therapy methods, such as anti-cancer agents. An "anti-cancer" agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with the peptide compositions described herein and the agent(s) or multiple factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the expression construct and the other includes the second agent(s).

[0083] One goal of cancer research is to find ways to improve the efficacy of chemo- and radiotherapy. In the context of the present invention, it is contemplated that the peptides or mimetics described herein could be used in conjunction with chemotherapeutic, radiotherapeutic, or immunotherapeutic intervention, in addition to other anti-angiogenic agents. Administration of the described compositions can precede or follow a second anticancer therapy or agent by intervals ranging from minutes to weeks. In embodiments where herapy or agent is applied separately to the cell or subject, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the treatments would still be able to exert an advantageously combined effect on the cell, tumor, or subject. In such instances, it is contemplated that one may contact the cell with or administer both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

[0084] Various combinations may be employed, the peptide or peptide mimetic is "A" and the secondary agent, such as radio- or chemotherapy, is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

[0085] Administration of the therapeutic compositions of the present invention to a patient will follow general protocols of administration, taking into account the toxicity, if any. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described therapy.

1. Chemotherapy

[0086] Cancer therapies also include a variety of combination therapies with both chemical and radiation based treatments. Combination chemotherapies include, for example, paclitaxel, ABRAXANE™, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, famesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin, methotrexate, or any analog or derivative variant of the foregoing.

2. Radiotherapy

[0087] Other factors that cause DNA damage and have been used extensively include what y known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[0088] The terms "contacted" and "exposed," when applied to a cell, tumor, or subject, are used herein to describe the process by which a therapeutic or a chemotherapeutic or a radiotherapeutic agent are delivered to a target or are placed in direct juxtaposition with the target cell. To achieve cell killing or stasis, both agents are delivered in a combined amount effective to treat a tumor or other condition.

3. Immunotherapy

[0089] Immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0090] Immunotherapy, thus, could be used as part of a combined therapy, in conjunction with a therapy described herein. Generally, a tumor cell target bears some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55. 4. Surgery

[0091] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.

[0092] Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancerous lesions, or incidental amounts of normal tissue.

[0093] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well. 5. Other agents

[0094] It is contemplated that other agents may be used in combination with the present invention to improve the therapeutic efficacy of treatment. These additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, or agents that increase the sensitivity of the cancer cells or cells associated with neovasculature to apoptotic inducers. Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, ΜΓΡ- lbeta, MCP-1, RANTES, and other chemokines. It is further contemplated that the upregulation of cell surface receptors or their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL would potentiate the apoptotic inducing abilities of the present invention by it of an autocrine or paracrine effect on target cells. Increases intercellular signaling by elevating the number of GAP junctions would increase the anti-proliferative effects on the neighboring target cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with the present invention to improve the efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a target cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve treatment efficacy.

[0095] Hormonal therapy may also be used in conjunction with the present invention or in combination with any other cancer therapy previously described. The use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases. [0096] Other methods and compositions further include administering to the patient the SPARC peptides of the invention in conjunction with other anti-angiogenic agents. These include are but are not limited to Nexavar, Thalomid, Avastin, Cilengitide, Exherin, WX- UK1, Combretastatin A-4 phosphate, GCS-100LE, PTK/ZK, AS-1404, Phosphomannopentose sulfate, Squalamine, talactoferrin alfa, ZD-6474, AP-23573, Volociximab, XL-999, or any analog or derivative variant thereof.

E. Cancers and hyperproliferative conditions

[0097] In some methods of the invention, the cancer cell is a tumor cell. Furthermore, the cell may be administered compositions of the invention in vitro, in vivo, or ex vivo. Thus, the cancer cell may be in a patient. The patient may have a solid tumor. In such cases, embodiments may further involve performing surgery on the patient, such as by resecting all or part of the tumor. The peptide compositions described herein may be administered to the patient before, after, or at the same time as surgery. In additional embodiments, patients may also be administered directly, endoscopically, intratracheally, intratumorally, intravenously, intralesionally, intramuscularly, intraperitoneally, regionally, percutaneously, topically, intrarterially, intravesically, subcutaneously, infusion, or continuous infusion. The peptide ^rv,^cit^ _s may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, ore times, and they may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, 5 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months.

