Use of caveolin-1 scaffold domain peptides to treat diseases and disorders

Abstract

There remains a need in the art for compositions and methods for treating or preventing fibrosis, diseases and disorders involving microvascular leakage, and aging and aging-related diseases and disorders. CSD domain peptides and methods of use are disclosed for treating diseases or disorders involving fibrosis, diseases or disorders involving microvascular leakage, kidney disease, heart disease, and diseases or disorders associated with aging.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 046,106, filed June 30, 2020, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under R01AR062078 awarded by the National Institutes of Health and W81XWH-11-1-0508 awarded by the Department of Defense. The government has certain rights in this invention. [Background technology]

[0003] Background of the Invention Caveolin-1 (Cav-1) is a major structural component of caveolae organelles in smooth muscle cells, adipocytes, fibroblasts, epithelial cells, and endothelial cells (ECs). Caveolin-1 is a master regulatory protein that binds to kinases in several signaling cascades, thereby inhibiting kinase function or promoting kinase turnover (Tourkina et al., 2005, J Biol Chem, 280:13879-13887; Couet et al., 1997, J Biol Chem, 272:6525-6533; Le Saux et al., 2008, Am J Physiol Lung Cell Mol Physiol, 295:L1007-L1017; Oka et al., 1997, J Biol Chem, 272:33416-33421; Razani et al., 2001, J Biol Chem, 276:6727-6738; Rybin et al., 1999, Circ Res, 84:980-988; Wang et al, 2008, Am J Respir Crit Care Med, 178:583-591). Caveolin-1 is underexpressed in several cell types, including fibroblasts and monocytes, in SSc patients and animal models (Tourkina et al., 2005, J Biol Chem, 280:13879-13887; Lee et al., 2014, Front Pharmacol, 5:140; Lee et al., 2014, Am J Physiol Lung Cell Mol Physiol, 306:L736-L748; Del Galdo et al., 2008, Arthritis Rheum, 58:2854-2865; Kasper et al., 1998, Histochem Cell Biol, 109:41-48; Tourkina et al., 2010, Ann Rheum Dis, 69:1220-1226).This deficiency leads to overexpression of ColI by fibroblasts, hypermigration of monocytes toward several chemokines, and enhanced differentiation of monocytes into CD45+ / ColI+ / α-smooth muscle actin+ (ASMA+) fibroblasts (Tourkina et al., 2011, Fibrogenesis Tissue Repair, 4:15; Tourkina et al., 2005, J Biol Chem, 280:13879-13887; Reese et al., 2014, Front Pharmacol, 16:141; Lee et al., 2014, Front Pharmacol, 5:140; Tourkina et al., 2010, Ann Rheum Dis, 69:1220-1226). The effects of caveolin-1 deficiency in cells and animals can be reversed using the caveolin-1 scaffold domain peptide (CSD, amino acids 82–101 of caveolin-1) (Tourkina et al., 2008, Am J Physiol Lung Cell Mol Physiol, 294:L843–L861; Wang et al., 2006, J Exp Med, 203:2895–2906). CSD can enter cells (Tahir et al., 2009, Cancer Biol Ther, 8:2286-2296; Tahir et al., 2008, Cancer Res, 68:731-739) and function as a surrogate for full-length caveolin-1 by inhibiting kinases similar to full-length caveolin-1 (Bucci et al., 2000, Nat Med, 6:1362-1367; Bernatchez et al., 2005, Proc Natl Acad Sci USA, 102:761-766). In addition to the profibrotic effects of low caveolin-1 and their reversal by CSD in vitro, low caveolin-1 is also profibrotic in vivo.Caveolin-1 knockout mice exhibit pulmonary, cutaneous, and cardiac fibrosis (DelGaldo et al., 2008, Arthritis Rheum, 58:2854-65; Cohen et al., 2003, Amer Jour of Cell Phys, 284:C457-74; Drab et al., 2001, Science, 293:2449-52; Razani et al., 2001, J Biol Chem, 276:38121-38). CSD also inhibits lung, skin, and cardiac fibrosis in vivo (Tourkina et al., 2011, Fibrogenesis Tissue Repair, 4:15; Reese et al., 2014, Frontiers in Pharma, 5: epub; Tourkina et al., 2008, Amer Journal of Lung Cell Mol Phys, 294:L843-61; Pleasant-Jenkins et al., 2017, Lab Invest, 97:370-382). In contrast, in endothelial cells (which express high levels of caveolin-1), the beneficial effects of CSD may in some cases result from CSD acting as a competitor for caveolin-1 function (Chidlow et al., 2009, Gastroenterology, 136(2):575-84 e2; Tahir et al., 2009, Cancer biology & therapy, 8(23):2286-96).

[0004] There remains a need in the art for compositions and methods for treating or preventing fibrosis, diseases and disorders involving microvascular leakage, and aging and aging-related diseases and disorders. The present invention fulfills this unmet need. Summary of the Invention

[0005] Summary of the Invention In one embodiment, the invention relates to a method for treating or preventing microvascular leakage or a disease or disorder related thereto, kidney disease, heart disease, or a disease or disorder related to aging in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition comprising a CSD domain peptide, or a fragment or variant thereof, or a nucleic acid molecule encoding a CSD domain peptide, or a fragment or variant thereof, wherein the CSD domain peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0006] In one embodiment, the disease or disorder is an aging-related disease or disorder. In one embodiment, the disease or disorder is atherosclerosis, cardiovascular disease, microvascular leakage, cancer, arthritis, cataracts, osteoporosis, Alzheimer's disease and related neurodegenerative diseases, or hypertension.

[0007] In one embodiment, the disease or disorder is a renal disease. In one embodiment, the renal disease is renal inflammatory damage, renal dysfunction, chronic renal failure, or hypertension.

[0008] In one embodiment, the disease or disorder is cardiac disease. In one embodiment, the cardiac disease is cardiac hypertrophy, atherosclerosis, cardiomyopathy, stroke, or hypertension.

[0009] In one embodiment, the disease or disorder is associated with microvascular leakage. In one embodiment, the disease or disorder is congestive heart failure, scleroderma and interstitial lung disease in general, asthma, renal failure, neurodegenerative diseases including Alzheimer's disease and vascular dementia, cancer, venous thrombosis, diabetes and diabetic complications, sepsis, or acute respiratory distress syndrome (ARDS).

[0010] In one embodiment, the present invention relates to a method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition comprising a CSD domain peptide, or a fragment or variant thereof, or a nucleic acid molecule encoding the CSD domain peptide, or a fragment or variant thereof, wherein the CSD domain peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0011] In one embodiment, the disease or disorder is an aging-related disease or disorder selected from the group consisting of atherosclerosis, cardiovascular disease, microvascular leakage, cancer, arthritis, cataracts, osteoporosis, Alzheimer's disease and related neurodegenerative diseases, and hypertension.

[0012] In one embodiment, the disease or disorder is fibrosis or a fibrosis-related disease or disorder.

[0013] In one embodiment, the disease or disorder is microvascular leak or a disease or disorder associated with microvascular leak. In one embodiment, the disease or disorder is congestive heart failure, scleroderma and interstitial lung disease in general, asthma, renal failure, neurodegenerative diseases including Alzheimer's disease and vascular dementia, cancer, venous thrombosis, diabetes and diabetic complications, sepsis, or acute respiratory distress syndrome (ARDS).

[0014] In one embodiment, the disease or disorder is a renal disease. In one embodiment, the renal disease is renal inflammatory damage, renal dysfunction, chronic renal failure, or hypertension.

[0015] In one embodiment, the disease or disorder is cardiac disease. In one embodiment, the cardiac disease is cardiac hypertrophy, atherosclerosis, cardiomyopathy, stroke, or hypertension.

[0016] In one embodiment, the invention relates to a modified CSD domain peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, or a fragment or variant thereof.

[0017] In one embodiment, the invention relates to a composition comprising a modified CSD domain peptide, or a fragment or variant thereof, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier.

[0018] In one embodiment, the composition treats or prevents a disease or disorder in a subject. In one embodiment, the disease or disorder is an aging-related disease or disorder selected from the group consisting of atherosclerosis, cardiovascular disease, microvascular leakage, cancer, arthritis, cataracts, osteoporosis, Alzheimer's disease and related neurodegenerative diseases, and hypertension. In one embodiment, the disease or disorder is fibrosis or a fibrosis-related disease or disorder. In one embodiment, the disease or disorder is microvascular leakage or a microvascular leakage-related disease or disorder. In one embodiment, the disease or disorder is congestive heart failure, scleroderma and interstitial lung disease in general, asthma, renal failure, neurodegenerative diseases including Alzheimer's disease and vascular dementia, cancer, venous thrombosis, diabetes and diabetic complications, sepsis, or acute respiratory distress syndrome (ARDS). In one embodiment, the disease or disorder is renal disease. In one embodiment, the renal disease is renal inflammatory damage, renal dysfunction, chronic renal failure, tumor growth and metastasis, or hypertension. In one embodiment, the disease or disorder is heart disease. In one embodiment, the heart disease is cardiac hypertrophy, atherosclerosis, cardiomyopathy, stroke, or hypertension.

[0019] In one embodiment, the composition is formulated for administration by a delivery route selected from the group consisting of intranasal, oropharyngeal, and intraperitoneal.

[0020] In one embodiment, the present invention relates to a composition for treating or preventing microvascular leakage or a disease or disorder related thereto, kidney disease, heart disease, or a disease or disorder related to aging, the composition comprising a CSD domain peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, or a fragment or variant thereof.

[0021] In one embodiment, the disease or disorder is an aging-related disease or disorder. In one embodiment, the disease or disorder is atherosclerosis, cardiovascular disease, kidney disease, microvascular leakage, cancer, arthritis, cataracts, osteoporosis, AD and related neurodegenerative diseases, diabetic complications, or hypertension.

[0022] In one embodiment, the disease or disorder is a renal disease. In one embodiment, the renal disease is renal inflammatory damage, renal dysfunction, chronic renal failure, or hypertension.

[0023] In one embodiment, the disease or disorder is cardiac disease. In one embodiment, the cardiac disease is cardiac hypertrophy, atherosclerosis, cardiomyopathy, stroke, or hypertension.

[0024] In one embodiment, the disease or disorder is a disease or disorder associated with microvascular leak. In one embodiment, the disease or disorder associated with microvascular leak is congestive heart failure, scleroderma and interstitial lung disease in general, asthma, renal failure, neurodegenerative diseases including Alzheimer's disease and vascular dementia, cancer, venous thrombosis, diabetes and diabetic complications, sepsis, or acute respiratory distress syndrome (ARDS).

[0025] In one embodiment, the composition is formulated for administration by a delivery route selected from the group consisting of intranasal, oropharyngeal, and intraperitoneal.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS The following description of exemplary embodiments of the invention will be better understood when read in conjunction with the accompanying drawings, it being understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 provides an overview of experiments using full-length CSDs 82-89, 88-95, and 94-101 (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively) in a congestive heart failure (CHF) model. [Figure 2] FIG. 2 shows exemplary experimental data demonstrating that CSD suppresses the effects of AngII on HW / BW ratio, left ventricular (LV) mass, and posterior wall thickness (pWTh-d) in a CHF model. [Figure 3] FIG. 3 shows exemplary experimental data demonstrating that CSD inhibits the effects of AngII on ejection fraction (EF), fractional shortening (FS), and isovolumic relaxation time (IVRT) in a CHF model. [Figure 4] FIG. 4 shows exemplary experimental data demonstrating that CSD subdomains suppress the effects of AngII-induced fibrosis in the heart, as measured by elevated ColI deposition and HSP47 levels. [Figure 5] FIG. 5 shows exemplary experimental data demonstrating that Ang II induces cardiac microvascular leakage (as measured by tissue IgG heavy chain levels) that is almost completely inhibited by both 82-89 and 88-95. [Figure 6] FIG. 6 provides a summary of experiments using W82-89 (SEQ ID NO: 6) in a CHF model. [Figure 7] FIG. 7 shows exemplary experimental data demonstrating that W82-89 suppresses AngII-induced pathological increases in cardiac HW / BW ratio, microvascular leakage, and ColI levels. [Figure 8] Figure 8 shows an overview of valid domains. [Figure 9]FIG. 9 shows the experimental design of the experiment demonstrating that CSD suppresses aging-related pathological changes in the heart and kidney. [Figure 10] FIG. 10 shows exemplary experimental data demonstrating that CSD reverses the effects of aging on cardiac fibrosis and microvascular leakage. [Figure 11] FIG. 11 shows exemplary experimental data demonstrating that CSD reverses the effects of aging on renal fibrosis and microvascular leakage. [Figure 12] FIG. 12 shows exemplary experimental data showing representative examples of picrosirius red staining of heart and kidney tissue sections from young or old mice with or without CSD treatment. [Figure 13] FIG. 13 shows exemplary experimental data demonstrating the reversal of the effects of aging in the heart and kidney by CSD. [Figure 14] FIG. 14 shows exemplary experimental data demonstrating that CSD has a positive effect on fractional shortening (FS) and ejection fraction (EF), as well as isovolumic relaxation time (IVRT) in the aging heart. [Figure 15] FIG. 15 shows exemplary experimental data demonstrating that cardiomyocyte hypertrophy is elevated in aged mice and that this elevation is reversed by CSD. [Figure 16] FIG. 16 provides an overview of experiments using CSD (SEQ ID NO: 1) to examine the effects of CSD on brain aging. [Figure 17] FIG. 17 shows exemplary experimental data demonstrating that 18-month-old mice have much higher levels of microvascular leakage and fibrosis in the brain than young mice, and that systemic CSD treatment reduces these levels to approximately those observed in healthy 3-month-old mice. [Figure 18] FIG. 18 shows exemplary experimental data demonstrating that 18-month-old mice have much higher levels of activated tyrosine kinases in the brain than young mice, and that systemic CSD treatment reduces these levels to approximately those observed in healthy 3-month-old mice. [Figure 19]FIG. 19 shows the experimental design for experiments demonstrating inhibition of lung and skin fibrosis by unmodified CSD subdomains. [Figure 20] Figure 20 shows exemplary experimental data demonstrating the inhibition of pulmonary fibrosis by CSD and unmodified subdomains. (Top) Masson's trichrome-stained lung tissue sections demonstrate the extensive fibrosis caused by bleomycin and its inhibition by 82-89. (Bottom) Ashcroft scores were determined by a veterinary pathologist blinded to the identity of the samples (n=6 per group). *p<0.05 for Bleo+treatment vs. Bleo+vehicle. [Figure 21] Figure 21 shows data demonstrating the inhibition of skin fibrosis and intradermal fat loss by CSD and CSD subdomain peptides. Skin near the pump outlet was harvested for measurements of dermal thickness and intradermal fat. For saline / vehicle vs. bleo / vehicle, ∧∧∧p<0.001. For bleo / vehicle vs. bleo / peptide treatment, ***p<0.001, **p<0.01, *p<0.05. [Figure 22] FIG. 22 shows data demonstrating inhibition of monocyte migration by the CSD and unmodified subdomains. [Figure 23] FIG. 23 shows the experimental design for experiments demonstrating the inhibition of lung and skin fibrosis by a modified water-soluble version of CSD (WCSD). [Figure 24] Figure 24 shows survival and histological data demonstrating the inhibition of lung and skin fibrosis by modified water-soluble CSD. The bottom panel is a Masson's Trichrome stained tissue section. [Figure 25] FIG. 25 shows exemplary experimental data demonstrating that WCSD suppresses bleomycin-induced pulmonary fibrosis through its effects on fibrocytes, ECM proteins, myofibroblast markers, and microvascular leakage. [Figure 26] FIG. 26 shows data demonstrating inhibition of tumor growth by WCSD. [Figure 27]Figure 27 shows data demonstrating that nintedanib and modified water-soluble versions of CSD have distinct kinase inhibition profiles. [Figure 28] FIG. 28 presents data demonstrating that modified water-soluble versions of CSD are more active as kinase inhibitors than their parent unmodified forms. [Figure 29] FIG. 29 shows data demonstrating that intranasal (in) is a promising delivery route for modified water-soluble versions of CSD. [Figure 30] FIG. 30 shows data demonstrating uptake of fluorescent peptides by primary mouse lung fibroblast cultures. [Figure 31] FIG. 31 shows data demonstrating plasma levels of W82-89 after different routes of administration. [Figure 32] FIG. 32 shows data demonstrating that W82-89 uptake into plasma after intraperitoneal administration is much more effective than that of CSD or WCSD. DETAILED DESCRIPTION OF THE INVENTION

