Modified peptide fragments of the CAV-1 protein and their use in the treatment of fibrosis

Abstract

To provide modified peptide fragments of CAV-1 protein and use thereof in treatment of fibrosis.SOLUTION: Provided herein are compositions comprising modified caveolin-1 (Cav-1) peptides. Further provided are methods of using the modified Cav-1 peptides for the treatment of lung infections or acute or chronic lung injury, particularly lung fibrosis.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 728,997, filed September 10, 2018, the entire contents of which are incorporated herein by reference.

[0002] This invention was made as a result of activities carried out within the scope of a collaborative research agreement in effect at the time this invention was made. Parties to said collaborative research agreement are the Board of Regents of the University of Texas System and Lung Therapeutics. [Background technology]

[0003] 1.Technical Field The present invention relates generally to the fields of molecular biology and medicine. More particularly, it relates to compositions and methods for delivery of therapeutic polypeptide compositions to a subject, such as by delivery to the respiratory system.

[0004] 2. Description of Related Technology During lung injury, p53 expression increases and plasminogen activator inhibitor-1 (PAI-1) is induced, while the expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) is inhibited, resulting in apoptosis of lung epithelial cells (LECs). The mechanism of injury involves cell surface signaling interactions between uPA, uPAR, caveolin-1 ("Cav-1"), and β1-integrin (Shetty et al., 2005). Compositions that modulate these interactions can be used in methods for inhibiting apoptosis of damaged or injured lung epithelial cells, as well as in methods for treating acute lung injury and the resulting pulmonary fibrosis. Therefore, there is a need for polypeptides that can be used to prevent or treat lung injury, particularly formulations and methods for the therapeutic delivery of such polypeptides. Summary of the Invention

[0005] According to the present disclosure, a peptide is provided that includes the amino acid sequence of SEQ ID NO: 2, wherein the peptide includes at least one N- or C-terminal addition. The N- or C-terminal addition may be a standard amino acid, a non-standard amino acid, or a chemical modification. Peptide multimers of the peptides of the present disclosure are provided. Pharmaceutical compositions of the peptides are also provided. The peptides of the present disclosure can be used to treat lung injury, infection, or disease. In a further aspect, the peptides of the embodiments can be used to treat fibrotic conditions, such as organ fibrosis, or inflammation.

[0006] In some embodiments, the present disclosure provides a peptide comprising the amino acid sequence ASFTTFTVT (SEQ ID NO: 3), wherein the peptide comprises at least one N- or C-terminal addition that lacks identity with SEQ ID NO: 1. In some aspects, the peptide comprises at least one amino acid added to the N-terminus. In some aspects, the peptide comprises at least one amino acid added to the C-terminus. In some aspects, the peptide comprises at least one amino acid added to the N- and C-terminus. In some aspects, the peptide maintains the biological activity of caveolin-1 (Cav-1). In further aspects, the peptides of the embodiments may comprise one or more deuterated residues.

[0007] In some embodiments, the peptide comprises L-amino acids. In some embodiments, the peptide comprises D-amino acids. In some embodiments, the peptide comprises both L- and D-amino acids.

[0008] In some embodiments, the peptide comprises at least one non-standard amino acid. In some embodiments, the peptide comprises two or more non-standard amino acids. In some embodiments, the peptide comprises four or more non-standard amino acids. In some embodiments, the non-standard amino acid is ornithine. In some embodiments, the non-standard amino acid is D-alanine.

[0009] In some embodiments, the peptide comprises an N- or C-terminal modification. In some embodiments, the peptide comprises an N-terminal modification. In some embodiments, the peptide comprises a C-terminal modification. In some embodiments, the peptide comprises an N- and C-terminal modification. In some embodiments, the N-terminal modification is an acylation. In some embodiments, the C-terminal modification is an amidation.

[0010] In some embodiments, the peptide comprises the amino acid sequence KASFTTFTVTKGS (SEQ ID NO:4). In some embodiments, the peptide comprises the amino acid sequence aaEGKASFTTFTVTKGSaa (SEQ ID NO:6). In other embodiments, the peptide comprises the amino acid sequence OASFTTFTVTOS (SEQ ID NO:9). In other embodiments, the peptide comprises the amino acid sequence aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO:7). In yet other embodiments, the peptide comprises the amino acid sequence Ac-aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO:8). In other embodiments, the peptide comprises the amino acid sequence OASFTTFTVTOS-NH2 (SEQ ID NO:10).

[0011] In some aspects, the peptide further comprises a cell penetrating peptide (CPP). In some embodiments, the CPP comprises an amino acid sequence selected from the group including GRKKRRQRRRPPQ (SEQ ID NO: 21), RQIKIWFQNRRMKWKK (SEQ ID NO: 22), and GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 23).

[0012] In some embodiments, the present disclosure provides a peptide multimer comprising at least two peptides as disclosed herein.In some embodiments, the first peptide of the at least two peptides is essentially identical to the second peptide of the at least two peptides.In other embodiments, the first peptide of the at least two peptides is not identical to the second peptide of the at least two peptides.

[0013] In some embodiments, the present disclosure provides compositions comprising the peptides disclosed herein. In some embodiments, the peptides are substantially pure. In some embodiments, the peptides are at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, or at least 99% pure.

[0014] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the peptide, a peptide as disclosed herein, and a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition is formulated for oral, intravenous, intraarticular, parenteral, enteral, topical, subcutaneous, intramuscular, buccal, sublingual, rectal, vaginal, intrapenile, intraocular, epidural, intracranial, or inhalation administration. In some aspects, the pharmaceutical composition is formulated for pulmonary instillation. In some aspects, the pharmaceutical composition is formulated as a nebulized solution.

[0015] In some embodiments, the present disclosure provides a polynucleotide comprising a nucleic acid sequence encoding a peptide as described herein.

[0016] In certain aspects, the peptide compositions of the embodiments can be used in methods for treating or preventing a disease in a subject. In some aspects, the disease is a fibrotic disease or an inflammatory disease. For example, the fibrotic disease can be an organ fibrotic disease, such as fibrosis of the kidney, liver, lung, or heart. In some aspects, the inflammatory disease is an inflammatory eye disease. The compositions of the embodiments can be administered systemically or locally (e.g., to the site of the diseased tissue).

[0017] In some embodiments, the present disclosure provides a method for treating or preventing acute lung injury, lung infection, or lung disease in a subject, comprising administering to the subject an effective amount of a peptide described herein. In some embodiments, the subject has pulmonary inflammation. In some embodiments, the subject is undergoing chemotherapy or radiation therapy. In some embodiments, the subject is suffering from acute lung injury or infection. In some embodiments, the subject has chemical-induced lung injury. In some embodiments, the subject has plastic bronchitis, chronic obstructive pulmonary disease, bronchitis, bronchiolitis, bronchiolitis obliterans, asthma, acute respiratory distress syndrome (ARDS), or inhalation smoke-induced acute lung injury (ISALI). In some embodiments, the lung disease is a pulmonary fibrotic condition. In some embodiments, the lung disease is interstitial lung disease. In some embodiments, the lung disease is idiopathic pulmonary fibrosis (IPF) or lung scarring. In some embodiments, administering comprises nebulizing a solution comprising the peptide. In some embodiments, the method further comprises administering at least one additional antifibrotic therapeutic agent. In some embodiments, the at least one additional anti-fibrotic agent is an NSAID, a steroid, a DMARD, an immunosuppressant, a biological response modulator, or a bronchodilator. In some embodiments, the subject is a human.

[0018] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0019] The following drawings form part of the present specification and are included to further support certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0020] [Figure 1] Western blot of SMA and tubulin idiopathic pulmonary fibrosis cells treated with Cav-1 peptides. IPF cells were treated with the following: 1: untreated, 2: 10 μM LTI-03, 3: 90 μM LTI-03, 4: 10 μM APi2350, 5: 10 μM APi2354, 6: 10 μM APi2355, 7: 10 μM APi2356, and 8: DMSO, and SMA and tubulin expression was assessed by Western blot. [Figure 2] Treatment with Cav-1 peptides increases SMA relative to tubulin in IPF cells. Graphical representation of the ratio of SMA to tubulin in cells receiving the indicated treatments. DETAILED DESCRIPTION OF THE INVENTION

[0021] Description of exemplary embodiments The present disclosure overcomes the challenges associated with current technology by providing modified caveolin-1 (Cav-1) peptides and their uses for the treatment and prevention of diseases, particularly pulmonary fibrosis. In some embodiments, pharmaceutical formulations of the modified Cav-1 peptides are provided. For example, in some embodiments, the peptides are formulated for delivery to the respiratory system. For example, the peptides can be prepared for administration to the respiratory tract of a subject by formulating them in an aqueous solution and spraying the solution using a nebulizer. In other embodiments, the peptides can be formulated for injection. Also provided herein are methods for treating lung injury and disease by administering a therapeutically effective amount of a modified Cav-1 peptide to a subject (e.g., via the respiratory tract).

[0022] I. Definition As used herein, "essentially free" with respect to a particular component means that none of the particular component is intentionally incorporated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the particular component resulting from unintentional contamination of the composition is well below 0.01%. Most preferred are compositions in which the amount of the particular component is undetectable by standard analytical methods.

[0023] As used herein, "a" or "an" may mean one or more. As used herein in the claims, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more.