[0098] In certain aspects, a peptide is provided to a patient intravenously. In certain embodiments, the infusion rate is initially from about 0.1, 1, 2, 3, 4, 5, pg, ng or μg /kg/min to about 2, 3, 4, 5, 10 pg, ng or μg/kg/min, including all ranges and values there between. The infusion rate may be modified about every 1, 5, 15, 20, 25, 30, 40, 50, 100 minutes or so. The increase in rate of administration is limited by side effects (flushing, diarrhea, leg pain). In certain embodiments, the infusion rate is modified less frequently than every 15 minutes. In other embodiments, the infusion rate is modified more frequently than about every 15 minutes. Due to mobile intravenous pumps, a patient may receive peptide intravenously for extended periods of time. The length of time of infusion and/or the rate of infusion may be modified based upon the response of the patient to the treatment. In certain embodiments of the present invention, the age and physical condition of the patient may warrant a reduction of the rate of infusion. In other embodiments, when the patient is not suffering any side effects from the treatment, the infusion rate is raised. In certain aspects, an Azlet® osmotic pump (e.g., model 2ML1, 2ML2, 2ML4) can be used to infuse a patient with a peptide.

[0099] Methods of treating cancer may further include administering to the patient chemotherapy or radiotherapy, which may be administered more than one time. Chemotherapy includes, but is not limited to, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, taxotere, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastin, methotrexate, gemcitabine, oxaliplatin, irinotecan, topotecan, or any analog or derivative variant thereof. Chemotherapy may also include administration of a Receptor Tyrosine Kinase Inhibitor (RTKi) which include but are not limited to, Tarceva, Gefitinib, Fluro-Sorafenib, Sorafenib, PF-2341066, or any analog or derivative variant thereof. Radiation therapy includes, but is not limited to, X-ray irradiation, UV-irradiation, γ-irradiation, electron-beam radiation, or microwaves. Moreover, a cell or a patient may be administered a microtubule stabilizing agent, including, but not limited to, taxane, as part of methods of the invention. Furthermore, a cell or a patient may be administered the SPARC peptides of the invention in conjunction with other anti-angiogenic agents, including but not exavar, Thalomid, Avastin, Cilengitide, Exherin, WX-UK1, Combretastatin A-4 phosphate, GCS-100LE, PTK/ZK, AS- 1404, Phosphomannopentose sulfate, Squalamine, talactoferrin alfa, ZD-6474, AP-23573, Volociximab, XL-999, or any analog or derivative variant thereof. It is specifically contemplated that any of the compounds or derivatives or analogs, can be used with these combination therapies. [00100] In some embodiments, the cancer cell that is administered the peptide compositions described herein may be a bladder, blood, bone, bone marrow, brain, breast, colorectal, esophagus, gastrointestine, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testicular, tongue, or uterus cell.

[00101] Cancers that may be treated by methods and compositions of the invention include cells and cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, sympathetic nerve, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal

, ^-gnant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. Moreover, the methods and compositions of the invention may be used to treat pre-cancers, such as metaplasia, dysplasia, and hyperplasia. [00102] The therapeutic peptides may also be used for decreasing overproliferation of normal, vascularized tissues, such as adipose tissue, benign polyps, hypertrophied cardiac tissue, hypertrophied renal tissue, hypertrophied prostatic tissue, tissue containing amyloid i uterine fibroids. The therapeutics may be administered in an amount effective to reduce the vascular supply to the tissue, or to decrease the size or growth of the vascularized tissue, such as adipose tissue, polyps (e.g., intestinal or nose polyps), and muscle (including cardiac) tissue. Accordingly, subjects that may be treated include a subject having polypsis, and enlarged prostate, cardiac or renal hypertrophy or is obese or overweight. The peptide therapeutics may be administered in an amount and time period which results in blood levels regulating the size and/or growth of the vascularized tissue to be treated.