[0028] Detailed Description The present invention is based, in part, on experiments demonstrating that CSD domain peptides are effective in inhibiting fibrosis and microvascular leakage, as well as other aspects of angiotensin II (Ang II)-induced congestive heart failure (CHF) and renal disease. Furthermore, CSD domain peptides have been effective in treating aging-related pathological changes in the heart, kidney, and brain. Thus, in some embodiments, the present invention relates to methods for treating fibrosis, microvascular leakage, and associated diseases and disorders, as well as aging and aging-related diseases and disorders, cardiac disease, and renal disease in a subject, comprising administering a CSD domain peptide of the invention.

[0029] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0030] As used herein, each of the following terms has the meaning associated with it in this section.

[0031] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0032] As used herein, "about" when referring to a measurable value such as an amount, duration, or the like is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.1%, less than ±0.1%, or any percentage therebetween from the specified value, as such variations are appropriate for performing the disclosed methods.

[0033] The term "abnormal," when used in the context of an organism, tissue, cell, or component thereof, refers to an organism, tissue, cell, or component thereof that differs in at least one observable or detectable characteristic (e.g., age, treatment, time) from an organism, tissue, cell, or component thereof that exhibits "normal" (expected) respective characteristics. A characteristic that is normal or expected for one cell or tissue type may be abnormal for another cell or tissue type.

[0034] The term "in combination with" is used herein to mean that the indicated therapies are administered simultaneously, or that a first treatment is administered sequentially with one or more additional therapies.

[0035] A "disease" is a state of health in an animal where the animal is unable to maintain homeostasis and where, if the disease is not ameliorated, the animal's health continues to deteriorate.

[0036] In contrast, an animal "disorder" is a state of health in which the animal is able to maintain homeostasis, but the animal's health state is less favorable than it would be in the absence of the disorder. Left untreated, the illness does not necessarily result in further deterioration of the animal's health state.

[0037] A disease or disorder is "alleviated" if the severity of a symptom of the disease or disorder, the frequency with which the patient experiences such symptoms, or both, is reduced.

[0038] An "effective amount" or "therapeutically effective amount" of a compound is the amount of the compound sufficient to provide a beneficial effect to the subject to which it is administered. An "effective amount" of a delivery vehicle is the amount sufficient to effectively bind or deliver the compound.

[0039] As used herein, the term "fusion protein" refers to two or more peptides, polypeptides, or proteins operatively linked to each other.

[0040] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" or grammatical equivalents refer to at least two nucleotides covalently linked to each other. The term "nucleic acid" includes single-stranded, double-stranded, or multi-stranded DNA, RNA, and analogs (derivatives) thereof. Oligonucleotides are typically about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50, or more nucleotides in length, up to about 100 nucleotides in length. Nucleic acids and polynucleotides are polymers of any length, including longer lengths, for example, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. In certain embodiments, the nucleic acids herein contain phosphodiester bonds. Other embodiments include nucleic acid analogs that may have alternative backbones, including peptide backbones containing, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoramidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), as well as peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive, non-ionic, and non-ribose backbones, such as those described in U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research," Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included in one definition of nucleic acid. Modifications of the ribose monophosphate backbone can be made for a variety of reasons, such as to increase the stability and half-life of such molecules in physiological environments or as probes on biochips. Mixtures of naturally occurring nucleic acids and analogs can be made, or mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs can be made.

[0041] A nucleotide sequence is "operably linked" when it is placed into a functional relationship with another nucleotide sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are near each other and, in the case of a secretory leader, contiguous and in reading phase; however, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are conventionally used.

[0042] The term "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, means that the sequences are identical or a specified percent identical (i.e., the sequences are identical when compared and aligned for maximum correspondence over a comparison window or designated region) as determined using the BLAST or BLAST 2.0 sequence comparison algorithm with the default parameters described below, or by manual alignment and visual inspection (see, e.g., the NCBI website). "Identical" refers to two or more sequences or subsequences that have amino acid residues or nucleotides with about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 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% or more identity for each sequence. Such sequences are said to be "substantially identical." This definition also refers to, or can apply to, the complement of a test sequence. This definition also includes sequences that have deletions and / or additions, as well as sequences that have substitutions. As explained below, preferred algorithms can account for gaps, etc. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10 to 50 amino acids or 20 to 50 nucleotides in length. As used herein, percent (%) amino acid sequence identity is defined as the percent of amino acids in a candidate sequence that are identical to those in a reference sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity. Alignment to determine percent sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared, can be determined by known methods.

[0043] In sequence comparison, one sequence usually serves as a reference sequence, and test sequences are compared to it.When using sequence comparison algorithm, test sequences and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Preferably, default program parameters are used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the program parameters.

[0044] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence.

[0045] Twenty amino acids are commonly found in proteins. These amino acids can be classified into nine classes or groups based on the chemical properties of their side chains. Substitution of one amino acid residue for another within the same class or group is referred to herein as a "conservative" substitution. Conservative amino acid substitutions can frequently be made in proteins without significantly altering the conformation or function of the protein. Substitution of one amino acid residue with another amino acid from a different class or group is referred to herein as a "non-conservative" substitution. In contrast, non-conservative amino acid substitutions tend to alter the conformation and function of a protein.

[0046] In some embodiments, conservative amino acid substitutions include the following: substitution of any of these aliphatic amino acids with glycine (G), alanine (A), isoleucine (I), valine (V), and leucine (L); substitution of threonine (T) with serine (S), or vice versa; substitution of glutamic acid (E) with aspartic acid (D), or vice versa; substitution of asparagine (N) with glutamine (Q), or vice versa; substitution of arginine (R) with lysine (K), or vice versa; substitution of any other of these aromatic amino acids with phenylalanine (F), tyrosine (Y), tryptophan (W); and substitution of cysteine ​​(C) with methionine (M), or vice versa. Other substitutions can also be considered conservative, depending on the environment and role of the particular amino acid in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) are often interchangeable, as are alanine (A) and valine (V). The relatively hydrophobic methionine (M) can frequently be interchanged with leucine, isoleucine, and sometimes valine. Lysine (K) and arginine (R) are often interchangeable at positions where the important feature of the amino acid residue is its charge and the difference in pK between these two amino acid residues is not significant. Moreover, other changes may be considered "conservative" in certain circumstances (see, e.g., BIOCHEMISTRY at pp. 13-15, 2nd ed. Lubert Stryer ed. (Stanford University); Henikoff et al., Proc. Nat'l Acad. Set USA (1992) 89: 10915-10919; Lei et al., J. Biol. Chem. (1995) 270(20): 1 1882-1 1886).

[0047] In some embodiments, non-conservative amino acid substitutions include substitution of any of serine (S), threonine (T), aspartic acid (D), glutamic acid (E), glutamine (Q), asparagine (N), lysine (K), arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W), methionine (M), cysteine ​​(C), histidine (H), and proline (P) with any of glycine (G), alanine (A), isoleucine (I), valine (V), and leucine (L). In some embodiments, non-conservative amino acid substitutions include substitution of any of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), aspartic acid (D), glutamic acid (E), glutamine (Q), asparagine (N), lysine (K), arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W), methionine (M), cysteine ​​(C), histidine (H), and proline (P) with any of serine (S) and threonine (T). In some embodiments, non-conservative amino acid substitutions include substitution of any of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), serine (S), threonine (T), glutamine (Q), asparagine (N), lysine (K), arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W), methionine (M), cysteine ​​(C), histidine (H), and proline (P) with any of aspartic acid (D) and glutamic acid (E). In some embodiments, non-conservative amino acid substitutions include substitution of any of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D), glutamic acid (E), lysine (K), arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W), methionine (M), cysteine ​​(C), histidine (H), and proline (P) with any of glutamine (Q) and asparagine (N).In some embodiments, non-conservative amino acid substitutions include substitution of any of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D), glutamic acid (E), glutamine (Q), asparagine (N), phenylalanine (F), tyrosine (Y), tryptophan (W), methionine (M), cysteine ​​(C), histidine (H), and proline (P) with any of lysine (K) and arginine (R). In some embodiments, non-conservative amino acid substitutions include substitution of any of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D), glutamic acid (E), glutamine (Q), asparagine (N), lysine (K), arginine (R), methionine (M), cysteine ​​(C), histidine (H), and proline (P) with any of phenylalanine (F), tyrosine (Y), and tryptophan (W). In some embodiments, non-conservative amino acid substitutions include substitution of any of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D), glutamic acid (E), glutamine (Q), asparagine (N), lysine (K), arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), and proline (P) with any of methionine (M) and cysteine ​​(C). In some embodiments, non-conservative amino acid substitutions include substitution of histidine (H) for any of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D), glutamic acid (E), glutamine (Q), asparagine (N), lysine (K), arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W), methionine (M), cysteine ​​(C), and proline (P).In some embodiments, non-conservative amino acid substitutions include substitution of any of glycine (G), alanine (A), isoleucine (I), valine (V), leucine (L), serine (S), threonine (T), aspartic acid (D), glutamic acid (E), glutamine (Q), asparagine (N), lysine (K), arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W), methionine (M), cysteine ​​(C), and histidine (H) with proline (P).

[0048] "Polypeptide," "peptide," and "protein" are used interchangeably herein and refer to any peptide-linked chain of amino acids, regardless of length or post-translational modification. As indicated below, a polypeptide described herein can be, for example, a wild-type protein, a biologically active fragment of a wild-type protein, or a variant of the wild-type protein or fragment. A variant can include amino acid substitutions, deletions, or insertions in accordance with the present disclosure. Substitutions can be conservative or non-conservative. In some embodiments, conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine, and threonine; lysine, histidine, and arginine; and phenylalanine and tyrosine.

[0049] Following expression, the protein (e.g., a CSD domain peptide) can be isolated. The terms "purified" or "isolated," as applied to any protein described herein (e.g., a conjugate described herein, an antibody or antigen-binding fragment thereof described herein), refer to a polypeptide that has been separated or purified from components (e.g., proteins or other naturally occurring biological or organic molecules) that naturally accompany it, e.g., other proteins, lipids, and nucleic acids, e.g., in the prokaryote that expresses the protein. Typically, a polypeptide is purified when it constitutes at least 60% by weight (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99% by weight) of the total protein in a sample.

[0050] A "label" or "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means. For example, useful detectable moieties include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., those commonly used in ELISAs), biotin, digoxigenin, paramagnetic molecules, paramagnetic nanoparticles, ultrasmall superparamagnetic iron oxide ("USPIO") nanoparticles, USPIO nanoparticle aggregates, superparamagnetic iron oxide ("SPIO") nanoparticles, SPIO nanoparticle aggregates, standard superparamagnetic iron oxide ("SSPIO"), SSPIO nanoparticle aggregates, polydisperse superparamagnetic iron oxide ("PSPIO"), PSPIO nanoparticle aggregates, single crystal SPIO, single crystal SPIO aggregates, single crystal iron oxide nanoparticles, single crystal iron oxide, other nanoparticle contrast agents, liposomes or other delivery vehicles containing gadolinium chelate ("Gd chelate") molecules, gadolinium, radioisotopes, radionuclides (e.g., carbon-11, nitrogen-13, oxygen-15, fluorine-18, rubidium-82), fluorodeoxyglucose (e.g., fluorine-18 labeled), any These include gamma-emitting radionuclides, positron-emitting radionuclides, radiolabeled glucose, radiolabeled water, radiolabeled ammonia, biocolloids, microbubbles (e.g., microbubble shells comprising albumin, galactose, lipids, and / or polymers; microbubble gas cores comprising air, heavy gases, perfluorocarbons, nitrogen, octafluoropropane, perflexan lipid microspheres, perflutren, etc.), iodinated contrast agents (e.g., iohexol, iodixanol, ioversol, iopamidol, ioxilan, iopromide, diatrizoate, metrizoate, ioxaglate), barium sulfate, thorium dioxide, gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores, two-photon fluorophores, or haptens and proteins, or other substances that can be made detectable (e.g., by incorporating the radiolabel into a peptide or antibody that specifically reacts with the target peptide). Detectable moieties include any of the above compositions encapsulated in nanoparticles, particles, aggregates, coated with additional compositions, and derivatized to bind to a target substance (e.g., an antibody or antigen-binding fragment).Any method known in the art for binding an antibody to a label can be used, for example, the method described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

[0051] As used herein, the term "pharmaceutically acceptable" is used interchangeably with "physiologically acceptable" and "pharmacologically acceptable." Pharmaceutical compositions generally include buffers and storage preservatives, and depending on the route of administration, may include buffers and carriers for appropriate delivery. The term "diagnostically acceptable" is used interchangeably with "physiologically acceptable" and "pharmacologically acceptable" and refers to a diagnostic composition.