[0024] Use of the term "or" in the claims is used to mean "and / or," unless expressly indicated to refer to alternatives only or where the alternatives are mutually exclusive, but this disclosure supports the definition to refer to alternatives only and "and / or," and as used herein, "another" can mean at least one second or more. As used herein, "another" can mean at least a second or more.

[0025] Throughout this specification, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among test subjects.

[0026] The term "peptide" as used herein refers to a sequence of amino acids, typically composed of a single chain of amino acids linked by peptide bonds. Generally, a peptide contains at least two amino acid residues and is less than about 50 amino acids in length, unless otherwise defined.

[0027] A "biologically active" caveolin-1 (Cav-1) peptide refers to a peptide that increases p53 protein levels, decreases urokinase plasminogen activator (uPA) and uPA receptor (uPAR), and / or increases plasminogen activator inhibitor-1 (PAI-1) expression in cells, such as fibrotic lung fibroblasts. In some embodiments, a biologically active peptide has at least 20% of the biological or biochemical activity of a native Cav-1 polypeptide of SEQ ID NO: 1 (e.g., as measured by in vitro or in vivo assays). In some embodiments, a biologically active peptide has increased biological or biochemical activity compared to a native Cav-1 polypeptide.

[0028] The term "identity" or "homology" should be interpreted to mean the percentage of amino acid residues in a candidate sequence that are identical with the residues of the corresponding sequence to which it is compared, after aligning the sequences and introducing gaps (if necessary to achieve the maximum percent identity for the overall sequence), and without taking into account any conservative substitutions as part of the sequence identity. Neither N- nor C-terminal extensions nor insertions should be construed as reducing identity or homology. Methods and computer programs for alignment are well known in the art. Sequence identity can be measured using sequence analysis software.

[0029] The terms "polypeptide" or "protein" are used in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In alternative embodiments, the subunits may be linked by other bonds, such as esters, ethers, etc. As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acid, including glycine and both its D or L optical isomers, as well as amino acid analogs and peptidomimetics. The term "peptidomimetic" or "peptido mimic" means that the peptides of the present invention are modified to include at least one non-peptide bond, such as a urea bond, a carbamate bond, a sulfonamide bond, a hydrazine bond, or any other covalent bond. When the peptide chain is short, a peptide of three or more amino acids is generally called an oligopeptide. When the peptide chain is long, the peptide is generally called a polypeptide or a protein.

[0030] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to an animal, e.g., a human or non-human animal (e.g., a mammal), receiving treatment, including prophylactic treatment, with a pharmaceutical composition as disclosed herein. As used herein, the term "subject" refers to a human and a non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals such as non-human primates (especially higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, and cows, and non-mammals such as chickens, amphibians, and reptiles. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal surrogate for a disease model. Non-human mammals include mammals such as non-human primates (especially higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, and cows. In some embodiments, the non-human animal is a companion animal, such as a dog or cat.

[0031] "Treating" a disease or condition in a subject or a patient with a disease or condition refers to administering pharmaceutical treatment to an individual, e.g., administering a drug, such that at least one symptom or condition of the disease is reduced or stabilized. Typically, when a peptide is administered therapeutically as a treatment, it is administered to a subject who exhibits one or more symptoms of lung injury or pulmonary fibrosis.

[0032] By "isolated" it is meant that the polypeptide is separated from any natural environment, such as a body fluid, e.g., blood, and is separated from components that naturally accompany the polypeptide.

[0033] Isolated and "substantially pure" refers to a polypeptide that has been separated and purified to at least some extent from components that naturally accompany it. Typically, a polypeptide is substantially pure when it is at least about 60%, or at least about 70%, at least about 80%, at least about 90%, at least about 95%, or even at least about 99%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. For example, a substantially pure polypeptide may be obtained by extraction from a natural source, by expression of a recombinant nucleic acid in a cell that does not normally express the protein, or by chemical synthesis.

[0034] As used herein, the term "variant" refers to a polypeptide or nucleic acid that differs from the polypeptide or nucleic acid by one or more amino acid or nucleic acid deletions, additions, substitutions, or side chain modifications, but retains one or more specific functions or biological activities of the naturally occurring molecule. Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally occurring or non-conventional amino acid residue. Such substitutions can be classified as "conservative," in which an amino acid residue present in a polypeptide is replaced with another naturally occurring amino acid of similar characteristics in terms of either polarity, side chain functionality, or size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present invention can also be "non-conservative," in which an amino acid residue present in the peptide is replaced with an amino acid with different properties, such as a naturally occurring amino acid from a different group (e.g., replacing a charged or hydrophobic amino acid with alanine), or a naturally occurring amino acid is replaced with a non-conventional amino acid. In some embodiments, the amino acid substitution is conservative. Also included in the term variant when used in reference to a polynucleotide or polypeptide, refers to a polynucleotide or polypeptide that may differ in primary, secondary, or tertiary structure compared to a reference polynucleotide or polypeptide (e.g., compared to a wild-type polynucleotide or polypeptide), respectively.

[0035] The term "insertion" or "deletion" typically ranges from about 1 to 5 amino acids. The variation allowed can be determined experimentally by using recombinant DNA technology to synthetically produce the peptide while systematically inserting, deleting, or substituting nucleotides within the sequence.

[0036] The term "substitution" when referring to a peptide refers to a change of amino acid for a different entity, e.g., another amino acid or amino acid moiety. The substitution may be a conservative or non-conservative substitution.

[0037] An "analog" of a molecule such as a peptide refers to a molecule similar in function to either the entire molecule or a fragment thereof. The term "analog" is also intended to include allelic species and induced variants. Analogs typically differ from naturally occurring peptides at one or several positions, often due to conservative substitutions. Analogs typically exhibit at least 80 or 90% sequence identity with the native peptide. Some analogs also include unnatural amino acids or N- or C-terminal amino acid modifications. Examples of unnatural amino acids include, but are not limited to, disubstituted amino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, and σ-N-methylarginine. Fragments and analogs can be screened for prophylactic or therapeutic efficacy in transgenic animal models, as described below.

[0038] "Covalently linked" means linked directly or indirectly (e.g., via a linker) by a covalent chemical bond. In some aspects of all embodiments of the present invention, the fusion peptide is covalently linked.

[0039] As used herein, the term "fusion protein" refers to a recombinant protein of two or more proteins. Fusion proteins, for example, involve the ligation of a nucleic acid sequence encoding one protein with a nucleic acid sequence encoding another protein, resulting in a single open reading frame that can be translated in a cell into a single polypeptide containing all the intended proteins. The order of the proteins can vary. Fusion proteins can also contain epitope tags or half-life extenders. Epitope tags include biotin, FLAG tag, c-myc, hemagglutinin, His6, digoxigenin, FITC, Cy3, Cy5, green fluorescent protein, V5 epitope tag, GST, β-galactosidase, AU1, AU5, and avidin. Half-life extenders include Fc domains and serum albumin.

[0040] The term "respiratory tract," as used herein, refers to any portion of the respiratory tract, including the upper respiratory tract, airways, and lungs. The upper respiratory tract includes the nose and nasal passages, mouth, and throat. The respiratory tract includes the larynx, trachea, bronchi, and bronchioles. The lungs include respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli.

[0041] The terms "inhalation smoke-induced acute lung injury" and "ISALI" are used interchangeably herein and refer to forms of acute lung injury (ALI) caused by smoke inhalation. ALI is also known as "mild acute respiratory distress syndrome (ARDS)." ARDS may be defined by the presence of one or more of the following conditions in a subject: 1) bilateral pulmonary infiltrates on chest x-ray, 2) pulmonary artery occlusion pressure less than 18 mmHg (2.4 kPa) as measured by right heart catheterization as clinically indicated, and 3) PaO2 / FiO2 < 300 mmHg (40 kPa). In some embodiments, treatment of ISALI includes treatment of one or more of the following conditions: reduced oxygenation, airway obstruction (including severe airway obstruction), fibrous airway casts or debris, and alveolar fibrin deposition.

[0042] The terms "atomization," "nebulization," and other grammatical variations thereof, as used herein, refer to the process of converting a liquid into small aerosol droplets. In some embodiments, the aerosol droplets have a median diameter of about 2-10 μm. In some embodiments, the aerosol droplets have a median diameter of about 2-4 μm.

[0043] II. Caveolin-1 Peptides Embodiments of the present disclosure provide peptide variants of the caveolin-1 (Cav-1) protein. The caveolin-1 (Cav-1) scaffold domain or polypeptide disrupts Cav-1 interaction with Src kinase and mimics the combined effect of uPA and anti-β1-integrin antibodies. Native human Cav-1 is 178 amino acids in length and has a molecular weight of 22 kDa. The amino acid sequence of Cav-1 is shown below (SEQ ID NO: 1). 1 MSGGKYVDSE GHLYTVPIRE QGNIYKPNNK AMADELSEKQ VYDAHTKEID LVNRDPKHLN 61 DDVVKIDFED VIAEPEGTHS FDGIWKAS FT TFTVT KYWFY RLLSALFGIP MALIWGIYFA 121 ILSFLHIWAV VPCIKSFLIE IQCISRVYSI YVHTVCDPLF EAVGKIFSNV RINLQKEI

[0044] In some embodiments, the peptide is a scaffold domain peptide comprising an amino acid sequence at least about 40%, 50%, 60%, 70%, 80%, 85%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, FTTFTVT. The peptide may contain one, two, three, four, or more amino acid substitutions, deletions, or insertions relative to the sequence of SEQ ID NO: 1 to derive a polypeptide of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 residues. In certain embodiments, the peptide is a truncated version of a native Cav-1 polypeptide, such as the exemplary polypeptides set forth in Table 1.