II. EXAMPLES

[00103] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

A. Results

[00104] Peptide design. The inventors previously demonstrated that peptide FS-E, representing the N-terminal EGF-like module of SPARC follistatin domain, potently inhibits angiogenesis (Chlenski et ah, 2004). This module of SPARC is a /3-hairpin, highly twisted by disulfide bonds that link cysteine 1 to cysteine 3 and cysteine 2 to cysteine 4. The crystal structure of peptide FS-E shows the two central cysteines are closely located (FIG. 1). By linking cysteine 4 with cysteine 3 in lieu of cysteine 2, separate N- and C-terminal loops of the peptide can be produced without disturbing the native structure. Using this strategy, the inventors synthesized the N- and C-terminal loops of the peptide FS-E as two separate peptides, FSEN and FSEC, as detailed in FIG. 1. Both peptides were folded into their native conformation by linking the cysteines that were placed at both ends. In the FSEN peptide, the unpaired cysteine was substituted with alanine. Corresponding scrambled control peptides, scFSEN and scFSEC that were designed to contain the same amino acids as peptides FSEN and FSEC in a random order, were synthesized without special modifications. [00105] Peptides FSEN and FSEC inhibit angiogenesis in vitro and in vivo. Migration assays were performed with Human Umbilical Vein Endothelial cells (HUVEC) and each of the synthetic SPARC peptides to characterize their anti-angiogenic properties in vitro and to compare their potencies with the original peptide FS-E. As shown in FIG. 2, the C-terminal loop peptide FSEC inhibited bFGF-stimulated endothelial cell migration with an EC₅₀ of ~ 1 pM, which was even lower than EC₅₀ of ~ 10 pM of the original peptide FS-E. The N- terminal loop peptide FSEN showed weaker inhibition of bFGF-stimulated endothelial cell migration, with an EC₅₀ of ~ 2 nM.

[00106] The anti-angiogenic properties of the peptides were further characterized in vivo using the Matrigel plug assay. The Matrigel plugs containing bFGF and either SPARC peptides FSEN or FSEC were less hemorrhagic than the positive control Matrigel plugs with bFGF alone and the plugs with bFGF and the scrambled SPARC peptides (FIG. 3A). To quantify the anti-angiogenic effects of the peptides, the endothelial cells were stained in paraffin sections of the Matrigel plugs with green fluorescence using anti-CD31 antibody and the perivascular pericytes were visualized with red fluorescence using anti-a-SMA antibody. The area occupied by each type of cells was quantified at low magnification in duplicate fields in each sample using the ImagePro software. As shown in FIG. 3, the quantity of CD31 positive endothelial cells was statistically significantly decreased in the SPARC peptide- treated Matrigel plugs compared to the positive control with bFGF alone (p<0.005) . Similarly, the number of SMA-positive pericytes in the Matrigel plugs containing the SPARC peptides was statistically significantly lower compared to the positive controls (p<0.005). In the Matrigels treated with scrambled peptides, no differences in endothelial cell number (p>0.7) or pericyte coverage (p>0.3) were detected compared to the positive control with bFGF alone. [00107] SPARC peptide FSEC potently inhibits neuroblastoma tumor growth. To examine the anti-tumor activity of the SPARC peptides, mice with subcutaneous neuroblastoma xenografts were treated with either FSEN, FSEC, scrambled peptide scFSEN, or PBS 5 days/week for 2.5 weeks. Compared to control experiments, statistically significant inhibition of tumor growth was seen in the animals treated with the FSEC peptide (FIG. 4A). In the FSEC-treated animals, the average tumor weight at the end of the experiment was 26% of the weight of the control tumors (0.38±0.42 g vs 1.48±1.24 g, respectively; p=0.01). In the