[0052] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to substances that aid in the administration and absorption of an active agent by a subject and can be included in the compositions of the present invention without causing significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates (e.g., lactose, amylose, or starch), fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and dyes. Such preparations are sterilized and, if necessary, can be mixed with auxiliary substances such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents, and / or aromatic substances that do not adversely react with the compounds of the present invention. Those skilled in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0053] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cells thereof suitable for the methods described herein, whether in vitro or in situ. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0054] "Treatment," "treat," or "treating" refers to a method of reducing the effects of a disease or condition. Treatment can refer to a method of reducing the disease or condition itself, not just symptoms. Treatment can be any reduction from natural levels, including, but not limited to, complete elimination of the disease, condition, or symptoms of the disease or condition. Thus, in the disclosed methods, "treatment" can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or disease progression. For example, the disclosed methods for reducing the effects of a disease or disorder are considered therapeutic if, in a subject with the disease, one or more symptoms of the disease are reduced by 10% compared to the natural levels in the same subject or a control subject. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount in between, compared to natural or control levels. It is understood and contemplated herein that "treatment" does not necessarily mean curing a disease or condition, but rather means improving the outlook for a disease or condition.

[0055] As used herein, the terms "treat" and "prevent" refer to delaying onset, reducing the frequency or severity of symptoms, ameliorating symptoms, improving patient comfort or function (e.g., joint function), reducing the severity of a medical condition, and the like. The effect of treatment can be compared to an individual or population of individuals not receiving the given treatment, or to the same patient before or after treatment has ceased. The term "prevent" generally refers to a reduction in the occurrence of a given disease (e.g., an autoimmune disease, an inflammatory autoimmune disease, cancer, an infectious disease, an immune disorder, or other disease) or disease symptom in a patient. As noted above, prevention may be complete (no detectable symptoms) or partial, such that fewer symptoms will be observed than would occur without treatment.

[0056] A "vector" is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

[0057] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6, etc. This applies regardless of the breadth of the range.

[0058] explanation Endothelial cells are directly involved in many diseases and disorders, including, but not limited to, microvascular leakage, peripheral vascular disease, stroke, heart disease, diabetes, insulin resistance, chronic renal failure, tumor growth and metastasis, venous thrombosis, asthma, diabetic retinopathy and other complications, ARDS (e.g., induced by viral infection or lung injury), sepsis, and severe viral infections. Furthermore, endothelial dysfunction is associated with diseases and disorders, including Alzheimer's disease (AD).

[0059] The present invention is based, in part, on the discovery that administration of CSD domain peptides could inhibit microvascular leakage and suppress the pathological effects of AngII and bleomycin and aging.

[0060] Thus, the compositions of the present invention can be used to treat fibrosis, microvascular leakage, aging and aging-related diseases and disorders, cardiac disease, and renal disease.

[0061] In various embodiments, the compositions and methods of the present invention can be used to treat fibrotic diseases or disorders. Fibrosis can occur in many tissues in the body, including, but not limited to, the lungs, liver, heart, kidneys, brain, joints, skin, and bone marrow. Non-limiting examples of fibrotic diseases and disorders that can be treated using the compositions and methods described herein include, but are not limited to, interstitial lung disease, idiopathic pulmonary fibrosis, pulmonary fibrosis, asthma, chronic obstructive pulmonary disease (COPD), Raynaud's phenomenon, pulmonary fibrosis, cirrhosis, atrial fibrosis, endocardial fibrosis, arthrofibrosis, Crohn's disease, mediastinal fibrosis, myelofibrosis, tubulointerstitial fibrosis, hepatic fibrosis, premacular fibrosis, retinal fibrosis, dermal fibrosis, wound-associated fibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive mass fibrosis, retroperitoneal fibrosis, fibromas, scleroderma, and radiation-induced fibrosis, particularly from radiation therapy.

[0062] Non-limiting examples of aging-related diseases and disorders that can be treated using the compositions and methods described herein include, but are not limited to, atherosclerosis, cardiovascular disease, kidney disease, microvascular leakage, cancer, arthritis, cataracts, osteoporosis, AD and related neurodegenerative diseases, diabetic complications, and hypertension.

[0063] Non-limiting examples of diseases and disorders involving microvascular leakage that can be treated using the compositions and methods described herein include, but are not limited to, congestive heart failure, scleroderma and interstitial lung disease in general, asthma, renal failure, neurodegenerative diseases including Alzheimer's disease and vascular dementia, cancer, venous thrombosis, diabetes and complications of diabetes, sepsis, and acute respiratory distress syndrome (ARDS).

[0064] Non-limiting examples of cardiac and renal diseases and disorders that can be treated using the compositions and methods described herein include, but are not limited to, cardiac hypertrophy, atherosclerosis, cardiomyopathy, stroke, renal inflammatory damage, renal dysfunction, chronic renal failure, and hypertension.

[0065] Those skilled in the art will appreciate that the term treatment as used herein includes repair, replacement, augmentation, amelioration, prevention of occurrence or recurrence, rescue, repopulation, or regeneration.

[0066] Caveolin-1 scaffold domain peptide (CSD) In one embodiment, the method comprises administering a CSD domain peptide to a subject in need thereof for treatment of a disease or disorder.

[0067] Caveolin-1 is the major coat protein of caveolae. Caveolae were originally observed in electron microscopy images as flask-shaped invaginations of the plasma membrane. These organelles, rich in cholesterol and sphingolipids, function in endocytosis, vesicle trafficking, and compartmentalization of specific signaling cascades. The caveolin family of caveolae coat proteins includes three members, of which caveolin-1 and caveolin-2 are abundantly expressed in adipocytes, endothelial cells, and fibroblasts. Caveolins function as scaffolds for signaling molecules, including members of the MAP kinase family, PKC isoforms, Akt, G proteins, Src family kinases, and growth factor receptors. The ability of caveolin-1 to bind to various kinases and thus inhibit their activity has been mapped to a sequence known as the caveolin-1 scaffold domain (CSD, amino acids 82–101 of caveolin-1; DGIWKASFTTFTVTKYWFYR (SEQ ID NO: 1)).

[0068] In one embodiment, a subdomain of a CSD domain peptide comprises at least six consecutive amino acid residues of the CSD domain peptide, hi one embodiment, the subdomain comprises the amino acid sequence DGIWKASF (SEQ ID NO: 2), SFTTFTVT (SEQ ID NO: 3), or VTKYWFYR (SEQ ID NO: 4).

[0069] In one embodiment, the CSD domain peptide further comprises at least one additional amino acid residue. In various embodiments, the at least one additional amino acid residue modifies the peptide to 1) increase the water solubility of the peptide, 2) protect the peptide from proteolysis by exoproteases, 3) transport the peptide across the plasma membrane where it would not normally do so, or any combination thereof.

[0070] In one embodiment, the CSD domain peptide further comprises at least 1, 2, 3, 4, 5, or more than 5 D-lysine residues at the C-terminus or N-terminus of the peptide. In one embodiment, the CSD domain peptide further comprises at least 1, 2, 3, 4, 5, or more than 5 D-lysine residues at each of the C-terminus and N-terminus of the peptide. In one embodiment, the CSD domain peptide further comprises two D-lysine residues at the C-terminus and two D-lysine residues (k) at the N-terminus. In one embodiment, the subdomain comprises the amino acid sequence kkDGIWKASFTTFTVTKYWFYRkk (SEQ ID NO: 5), kkDGIWKASFkk (SEQ ID NO: 6), kkSFTTFTVTkk (SEQ ID NO: 7), or kkVTKYWFYRkk (SEQ ID NO: 8).

[0071] In one embodiment, the CSD domain peptide further comprises at least one protein modification, hi one embodiment, the CSD domain peptide comprises at least one of an N-terminal acetylation and a C-terminal amide.

[0072] It is understood, and contemplated herein, that there are many variations of the CSD or subdomains thereof that can be used in the disclosed therapeutic methods. Specifically contemplated herein are modifications or mutations made to the CSD or subdomains thereof that do not inhibit target binding but can assist the peptide, for example, in avoiding proteolysis. Modifications and mutations of the CSD or subdomains thereof that can be made include those described in detail in U.S. Patent Application No. 8,058,227 B2, which is incorporated herein in its entirety.

[0073] It is further understood that CSD domain peptides or subdomains thereof can be modified to aid in cell entry. Accordingly, contemplated herein are any known modifications that can be made to a CSD or subdomain thereof that can aid in cell entry. Also contemplated herein are modifications to a CSD or subdomain thereof that aid in cell entry based on empirical data. Additionally, contemplated herein are methods for treating fibrosis, microvascular leakage, aging and aging-related diseases and disorders, cardiac disease, and renal disease, comprising contacting a subject with a composition comprising a fusion peptide comprising a CSD or subdomain thereof.

[0074] It is understood and contemplated herein that CSD domain peptides treat fibrosis, microvascular leakage, senescence and senescence-related diseases and disorders, cardiac disease, and renal disease through binding to the caveolin-1 binding domain. It is further understood that any variant of the CSD, such as a derivative or analog of the CSD, or an agent capable of binding to the caveolin-1 target, is also effective in treating fibrosis, microvascular leakage, senescence and senescence-related diseases and disorders, cardiac disease, and renal disease. The identity of such agents, analogs or derivatives of the CSD or its subdomains, can be determined by their beneficial effects on in vivo and in vitro disease models compared to other versions of the CSD.

[0075] The present invention should also be construed to include any form of peptide variant having substantial homology to the amino acid sequences disclosed herein, in one embodiment, the peptide variant is at least about 50%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequences disclosed herein.

[0076] The present invention should also be construed to include any form of fragment having a substantial length of the amino acid sequences disclosed herein, hi one embodiment, the fragment is at least about 50%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the amino acid sequences disclosed herein.

[0077] The present invention should also be construed to include any form of fragment of a peptide variant that has both substantial homology and substantial length to the amino acid sequences disclosed herein. In one embodiment, a fragment of a peptide variant is at least about 50%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the amino acid sequences disclosed herein and is at least about 50%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the amino acid sequences disclosed herein.

[0078] Alternatively, the peptide may be produced by recombinant means or by cleavage from a longer peptide. The peptide may be verified by amino acid analysis or sequencing.

[0079] Variants of the peptides of the present invention can be (i) those in which one or more amino acid residues have been replaced with a conserved or non-conserved amino acid residue (e.g., a conserved amino acid residue), where such substituted amino acid residues may or may not be encoded by the genetic code; (ii) those with one or more modified amino acid residues, e.g., those with residues modified by the attachment of a substituent group; (iii) fragments of the peptides or domains described herein; and / or (iv) those fused to another peptide, i.e., a peptide such as a leader sequence or secretion sequence, or a sequence used for purification (e.g., a His tag) or detection (e.g., an Sv5 epitope tag). Fragments include peptides or peptides generated via proteolytic cleavage (including multisite proteolysis) of the original sequence. Variants can also be post-translationally or chemically modified. Such variants are considered to be within the scope of one skilled in the art in light of the teachings herein.

[0080] As known in the art, "similarity" between two peptides is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutes with the sequence of a second polypeptide. A variant is defined to include peptide sequences that differ from the original sequence, e.g., by fewer than 40% of the residues per segment of interest, fewer than 25% of the residues per segment of interest, fewer than 10% of the residues per segment of interest, or by only a few residues per segment of interest, while still being sufficiently homologous to the original sequence to retain the function of the original sequence. The present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two polypeptides can be determined using computer algorithms and methods well known to those skilled in the art. The identity between two amino acid sequences can be determined using the BLASTP algorithm (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)).

[0081] The peptides of the present invention may or may not be post-translationally modified. For example, post-translational modifications within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, peptide folding, and proteolytic processing. Some modifications or processing events require the introduction of additional biological mechanisms. For example, processing events such as signal peptide cleavage and core glycosylation can be examined by adding dog microsomal membranes or Xenopus egg extract (U.S. Patent No. 6,103,489) to a standard translation reaction. The polypeptides or peptides of the present invention can be phosphorylated using conventional methods, such as those described by Reedijk et al. (The EMBO Journal 11(4):1365, 1992).

[0082] The peptides of the invention can include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation. A variety of approaches are available for introducing unnatural amino acids during translation of a polypeptide.

[0083] The peptides of the present invention can be conjugated to other molecules, such as polyethylene glycol (PEG). This can be achieved by inserting cysteine ​​mutations or unnatural amino acids that can be modified with chemically reactive PEG derivatives. In one embodiment, peptides are conjugated to other peptides to prepare fusion peptides. This can be achieved, for example, by synthesizing N- or C-terminal fusion peptides, provided that the resulting fusion peptides retain the function of the peptides described herein.