[0045] [Table 1] (a=D-alanine, O=ornithine)

[0046] The peptides provided herein are biologically active derivatives that possess the activity of native CAV-1 polypeptides in in vitro or in vivo assays of binding or biological activity. In certain embodiments, the peptides inhibit or prevent BLM-induced apoptosis of LECs in vitro or in vivo with at least about 20%, or at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, about 95%, 97%, 99%, and any range derivable therein, e.g., about 70% to about 80%, more preferably about 81% to about 90%, or even more preferably about 91% to about 99% of the activity of the native CAV-1 polypeptide. The peptides may be 100% or more active than the native CAV-1 polypeptide. Assays for testing biological activity, such as anti-fibrotic activity, the ability to affect the expression of uPA, uPAR and PAI-1 mRNA, or the ability to inhibit the proliferation of pulmonary fibroblasts, are well known in the art.

[0047] The peptides of the present disclosure are peptides of native Cav-1 polypeptides or modified versions thereof. Peptides can be synthetic, recombinant, or chemically modified peptides isolated or produced using methods well known in the art. Modifications can be made to the N-terminus, C-terminus, or internal amino acids. N-terminal modifications can be, for example, but not limited to, acylation, acetylation, or C-terminal amidation. Peptides can contain conservative or non-conservative amino acid changes, as described below. Polynucleotide changes can result in amino acid substitutions, additions, deletions, fusions, and truncations of the polypeptide encoded by the reference sequence. Peptides can also contain amino acid insertions, deletions, or substitutions, including insertions and substitutions of amino acids (and other molecules) that do not normally occur in the peptide sequence underlying the modified variant, such as, but not limited to, insertions of L-amino acids or non-standard amino acids (not normally found in human proteins), such as ornithine. The term conservative substitution, when describing a peptide, refers to a change in the amino acid composition of the peptide that does not substantially alter the peptide's activity. For example, a conservative substitution refers to the substitution of one amino acid residue for a different amino acid residue having similar chemical properties, such as replacing leucine with isoleucine or valine, aspartic acid with glutamate, or threonine with serine.

[0048] Conservative amino acid substitutions result from replacing one amino acid with another amino acid with similar structural and / or chemical properties, such as replacing leucine with isoleucine or valine, aspartic acid with glutamate, or threonine with serine. Thus, conservative substitutions of a particular amino acid sequence refer to the substitution of an amino acid that is not essential to the activity of the polypeptide, or the substitution of an amino acid with another amino acid with similar properties (e.g., acidic, basic, positively or negatively charged, polar or nonpolar, etc.), such that even the substitution of an essential amino acid does not reduce the activity of the peptide. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups contain amino acids that are conservative substitutions for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W). (See also Creighton, Proteins, W.H. Freeman and Company (1984), incorporated by reference in its entirety.) In some embodiments, individual substitutions, deletions, or additions that change, add, or remove a single amino acid or a small number of amino acids can also be considered conservative substitutions if the changes do not reduce the activity of the peptide. Insertions or deletions typically range from about 1 to 5 amino acids. Conservative amino acids may be selected based on the position of the amino acid to be substituted within the peptide, e.g., whether the amino acid is on the exterior of the peptide and exposed to solvent, or on the interior and not exposed to solvent.

[0049] In alternative embodiments, amino acids to replace existing amino acids may be selected based on the position of the existing amino acid, i.e., its solvent exposure (i.e., whether the amino acid is solvent exposed or present on the exterior of the peptide or polypeptide compared to an amino acid located in the interior that is not solvent exposed). The selection of such conservative amino acid substitutions is well known in the art, for example, as disclosed in Dordo et al., J. Mol. Biol., 1999, 217, 721-739 and Taylor et al., J. Theor. Biol. 119 (1986); 205-218, and S. French and B. Robson, J. Mol. Evol. 19 (1983) 171. Thus, suitable conservative amino acid substitutions may be selected for amino acids on the exterior of the protein or peptide (i.e., amino acids exposed to solvent), for example, but not limited to, the following substitutions may be used: Y for F, T for S or K, P for A, E for D or Q, N for D or G, R for K, G for N or A, T for S or K, D for N or E, I for L or V, F for Y, S for T or A, R for K, G for N or A, K for R, A for S, K or P.

[0050] In alternative embodiments, conservative amino acid substitutions may be selected that are appropriate for amino acids within the interior of a protein or peptide, for example, suitable conservative substitutions of amino acids that are within the interior of a protein or peptide (i.e., amino acids that are not exposed to solvent) may be used, for example, but not limited to, the following conservative substitutions may be used: where Y is substituted with F, T is A or S, I is L or V, W is Y, M is L, N is D, G is A, T is A or S, D is N, I is L or V, F is Y or L, S is A or T, and A is S, G, T, or V. In some embodiments, non-conservative amino acid substitutions are also included in the term variant.

[0051] In some embodiments, the polypeptide is a derivative of a native Cav-1 polypeptide. As used herein, the term "derivative" refers to a peptide that has been chemically modified, for example, but not limited to, by techniques such as acetylation, ubiquitination, labeling, pegylation (derivatization with polyethylene glycol), lipidation, glycosylation, amidation, or the addition of other molecules. A molecule is also a "derivative" of another molecule if it contains additional chemical moieties not normally part of the molecule. Such moieties may alter the pH or improve the solubility, absorption, biological half-life, etc. of the molecule. Alternatively, the moiety may reduce the toxicity of the molecule, eliminate or mitigate any undesirable side effects of the molecule, etc. Moieties that can mediate such effects are disclosed in Remington's Pharmaceutical Sciences, 18th edition, A.R. Gennaro, Ed., MackPubl., Easton, PA (1990), which is incorporated herein by reference in its entirety.

[0052] The term "functional" when used in conjunction with "derivative" or "variant" refers to a polypeptide of the invention (of which it is a functional derivative or functional variant) that has a biological activity (either functional or structural) that is substantially similar to that of the entity or molecule. The term functional derivative is intended to include fragments, analogs, or chemical derivatives of a molecule.

[0053] In some embodiments, amino acid substitutions may be made in a polypeptide at one or more positions, with the substitution being for an amino acid with similar hydrophilicity. The importance of the amino acid hydropathic index in conferring interactive biological function to a protein is generally understood in the art (Kyte and Doolittle, 1982). It is recognized that the relative hydropathic characteristics of amino acids contribute to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Thus, such conservative substitutions may be made in polypeptides and are likely to have only a minor effect on their activity. As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values ​​have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (0.5); histidine -0.5; cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). These values ​​can be used as a guide, with substitution of amino acids whose hydrophilicity values ​​are within ±2 being preferred, those within ±1 being particularly preferred, and those within ±0.5 being even more particularly preferred. Thus, any of the polypeptides described herein may be modified by substituting an amino acid for a different but homologous amino acid having a similar hydrophilicity value. Amino acids whose hydrophilicity values ​​are within ±1.0 or ±0.5 points are considered homologous.

[0054] Modified Cav-1 peptides may include co-translational and post-translational (C-terminal peptide cleavage) modifications such as, for example, disulfide bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., cleavage by furin or metalloproteases), to the extent that such modifications do not affect the anti-inflammatory properties of the isolated peptides or their ability to improve glycemic control.

[0055] In some embodiments, the modified Cav-1 peptide contains non-naturally occurring amino acids. The polypeptide may contain a combination of naturally occurring and non-naturally occurring amino acids, or may contain only non-naturally occurring amino acids. Non-naturally occurring amino acids may be synthetic non-natural amino acids, substituted amino acids, or one or more D-amino acids in the peptide (or other components of the composition, excluding the protease recognition sequence), which may be desirable in certain situations. D-amino acid-containing peptides exhibit increased stability in vitro or in vivo compared to L-amino acid-containing forms. Thus, constructing peptides incorporating D-amino acids may be particularly useful when greater in vivo or intracellular stability is desired or required. More specifically, D-peptides are resistant to endogenous peptidases and proteases, thereby providing better oral transepithelial and transdermal delivery of linked drugs and conjugates, improved bioavailability of membrane-permanent complexes (see below for further discussion), and prolonged intravascular and interstitial longevity (where such properties are desirable). The use of D-isomer peptides can also enhance transdermal and oral transepithelial delivery of linked drugs and other cargo molecules. Furthermore, D-peptides cannot be efficiently processed for major histocompatibility complex class II restricted presentation to T helper cells, making them less likely to induce humoral immune responses in the whole organism. Therefore, peptide conjugates can be constructed using, for example, a D-isomer form of the cell-penetrating peptide sequence, an L-isomer form of the cleavage site, and a D-isomer form of the therapeutic peptide.