" imals treated with the peptide FSEN, the average weight of tumors was 88% of the controls, however the reduction observed in this experimental group did not reach statistical significance in this experiment (1.30±1.77 g, p=0.83). The average weight of tumors resected from animals treated with the scrambled peptide was not different from the PBS controls (1.51±1 g, p=0.97). [00108] Vascular architecture was initially evaluated on H&E stained sections. In the control tumors, the blood vessels were structurally abnormal with dilated, tortuous appearance and prominent MVP (FIG. 4C). In xenografts treated with scrambled peptide, a similar architecture was noted with disorganized layers of endothelial and perivascular cells. Multiple areas of hemorrhage were noted in the control and scrambled peptide-treated tumors. In the SPARC peptide-treated tumors, the blood vessel architecture was more normal, and there was no evidence of MVP. Immunofluorescent analysis of the tumors demonstrated a significant reduction in the quantity of endothelial cells in the SPARC peptide-treated xenografts compared to controls (FIG. 5). The average area occupied by the endothelial cells in FSEC-treated xenografts was 13±6% of the blood vessel area in the control xenografts (5.9±2.6 pixels xlO³ vs 44.9±6.6 pixels xlO³, respectively; pO.001). Peptide FSEN also inhibited angiogenesis in the xenografts, although less potently. The average area occupied by endothelial cells in the FSEN-treated tumors was 33±9% of the control (15.0±4.0 pixels xlO³; pO.001). In contrast, no significant difference in the quantity of endothelial cells in the xenografts treated with scrambled peptide or control vehicle was detected (42.4±13.1 pixels xlO³, p=0.67).

[00109] The quantity of perivascular cells in each group corresponded to the level of angiogenesis. The average area occupied by the pericytes in the tumors treated with peptides FSEC vs control tumors was 16±10% (6.2±4.0 pixels xlO³ vs 38.6±10.4 pixels xlO³, respectively; pO.001) and 33±16% (12.8±6.2 pixels xlO³; p=0.004) for peptide FSEN. No difference in the quantity of pericytes in tumors treated with the scrambled peptide and controls was detected (40.1±13.1 pixels xlO³; p=0.84). Although pericyte-to-endothelial cell ratio was not affected by the treatment, differences in the blood vessel architecture were apparent between the control and SPARC peptide-treated tumors. In the xenografts treated with the vehicle or scrambled peptide the blood vessels exhibited abnormal architecture with multiple layers of hypertrophic endothelial cells which often lacked co-localized pericytes. In contrast, blood vessels in the SPARC peptide-treated tumors had more normal architecture, mainly consisting of a single layer of spindle-shaped endothelial cells, enveloped by a layer of pericytes (FIG. 6).

B. Materials and Methods

[00110] Peptide synthesis. SPARC peptides FSEN (CQNHHAKHGKVC)(SEQ ID NO:3) and FSEC (CELDENNTPMC) (SEQ ID NO:4) were synthesized as cyclic cysteine-linked molecules at Alpha Diagnostics International (San Antonio, TX) using fmoc/tboc chemistry. The purity of synthesis was assessed by high-performance liquid chromatography. The molecular mass of the peptides was checked by mass spectrometry. Control scrambled peptides scFSEN (KCGHKHQCAVHN) (SEQ ID NO:5) and scFSEC (MEPECNLNCTD) (SEQ ID NO:6), which contain the same amino acids as peptides FSEN and FSEC in a random order, were made without special modifications.

[00111] Cell lines. HUVEC cells (VEC Technologies, Rensselaer, NY) were maintained at 5% C0₂ in EGM media (Lonza, Walkersville, MD) supplemented with 5% FBS (Life Technologies, Carlsbad, CA). Neuroblastoma cell line SMS-KCNR (KCNR) was grown at 5% C0₂ in RPMI 1640 (Life Technologies) supplemented with 10% FBS and 1% penicillin/streptomycin.

[00112] Endothelial cell migration assay. To characterize the anti-angiogenic properties of the peptides in vitro, migration assays were performed with HUVEC cells and each of the synthetic SPARC peptides in EBM media (Cambrex Corporation, East Rutherford, NJ) containing 0.01% BSA. Serial dilutions of the peptides starting at 100 μηιοΙ/L were assayed with or without 10 ng/mL bFGF (National Cancer Institute Preclinical Repository, Frederick, MD). At least three independent experiments were performed for each peptide. To generate dose-response curves, the data were normalized as the percentage of maximum migration using the difference between bFGF-induced migration and background migration in EBM alone as 100% control.