[0084] Cyclic derivatives of the peptides of the present invention are also part of the present invention. Cyclization allows peptides to adopt a conformation suitable for conjugation with other molecules. Cyclization can be achieved using techniques known in the art. For example, a disulfide bond can be formed between two appropriately spaced components with free sulfhydryl groups, or an amide bond can be formed between an amino group of one component and a carboxyl group of another component. Cyclization can also be achieved using azobenzene-containing amino acids, as described by Ulysse, L., et al., J. Am. Chem. Soc. 1995, 117, 8466-8467. The bond-forming component can be an amino acid side chain, a non-amino acid component, or a combination of the two. In certain embodiments of the present invention, cyclic peptides can contain a beta-turn at the correct position. A beta-turn can be introduced into a peptide of the present invention by adding the amino acids Pro-Gly at the correct position.

[0085] It may be desirable to generate cyclic peptides that are more flexible than those containing the peptide bond linkages described above. A more flexible peptide can be prepared by introducing cysteines at the left and right positions of the peptide and forming a disulfide bridge between the two cysteines. The two cysteines are positioned so as not to distort the beta sheet and turn. The peptide is more flexible as a result of the length of the disulfide bonds and the fewer hydrogen bonds in the beta sheet portion. The relative flexibility of cyclic peptides can be determined by molecular dynamics simulations.

[0086] The present invention also relates to the peptides described herein fused to or incorporated into a target protein or a targeting domain that can direct the resulting protein to a desired cellular component or cell type or tissue. Chimeric or fusion proteins may also contain additional amino acid sequences or domains. Chimeric or fusion proteins are recombinant in the sense that the various components are derived from different sources and are therefore not found together in nature (i.e., heterologous).

[0087] In one embodiment, the targeting domain can be a transmembrane domain, a membrane-binding domain, or a sequence that directs the protein to bind to, for example, a vesicle or a cell surface. In one embodiment, the targeting domain can target the protein to a specific cell type or tissue. For example, the targeting domain can be a cell surface ligand or antibody against a cell surface antigen of the target tissue. The targeting domain can target the protein of the present invention to a cellular component.

[0088] The proteins of the present invention can be synthesized by conventional techniques. For example, proteins can be synthesized by chemical synthesis using solid-phase peptide synthesis. These methods use either solid-phase or solution-phase synthesis techniques (e.g., for solid-phase synthesis, see JM Stewart, and JD Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford Ill. (1984) and G. Barany and RB Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254 for solid phase synthesis techniques; and for classical solution synthesis, see M Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, suprs, Vol 1). By way of example, polypeptides of the invention can be synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) solid phase chemistry to directly incorporate phosphothreonine as the N-fluorenylmethoxy-carbonyl-O-benzyl-L-phosphothreonine derivative.

[0089] N- or C-terminal fusion proteins comprising the peptides or proteins of the present invention linked to at least one other molecule can be prepared by recombinantly fusing the N- or C-terminus of the peptide or protein with the sequence of a selected protein or selectable marker having a desired biological function. The resulting fusion protein contains a CSD domain peptide fused to the selected protein or marker protein described herein. Examples of proteins that can be used to prepare fusion proteins include immunoglobulins and regions thereof, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc.

[0090] The peptides of the present invention can be developed using biological expression systems. These systems allow for the creation of large libraries of random sequences and screening of these libraries for sequences that bind to specific peptides. Libraries can be produced by cloning synthetic DNA encoding random peptide sequences into appropriate expression vectors (see Christian et al. 1992, J. Mol. Biol. 227:711; Devlin et al. 1990 Science 249:404; Cwirla et al. 1990, Proc. Natl. Acad. Sci. USA, 87:6378). Libraries can also be constructed by simultaneous synthesis of overlapping peptides (see U.S. Pat. No. 4,708,871).

[0091] The peptides of the present invention can be converted into pharmaceutical salts by reaction with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, and the like, or with organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benzenesulfonic acid, and toluenesulfonic acid.

[0092] The present invention further encompasses fusion peptides in which a peptide of the present invention or a fragment thereof is recombinantly fused or chemically conjugated (including both covalent and non-covalent bonds) to a heterologous peptide (i.e., an unrelated peptide or portion thereof, e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, or at least 500 amino acids of a polypeptide) to produce the peptide. The fusion need not necessarily be direct, but may be via a linker sequence.

[0093] In one example, a peptide of the present invention or a fragment thereof can be fused to sequences derived from various types of immunoglobulins. For example, as described herein, a polypeptide of the present invention can be fused to the constant region (e.g., hinge, CH2, and CH3 domains) of a human IgG or IgM molecule, making the fused peptide or fragment thereof more soluble and stable in vivo. In another embodiment, such a fusion peptide can be administered to a subject to inhibit the interaction between a ligand and its receptor in vivo. Inhibition of such interaction blocks or suppresses signal transduction that triggers a specific cellular response.

[0094] In one embodiment, a fusion peptide comprises a polypeptide of the present invention fused at its N- or C-terminus to a heterologous sequence. In another embodiment, the peptide of the present invention can be fused to a tag sequence such as a hexahistidine peptide, particularly a tag (many of which are commercially available), such as a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311). As described in Gentz, et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for example, hexahistidine provides for convenient purification of the fusion protein. Other examples of peptide tags are the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, et al., 1984, Cell 37:767), and the "Flag" tag (Knappik, et al., 1994, Biotechniques 17(4):754-761). These tags are particularly useful for purifying recombinantly produced peptides of the invention.

[0095] Methods for introducing and expressing genes into cells are known in the art. In one embodiment, peptides can be encapsulated in microcapsules prepared by coacervation techniques or interfacial polymerization, for example, by using hydroxymethylcellulose or gelatin microcapsules, or poly(methyl methchlorate) microcapsules, respectively, or in colloidal systems. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes).

[0096] In one embodiment, the invention provides an implantable scaffold or device comprising a CSD domain peptide or a nucleic acid molecule encoding a CSD domain peptide. For example, in some embodiments, the invention provides tissue-engineered scaffolds, including but not limited to, hydrogels, electrospun scaffolds, polymer matrices, etc., comprising a CSD domain peptide or a nucleic acid molecule encoding a CSD domain peptide in or on the scaffold.

[0097] nucleic acid molecule In one embodiment, the method of the invention comprises administering a composition comprising a nucleic acid molecule encoding a CSD domain peptide or a subdomain thereof, hi one embodiment, the nucleic acid molecule encodes the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

[0098] Furthermore, the nucleic acid molecule encodes a peptide having substantial homology to the CSD domain peptides disclosed herein. In some embodiments, the isolated nucleic acid sequence encodes a CSD domain peptide comprising an amino acid sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0099] Isolated nucleic acids can include any type of nucleic acid, including, but not limited to, DNA, cDNA, and RNA. For example, in one embodiment, a composition includes an isolated DNA molecule, including, for example, an isolated cDNA molecule, encoding a CSD domain peptide. In one embodiment, a composition includes an isolated RNA molecule encoding a CSD domain peptide.

[0100] The nucleic acid molecules of the present invention can be modified to improve their stability in serum or cell culture growth medium. Modifications can be made to enhance the stability, functionality, and / or specificity of the nucleic acid molecules of the present invention and minimize their immunostimulatory properties. For example, to enhance stability, 3'-residues can be stabilized against degradation, e.g., selected to consist of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides with modified analogs, e.g., substitution of uridine with 2'-deoxythymidine, is tolerated and does not affect the function of the molecule.

[0101] Nucleic acids can be produced using a variety of standard cloning and chemical synthesis techniques, including, but not limited to, nucleic acid amplification, such as polymerase chain reaction (PCR) with genomic DNA or cDNA targets using primers (e.g., degenerate primer mixtures) that can anneal to CSD coding sequences. Nucleic acids can also be produced by chemical synthesis (e.g., solid-phase phosphoramidite synthesis) or transcription from a gene. The resulting sequences can then be translated in vitro or cloned into a plasmid for propagation and subsequent expression in cells (e.g., host cells such as yeast or bacteria, eukaryotic organisms, e.g., animal or mammalian cells, or plants).

[0102] Cell transfection typically uses a vector, and thus the nucleic acid can be contained within a vector. The term "vector" refers to, for example, a plasmid, a virus such as a viral vector, or other vehicle known in the art that can be used for genetic manipulation (i.e., a "cloning vector") or to transcribe or translate an inserted polynucleotide (i.e., an "expression vector"). Such vectors are useful for introducing a polynucleotide in operative linkage with a nucleic acid and expressing the transcribed encoded protein in cells in vitro, ex vivo, or in vivo.

[0103] A vector generally contains at least an origin of replication for propagation within a cell. Control elements, including expression control elements, present in a vector are included to facilitate transcription and translation. The term "control element" is intended to include at least one or more components whose presence can affect expression, and can include, other than or in addition to promoters or enhancers, for example, leader sequences and fusion partner sequences, ribosome binding site (IRES) elements for creating multiple genes or polycistronic messages, splicing signals for introns, maintaining the correct reading frame of the gene to allow in-frame translation of mRNA, polyadenylation signals to provide for proper polyadenylation of the transcript of the gene of interest, and stop codons.

[0104] Included vectors are those based on viral vectors, such as retroviruses (lentiviruses for infecting dividing and non-dividing cells), foamy viruses (U.S. Pat. Nos. 5,624,820, 5,693,508, 5,665,577, 6,013,516, and 5,674,703; WO92 / 05266, and WO92 / 14829), adenoviruses (U.S. Pat. Nos. 5,700,477, 5,700,478, 5,700,479 ... 0, 5,731,172, and 5,928,944), adeno-associated virus (AAV) (U.S. Pat. No. 5,604,090), herpes simplex virus vectors (U.S. Pat. No. 5,501,979), vectors based on cytomegalovirus (CMV) (U.S. Pat. No. 5,561,063), reovirus, rotavirus genome, simian virus 40 (SV40), or papillomavirus (Cone et al., Proc. Natl. Acad. Sci. USA 81:6349 (1984); Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982; Sarver et al., Mol. Cell. Biol. 1:486 (1981); U.S. Pat. No. 5,719,054). Adenoviruses efficiently infect slowly replicating and / or terminally differentiated cells and can be used to target slowly replicating and / or terminally differentiated cells. Simian virus 40 (SV40) and bovine papillomavirus (BPV) have the ability to replicate as extrachromosomal elements (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982; Sarver et al., Mol. Cell. Biol. 1:486 (1981)).Additional viral vectors useful for expression include reoviruses, parvoviruses, Norwalk viruses, coronaviruses, paramyxoviruses, and rhabdoviruses, togaviruses (e.g., Sindbis virus and Semliki Forest virus), and vesicular stomatitis virus (VSV) for introducing and directing expression of a polynucleotide or transgene in pluripotent stem cells or their progeny (e.g., differentiated cells).

[0105] A vector containing a nucleic acid can be expressed when the nucleic acid is operably linked to expression control elements. As used herein, the term "operably linked" refers to a physical or functional relationship between the referenced elements that permits them to function in their intended manner. Thus, an expression control element "operably linked" to a nucleic acid means that the control element controls transcription of the nucleic acid and, optionally, translation of the transcript.

[0106] The term "expression control element" refers to a nucleic acid that affects the expression of an operably linked nucleic acid. Promoters and enhancers are specific, non-limiting examples of expression control elements. A "promoter sequence" is a DNA regulatory region capable of initiating transcription of a downstream (3') sequence. A promoter sequence contains nucleotides that facilitate transcription initiation. Enhancers also regulate gene expression, but can function at locations distant from the transcription start site of an operably linked gene. Enhancers function at either the 5' or 3' end of a gene, as well as within a gene (e.g., in an intron or coding sequence). Additional expression control elements include leader and fusion partner sequences, internal ribosome binding site (IRES) elements for creating multiple genes or polycistronic messages, splicing signals for introns, maintaining the correct reading frame of a gene to allow in-frame translation of mRNA, polyadenylation signals to provide for proper polyadenylation of the transcript of interest, and stop codons.

[0107] Expression control elements include "constitutive" elements, in which transcription of an operably linked nucleic acid occurs without the presence of a signal or stimulus. For expression in mammalian cells, constitutive promoters of viral or other origins can be used. For example, SV40 or viral long terminal repeats (LTRs), or inducible promoters derived from the genomes of mammalian cells (e.g., metallothionein IIA promoter; heat shock promoters, steroid / thyroid hormone / retinoic acid response elements), or mammalian viruses (e.g., adenovirus late promoter; mouse mammary tumor virus LTR) can be used.

[0108] Expression control elements that confer expression in response to a signal or stimulus that either increases or decreases expression of an operably linked nucleic acid are "regulatable." Regulatable elements that increase expression of an operably linked nucleic acid in response to a signal or stimulus are called "inducible elements." Regulatable elements that decrease expression of an operably linked nucleic acid in response to a signal or stimulus are called "repressive elements" (i.e., the signal decreases expression; when the signal is removed or absent, expression increases).

[0109] Expression control elements include elements that are active in particular tissues or cell types, referred to as "tissue-specific expression control elements." Tissue-specific expression control elements are typically more active in particular cell or tissue types because they are recognized by a transcriptional activator protein or other transcriptional regulator that is active in that particular cell or tissue type relative to other cell or tissue types.

[0110] The nucleic acid or protein can be stably or transiently transfected (expressed) in the cell and its progeny. The cell can be grown to transcribe the introduced nucleic acid and express the protein. The progeny of a transfected cell may not be identical to the parent cell because mutations may occur during replication.

[0111] Methods for introducing and expressing genes in cells are known in the art. With respect to expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, the expression vector can be introduced into the host cell by physical, chemical, or biological means.

[0112] Physical methods for introducing nucleic acid molecules encoding peptides or proteins into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0113] Biological methods for introducing nucleic acid molecules encoding peptides or proteins of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0114] Chemical means for introducing nucleic acid molecules encoding peptides or proteins into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0115] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid can be associated with a lipid. Lipid-associated nucleic acids can be encapsulated within the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, bound to a liposome via a linking molecule attached to both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing lipids, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained in or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to a particular structure in solution. For example, they may exist in a bilayer structure, as micelles, or in a "collapsed" structure. They may also simply be interspersed in a solution, forming aggregates that are not uniform in size or shape. Lipids are fatty substances that can be natural or synthetic lipids. For example, lipids include the lipid droplets that occur naturally in the cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes, and their derivatives.

[0116] The vector of the present invention can also be used for nucleic acid gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466 (which are incorporated herein by reference in their entirety). In another embodiment, the present invention provides a gene therapy vector.