[0056] In addition to the 20 "standard" L-amino acids, D-amino acids well-defined in the art or non-standard modified or unusual amino acids are also contemplated for use in this disclosure. Phosphorylated amino acids (Ser, Thr, Tyr), glycosylated amino acids (Ser, Thr, Asn), β-amino acids, GABA, ω-amino acids are further contemplated for use in this disclosure. These include, for example, β-alanine (β-Ala) and other ω-amino acids such as 3-aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid, etc.; α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly); ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (MeIle); phenylglycine (Phg); norleucine (Nle); 4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F)); 3 -fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); homoarginine (hArg); N-acetyllysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,4-diaminobutyric acid (Dab); p-aminophenylalanine (Phe(pNH2)); N-methylvaline (MeVal); homocysteine ​​(hCys), homophenylalanine (hPhe), and homoserine (hSer); hydroxyproline (Hyp), homoproline (hPro), N-methylated amino acids, and peptoids (N-substituted glycines).

[0057] Carboxylic acid terminal modifications include acylation with carboxylic acids: formic acid, acetic acid, propionic acid, fatty acids (myristic acid, palmitic acid, stearic acid), succinic acid, benzoic acid, carbobenzoxy (Cbz); acetylation, and biotinylation. Amino-terminal modifications include: (i) acylation with carboxylic acids: formic acid, acetic acid, propionic acid, fatty acids (such as myristic acid, palmitic acid, and stearic acid), succinic acid, benzoic acid, and carbobenzoxy (Cbz); (ii) biotinylation; (iii) amidation; (iv) attachment of dyes such as fluorescein (FITC, FAM, and the like), 7-hydroxy-4-methylcoumarin-3-acetic acid, 7-hydroxycoumarin-3-acetic acid, 7-methoxycoumarin-3-acetic acid, and other coumarins; rhodamine (5-carboxyrhodamine 110 or 6G, 5(6)-TAMRA, ROX); N-[4-(4-dimethylamino)phenylazo]benzoic acid (Dabcyl), 2,4-dinitrobenzene (Dnp), 5-dimethylaminonaphthalene-1-sulfonic acid (Dansyl), and other dyes; and (v) polyethylene glycol.

[0058] Polypeptides may be capped at their N- and C-termini with acyl (abbreviated "Ac") and amide (abbreviated "Am") groups, respectively, e.g., acetyl (CHCO-) ​​at the N-terminus and amide -NH) at the C-terminus. A wide range of N-terminal capping functions are contemplated, preferably attached to the terminal amino group, e.g., formyl; Alkanoyl having 1 to 10 carbon atoms, such as acetyl, propionyl, butyryl; Alkenoyl having 1 to 10 carbon atoms, such as hex-3-enoyl; Alkynoyl having 1 to 10 carbon atoms, such as hex-5-ynoyl; aroyl, such as benzoyl or 1-naphthoyl; heteroaroyl, such as 3-pyrroyl or 4-quinoloyl; alkylsulfonyl such as methanesulfonyl; arylsulfonyl such as benzenesulfonyl or sulfanilyl; heteroarylsulfonyl such as pyridine-4-sulfonyl; substituted alkanoyl having 1 to 10 carbon atoms, such as 4-aminobutyryl; substituted alkenoyl having 1 to 10 carbon atoms, such as 6-hydroxy-hex-3-enoyl; substituted alkynoyl having 1 to 10 carbon atoms, such as 3-hydroxy-hex-5-ynoyl; substituted aroyls such as 4-chlorobenzoyl or 8-hydroxy-naphth-2-oyl; substituted heteroaroyls such as 2,4-dioxo-1,2,3,4-tetrahydro-3-methyl-quinazolin-6-oyl; substituted alkylsulfonyls such as 2-aminoethanesulfonyl; substituted arylsulfonyls such as 5-dimethylamino-1-naphthalenesulfonyl; substituted heteroarylsulfonyl such as 1-methoxy-6-isoquinolinesulfonyl; carbamoyl or thiocarbamoyl; substituted carbamoyl (R'-NH-CO) or substituted thiocarbamoyl (R'-NH-CS), where R' is alkyl, alkenyl, alkynyl, aryl, heteroaryl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted aryl, or substituted heteroaryl; Substituted carbamoyl (R'-NH-CO) and substituted thiocarbamoyl (R'-NH-CS) where R' is alkanoyl, alkenoyl, alkynoyl, aroyl, heteroaroyl, substituted alkanoyl, substituted alkenoyl, substituted alkynoyl, substituted aroyl, or substituted heteroaroyl (all as defined above). The C-terminal capping function can be either an amide or ester bond with the terminal carboxyl. The capping function that provides the amide bond is NR 1 R 2 where R 1 and R 2 may be independently drawn from the following group: hydrogen; alkyl preferably having 1 to 10 carbon atoms, such as methyl, ethyl, isopropyl, etc.; Alkenyl preferably having 1 to 10 carbon atoms, such as prop-2-enyl; Alkynyl preferably having 1 to 10 carbon atoms, such as prop-2-ynyl; substituted alkyls having 1 to 10 carbon atoms, such as hydroxyalkyl, alkoxyalkyl, mercaptoalkyl, alkylthioalkyl, halogenoalkyl, cyanoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkanoylalkyl, carboxyalkyl, and carbamoylalkyl; substituted alkenyls having 1 to 10 carbon atoms, such as hydroxyalkenyl, alkoxyalkenyl, mercaptoalkenyl, alkylthioalkenyl, halogenoalkenyl, cyanoalkenyl, aminoalkenyl, alkylaminoalkenyl, dialkylaminoalkenyl, alkanoylalkenyl, carboxyalkenyl, and carbamoylalkenyl; substituted alkynyl having 1 to 10 carbon atoms, such as hydroxyalkynyl, alkoxyalkynyl, mercaptoalkynyl, alkylthioalkynyl, halogenoalkynyl, cyanoalkynyl, aminoalkynyl, alkylaminoalkynyl, dialkylaminoalkynyl, alkanoylalkynyl, carboxyalkynyl, and carbamoylalkynyl; aroylalkyl having up to 10 carbon atoms, such as phenacyl or 2-benzoylethyl; aryl, such as phenyl or 1-naphthyl; heteroaryls such as 4-quinolyl; alkanoyl having 1 to 10 carbon atoms, such as acetyl, or butyryl; aroyls such as benzoyl; heteroaroyl, such as 3-quinoloyl; OR' or NR'R'' (wherein R' and R'' are independently hydrogen, alkyl, aryl, heteroaryl, acyl, aroyl, sulfonyl, or sulfinyl), or SO2-R''' or SO-R''' (wherein R''' is substituted or unsubstituted alkyl, aryl, heteroaryl, alkenyl, or alkynyl).

[0059] Capping functions providing an ester bond are designated as OR, where R can be alkoxy; aryloxy; heteroaryloxy; aralkyloxy; heteroaralkyloxy; substituted alkoxy; substituted aryloxy; substituted heteroaryloxy; substituted aralkyloxy; or substituted heteroaralkyloxy.

[0060] Either the N-terminal or C-terminal capping function, or both, may be structured such that the capped molecule functions as a prodrug (a pharmacologically inactive derivative of the parent drug molecule) that undergoes spontaneous or enzymatic conversion in the body to release the active drug, thereby providing improved delivery properties over the parent drug molecule (Bundgaard H, Ed: Design of Prodrugs, Elsevier, Amsterdam, 1985).

[0061] Judicious selection of capping groups allows for the addition of other activities to the peptides, for example, the presence of a sulfhydryl group attached to the N- or C-terminal cap allows for the conjugation of the derivatized peptide to other molecules.

[0062] In yet another embodiment, the peptide, or fragment or derivative thereof, may be a "retro-inverso peptide." A "retro-inverso peptide" refers to a peptide in which the direction of the peptide bond is inverted at at least one position, i.e., the amino and carboxy termini are inverted with respect to the amino acid side chains. Thus, a retro-inverso analog has an inverted terminus and inverted direction of the peptide bond while largely maintaining the side chain topology of the native peptide sequence. Retro-inverso peptides may contain L- or D-amino acids, or a mixture of L- and D-amino acids, with up to all amino acids being D-isomers. A partial retro-inverso peptide analog is a polypeptide in which only a portion of the sequence is inverted and replaced with enantiomeric amino acid residues. Because the inverted portion of such analogs has inverted amino and carboxy termini, the amino acid residues adjacent to the inverted portion are replaced with side chain-like α-substituted geminal diaminomethane and malonate, respectively. Retro-inverso forms of cell-penetrating peptides have been found to function as efficiently as the native forms at translocating across membranes. The synthesis of retro-inverso peptide analogs is described in Bonelli, F. et al., Int J Pept Protein Res. 24(6):553-6 (1984); Verdini, A and Viscomi, G.C., J. Chem. Soc. Perkin Trans. 1:697-701 (1985); and U.S. Patent No. 6,261,569, which are incorporated herein by reference in their entireties. A process for the solid-phase synthesis of partial retro-inverso peptide analogs has been described (EP 97994-B), which is also incorporated herein by reference in its entirety.

[0063] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (e.g., 80%, 85%, 90%, or 95%) of "sequence identity" or "homology" to another sequence, meaning that, when aligned, the two sequences compare with the same percentage of bases (or amino acids). This alignment and percent homology or sequence identity can be determined using software programs known in the art, such as the programs described in Current Protocols in Molecular Biology (F.M.A.usubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for the alignment. A preferred alignment program is BLAST, using default parameters. Particularly preferred programs are BLASTN and BLASTP using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expectation=10; matrix=BLOSUM62; description=50 sequences; sort=high score; database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBankCDStranslation+SwissProtein+SPupdate+PIR.