[00113] Matrigel assay. To characterize the anti-angiogenic properties of the peptides in vivo, Matrigel assays were performed in 4- to 6-week-old homozygous athymic nude mice (Harlan, Madison, WI). SPARC peptides FSEN, FSEC and corresponding scrambled peptides scFSEN and scFSEC were mixed with 0.4 mL of growth factor-reduced Matrigel (Discovery Labware, Bedford, MA) containing 10 units/mL heparin, 50 ng/mL bFGF. At least 5 animals aneously injected with Matrigel plugs containing bFGF and each SPARC peptide. Matrigel plugs with bFGF and no peptides served as positive controls. Matrigels without either bFGF or peptides were used as negative controls. Mice were sacrificed 7 days after the Matrigel injections, gels were recovered by dissection, photographed, fixed in formaldehyde, and embedded in paraffin. [00114] Xenograft model. A neuroblastoma xenograft model was used to examine the antitumor activity of peptides FSEN and FSEC. Briefly, 4-6 week old athymic nude mice were injected subcutaneously with 0.2 ml PBS containing 5 x 10⁶ KCNR neuroblastoma tumor cells. Once tumors were palpable (-70 mm³), animals were randomized into four treatment groups with at least 6 animals per group. Experimental animals received intraperitoneal injection of 10 mg/kg/day of the SPARC peptides FSEN or FSEC, or scrambled peptide scFSEN (0.3 mg of the peptide in 150 ul of PBS) 5x/week for 2.5 weeks. Animals in the control group were injected with PBS without the peptide. The size of the tumors was determined every 2-3 days by external measurements with a caliper. At the end of treatment animals were euthanized using C0₂ followed by cervical dislocation. Tumors were removed, measured, weighed, and photographed. Half of each tumor was immediately frozen in liquid nitrogen for RNA and protein isolation, the other half was fixed with 10% buffered formalin, embedded in paraffin and 5 μιη-thick sections were prepared for histologic evaluation. All animal studies were approved by the AICUC at the University of Chicago.

[00115] Histological analysis and immunofluorescence. Four^m-thick sections were stained with hematoxylin and eosin for histological evaluation. The entire tissue section was evaluated for vascular morphology. Vessels with thickened walls containing complete layers of hypertrophied endothelial cells plus additional layers of vascular mural cells were classified as positive for MVP as previously described (Peddinti et al., 2007). Vessels with normal vessel architecture and no more than a single layer of flat, spindle shaped endothelial cells were characterized as MVP negative. For quantitative analysis of angiogenesis 4-μιη- thick sections were stained with anti-CD31 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) at a 1:50 dilution followed by FITC-labeled secondary antibody. To characterize the blood vessel architecture, pericytes were visualized with red fluorescence using anti-a- SMA antibody (Sigma- Aldrich, St. Louis, MO) at 1:100 dilution. The area occupied by each cell type was quantified at xlOO magnification in duplicate fields in each sample using the ImagePro software. [00116] Statistical analysis. All in vitro experiments were repeated at least in triplicate and standard deviations were calculated. All animal studies had at least 5 mice per group and mean values of the tumor volumes, weights, and vessel densities were compared. All the quantitative values obtained in the experiments were evaluated using paired Student's t-test. A p-value of 0.05 was required to ascertain statistical significance.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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Claims

WHAT IS CLAIMED IS:

1. A cyclic peptide of 5 to 20 amino acids comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO.l or SEQ ID NO:2.

2. The peptide of claim 1, wherein the peptide comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 1.

3. The peptide of claim 1, wherein the peptide comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:2.

4. The peptide of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: l .

5. The peptide of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:2.

6. An anti-angiogenic composition comprising at least one cyclic peptide of 5 to 20 amino acids comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: l or SEQ ID NO:2.

7. The anti-angiogenic composition of claim 6, comprising a first cyclic peptide of 5 to 20 amino acids comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: l and a second cyclic peptide of 5 to 20 amino acids comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:2.

8. An anti-angiogenic agent comprising a cyclic peptide having the formula:

X] QNHHAKHGKVX₂ or

X i ELDENNTPMX₂ ; wherein X_\ is coupled to X₂ forming a cyclic peptide.

9. The anti-angiogenic agent of claim 8, wherein Xj and X₂ are coupled by a disulfide bond.

10. The anti-angiogenic agent of claim 8, wherein X_! and X₂ are independently selected from mercaptopropionyl (Mpr), mercaptovaleryl (Mvl), cysteine, penicillamine (Pen), βι,β- pentamethylene^-mercaptopropionic acid (Pmp), or amino- i, -pentamethylene-P- mercaptopropionic acid (Pmc).