[0117] Treatment planning In one embodiment, a composition comprising a CSD domain peptide of the present invention or a nucleic acid molecule encoding the same is administered to a subject. In one embodiment, a treatment regimen can include a single administration of a composition comprising a CSD domain peptide of the present invention or a nucleic acid molecule encoding the same, or multiple administrations of a composition comprising a CSD domain peptide of the present invention or a nucleic acid molecule encoding the same. Multiple administrations of at least one composition of the present invention can be administered sequentially over a period of time selected by the attending physician. Methods for evaluating the course of treatment are within the skill of the attending physician.

[0118] The need for treatment is typically assessed by a medical history and physical examination consistent with the disease or disorder in question. Subjects identified as needing treatment include those diagnosed with fibrosis, microvascular leak, aging and aging-related diseases and disorders, cardiac disease, and renal disease. Causes of fibrosis, microvascular leak, aging and aging-related diseases and disorders, cardiac disease, and renal disease include, but are not limited to, genetic disease, autoimmune disease, inflammation, injury or trauma, damage from radiation or oxidative free radicals, or exposure to environmental or medical substances.

[0119] In one embodiment, the subject in need of treatment with the methods described herein has been diagnosed with or is at risk of developing fibrosis, microvascular leakage, aging and aging-related diseases and disorders, heart disease, and kidney disease. In one embodiment, the subject is an animal, including, but not limited to, mammals (e.g., horses, cows, dogs, cats, sheep, pigs, and humans), reptiles, and birds (e.g., chickens).

[0120] It should be appreciated that the methods of the present invention can be readily implemented in conjunction with existing therapies to effectively treat or prevent disease. The methods and compositions of the present invention can include simultaneous or sequential treatment with non-biologic and / or biologic agents.

[0121] The compositions of the present invention can be administered by several routes, including systemic administration (e.g., intravenous injection) or direct administration to the site where it is believed to be beneficial. The compositions of the present invention can be administered using any known route of administration, including, but not limited to, topical, colonic (rectal), local, intranasal, and parenteral (intraperitoneal, subcutaneous, intravenous, intradermal, or intramuscular injection), systemic, parenteral, or topical, such as oral formulations, inhalation formulations, including solid or aerosol, and other formulations, including formulations for transdermal, buccal, or sublingual administration. In some embodiments, the compositions are formulated for intranasal (in), oropharyngeal (op), or intraperitoneal (ip) administration.

[0122] In one embodiment, the invention relates to a method for inhibiting at least one kinase, comprising administering a CSD domain peptide of the invention or a nucleic acid molecule encoding a CSD domain peptide of the invention, hi one embodiment, the kinase is at least one of TGFβR2, PKCα, PKCε, cMet, VEGFR2, TGFβR1, and Src.

[0123] Pharmaceutical Formulation and Administration In one embodiment, the invention relates to a method of administering a composition comprising a CSD domain peptide of the invention or a nucleic acid molecule encoding a CSD domain peptide of the invention to a subject having a disease or disorder described herein. For example, in certain embodiments, the subject is diagnosed with interstitial lung disease, idiopathic pulmonary fibrosis, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), Raynaud's phenomenon, pulmonary fibrosis, cirrhosis, atrial fibrosis, endocardial fibrosis, arthrofibrosis, Crohn's disease, mediastinal fibrosis, myelofibrosis, tubulointerstitial fibrosis, hepatic fibrosis, premacular fibrosis, retinal fibrosis, dermal fibrosis, wound-associated fibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive mass fibrosis, retroperitoneal fibrosis, fibroma, scleroderma, In particular, radiation for the treatment of radiation-induced fibrosis, atherosclerosis, cardiovascular disease, kidney disease, microvascular leakage, arthritis, cataracts, osteoporosis, hypertension, congestive heart failure, interstitial lung disease, asthma, renal failure, neurodegenerative diseases including Alzheimer's disease and vascular dementia, cancer, venous thrombosis, diabetes and diabetic complications, sepsis, acute respiratory distress syndrome (ARDS), cardiac hypertrophy, cardiomyopathy, stroke, renal inflammatory damage, chronic renal failure, and renal dysfunction.

[0124] In one embodiment, the sample is isolated from a subject with or at risk of cancer. In one embodiment, the cancer is breast cancer, although the present invention is not limited to the detection of breast cancer. The following are non-limiting examples of cancers that can be diagnosed or treated by the disclosed methods and compositions: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors, brainstem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumor, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cerebral astrocytoma / malignant glioma, cervical cancer, pediatric visual pathway tumor. , chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, skin cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, Ewing's family of tumors, extracranial cancer, extragonadal germ cell tumor, extrahepatic bile duct cancer, extrahepatic cancer, eye cancer, fungoides, gallbladder cancer, gastric (stomach) cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (gist), germ cell tumor, gestational cancer, gestational trophoblastic tumor, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) carcinoma, histiocytosis, hominis Muscle lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, hypothalamic tumors, intraocular (eye) cancer, intraocular melanoma, pancreatic islet cell tumors, Kaposi's sarcoma, kidney (renal cell) cancer, Langerhans cell carcinoma, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, macroglobulinemia, malignant osteofibrous histiocytoma and osteosarcoma, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of the cervix, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycoses, myelodysplastic syndrome, bone marrow Dysplastic / myeloproliferative disorders, myeloid leukemia, myeloma, myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cavity cancer, oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma, osteosarcoma and malignant fibrous histiocytoma, ovary, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, paraganglioma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell tumor,Plasma cell neoplasms / multiple myeloma, pleuropulmonary blastoma, primary central nervous system cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer, respiratory tract cancer involving the Nat gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer (melanoma), skin cancer (non-melanoma), skin cancer, small cell lung cancer, small intestine cancer, soft tissue cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell neck cancer, gastric (stomach) cancer, ten supratentorial primitive neuroectodermal tumor, supratentorial primitive neuroectodermal tumor and pineoblastoma, T-cell lymphoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrolem's macroglobulinemia, and Wilms' tumor [[Inventor, please review this list or add additional cancers as needed.]],

[0125] Administration of one or more compositions of the present invention to a subject can be carried out using known procedures at an effective dosage and for an effective period of time to prevent or treat microvascular leakage, kidney disease, heart disease, and aging-related diseases or disorders in the subject. The effective amount of one or more therapeutic compositions required to achieve a therapeutic effect can vary depending on factors such as the state of the disease or disorder in the subject, as well as the age, sex, and weight of the subject. The dosing regimen can affect what constitutes an effective amount.

[0126] The dosage of one or more of the compositions may be increased or decreased proportionally to the exigencies of the therapeutic situation. One of ordinary skill in the art would be able to examine the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0127] Actual dosage levels of the active ingredients in one or more pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient effective to achieve a desired therapeutic response without toxicity to the subject for a particular subject, composition, and mode of administration.

[0128] In particular, the selected dose level will depend on a variety of factors, including the activity of the particular composition(s) used, the time of administration, the rate of excretion of the composition(s), the duration of treatment, other drugs, compounds or materials used in combination with the composition(s), the age, sex, weight, condition, general health, and past medical history of the subject being treated, and similar factors well known in the medical arts.

[0129] A physician, such as a physician or veterinarian, skilled in the art can readily determine and prescribe the effective amount of one or more pharmaceutical compositions required. For example, the physician or veterinarian can start the dosage of one or more compounds of the present invention used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0130] Typically, dosages that can be administered to a subject in the methods of the present invention range from 0.5 ng to about 50 mg per kg of the subject's body weight, although the exact dosage administered will vary depending on any number of factors, including, but not limited to, the type of subject and type of disease state being treated, the subject's age, and the route of administration. In one embodiment, the dosage of the compound ranges from about 1 ng to about 10 mg per kilogram of the subject's body weight. In one embodiment, the dosage ranges from about 3 ng to about 1 mg per kilogram of the subject's body weight.

[0131] To improve bioavailability and reduce complications associated with repeated injections, the present invention contemplates sustained delivery of the compositions of the present invention, alone or in combination with other agents. Sustained delivery in the present invention can be achieved via many different delivery systems, including but not limited to polymer gels, colloidal systems including liposomes and nanoparticles, cyclodextrins, collagen shields, diffusion chambers, flexible carrier strips, and implants.

[0132] The composition of the present invention can be in the form of an ointment. Ointments have the advantage of extending the contact time of the drug with the surface. Ointments generally contain a base, for example, white petrolatum and mineral oil, and often contain anhydrous lanolin, polyethylene mineral oil gel, and other substances that are recognized by formulation chemists as non-irritating, allow drug diffusion, and maintain the drug's activity for a reasonable period under storage conditions.

[0133] Therapeutic amounts of the compositions of the present invention can be administered orally. For these oral dosage forms, the compositions can be formulated with pharmaceutically acceptable solid or liquid carriers. Solid formulations include powders, tablets, pills, capsules, cachets, and dispersible granules. The concentration or effective amount of the composition administered per dose will vary widely depending on the actual composition. However, the total oral daily dose will generally range from about 50 mg to 30 g, and in certain embodiments, from about 250 mg to 25 g. A solid carrier can be one or more substances which may also function as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegration aids, or encapsulating materials. Suitable carriers include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, low-melting waxes, cocoa butter, and the like. The term "formulation" is intended to include formulations of the active compound with an encapsulating material as a carrier, providing a capsule in which the active ingredient, with or without other carriers, is surrounded by, and thus bound to, the carrier. Cachets and lozenges are also included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0134] For administration by inhalation, the compounds according to the present invention are conveniently delivered from an insufflator, nebulizer, or pressurized pack, or other convenient means for delivering an aerosol spray. The pressurized pack can contain a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Alternatively, for administration by inhalation or insufflation, the compounds according to the present invention can be in the form of a dry powder composition, for example, a powder mix of the compound and a suitable powder base, such as lactose or starch. The powder composition can be presented in unit dosage form, for example, in capsules or cartridges, or in gelatin or blister packs, from which the powder can be administered using an inhaler or insufflator.

[0135] It is understood that the unit content of one or more active ingredients contained in an individual aerosol dose of each dosage form need not itself constitute an effective amount for treating a particular indication or disease, since the required effective amount is achieved by administering multiple dosage units. Moreover, an effective amount can be achieved using smaller amounts than the doses in the dosage form, either individually or in a series of administrations.

[0136] Formulations suitable for buccal administration may include a powder or an aerosolized or atomized solution or suspension comprising the active ingredient. Such powdered, aerosolized, or aerosolized formulations, when dispersed, preferably have an average particle or droplet size in the range of about 0.1 nanometers to about 2000 micrometers, and may further include one or more of the additional ingredients described herein.

[0137] The composition can also be contained in an inert matrix for either direct application to a subject or injection. As an example of an inert matrix, liposomes can be prepared from dipalmitoylphosphatidylcholine (DPPC), or, for example, from egg phosphatidylcholine (PC) because this lipid has a low thermal transition. Liposomes are prepared using standard procedures known to those skilled in the art. A nanogram to microgram amount of the composition is added to a solution of egg PC, and the lipophilic drug is bound to the liposomes.

[0138] Sustained-release drug delivery systems can be used to provide sustained release of an active agent (e.g., a CSD domain peptide or subdomain thereof) over a period of time. Sustained-release formulations can be in the form of capsules of polymers (e.g., polycaprolactone, poly(glycolic) acid, poly(lactic) acid, polyanhydride) or lipids, which can be formulated as microspheres. Liposome-bound compositions can be applied directly as drops or aqueous-based creams, or can be injected. In formulations for direct application, the drug is slowly released over time as the liposomal capsule degrades due to wear and tear. In formulations for injection, the liposomal capsule is degraded by cellular digestion. Both of these formulations offer the advantages of sustained-release drug delivery systems, allowing subjects to be continuously exposed to the drug over long periods of time.

[0139] In sustained-release formulations, microspheres, capsules, liposomes, etc., may contain concentrations of the composition that would be toxic if administered as a bolus dose. However, sustained-release administration is formulated so that the concentration released over any given period does not exceed toxic amounts. This can be achieved, for example, by various formulations of the vehicle (coated or uncoated microspheres, coated or uncoated capsules, lipid or polymer components, single- or multi-layer structures, and combinations of the above, etc.). Other variables may include the subject's pharmacokinetic-pharmacodynamic parameters (e.g., body weight, sex, plasma clearance rate, liver function, etc.). The formation and loading of microspheres, microcapsules, liposomes, etc., and their implantation are standard techniques known to those skilled in the art.

[0140] The compositions of the present invention can be administered simultaneously, separately, or spaced apart over a period of time to maximize the effectiveness of the combination. The duration of each administration can vary, from rapid administration to continuous infusion. Consequently, for purposes of this invention, a combination is not limited to one achieved by physical association of the components, but also applies to one that allows for separate administration, which can be performed simultaneously or at intervals of a certain period of time.

[0141] Administration of nucleic acids encoding the CSD domain peptides of the invention or subdomains thereof to a subject can be achieved using gene therapy, which relies on the insertion of a therapeutic gene into a cell by ex vivo or in vivo techniques. Suitable vectors and methods for in vitro or in vivo gene therapy have been described and are known in the art. See, for example, Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res 77 (1995), 1077-1086; Wang, Nature Medicine 2 (1996), 714-716; WO94 / 29469; WO97 / 00957, or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640, and the references cited therein. Polynucleotides, i.e., polynucleotides encoding the peptides of the present invention, can be engineered for direct insertion into cells or via liposomes or viral vectors (e.g., adenovirus or retrovirus vectors). Suitable gene delivery systems that can be used in accordance with the present invention include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, adenoviruses, and adeno-associated viruses. Delivery of nucleic acids to specific sites within the body for gene therapy can also be achieved using biolistic delivery systems, such as those described by Williams (Proc. Natl. Acad. Sci. USA, 88 (1991), 2726-2729). Standard methods for transfecting cells with recombinant DNA are well known to those skilled in the art of molecular biology. See, for example, WO 94 / 29469. Gene therapy can be performed by directly administering the recombinant DNA molecules or vectors of the present invention to a patient.