[0064] A. Multimeric Polypeptides Embodiments of the present disclosure also include longer polypeptides constructed from repeating units of modified Cav-1 variant polypeptides. Polypeptide multimers may contain different combinations of polypeptides. Such multimeric polypeptides can be produced by chemical synthesis or by recombinant DNA technology, as discussed herein. When produced by chemical synthesis, oligomers preferably have 2-5 repeats of the core polypeptide sequence, and the total number of amino acids in the multimer should not exceed about 160 residues, preferably 100 or less (or their equivalent, if including linkers or spacers).

[0065] B. Peptide Mimetics Modified Cav-1 peptides can be peptidomimetic compounds that mimic the biological effects of native Cav-1 polypeptides. Peptidomimetics can be non-natural peptides or non-peptide agents that mimic the spatial properties of the binding elements of native Cav-1 polypeptides so as to have the binding activity and biological activity of the native Cav-1 polypeptides. Like native Cav-1 polypeptides or polypeptide multimers, peptidomimetics have a binding face (which interacts with any ligand that native Cav-1 binds) and a non-binding face.

[0066] In some embodiments, the present disclosure also includes compounds that retain partial peptide characteristics. For example, any proteolytically labile bond within a peptide of the invention may be selectively replaced by a non-peptide element, such as an isostere (N-methylation; D-amino acid) or reduced peptide bond, while the remainder of the molecule retains its peptidic character.

[0067] Peptidomimetic compounds, either agonists, substrates or inhibitors, have been described for many biologically active peptides / polypeptides, such as opioid peptides, VIP, thrombin, HIV protease, etc. Methods for designing and preparing peptidomimetic compounds are known in the art (Hruby, VJ, Biopolymers 33:1073-1082 (1993); Wiley, RA et al., Med. Res. Rev. 13:327-384 (1993); Moore et al., Adv. in Pharmacol 33:91-141 (1995); Giannis et al., Adv. in Drug Res. 29:1-78 (1997)). Specific mimetics that mimic secondary structure are described in Johnson et al., In: Biotechnology and Pharmacy, Pezzuto et al., Chapman and Hall (Eds.), NY, 1993. These methods are used to generate peptidomimetics that retain at least the binding ability and specificity of the native Cav-1 polypeptide, and preferably also retain biological activity. Knowledge of peptide chemistry and general organic chemistry available to those skilled in the art is sufficient for the synthesis and synthesis of such compounds in light of the present disclosure.

[0068] For example, such peptidomimetics can be identified by examining the three-dimensional structure of the polypeptide of the present invention, either free or complexed with a ligand (e.g., soluble uPAR or its fragment). Alternatively, the structure of the polypeptide of the present invention bound to its ligand can be obtained by nuclear magnetic resonance spectroscopy techniques. A deeper understanding of the stereochemistry of the interaction between the peptide and its ligand or receptor will enable the rational design of such peptidomimetics. The structure of the peptide or polypeptide of the present invention in the absence of a ligand can also provide a scaffold for the design of mimetic molecules.

[0069] C. PEGylation The modified Cav-1 peptide can be conjugated with a heterologous polypeptide segment or polymer, such as polyethylene glycol. The polypeptide can be linked to PEG to increase the hydrodynamic radius of the enzyme, thereby increasing serum persistence. The polypeptide can also be conjugated to any targeting agent, such as a ligand that can specifically and stably bind to an external receptor (US Patent Publication No. 2009 / 0304666).

[0070] In certain aspects, methods and compositions of the disclosed embodiments relate to PEGylation of polypeptides. PEGylation is the process of covalently attaching a poly(ethylene glycol) polymer chain to another molecule, usually a drug or therapeutic protein. PEGylation is conventionally achieved by incubating a reactive derivative of PEG with a target macromolecule. Covalent attachment of PEG to a drug or therapeutic protein can "mask" the drug from the host's immune system (reducing immunogenicity and antigenicity) or increase the drug's hydrodynamic size (size in solution), prolonging circulation time by reducing renal clearance. PEGylation can also make hydrophobic drugs and proteins water-soluble.

[0071] The first step in PEGylation is the appropriate functionalization of the PEG polymer at one or both termini. PEG activated at each terminus with the same reactive moiety is known as "homobifunctional," whereas if the functional groups present are different, the PEG derivative is termed "heterobifunctional" or "heterofunctional." Chemically active or activated derivatives of PEG polymers are prepared to attach PEG to desired molecules.

[0072] The selection of an appropriate functional group for the PEG derivative is based on the type of available reactive group on the molecule to be attached to PEG. In the case of proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. N-terminal amino groups and C-terminal carboxylic acids may also be used.

[0073] The technique used to form first-generation PEG derivatives generally involves reacting PEG polymers with groups reactive with hydroxyl groups, typically anhydrides, acid chlorides, chloroformates, and carbonates. In second-generation PEGylation chemistries, more efficient functional groups, such as aldehydes, esters, and amides, become available for conjugation.

[0074] As the applications of PEGylation become more sophisticated and sophisticated, the need for heterobifunctional PEGs for conjugation is growing. These heterobifunctional PEGs are very useful for linking two entities that require a hydrophilic, flexible, and biocompatible spacer. Preferred terminal groups for heterobifunctional PEGs are maleimide, vinyl sulfone, pyridyl disulfide, amine, carboxylic acid, and NHS ester.

[0075] The most common modifiers, or linkers, are based on methoxy PEG (mPEG) molecules. Their activity relies on the addition of protein-modifying groups to the alcohol termini. In some cases, polyethylene glycol (PEG diol) is used as a precursor molecule. The diol is subsequently modified at both ends to create hetero- or homodimeric PEG-linked molecules.

[0076] Proteins are commonly PEGylated at nucleophilic sites such as unprotonated thiols (cysteine ​​residues) or amino groups. Examples of cysteinyl-specific modification reagents include PEG maleimide, PEG iodoacetate, PEG thiol, and PEG vinyl sulfone. All four are strongly cysteine-specific under mild conditions and at neutral to slightly alkaline pH, but each has some drawbacks. The thioether formed with maleimide can be somewhat unstable under alkaline conditions, which may limit formulation options using this linker. The carbamothioate bond formed with iodoPEG is more stable, but free iodine can modify tyrosine residues under some conditions. PEG thiol forms disulfide bonds with protein thiols, but this bond can also be unstable under alkaline conditions. The reactivity of PEG-vinyl sulfone is relatively slow compared to maleimide and iodoPEG. However, the thioether bond formed is very stable. The slow reaction rate may also facilitate control of the PEG-vinyl sulfone reaction.

[0077] Site-specific PEGylation at natural cysteine ​​residues is rarely performed because these residues are usually in the form of disulfide bonds or are required for biological activity. Alternatively, site-directed mutagenesis may be used to incorporate cysteine ​​PEGylation sites for thiol-specific linkers. The cysteine ​​mutation must be designed to be accessible to PEGylation reagents and remain biologically active after PEGylation.

[0078] Amine-specific modifiers include PEG NHS ester, PEG tresylate, PEG aldehyde, PEG isothiocyanate, and several others. All react under mild conditions and are highly specific for amino groups. PEG NHS ester is perhaps one of the more reactive agents. However, its high reactivity can make the PEGylation reaction difficult to control on a large scale. PEG aldehyde forms an imine with the amino group, which is then reduced to a secondary amine with sodium cyanoborohydride. Unlike sodium borohydride, sodium cyanoborohydride does not reduce disulfide bonds. However, this chemical is highly toxic and must be handled with care, especially at low pH, where it becomes volatile.

[0079] Due to the multiple lysine residues on most proteins, site-specific PEGylation can be challenging. Fortunately, because these reagents react with unprotonated amino groups, it is possible to target PEGylation to lower pK amino groups by performing the reaction at a lower pH. Generally, the pK of alpha-amino groups is 1–2 pH units lower than the epsilon-amino groups of lysine residues. High N-terminal selectivity can frequently be achieved by PEGylating molecules at pH 7 or below. However, this is only feasible if the N-terminal portion of the protein is not required for biological activity. Nevertheless, the pharmacokinetic benefits of PEGylation often outweigh the significant loss in in vitro bioactivity, resulting in products with significant in vivo bioactivity, regardless of the PEGylation chemistry.

[0080] There are several parameters to consider when developing a PEGylation procedure. Fortunately, there are usually no more than four or five critical parameters. A "design of experiments" approach to optimizing PEGylation conditions can be very useful. For thiol-specific PEGylation reactions, parameters to consider include protein concentration, PEG-to-protein ratio (on a molar basis), temperature, pH, reaction time, and, in some cases, the exclusion of oxygen. (Oxygen can contribute to intermolecular disulfide formation with the protein, reducing the yield of PEGylated product.) For amine-specific modifications, the same factors (except oxygen) must be considered, except that pH may be even more important, especially when targeting N-terminal amino groups.

[0081] For both amine- and thiol-specific modifications, reaction conditions can affect protein stability. This can limit temperature, protein concentration, and pH. Furthermore, the reactivity of the PEG linker must be known before initiating the PEGylation reaction. For example, if the PEGylation agent is only 70% active, the amount of PEG used must ensure that only active PEG molecules are counted in the protein-PEG reaction stoichiometry.