11. The anti-angiogenic agent of claim 8, wherein X_! and X₂ are cysteine.

12. An anti-angiogenic agent comprising a cyclic polymer having the formula: XjQNHHAKHGKVXz or X i ELDENNTPMX₂ ; wherein Xj is coupled to X₂ forming a cyclic polymer.

13. The anti-angiogenic agent of claim 12, wherein Xi and X₂ are coupled by a disulfide bond.

14. The anti-angiogenic agent of claim 12, wherein Xi and X₂ are independently selected from mercaptopropionyl (Mpr), mercaptovaleryl (Mvl), cysteine, penicillamine (Pen), ^p-pentamethylene- -mercaptopropionic acid (Pmp), or amino- -pentamethylene-P- mercaptopropionic acid (Pmc).

15. The anti-angiogenic agent of claim 12,

and X₂ are cysteine.

16. A method of inhibiting pathological vascularization in a subject comprising contacting a tissue or tumor having or suspected of having pathological vascularization with an effective amount of cyclic peptide of 5-20 amino acids comprising an amino acid sequence that is at least 70%, 80%, or 90% identical to the amino acid sequence of SEQ ID NO:l or SEQ ID NO:2.

The method of claim 16, wherein pathological vascularization is associated with a tumor or an ophthalmologic disorder.

The method of claim 17, wherein the tumor is a sarcoma, carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell, carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, ganglioneuroblastoma, neuroblastoma, or retinoblastoma.

The method of claim 17, wherein the tumor is a neuroblastoma.

The method of claim 17, wherein the ophthalmologic disorder is neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasias, uveitis, retinopathy of prematurity, macular degeneration, or corneal graft neovascularization.

The method of claim 16, wherein the cyclic peptide is administered systemically.

The method of claim 16, wherein the cyclic peptide is administered orally, intravascularly, intraocularly, or intratumorally.

The method of claim 16, wherein the cyclic peptide is administered at a dose of 0.001 mg/kg/day to 100 mg/kg/day.

The method of claim 17, further comprising a second anti-tumor therapy.

25. The method of claim 24, wherein the second anti-tumor therapy is a chemotherapy, a radiotherapy, an immunotherapy, an anti-angiogenic therapy, or surgery.

26. The method of claim 24, wherein the second anti-tumor therapy is chemotherapy.

27. The method of claim 26, wherein the chemotherapy is fluorouracil, irinotecan, vitamin D, taxol, doxorubicin, etoposide, Tarceva, Gefitinib, Fluoro-Sorafenib, Sorafenib, or PF-

2341066.

28. A method of treating cancer comprising administering to a subject having cancer or at risk for developing cancer or at risk for recurrence of cancer an effective amount of a peptide composition comprising a cyclic peptide having an amino acid sequence of SEQ ID NO:l or SEQ ID NO:2.

29. The method of claim 28, wherein the cancer is a sarcoma, carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell, carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, Kaposi's sarcoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, ganglioneuroblastoma, neuroblastoma, or retinoblastoma.

30. The method of claim 29, wherein the cancer is neuroblastoma.

31. The method of claim 28, wherein the cyclic peptide is administered systemically.

32. The method of claim 28, wherein the cyclic peptide is administered topically, orally, intravascularly, locally, or intratumorally.

33. The method of claim 28, wherein the cyclic peptide is administered at a dose of 0.1 mg/kg/day to 20 mg/kg/day.

34. The method of claim 28, further comprising a second anti-tumor therapy.

35. The method of 34, wherein the second anti-tumor therapy is a chemotherapy, a radiotherapy, an immunotherapy, an anti-angiogenic therapy, or surgery.

36. The method of claim 34, wherein the second anti-tumor therapy is chemotherapy.

37. The method of claim 36, wherein the chemotherapy is fluorouracil, irinotecan, vitamin D, taxol, doxorubicin, etoposide, Tarceva, Gefitinib, Fluoro-Sorafenib, Sorafenib, or PF-

2341066.