[0142] Cancer treatment drugs In one embodiment, the invention provides a method for treating cancer metastasis, comprising administering a CSD domain peptide of the invention to a subject in need thereof. In some embodiments, the CSD domain peptide can be administered in combination with a complementary cancer treatment, such as surgery, chemotherapy, a chemotherapeutic agent, radiation therapy, or hormone therapy, or a combination thereof.

[0143] Chemotherapeutic agents include cytotoxic agents (e.g., 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucin sodium phosphate, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon alpha-2 a recombinant, paclitaxel, teniposide, and streptozotocin), cytotoxic alkylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylsulfonic acid), alkylating agents (e.g., azalay, AZQ, BCNU, busulfan, bisulfan, carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cisplatinum, clomezone, cyanomorpholinodoxorubicin, cyclodizone, cyclophosphamide, dianhydrogalactitol, fluorodopan, hepsul anti-mitotic agents (e.g., allocolchicine, halichondrin M, colchicine, colchicine derivatives, dolastatin 10, maytansine, rhizoxin, paclitaxel, benzodiazepine, benzophenone, benzocaine, benzodiazepine ... derivatives, paclitaxel, thiocolchicine, trityl cysteine, vinblastine sulfate, and vincristine sulfate), plant alkaloids (e.g., actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mithramycin, mitomycin, daunorubicin, VP-16-213, VM-26, navelbine, taxotere), biological agents (alpha interferon, BCG, G-CSF, GM-CSF, interleukin 2), topoisomerase I inhibitors (e.g., camptothecin,camptothecin derivatives, morpholinodoxorubicin), topoisomerase II inhibitors (e.g., mitoxantrone, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene HCl, daunorubicin, deoxydoxorubicin, menogaril, N,N-dibenzyldaunomycin, oxantrazole, rubidazone, VM-26, and VP-16), and synthetic agents (e.g., hydroxyurea, procarbazine, o,p'-DDD, dacarbazine, CCNU, BCNU, cis-diamminedichloroplatinum, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, gliadel, and porfimer sodium).

[0144] Antiproliferative agents are compounds that reduce cell proliferation. Antiproliferative agents include alkylating agents, antimetabolites, enzymes, biological response modifiers, other drugs, hormones and antagonists, androgen inhibitors (e.g., flutamide and leuprolide acetate), antiestrogens (e.g., tamoxifen citrate and its analogs, toremifene, droloxifene, and roloxifene). Further examples of specific antiproliferative agents include, but are not limited to, levamisole, gallium nitrate, granisetron, sargramostim strontium-89 chloride, filgrastim, pilocarpine, dexrazoxane, and ondansetron.

[0145] The CSD domain peptides of the present invention can be administered alone or in combination with other anti-tumor agents, including cytotoxic / anti-tumor agents and anti-angiogenic agents. Cytotoxic / anti-tumor agents are defined as agents that attack and kill cancer cells. Some cytotoxic / anti-tumor agents are alkylating agents that alkylate the genetic material of tumor cells, such as cisplatin, cyclophosphamide, nitrogen mustard, trimethylenethiophosphoramide, carmustine, busulfan, chlorambucil, verstine, uracil mustard, chromafazine, and dacabazine. Other cytotoxic / anti-tumor agents are tumor cell antimetabolites, such as cytosine arabinoside, fluorouracil, methotrexate, mercaptopurine, azathioprime, and procarbazine. Other cytotoxic / antitumor agents are antibiotics, such as doxorubicin, bleomycin, dactinomycin, daunorubicin, mithramycin, mitomycin, mitomycin C, and daunomycin. There are numerous liposomal formulations commercially available for these compounds. Still other cytotoxic / antitumor agents are mitotic inhibitors (vinca alkaloids). These include vincristine, vinblastine, and etoposide. Other cytotoxic / antitumor agents include taxol and its derivatives, L-asparaginase, antitumor antibodies, dacarbazine, azacitidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone, and vindesine.

[0146] Antiangiogenic agents are well known to those skilled in the art. Antiangiogenic agents suitable for use in the methods and compositions of the present disclosure include anti-VEGF antibodies, including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligonucleotides. Other known inhibitors of angiogenesis include angiostatin, endostatin, interferon, interleukin 1 (including alpha and beta), interleukin 12, retinoic acid, and tissue inhibitors of metalloproteinases-1 and -2 (TIMP-1 and -2). Small molecules that inhibit topoisomerase II, such as razoxane, which has antiangiogenic activity, can also be used.

[0147] Other anti-cancer agents that can be used in combination with the disclosed compounds include, but are not limited to, acurbicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; amethanthrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacytidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; baizelesin; bleomycin sulfate Benzene;Brequinar sodium;Bropirimine;Busulfan;Cactinomycin;Calsterone;Caracemide;Carbetimer;Carboplatin;Carmustine;Carubicin hydrochloride;Carzelesin;Cedefingol;Chlorambucil;Ciloremycin;Cisplatin;Cladribine;Crisnatol mesylate;Cyclophosphamide;Cytarabine;Dacarbazine;Dactinomycin;Daunorubicin hydrochloride;Decitabine;Dexormaplatin;Dezaguanine;Dezaguanine mesylate;Diazicon;Docetaxel;Doxorubicin;Doxorubicin hydrochloride;Droloxime Fen;Droloxifene citrate;Dromostanolone propionate;Duazomycin;Edatrexate;Eflornithine hydrochloride;Elsamitrucin;Enloplatin;Enpromate;Epipropizine;Epirubicin hydrochloride;Elbrozole;Esorubicin hydrochloride;Estramustine;Estramustine sodium phosphate;Etanidazole;Etoposide;Etoposide phosphate;Etoprine;Fadrozole hydrochloride;Fazarabine;Fenretinide;Floxuridine;Fludarabine phosphate;Fluorouracil;Fluorocitabine;Fosquidone;Fostriec rinotecan sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon alpha-2a; interferon alpha-2b; interferon alpha-n1; interferon alpha-n3; interferon beta-Ia; interferon gamma-Ib; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium;Lomustine;Losoxantrone hydrochloride;Masoprocol;Maytansine;Mechlorethamine hydrochloride;Megestrol acetate;Melengesterol acetate;Melphalan;Menogaril;Mercaptopurine;Methotrexate;Methotrexate sodium;Metoprine;Methodepa;Mitindomide;Mitocalcine;Mitochromin;Mitogillin;Mitomarcin;Mitomycin;Mitospar;Mitotane;Mitoxantrone hydrochloride;Mycophenolic acid;Nocodazole;Noga Ramicycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Periomycin; Pentamustine; Peplomycin sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone hydrochloride; Plicamycin; Promestane; Porfimer sodium; Porfiromycin; Prednimustine; Procarbazine hydrochloride; Puromycin; Puromycin hydrochloride; Pirazofurin; Ribopurin; Rogletimide; Safingol; Safi hydrochloride Ngol; Semustine; Simtrazen; Sparfosate sodium; Sparsomycin; Spirogermanium hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Surofenol; Tallysomycin; Tecogalan sodium; Tegafur; Teroxantrone hydrochloride; Temoporfin; Teniposide; Teloxylon; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Toremifene citrate; Toremifene acetate Stron; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tuburozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vimpidine sulfate; vinglisinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrocidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride. Other anticancer agents include, but are not limited to: 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecipenol; adzelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin;Amsacrine; Anagrelide; Anastrozole; Andrographolide; Angiogenesis inhibitors; Antagonist D; Antagonist G; Antarelix; Anti-dorsal morphogenetic protein-1; Antiandrogen, prostate cancer; Antiestrogens; Antineoplastons; Antisense oligonucleotides; Aphidicolin glycinate; Apoptosis gene modulators; Apoptosis regulators; Apurinic acid; ara-CDP-DL-PTBA; Arginine deaminase; Asulaculin; Atamestane; Atrimustine; Axinastatin 1; Axinastatin 2; Axinastatin 3;Azasetron;Azatoxin;Azatyrosine;Baccatin III derivatives;Balanol;Batimastat;BCR / ABL antagonists;Benzochlorins;Benzoylstaurosporine;Beta-lactam derivatives;Beta-arretin;Betaclamycin B;Betulinic acid;bFGF inhibitors;Bicalutamide;Bisantrene;Bisaziridinylspermine;Bisnafide;Bistraten A;Bizelesin;Brefurate;Bropirimine;Budotitanium;Buthionine sulfoximine;Calcpotriol;Calphostin C;Camptothecin derivatives;Canarypox IL-2;Cal Pecitabine; Carboxamido-amino-triazole; Carboxamidotriazole; Carrest M3; CARN700; Cartilage-derived inhibitor; Carzelesin; Casein kinase inhibitor (ICOS); Castanospermine; Cecropin B; Cetrorelix; Chlorin; Chloroquinoxaline sulfonamide; Cicaprost; cis-Porphyrin; Cladribine; Clomiphene analogues; Clotrimazole; Collismycin A; Collismycin B; Combretastatin A4; Combretastatin analogues; Conagenin; Crambesidin 816; Crisnatol; Chlorin Cryptophycin 8; Cryptophycin A derivatives; Curacin A; Cyclopentanthraquinone; Cycloplatin; Sipemycin; Cytarabine ocphosphate; Cytolytic factors; Cytostatin; Dacliximab; Decitabine; Dehydrodidemnin B; Deslorelin; Dexamethasone; Dexphosphamide; Dexrazoxane; Dexverapamil; Diazicon; Didemnin B; Didox; Diethylnorspermine; Dihydro-5-azacytidine; Dihydrotaxol, 9-; Dioxamycin; Diphenylspiromustine; Docetaxel; Docosanol;Dolasetron; Doxifluridine; Droloxifene; Dronabinol; Duocarmycin SA; Ebselen; Ecomustine; Edelfosine; Edrecolomab; Eflornithine; Elemene; Emiteflu; Epirubicin; Epristeride; Estramustine analogues; Estrogen agonists; Estrogen antagonists; Etanidazole; Etoposide phosphate; Exemestane; Fadrozole; Fazarabine; Fenretinide; Filgrastim; Finasteride; Flavopiridol; Flezelastine; Flusterone; Fludarabine; Fluorodauronil hydrochloride Syn; Forfenimex; Formestane; Fostriecin; Fotemustine; Gadolinium texaphyrin; Gallium nitrate; Galocitabine; Ganirelix; Gelatinase inhibitors; Gemcitabine; Glutathione inhibitors; Hepsulfam; Heregulin; Hexamethylene bisacetamide; Hypericin; Ibandronate; Idarubicin; Idoxifene; Idramanton; Ilmofosine; Ilomastat; Imidazoacridone; Imiquimod; Immunostimulating peptides; Insulin-like growth factor-1 receptor inhibitors; Interferon agonists; Interferons; Insulin Turleukin; Iobenguane; Iodoxorubicin; Ipomeanol, 4-; Ilopract; Irsogladine; Isobengazole; Isohomohalichondrin B; Itasetron; Jasplakinolide; Kahalalide F; Lamellarin-N triacetate; Lanreotide; Leinamycin; Lenograstim; Lentinan sulfate; Leptolstatin; Letrozole; Leukemia inhibitory factor; Leukocyte alpha interferon; Leuprolide + estrogen + progesterone; Leuprorelin; Levamisole; Liarozole; Linear polyamine analogs; Lipophilic disaccharide peptides; Parent Oil-soluble platinum compounds; lysocrine amide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; rhizophylline; lytic peptides; maytansine; mannostatin A; marimastat; massoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; mervalone; meterelin; methioninase; metoclopramide; MIF inhibitors; mifepristone; miltefosine; millimostim; mismatched double-stranded RNA;Mitoguazone; Mitolactol; Mitomycin analogues; Mitonafide; Mitotoxin fibroblast growth factor-saporin; Mitoxantrone; Mofalotene; Molgramostim; Monoclonal antibodies, human chorionic gonadotropin; Monophosphoryl lipid A + Myobacterium cell wall sk; Mopidamol; Multidrug resistance gene inhibitors; Multiple tumor suppressor 1-based therapies; Mustard anticancer drugs; Mycaperoxide B; Mycobacterial cell wall extract; Myriaporone; N-acetyldinal N-substituted benzamides; Nafarelin; Nagrestop; Naloxone + pentazocine; Napavine; Nafterpine; Nartograstim; Nedaplatin; Nemorubicin; Neridronic acid; Neutral endopeptidases; Nilutamide; Nisamycin; Nitric oxide modulators; Nitroxide antioxidants; Nitrulline; O6-benzylguanine; Octreotide; Oxenon; Oligonucleotides; Onapristone; Ondansetron; Ondansetron; Oracin; Oral cytokines Inducers; Ormaplatin; Osateron; Oxaliplatin; Oxaunomycin; Paclitaxel; Paclitaxel analogs; Paclitaxel derivatives; Palauamine; Palmitoylrhizoxin; Pamidronic acid; Panaxytriol; Panomyphen; Parabactin; Pazeliptin; Pegaspargase; Perdecin; Pentosan polysulfate sodium; Pentostatin; Pentrozole; Perflubron; Perfosfamide; Perillyl alcohol; Phenazinomycin; Phenylacine Tate; Phosphatase inhibitors; Picibanil; Pilocarpine hydrochloride; Pirarubicin; Piritrexim; Prasetin A; Prasetin B; Plasminogen activator inhibitors; Platinum complexes; Platinum compounds; Platinum triamine complexes; Porfimer sodium; Porfiromycin; Prednisone; Propylbisacridone; Prostaglandin J2; Proteasome inhibitors; Protein A-based immunomodulators; Protein kinase C inhibitors; Protein kinase C inhibitors, microalgae; Protein tyrosine phosphatase inhibitors; Purine nucleoside phosphorylase inhibitors; Purpurin; Pyrazoloacridines; Pyridoxylated hemoglobin polyoxyethylene conjugates; RAF antagonists; Raltitrexed; Ramosetron; Ras farnesyl protein transferase inhibitors; Ras inhibitors;ras-GAP inhibitors; reteriptin demethylation; rhenium Re186 etidronate; rhizoxin; ribozyme; RII retinamide; logletimide; rohitukin; romultid; roquinimex; rubidinone B1; ruboxil; safingol; saintpin; SarCNU; sarcophyte; Sorrel A; Sargramostim; Sdi1 mimetic; Semustine; Senescence-derived inhibitor 1; Sense oligonucleotide; Signal transduction inhibitor; Signal transduction modulator; Single-chain antigen-binding protein; Schizofuran; Sobuzoxane; Borocaptate sodium; Sodium phenylacetate; Sorvalol; Somatomedin-binding protein; Sonermin; Sparfosic acid; Spicamycin D; Spiromustine; Splenopentin; Spongistatin 1; Squalamine; Stem cell inhibitor; Stem cell division inhibitor; Stipiamide; Stromelysin inhibitor; Sulfinosine; Superactive vasoactive intestinal peptide antagonist; Sladista; Suramin; Swainsonine; Synthetic glycosaminoglycans; Talimustine; Tamoxifen methiodide; Tauromustine; Tazarotene; Tecogalan sodium; Tegafur; Terlapyrylium; Telomerase inhibitor; Temoporfin; Temozolomide; Teniposide; Tetrahydrofuran Chlordecaoxide;Tetrazomine;Taliblastine;Thiocoraline;Thrombopoietin;Thrombopoietin mimetics;Thymalfasin;Thymopoietin receptor agonists;Thymotrin;Thyroid-stimulating hormone;Tin ethyl etiopurpurin;Tirapazamine;Titanocene dichloride;Topsentin;Toremifene;Totipotent stem cell factor;Translation inhibitors;Tretinoin;Triacetyluridine;Triciribine;Trimetrexate;Triptorelin; Tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector systems, red blood cell gene therapy; veraresol; veramine; verdin; verteporfin; vinorelbine; vincalcin; vitaxin; vorozole; zanoteron; zeniplatin; zilascorub; and zinostatin stimalamer.