[0082] D. Fusion Proteins Certain embodiments of the present invention relate to fusion proteins of modified Cav-1 peptides. These molecules may have the polypeptides of the embodiments linked at the N- or C-terminus to a heterologous domain. For example, fusions may also use leader sequences from other species to enable recombinant expression of the protein in a heterologous host. The fusion protein may also include a half-life extender. Another useful fusion includes the addition of a protein affinity tag, such as a serum albumin affinity tag or six histidine residues, or an immunologically active domain, such as an antibody epitope, preferably a cleavable domain, to facilitate purification of the fusion protein. Non-limiting affinity tags include polyhistidine, chitin-binding protein (CBP), maltose-binding protein (MBP), and glutathione-S-transferase (GST).

[0083] In certain embodiments, the peptides of the embodiments may be linked to a peptide that extends in vivo half-life, such as an XTEN® polypeptide (Schellenberger et al., 2009), an IgG Fc domain, albumin, or an albumin-binding peptide.

[0084] Methods for generating fusion proteins are well known to those skilled in the art. Such proteins can be produced, for example, by de novo synthesis of an entire fusion protein, or by the attachment of a DNA sequence encoding a heterologous domain followed by expression of the intact fusion protein.

[0085] The production of fusion proteins that restore the functional activity of the parent proteins can be facilitated by connecting the genes with a bridging DNA segment encoding a peptide linker that is spliced ​​between the tandemly connected polypeptides, the linker being of sufficient length to allow proper folding of the resulting fusion protein.

[0086] 1. Linker In certain embodiments, the polypeptides of the embodiments may be chemically conjugated using bifunctional cross-linking reagents or fused at the protein level with peptide linkers.

[0087] Bifunctional cross-linking reagents have been widely used for a variety of purposes, including the preparation of affinity matrices, the modification and stabilization of various structures, the identification of ligand and receptor binding sites, and structural studies. An appropriate peptide linker may be used to link the polypeptides of the embodiments, such as a Gly-Ser linker.

[0088] Homobifunctional reagents, which contain two identical functional groups, have proven highly effective in inducing crosslinks between identical and different macromolecules or subunits of macromolecules and linking polypeptide ligands to their specific binding sites. Heterobifunctional reagents contain two different functional groups. By exploiting the differential reactivity of the two different functional groups, crosslinking may be selectively and sequentially controlled. Bifunctional crosslinking reagents can be classified according to the specificity of their functional groups, e.g., amino-, sulfhydryl-, guanidine-, indole-, and carboxyl-specific groups. Of these, reagents directed at free amino groups have become particularly popular due to their commercial availability, ease of synthesis, and the mild reaction conditions under which they can be applied.

[0089] Most heterobifunctional cross-linking reagents contain a primary amine-reactive group and a thiol-reactive group. In another example, heterobifunctional cross-linking reagents and methods for using the cross-linking reagents have been described (U.S. Pat. No. 5,889,155, specifically incorporated herein by reference in its entirety). The cross-linking reagents combine a nucleophilic hydrazide residue with an electrophilic maleimide residue, allowing, in one example, the coupling of an aldehyde to a free thiol. The cross-linking reagents may be modified to cross-link various functional groups.

[0090] Additionally, any other linking / coupling agent and / or mechanism known to one of skill in the art may be used to combine the polypeptides of the embodiments, such as, for example, antibody-antigen interactions, avidin-biotin bonds, amide bonds, ester bonds, thioester bonds, ether bonds, thioether bonds, phosphoester bonds, phosphoramide bonds, anhydride bonds, disulfide bonds, ionic and hydrophobic interactions, bispecific antibodies and antibody fragments, or combinations thereof.

[0091] It is preferable to use a crosslinker that has reasonable stability in blood.Many types of disulfide bond-containing linkers are known that can be used successfully to conjugate targeting agents and treatment / prevention agents.The linker that contains sterically hindered disulfide bonds may prove to increase in vivo stability.Therefore, these linkers are one group of linking agents.

[0092] In addition to hindered cross-linkers, unhindered linkers may also be used in accordance with the present invention. Other useful cross-linkers that are not believed to contain or generate protected disulfides include SATA, SPDP, and 2-iminothiolane (Wawrzynczak and Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.

[0093] Once chemically conjugated, the peptides are generally purified to separate the conjugate from unconjugated agents and other contaminants. Numerous purification techniques are available for use in providing conjugates of sufficient purity to make them clinically useful.

[0094] Purification methods based on size separation, such as gel filtration, gel permeation, or high-performance liquid chromatography, are generally most useful. Other chromatographic techniques, such as blue-Sepharose separation, may also be used. Traditional methods for purifying fusion proteins from inclusion bodies may be useful, such as using mild detergents such as sodium N-lauroyl-sarcosine (SLS).

[0095] 2. Cell-permeable and membrane-translocating peptides Furthermore, in certain embodiments, the modified Cav-1 peptide may further comprise a cell-binding domain or a cell-penetrating peptide (CPP). As used herein, the terms "cell-penetrating peptide" and "membrane translocation domain" are used interchangeably and refer to a segment of a polypeptide sequence that allows the polypeptide to cross a cell membrane (e.g., the plasma membrane in the case of eukaryotic cells). Examples of CPP segments include, but are not limited to, segments derived from HIV Tat (e.g., GRKKRRQRRRPPQ (SEQ ID NO: 21)), herpesvirus VP22, Drosophila Antennapedia homeobox gene product, protegrin I, penetratin (RQIKIWFQNRRMKWKK (SEQ ID NO: 22)), or melittin (GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 23)). In certain embodiments, the CPP comprises a T1 (TKIESLKEHG (SEQ ID NO: 24)), T2 (TQIENLKEKG (SEQ ID NO: 25)), T26 (AALEALAEALEALAEALEALAEAAAA (SEQ ID NO: 26)), or INF7 (GLFEAIEGFIENGWEGMIEGWYGCG (SEQ ID NO: 27)) CPP sequence.

[0096] III.How to use One aspect of the present invention relates to the use of the polypeptides described herein and mutants, variants, analogs or derivatives thereof. Specifically, these methods relate to administering to a subject any one of the polypeptides described herein, or a pharmaceutically acceptable modification thereof, in a pharmaceutically acceptable carrier, and compositions for use in the treatment of treating or preventing lung disease, injury or infection (e.g., a fibrotic condition of the lung), the composition comprising a polypeptide of the embodiments in a pharmaceutically acceptable carrier.

[0097] A. Pharmaceutical Compositions It is contemplated that modified Cav-1 peptides can be administered systemically or locally to inhibit cell apoptosis and to treat and prevent damage to lung tissue. They may be administered intravenously, intrathecally, and / or intraperitoneally. In certain embodiments, the polypeptides are delivered locally to the airways, such as by administering an aerosolized or dry powder formulation for inhalation. They may be administered alone or in combination with an anti-fibrotic compound.

[0098] The modified Cav-1 peptide may be administered in combination with, simultaneously with, or sequentially with at least one additional therapeutic agent for pulmonary fibrosis. The additional therapeutic agent may be an NSAID, steroid, DMARD, immunosuppressant, biological response modulator, bronchodilator, or antifibrotic agent, such as pirfenedone (the mechanism of action of which is not fully understood but may involve blocking TGF-beta, nintedanib, broad-spectrum tyrosine kinase blockers, or any other antifibrotic agent). Suitable NSAIDS include the non-selective COX inhibitors acetylsalicylic acid, mesalazine, ibuprofen, naproxen, flurbiprofen, fenoprofen, fenbufen, ketoprofen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, tiaprofenic acid, fluprofen, indomethacin, sulindac, tolmetin, zomepirac, nabumetone, diclofenac, fenclofenac, alclofenac, bromfenac, ibufenac, aceclofenac, acemetacin, fentiazac, clidanac, etodolac, and oxypinac. The antihistamine is selected from mefenamic acid, meclofenamic acid, flufenamic acid, niflumic acid, tolfenamic acid, diflunisal, flufenisal, piroxicam, tenoxicam, lonoxicam, and nimesulide, and their pharmaceutically acceptable salts; selective COX 2 inhibitors meloxicam, celecoxib, and rofecoxib, and their pharmaceutically acceptable salts. Suitable steroids include prednisone, prednisolone, methylprednisolone, dexamethasone, budenoside, fluocortolone, and triamcinolone. Suitable DMARDs include sulfasalazine, olsalazine, chloroquine, gold derivatives (auranofin), D-penicillamine, and cytostatics such as methotrexate and cyclophosphamide. Suitable immunosuppressants include cyclosporin A and its derivatives, mycophenolate mofetil, FK 506, OKT-3, ATG, 15-desoxyspergualin, mizoribine, misoprostol, rapamycin, leflunomide, and azathioprine. Suitable biological response modifiers include interferon beta, anti-TNF-α (etanercept), IL-10, anti-CD3, or anti-CD25. Suitable bronchodilators include ipratropium bromide, oxitropium bromide, tiotropium bromide, epinephrine hydrochloride, salbutamol, terbutamol sulfate, fenoterol bromide, salmeterol, and formoterol. In such combinations, each active ingredient can be administered either within its usual dosage range or at a dose lower than its usual dosage range. The combined dose of an NSAID, steroid, DMARD, immunosuppressant, and biological response modifier is suitably 1 / 50 of the normally recommended minimum dose to 1 / 1 of the normally recommended dose, preferably 1 / 20 to 1 / 2, and more preferably 1 / 10 to 1 / 5. The normally recommended dose of the concomitant medication should be understood to be the dose disclosed, for example, in RoteListe® 2002, Editio Cantor Verlag Aulendorf, Germany, or the Physician's Desk Reference.