[0148] Experimental Example The present invention will be described in further detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but rather as embracing any and all variations that become apparent as a result of the teachings provided herein.

[0149] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following examples therefore specifically point out exemplary embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0150] Example 1: CSD domains in the treatment of angiotensin II-induced cardiac disease The data presented herein demonstrate that CSD domain peptides (Table 1) have significant activity in suppressing cardiac disease and microvascular leakage in mice in which these pathologies were induced by treatment with angiotensin II.

[0151] FIG. 1 provides an overview of experiments using full-length CSDs, 82-89, 88-95, and 94-101 (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively).

[0152] Ang II induces highly significant changes in heart weight / body weight (HW / BW), left ventricular (LV) weight, and pWTh (posterior wall thickness) (Figure 2). Ang II also induces pathological changes in ejection fraction (EF), fractional shortening (FS), and isovolumic relaxation time (IVRT) (Figure 3). For each of these parameters, 88-95 is the most effective CSD subdomain in inhibiting the effects of Ang II. However, 82-89 are essentially as effective as 88-95 in inhibiting the effects of Ang II on HW / BW and have significant beneficial effects on EF, FS, and IVRT.

[0153] Figures 2 and 3 provide data showing that CSD and its two subdomains suppress AngII-induced increases in HW / BW ratio and pathological changes in ventricular function. Young mice (3 months old) infused with AngII or vehicle for 2 weeks were administered daily intraperitoneal injections of CSD, the indicated subdomains, or scrambled CSD (0.8 μmol / kg). Mice were evaluated by echocardiography for changes in posterior wall thickness (pWTh), fractional shortening (FS), cardiac output (CO), ejection fraction (EF), and isovolumic relaxation time (IVRT). Sham + Veh vs. AngII + Veh. *** Significant changes of p<0.001 are indicated. There was a significant inhibition of the change by AngII for AngII+Veh vs. AngII+CSD or AngII+CSD subdomains. ∧ p<0.05, ∧∧ p<0.01, ∧∧∧ Shown as p<0.001. The number of mice used for each treatment is shown in the HW / BW data.

[0154] In the heart, Ang II induces fibrosis, as measured by increased ColI deposition and HSP47 levels (Figure 4). For both parameters, 88-95 is the most effective CSD subdomain at inhibiting the effects of Ang II, but 82-89 also has a significant beneficial effect. Ang II induces cardiac microvascular leakage (as measured by tissue IgG heavy chain levels), which is almost completely inhibited by both 82-89 and 88-95 (Figure 5).

[0155] Figures 4 and 5 show that two CSD subdomains significantly suppress AngII-induced microvascular leakage and fibrosis in the heart. Young mice (3 months old) infused with AngII or vehicle for 2 weeks received daily intraperitoneal injections of the indicated subdomains of the CSD. Results of a typical experiment for ColI and HSP47 (Figure 4) and microvascular leakage (Figure 5) are shown by Western blot (2-3 mice per group). Data are quantified below (n=4). Significant changes are noted for Sham + Veh vs. AngII + Veh. * p<0.05 and **p<0.01. Significant subdomain inhibition of AngII-induced changes was observed for the AngII+Veh vs. AngII+CSD subdomains. ∧ p<0.05, ∧∧ p<0.01, and ∧∧∧ Shown as p<0.001.

[0156] Figure 6 provides an overview of experiments using W82-89 (SEQ ID NO: 6). As noted above, Ang II induces highly significant changes in HW / BW ratio, microvascular leakage, and fibrosis (Figure 7). All of these Ang II-induced pathologies are highly significantly inhibited by W82-89.

[0157] Figure 7 shows that W82-89 suppresses AngII-induced pathological increases in cardiac HW / BW ratio, microvascular leakage, and ColI levels. Young mice (3 months old) infused with AngII or vehicle for 2 weeks received daily intraperitoneal injections of W82-89. IgG heavy chain (IgGH) leakage and ColI accumulation are shown by Western blot. Because IgGH was analyzed in the supernatant fraction of cardiac homogenates, while ColI was found in the pellet fraction, actin loading controls are duplicated. Quantitation (n=4) revealed no significant changes for Sham + Veh vs. AngII + Veh. ** p<0.01 and *** p<0.001 for the reversal of Ang II-induced changes by W82-89. ∧∧ p<0.01 and ∧∧∧ Shown as p<0.001.

[0158] Figure 8 summarizes the data from Figures 1-7. 88-95 is the most effective dimethyl sulfoxide (DMSO)-solubilized domain tested. However, modifying 82-89 to make it water-soluble (W82-89) makes it more effective than 82-89 and at least as effective as 88-95. Thus, both 82-89 and 88-95 are active domains within the CSD.

[0159] [Table 1]

[0160] Lowercase letters indicate D-amino acids that are not part of caveolin-1. For injection, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 are dissolved in a very small amount of dimethyl sulfoxide (DMSO) and then diluted 100-fold with saline. SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 are water-soluble and therefore dissolved directly in saline. Because they are water-soluble, they are sometimes referred to as WCSD (SEQ ID NO:5), W82-89 (SEQ ID NO:6), W88-95 (SEQ ID NO:7), and W94-101 (SEQ ID NO:8).

[0161] Example 2: CSD reverses aging-associated pathological changes in the heart and kidney The data presented herein demonstrate that in mice with aging-related cardiac and renal disease and microvascular leakage, CSD peptides (Table 1) are highly active in suppressing these pathologies.

[0162] FIG. 9 provides a summary of experiments using CSD (SEQ ID NO: 1).

[0163] The function of various organs in aging progressively declines. This is due, in part, to increased fibrosis and microvascular leakage. The experiments presented in this example investigated these processes in mice. The results show that treating 18-month-old mice (similar in age to a 65-year-old human) with daily CSD for 6 weeks reduced the levels of fibrosis and microvascular leakage in the heart and kidney to those observed in healthy 3-month-old mice (similar in age to a 20-year-old human).

[0164] Both cardiac fibrosis and microvascular leakage progress with aging from 3 to 9 to 18 months. CSD treatment of 18-month-old mice reverses the levels of fibrosis and microvascular leakage to approximately those observed in 3-month-old mice (Figure 10).

[0165] Both renal fibrosis and microvascular leakage progress with aging from 3 to 9 to 18 months of age. CSD treatment of 18-month-old mice nearly reverses the levels of fibrosis and microvascular leakage to those observed in 3-month-old mice (Figure 11).

[0166] To further verify the reduced fibrosis in CSD-treated aged mice, cardiac and renal tissue sections were stained with picrosirius red to determine the collagen volume fraction (Figure 12). Compared with young mice, aged mice showed high levels of picrosirius red staining in both the heart and kidney, which was significantly reduced by CSD treatment.

[0167] Figures 10, 11, and 12 show that aging-associated microvascular leakage and fibrosis in the heart and kidney are reversed by CSD. Young C57 / Bl6 mice (3 months), middle-aged C57 / Bl6 mice (9 months), and old C57 / Bl6 mice (18 months) were intraperitoneally injected daily with saline vehicle or CSD for 6 weeks. IgG heavy chain leakage and ColI accumulation were assessed by Western blot of heart (Figure 10) and kidney tissue (Figure 11). Actin was the loading control. Two to three mice per category are shown. Quantitation (n=4) revealed significant changes between young and old mice. *** p<0.001, and in the CSD-treated aged mice, ∧∧∧ p<0.001. Figure 12 shows representative examples of picrosirius red staining of heart and kidney tissue sections from the indicated mice. Data were quantified in terms of collagen volume fraction (n=3). No significant changes were observed between young and old mice. *** p<0.001 and ** p<0.01 for the reduction in CSD-treated aged mice. ∧ Shown as p<0.05.

[0168] Because tyrosine kinase activation (phosphorylation) is involved in vascular hyperpermeability through its effects on junctional proteins, we examined the effects of CSD on receptor and nonreceptor tyrosine kinase activation (Figure 13). Similar to changes occurring in microvascular leakage and fibrosis, we observed a significant aging-associated increase in the activation of the receptor tyrosine kinase PDGFR and the nonreceptor tyrosine kinases c-Src and Pyk2 in both the heart and kidney. Again, these aging-associated changes were almost completely reversed by CSD.

[0169] Figure 13 shows that tyrosine kinase activation in the heart and kidney of aged mice is reversed by CSD. The same extracts used in Figures 10 and 11 were analyzed by Western blot for tyrosine kinase activation using antibodies specific for phosphorylated tyrosine residues in PDGFR (α-Y849 / β-Y857), c-SrcY426, and Pyk2Y402. Actin was a loading control. Quantitation revealed no significant changes between young and aged mice. *** p<0.001 and ** p<0.01 for the reduction in CSD-treated aged mice. ∧∧∧ p<0.001 and ∧∧ Shown as p<0.01.

[0170] Given the beneficial effects of CSD on microvascular leakage, fibrosis, and tyrosine kinase signaling in the heart, we assessed the effect of CSD on ventricular function by echocardiography. Although no significant differences were observed between young and old mice in terms of FS and SV, CSD had a positive effect on these parameters (Figure 14). Intravenous ventricle relaxation time (IVRT), a measure of diastolic function, was prolonged with aging, but this was reversed by CSD treatment (Figure 14). These observations suggest that cardiac function in these mice is similar to that of human patients with heart failure with preserved ejection fraction (HFpEF), the most common form of heart failure in the elderly.

[0171] Because young mice treated with AngII developed cardiomyocyte hypertrophy, which was reversed by CSD, we also evaluated this parameter in aged mice. Indeed, similar to AngII-treated mice, cardiomyocyte hypertrophy was elevated in aged mice, and this increase was reversed by CSD (Figure 15).

[0172] CSD improves ventricular function in aged mice Echocardiographic analysis was performed on the same mice used in Figures 10 and 11 before sacrifice. M-mode echocardiographic measurements in the parasternal short axis view (PSAX) were used to quantify changes in EF and FS. Tissue Doppler measurements of PSAX were used to measure IVRT. Values ​​are presented as mean ± SEM. Statistically significant changes were observed between young and old mice. *** p<0.001, the functional improvement of aged mice by CSD treatment was ∧ Shown as p<0.05 (Figure 14).

[0173] CSD reverses aging-associated cardiac hypertrophy Left ventricular tissue sections from the same mice used in Figure 10 were stained with hematoxylin and eosin, and the cross-sectional area of ​​cardiomyocytes was quantified by measuring at least 50 cardiomyocytes for each group (n=3) using SigmaScan Pro image analysis. Statistical significance was determined between young and old mice. *** p<0.001 for the reduction in hypertrophy in aged mice by CSD treatment. ∧ Shown as p<0.05 (Figure 15).

[0174] Example 3: CSD reverses aging-associated pathological changes in the brain The data presented herein demonstrate that in mice with aging-induced brain disease and microvascular leakage, the CSD peptide (Table 1) is highly active in suppressing these pathologies.

[0175] Figure 16 provides a summary of an experiment using CSD (SEQ ID NO: 1). This is similar to the experiment described in Figure 9, except that the displayed values ​​were performed on brain tissue rather than heart or kidney tissue. The results show that 18-month-old mice have much higher levels of microvascular leakage and fibrosis (Figure 17) and activated tyrosine kinases (Figure 18) in the brain than younger mice, and that systemic CSD treatment reduces these levels to approximately those observed in healthy 3-month-old mice.

[0176] Age-associated microvascular leakage, fibrosis, and tyrosine kinase activation in the brain are reversed by CSD Mice were treated as described in Figure 10, except that CSD infusion was for 4 weeks. Brain tissue extracts were analyzed by Western blot for microvascular leakage and fibrosis (Figure 17) and tyrosine kinase activation (Figure 18). Quantitative analysis (n=3) revealed that CSD treatment caused significant changes between young and old mice. *** p<0.001 and ** p<0.01, and for the decrease in aged mice, ∧∧∧ p<0.001 and ∧∧ Shown as p<0.01.

[0177] Example 4: Inhibition of lung and skin fibrosis by unmodified CSD subdomains Experiments were conducted to examine the effect of unmodified CSD subdomains on lung and skin fibrosis. Figure 19 provides details of the experimental design for experiments demonstrating the inhibition of lung and skin fibrosis by unmodified CSD subdomains. Pumps containing bleomycin or saline vehicle were implanted subcutaneously into mice. After one week, the pumps were emptied and replaced with pumps designed to empty in two weeks containing the indicated treatments (n=6 per group). After this two-week period, the mice were sacrificed.

[0178] Figure 20 shows data demonstrating the massive fibrosis caused by bleomycin and its inhibition by the 82-89, 88-95, and 94-101 subdomain peptides as measured by the Ashcroft score (n=6). *p<0.05 for Bleo+Veh vs. Bleo+CSD subdomains.

[0179] Figure 21 shows the inhibition of skin fibrosis and intradermal fat loss by CSD and CSD subdomain peptides. Skin samples were taken near the pump outlet, and the thickness of the dermis and intradermal fat were measured. For saline / vehicle vs. bleo / vehicle, ∧∧∧ p<0.001 for bleo / vehicle vs. bleo / peptide treatment. *** p<0.001, ** p<0.01, * p<0.05.

[0180] Recruitment of activated monocytes to injured lung tissue contributes to the progression of fibrosis and is inhibited in vivo by CSD (Figure 22). Bone marrow (BM) monocytes were isolated from control and bleomycin-treated mice. Significantly increased migration of BM monocytes was observed in bleomycin-treated mice compared to control mice. This enhanced migration was almost completely inhibited by treating mice in vivo with each of the unmodified subdomains prior to monocyte harvest. For Bleo / vehicle vs. Bleo / peptide treatment, *** p<0.001.

[0181] At all these readings, all three subdomains of the CSD were active. Overall, 82-89 was the most active of the subdomains tested.

[0182] Example 5: Inhibition of lung and skin fibrosis by a modified water-soluble version of CSD Experiments were also performed to examine the effect of modified water-soluble versions of the CSD subdomains on lung and skin fibrosis.

[0183] Figure 23 provides details regarding the experimental design of an experiment demonstrating the inhibition of lung and skin fibrosis by a modified water-soluble version of CSD (WCSD; SEQ ID NO: 5). Pumps containing bleomycin or saline vehicle were implanted subcutaneously into mice. After one week, the pumps were emptied. Mice were further injected intraperitoneally daily as indicated, and then all were sacrificed on day 22. All groups contained four mice.

[0184] Figure 24 provides survival and histology data demonstrating that WCSD has a significant beneficial effect on survival. Extensive pulmonary fibrosis and inflammatory cell infiltration caused by bleomycin are suppressed by WCSD, even when treatment is delayed until day 8. WCSD treatment beginning on day 8 also suppresses the near-complete loss of the skin's percutaneous fat layer induced by bleomycin.

[0185] Figure 25 demonstrates that WCSD suppresses bleomycin-induced pulmonary fibrosis through its effects on fibrocytes, ECM proteins, myofibroblast markers, and microvascular leakage. Fibrocytes (CD45+ / ColI+ cells) were quantified by flow cytometry. ECM proteins (ColI, tenascin-C), myofibroblast markers (HSP47, ASMA), and microvascular leakage (IgG heavy chains remaining in the tissue after perfusion) were quantified by Western blotting (n=4). Western blot data were quantified by densitometry after normalization to actin loading controls. Values ​​for saline-treated mice were set to 1.0 arbitrary unit. For saline / vehicle vs. bleomycin / vehicle, ∧∧ p<0.01, ∧ p<0.05; for Breo / vehicle vs. Breo / WCSD; * p<0.05.

[0186] All of these parameters were significantly elevated by bleomycin treatment and were almost completely suppressed by WCSD to the levels of control saline-treated mice.

[0187] Example 6: Inhibition of tumor growth by WCSD The function of tumor stromal cells (cancer-associated fibroblasts, endothelial cells) is modified by tumors to promote their growth. Several groups have observed a lack of caveolin-1 in tumor stromal cells in both humans and mice. Therefore, the ability of WCSD (SEQ ID NO: 5) to inhibit tumor growth was tested in syngeneic mice orthotopically injected with Met1 breast cancer cells. Mice received daily intraperitoneal injections of vehicle or WCSD (0.8 μmol / kg) starting the day after tumor cell injection (n=6 mice / group), resulting in 100% inhibition of tumor growth (Figure 26).

[0188] Example 7: Inhibition of purified kinases by water-soluble versions of CSD The water-soluble version of the CSD peptide inhibits TGFβR2, PKCα, PKCε, and cMet, whereas nintedanib has no effect on these kinases (Figure 27). Conversely, nintedanib inhibits VEGFR1, VEGFR3, PDGFRα, and PDGFRβ, whereas the water-soluble version of the CSD shows no direct inhibition. Both nintedanib and the water-soluble version of the CSD peptide inhibit VEGFR2, TGFβR1, and Src, but nintedanib worked at much lower concentrations. These observations, along with the fact that the water-soluble CSD peptide works at over 100-fold lower concentrations in vivo, suggest that nintedanib and the water-soluble CSD peptide must have different mechanisms of action. This supports the idea that water-soluble CSD candidates may have far less severe side effects than those known to occur with nintedanib.

[0189] The modified water-soluble CSD peptides are more active as kinase inhibitors than their parent unmodified forms (Figure 28).

[0190] Example 8: Optimization of delivery Experiments were conducted to evaluate different delivery routes for water-soluble CSD peptides. Figure 29 demonstrates that intranasal administration of water-soluble CSD peptides is a promising delivery route. Two mice received a single intraperitoneal injection of 0.8 μmol / kg fluoresceinated W82-89 in 100 μl of PBS. Two mice received intranasal administration of fluoresceinated W82-89 (4 μmol / kg in 30 μl of PBS (15 μl per nostril)). Using calibrated capillary tubes, 50 μl of blood was collected from the maxillary vein of one mouse of each pair at 3 minutes, 30 minutes, 2 hours, and 6 hours, and from the other mouse of each pair at 10 minutes, 1 hour, 4 hours, and 8 hours. The blood was immediately diluted with 200 μl of PBS / 10 mM EDTA and centrifuged to collect plasma. The W82-89 levels in plasma were determined using a fluorescent plate reader.

[0191] The intranasal (in) delivery described provided peak serum levels similar to those achieved by intraperitoneal delivery. Furthermore, intranasal delivery resulted in a prolonged plasma presence of W82-89, and the area under the curve for intranasal delivery was more than 1.5-fold higher than that for intraperitoneal administration. These results suggest that intranasal delivery may be an effective approach for delivering W82-89, as well as being preferable for patients.

[0192] Primary lung fibroblasts were cultured for 4 hours in complete medium (DMEM / 10% serum) supplemented with 5 μM of the indicated peptides synthesized with the N-terminal fluorescent dye FAM or free FAM. Free FAM was not taken up by the cells, but FAM-labeled W82-89 and FAM-labeled CSD were, with FAM-labeled WCSD being taken up to a much greater extent (Figure 30). This indicates that modifying CSD to make it water-soluble also significantly improves cellular uptake.

[0193] After a single dose of fluoresceinated W82-89 delivered by the indicated route (Figure 31), blood was collected from the maxillary vein at intervals, and the levels of W82-89 in the blood-derived plasma were determined using a fluorescent plate reader. Note that after intraperitoneal delivery, high levels of fluorescent peptide were present in the plasma. However, after topical delivery, oral gavage, or aerosol, almost no fluorescent peptide was present in the plasma. The intranasal and oropharyngeal routes, which are the preferred routes for treating patients, yielded relatively high levels of fluorescent peptide in the plasma. This is likely due to the possibility that intranasal and oropharyngeal administration may involve penetration via the lungs.

[0194] After a single intraperitoneal administration of the indicated fluorescent peptides, blood was collected from the maxillary vein at intervals, and the peptide levels in the blood-derived plasma were determined using a fluorescent plate reader. Uptake of W82-89 was significantly greater than that of CSD or WCSD (Figure 32).

[0195] It should be understood that the foregoing detailed description and accompanying examples are merely illustrative and do not limit the scope of the invention, which is defined solely by the appended claims and their equivalents.

[0196] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including but not limited to those relating to the chemical structures, substituents, derivatives, intermediates, synthetic methods, compositions, formulations, or methods of use of the invention, can be made without departing from the spirit and scope of the invention.

Claims

1. A method for treating or preventing microvascular leakage or a disease or disorder related thereto, kidney disease, heart disease, or a disease or disorder related to aging in a subject, comprising administering to a subject in need thereof an effective amount of a composition comprising a CSD domain peptide, or a fragment or variant thereof, or a nucleic acid molecule encoding a CSD domain peptide, or a fragment or variant thereof, wherein the CSD domain peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:

8.

2. 2. The method of claim 1, wherein the disease or disorder is an aging-related disease or disorder selected from the group consisting of atherosclerosis, cardiovascular disease, microvascular leakage, cancer, arthritis, cataracts, osteoporosis, Alzheimer's disease and related neurodegenerative diseases, and hypertension.

3. 2. The method of claim 1, wherein the renal disease is renal inflammatory damage, renal dysfunction, chronic renal failure, or hypertension.

4. 10. The method of claim 1, wherein the cardiac disease is cardiac hypertrophy, atherosclerosis, cardiomyopathy, stroke, or hypertension.

5. 2. The method of claim 1, wherein the disease or disorder associated with microvascular leakage is congestive heart failure, scleroderma and interstitial lung disease in general, asthma, renal failure, neurodegenerative diseases including Alzheimer's disease and vascular dementia, cancer, venous thrombosis, diabetes and diabetic complications, sepsis, or acute respiratory distress syndrome (ARDS).

6. A method for treating or preventing a disease or disorder in a subject, comprising administering to a subject in need thereof an effective amount of a composition comprising a CSD domain peptide, or a fragment or variant thereof, or a nucleic acid molecule encoding the CSD domain peptide, or a fragment or variant thereof, wherein the CSD domain peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:

8.

7. 7. The method of claim 6, wherein the disease or disorder is an aging-related disease or disorder selected from the group consisting of atherosclerosis, cardiovascular disease, microvascular leakage, cancer, arthritis, cataracts, osteoporosis, Alzheimer's disease and related neurodegenerative diseases, and hypertension.

8. 7. The method of claim 6, wherein the disease or disorder is fibrosis or a fibrosis-related disease or disorder.

9. 7. The method of claim 6, wherein the disease or disorder is microvascular leakage or a disease or disorder associated with microvascular leakage.

10. 10. The method of claim 9, wherein the disease or disorder is congestive heart failure, scleroderma and interstitial lung disease in general, asthma, renal failure, neurodegenerative diseases including Alzheimer's disease and vascular dementia, cancer, venous thrombosis, diabetes and diabetic complications, sepsis, or acute respiratory distress syndrome (ARDS).

11. 7. The method of claim 6, wherein the disease or disorder is a renal disease.

12. 12. The method of claim 11, wherein the renal disease is renal inflammatory damage, renal dysfunction, chronic renal failure, or hypertension.

13. 7. The method of claim 6, wherein the disease or disorder is a heart disease.

14. 14. The method of claim 13, wherein the cardiac disease is cardiac hypertrophy, atherosclerosis, cardiomyopathy, stroke, or hypertension.

15. A modified CSD domain peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, or a fragment or variant thereof.

16. A composition comprising the modified CSD domain peptide of claim 15.

17. 17. The composition of claim 16, further comprising a pharmaceutically acceptable carrier.

18. 17. The composition of claim 16, for treating or preventing a disease or disorder in a subject.

19. 19. The composition of claim 18, wherein the disease or disorder is an aging-related disease or disorder selected from the group consisting of atherosclerosis, cardiovascular disease, kidney disease, microvascular leakage, cancer, arthritis, cataracts, osteoporosis, AD and related neurodegenerative diseases, diabetic complications, and hypertension.

20. 19. The composition of claim 18, wherein the disease or disorder is fibrosis or a fibrosis-related disease or disorder.

21. 19. The composition of claim 18, wherein the disease or disorder is microvascular leakage or a disease or disorder associated with microvascular leakage.

22. 22. The composition of claim 21, wherein the disease or disorder is selected from the group consisting of congestive heart failure, scleroderma and interstitial lung disease in general, asthma, renal failure, neurodegenerative diseases including Alzheimer's disease and vascular dementia, cancer, venous thrombosis, diabetes and diabetic complications, sepsis, and acute respiratory distress syndrome (ARDS).

23. 19. The composition of claim 18, wherein the disease or disorder is a kidney disease.

24. 24. The composition of claim 23, wherein the renal disease is renal inflammatory damage, renal dysfunction, chronic renal failure, or hypertension.

25. 19. The composition of claim 18, wherein the disease or disorder is a heart disease.

26. 26. The composition of claim 25, wherein the cardiac disease is cardiac hypertrophy, atherosclerosis, cardiomyopathy, stroke, or hypertension.

27. 17. The composition of claim 16, wherein the composition is formulated for administration by a delivery route selected from the group consisting of intranasal, oropharyngeal, and intraperitoneal.

28. A composition for treating or preventing microvascular leakage or a disease or disorder related thereto, kidney disease, heart disease, or a disease or disorder related to aging, comprising a CSD domain peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, or a fragment or variant thereof.

29. 29. The composition of claim 28, wherein the disease or disorder is an aging-related disease or disorder selected from the group consisting of atherosclerosis, cardiovascular disease, kidney disease, microvascular leakage, cancer, arthritis, cataracts, osteoporosis, AD and related neurodegenerative diseases, diabetic complications, and hypertension.

30. 29. The composition of claim 28, wherein the renal disease is renal inflammatory damage, renal dysfunction, chronic renal failure, or hypertension.

31. 29. The composition of claim 28, wherein the cardiac disease is cardiac hypertrophy, atherosclerosis, cardiomyopathy, stroke, or hypertension.

32. 29. The composition of claim 28, wherein the disease or disorder associated with microvascular leakage is congestive heart failure, scleroderma and interstitial lung disease in general, asthma, renal failure, neurodegenerative diseases including Alzheimer's disease and vascular dementia, cancer, venous thrombosis, diabetes and diabetic complications, sepsis, or acute respiratory distress syndrome (ARDS).

33. 30. The composition of claim 28, wherein the composition is formulated for administration by a delivery route selected from the group consisting of intranasal, oropharyngeal, and intraperitoneal.

Images