[0099] Where clinical use is contemplated, it may be necessary to prepare pharmaceutical compositions containing proteins, antibodies, and drugs in a form appropriate for the intended use. Generally, pharmaceutical compositions may contain an effective amount of one or more polypeptides of the embodiments, or additional agents, dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction, as appropriate, when administered to an animal, e.g., a human. The preparation of pharmaceutical compositions containing at least one polypeptide of the embodiments isolated by the methods disclosed herein, or additional active ingredients, will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference. Furthermore, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biological Standards.

[0100] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, as known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference). Any conventional carrier is contemplated for use in pharmaceutical compositions, unless it is incompatible with the active ingredient.

[0101] Specific embodiments of the present invention may include different types of carriers depending on whether they are administered in solid, liquid, or aerosol form and whether they need to be sterile for routes of administration such as injection. The compositions may be administered intravenously, intrathecally, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly, subcutaneously, mucosally, orally, topically, locally, by inhalation (e.g., by inhalation of a nebulized or dry powder formulation), by injection, by infusion, by continuous infusion, by direct local perfusion of bathing target cells, via a catheter, via lavage, in lipid compositions (e.g., liposomes), or by other methods or any combination of the above, as known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference).

[0102] The modified polypeptide can be formulated into a composition in free base, neutral, or salt form. Pharmaceutically acceptable salts include acid addition salts, such as salts formed with the free amino groups of the proteinaceous composition, or salts formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Once formulated, the solution is administered in a therapeutically effective amount in a manner compatible with the dosage form. The formulations are easily administered in a variety of dosage forms, including those formulated for parenteral administration, such as injection solutions, or those formulated for pulmonary delivery by aerosols or digestive administration, such as drug-release capsules.

[0103] Furthermore, according to certain aspects of the present invention, compositions suitable for administration can be provided in a pharmaceutically acceptable carrier, with or without an inert diluent. Carriers must be assimilable and include liquid, semi-solid (i.e., paste), or solid carriers. Except insofar as any conventional vehicle, agent, diluent, or carrier is deleterious to the recipient or to the therapeutic effect of the composition contained therein, its use in administrable compositions for use in practicing the methods is appropriate. Examples of carriers or diluents include fats, oils, water, saline, lipids, liposomes, resins, binders, fillers, and the like, or combinations thereof. The compositions can also contain various antioxidants to retard oxidation of one or more components. Prevention of microbial action can also be achieved by preservatives, such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0104] According to certain aspects of the present invention, the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, mixing, encapsulation, absorption, etc. Such procedures are routine to those skilled in the art.

[0105] In a specific embodiment of the present invention, the composition is thoroughly combined or mixed with a semi-solid or solid carrier. Mixing can be performed by any convenient method, such as grinding. Stabilizers may be added during the mixing process to protect the composition from loss of therapeutic activity, i.e., from denaturation in the stomach. Examples of stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates, or cryoprotectants, such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

[0106] In some embodiments, the pharmaceutical formulation comprises one or more surfactants. Surfactants for use in accordance with the disclosed methods include ionic and non-ionic surfactants. Exemplary non-ionic surfactants include polysorbates, such as TWEEN®-20 and TWEEN-80® surfactants (ICI Americas, Bridgewater, NJ). Inc.); poloxamers (e.g., Poloxamer 188); TRITON® surfactants (Sigma, St. Louis, Missouri); sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; MONAQUAT™ surfactants (Mona Industries, Paterson, New Jersey). Inc.); polyethyl glycol; polypropylene glycol; block copolymers of ethylene and propylene glycol, e.g., PLURONIC® surfactants (BASF, Mount Olive, NJ); oligo(ethylene oxide) alkyl ethers; alkyl(thio)glucosides, alkyl maltosides; and phospholipids. For example, surfactants may be present in the formulation in an amount of about 0.01% to about 0.5% (weight of surfactant relative to the total weight of the other solid components of the formulation; "w / w"), about 0.03% to about 0.5% (w / w), about 0.05% to about 0.5% (w / w), or about 0.1% to about 0.5% (w / w). However, in further aspects, pharmaceutical formulations of embodiments are essentially free of nonionic surfactants or essentially free of all surfactants.

[0107] With respect to the therapeutic methods of the present invention, the administration of one or more peptides or mutants, variants, analogs, or derivatives thereof disclosed herein is not intended to be limited to a particular mode of administration, dosage, or frequency of administration; the present invention contemplates all modes of administration, including intramuscular, intravenous, intraperitoneal, intravesicular, intraarticular, intralesional, subcutaneous, or any other route sufficient to provide a dose sufficient to treat an inflammation-related disorder. The therapeutic agent may be administered to a patient in a single dose or in multiple doses. When multiple doses are administered, the doses may be separated from each other by, for example, 1 hour, 3 hours, 6 hours, 8 hours, 1 day, 2 days, 1 week, 2 weeks, or 1 month. For example, the therapeutic agent may be administered for, for example, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more weeks. It is understood that for any particular subject, specific dosing regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the composition. For example, the dosage of the therapeutic agent may be increased if a lower dose does not provide sufficient therapeutic activity.

[0108] While the attending physician will ultimately decide the appropriate amount and administration regimen, a therapeutically effective amount of one or more polypeptides disclosed herein, or mutants, variants, analogs, or derivatives thereof, can be provided in a dose of 0.0001, 0.01, 0.01 0.1, 1, 5, 10, 25, 50, 100, 500, or 1,000 mg / kg or g / kg. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test bioassays or systems.

[0109] The dosage for a particular patient or subject can be determined by one of ordinary skill in the art using conventional considerations (e.g., by appropriate conventional pharmacological protocols). A physician may, for example, prescribe a relatively low dose initially, and then increase the dose until an appropriate response is obtained. The dosage administered to a patient is sufficient to bring about a beneficial therapeutic response in the patient over time, or, for example, to reduce symptoms or other appropriate activity, depending on the application. The dosage is determined by the effectiveness of the particular formulation, as well as the activity, stability, or serum half-life of one or more polypeptides disclosed herein or mutants, variants, analogs, or derivatives thereof, and the patient's condition, as well as the body weight or surface area of ​​the patient to be treated.

[0110] In some embodiments, a subject is given a single dose, e.g., by infusion (by inhalation), of between about 0.2 mg / kg and 250 mg / kg, e.g., between about 10 mg / kg and about 50 mg / kg, given once daily to treat a subject, preferably a mammal, more preferably a human suffering from or susceptible to pulmonary fibrosis. Such doses may be administered daily for anywhere from about 3 days to a week or more. Chronic administration is also possible, although doses may need to be adjusted downward, as is well understood in the art. However, the foregoing ranges are only suggestive, as there are many variables in individual treatment regimes and significant deviations from these preferred values ​​are expected.

[0111] For example, for continuous administration via a pump system, such as the osmotic pump used in some of the experiments described below, the total dose over a time course of about 1 to 2 weeks is preferably in the range of 1 mg / kg to 1 g / kg, preferably 20 to 300 mg / kg, more preferably 50 to 200 mg / kg. After such a continuous administration regimen, the total concentration of active compound is preferably in the range of about 0.5 to about 50 μM, preferably about 1 to about 10 μM.

[0112] The effective concentration of active compound for inhibiting or preventing the inhibition of apoptosis in vitro is in the range of about 0.5 nM to about 100 nM, more preferably about 2 nM to about 20 nM. Effective doses and optimal dose ranges can be determined in vitro using the methods described herein.

[0113] B. Aerosol Dispersion and Nebulization Devices The formulations may be aerosolized using any suitable device, including, but not limited to, jet nebulizers, ultrasonic nebulizers, metered dose inhalers (MDIs), and devices for aerosolizing liquids by forcing them through a jet or nozzle (e.g., the AERX® drug delivery device by Aradigm, Hayward, California). Additionally, the compounds may be formulated as dry powders for delivery using dry powder inhaler devices. As further described herein below, to deliver the formulation to a subject, the pulmonary delivery device may also include a ventilator, optionally in combination with a mask, mouthpiece, mist inhaler, and / or platform that guides the user to inhale correctly and automatically delivers medication at the appropriate time of the breath. Representative aerosolization devices that can be used in accordance with the methods of the present invention include, but are not limited to, those described in U.S. Patent Nos. 6,357,671; 6,354,516; 6,241,159; 6,044,841; 6,041,776; 6,016,974; 5,823,179; 5,797,389; 5,660,166; 5,355,872; 5,284,133; and 5,277,175, and U.S. Patent Application Publication Nos. 20020020412 and 20020020409.

[0114] Using a jet nebulizer, compressed gas from a compressor or hospital airline is passed through a narrow constriction known as a jet. This creates a low-pressure region, causing the drug solution from the reservoir to be drawn up through the feed tube and fragmented into droplets by the airflow. Only the smallest droplets leave the nebulizer directly; most impact the baffles and walls and are returned to the reservoir. As a result, the time required to perform jet nebulization varies depending on, among other factors, the volume of the composition being nebulized, and such time can be easily adjusted by one skilled in the art.

[0115] Metered-dose inhalers (MDIs) may be used to deliver the compositions of the present invention in a more concentrated form than is typically delivered using nebulizers. To achieve optimal efficacy, MDI delivery systems require proper administration techniques, including coordinated aerosol delivery via inhalation, slow inhalation at approximately 0.5 to 0.75 liters per second, deep breathing to reach inspiratory capacity, and breath-holding for at least four seconds. Pulmonary delivery using MDIs is convenient and appropriate when treatment benefits from relatively short treatment times and low cost. Optionally, the formulation may be heated to about 25°C to about 90°C during nebulization to promote effective droplet formation and subsequent delivery. See, e.g., U.S. Patent No. 5,299,566.

[0116] The aerosol composition of the embodiment contains droplets of the composition of a size suitable for efficient delivery into the lungs. In some cases, the surfactant formulation is delivered to the pulmonary bronchi, more preferably the bronchioles, even more preferably the alveolar ducts, and even more preferably the alveoli. The aerosol droplets typically have a diameter of less than about 15 μm, less than about 10 μm, less than about 5 μm, or less than about 2 μm. For efficient delivery to the alveolar bronchi of a human subject, the aerosol composition may preferably contain droplets having a diameter of about 1 μm to about 5 μm.

[0117] Droplet size can be assessed using techniques known in the art, such as cascade, collision, laser diffraction, and optical patterning. See McLean et al. (2000) Anal Chem 72:4796-804, Fults et al. (1991) J Pharm Pharmacol 43:726-8, and Vecellio None et al. (2001) J Aerosol Med 14:107-14.

[0118] Protein stability after aerosolization can be assessed using techniques known in the art, including size exclusion chromatography; electrophoretic techniques; spectroscopic techniques such as UV spectroscopy and circular dichroism spectroscopy; and protein activity (measured in vitro or in vivo). To perform in vitro assays of protein stability, the aerosol composition may be collected, distilled, or absorbed into a filter. To perform in vivo assays or for pulmonary administration of the composition to a subject, the aerosolization device is adapted for inhalation by the subject. For example, protein stability can be assessed by determining the level of protein aggregation. Preferably, the aerosol compositions of the present invention are substantially free of protein aggregates. The presence of soluble aggregates can be qualitatively determined using DLS (DynaPro-801TC, ProteinSolutions Inc., Charlottesville, VA) and / or by UV spectrophotometry.

[0119] The term "vibrating mesh nebulizer" as used herein refers to any nebulizer that operates on the general principle of using a vibrating mesh or plate with multiple apertures (aperture plate) to generate a low-velocity aerosol of fine particles. Some nebulizers may include a mesh / membrane with 1,000 to 7,000 holes, which vibrates above a liquid reservoir (see, e.g., U.S. Patent Publication 20090134235 and Waldrep and Dhand 2008, each incorporated herein by reference). In some embodiments, the vibrating mesh nebulizer is an AERONEB® Professional Nebulizer, an OMRON MICROAIR®, a Pari EFLOW®, or an EZ Breathe Atomizer. In some embodiments, the vibrating mesh nebulizer has a vibration frequency of between about 50 to 250 kHz, 75 to 200 kHz, 100 to 150 kHz, or about 120 kHz. These devices are highly efficient at delivering aerosols to the lungs and the amount of liquid remaining in these devices is minimal, which is advantageous for expensive and potent compounds such as plasminogen activators.

[0120] In certain aspects, the nebulized composition of the embodiment is produced using a vibrating mesh nebulizer. For example, the composition can be produced using an active vibrating mesh nebulizer (e.g., Aeroneb® Professional Nebulizer System). Descriptions of such systems and their operation can be found, for example, in U.S. Patent Nos. 6,921,020; 6,926,208; 6,968,840; 6,978,941; 7,040,549; 7,083,112; 7,104,463; and 7,360,536 (each of which is incorporated herein by reference in its entirety). In yet another aspect, the composition of the embodiment can be produced using a passive vibrating mesh nebulizer, such as Omron MicroAir® or EZ Breathe Atomizer.

[0121] IV. Pulmonary Conditions for Treatment The modified peptides of the present invention can be used to treat various pulmonary conditions. The pulmonary conditions for treatment can be acute or chronic. Acute pulmonary conditions can be acute lung injury, infection, or chemical-induced. Chronic pulmonary conditions can be the result of injury, infection, or disease.

[0122] A. Lung injury In some embodiments, the subject has acute lung injury (ALI) or infectious or chemical-induced lung injury. In certain embodiments, the subject has acute respiratory distress syndrome (ARDS), inhalation smoke-induced acute lung injury (ISALI), bronchiectasis, inhaled toxin-induced airway disease (e.g., chlorine or other induced airway disease), exposure to mustard gas, exposure to particulate matter (e.g., silica dust), bronchiolitis obliterans, bronchiolitis obliterans organizing pneumonia, drug-induced lung disease, and accelerated pulmonary fibrosis (e.g., occurring after acute lung injury, including ARDS). Acute lung injury (ALI) is a serious medical problem among U.S. military personnel. ALI during combat can result from a wide variety of etiologies.

[0123] ALI due to inhalation injury has been treated with inhaled anticoagulants, steroids, beta-agonists, high-frequency ventilation, and extracorporeal membrane oxygenation, with variable and generally suboptimal outcomes. No effective preventative measures exist other than a respiratory mask barrier. While significant advances have been made in the management of ARDS, it remains largely supportive and involves watchful waiting for endogenous healing mechanisms to take effect; in-hospital mortality rates remain above 40% (Matthay et al., 2012). Survivors of ALI often suffer from chronic respiratory disease with reduced quality of life. Any procedure that can accelerate recovery and / or prevent later complications, such as chronic respiratory failure and pulmonary fibrosis, is highly desirable. Early diagnosis and, more importantly, improved prevention and treatment of ALI are desperately needed. The pathophysiology of ALI due to direct inhalation lung injury or ARDS caused by systemic diseases is highly complex and heterogeneous, involving systemic and local cardiopulmonary factors such as increased membrane permeability, influx of inflammatory cytokines, oxidative cell damage, compartment fluid shifts, and impaired ion channels (Matthay et al., 2012). Clearly, new therapeutic approaches are needed to treat and prevent lung injuries such as ALI.

[0124] In some embodiments, methods are provided for treating or preventing acute lung injury, pulmonary infection, or pulmonary disease in a subject, comprising administering to the subject an effective amount of a variant polypeptide comprising at least one amino acid substitution, insertion, or deletion relative to the amino acid sequence of FTTFTVT (SEQ ID NO: 2), wherein the variant polypeptide maintains the biological activity of caveolin-1 (Cav-1). In some aspects, methods of administering the pharmaceutical formulations of the embodiments comprise nebulizing a solution comprising the variant polypeptide. In certain aspects, the subject is human.

[0125] B. Pulmonary disease Pulmonary diseases include cystic fibrosis, chronic obstructive pulmonary disease (COPD), asthma, bronchiolitis obliterans, bronchiolitis plastica, and pulmonary infections, collagen-vascular lung diseases (e.g., from lupus, scleroderma, or mixed connective tissue disease), interstitial lung diseases (e.g., idiopathic pulmonary fibrosis or sarcoidosis), and acute and chronic lung injuries leading to fibrosis (Murray et al., 1997; Rabe et al., 2007; Tsushima et al., 2009). These diseases are the third leading cause of death worldwide.

[0126] Cystic fibrosis (CF) is a genetic disorder of the exocrine and exocrine sweat glands that primarily affects the digestive and respiratory systems. The disease is typically characterized by chronic respiratory infections, pancreatic insufficiency, abnormally thick mucus secretions, and premature death. Cystic fibrosis (CF) is characterized by progressive airflow obstruction. A subset of CF individuals also develops airway hyperresponsiveness to inhaled cholinergic agonists (Weinberger, 2002 and Mitchell et al., 1978) and reversible airflow limitation in response to bronchodilators (van Haren et al., 1991 and van Haren et al., 1992). The presence of bronchial hyperresponsiveness and airway obstruction suggests a possible shared etiology between CF and other airway narrowing diseases, such as asthma or chronic obstructive pulmonary disease (COPD), in which airway smooth muscle dysfunction is thought to contribute to the disease process.

[0127] The lung infection may be a bacterial infection. Infectious bacteria include Pseudomonas aeruginosa, Bacillus anthracis, Listeria monocytogenes, Staphylococcus aureus, Salmenellosis, Yersina pestis, Mycobacterium leprae, M.africanum, M.asiaticum, M.aviuin-intracellulaire, M.chelonei abscessus, M.fallax, M.fortuitum, M.kansasii, M.leprae, M.malmoense, M.shimoidei, M.simiae, M.szulgai, M.xenopi, M.tuberculosis, Brucella melitensis, Brucella suis, Brucella abortus, Brucella canis, Legionella pneumonophilia, Francisella tularensis, Pneurnocystis carinii, mycoplasma, or Burkholderia cepacia. Bacterial infections can result in pneumonia.

[0128] Chronic obstructive pulmonary disease (COPD) is a term used to classify two major airflow obstruction disorders: chronic bronchitis and emphysema. Approximately 16 million Americans have COPD, 80–90% of whom are lifetime smokers. COPD is the leading cause of death in the United States, accounting for 122,283 deaths in 2003. COPD cost the United States approximately $20.9 billion in direct medical costs in 2003. Chronic bronchitis is inflammation of the bronchial airways. The bronchial airways connect the trachea to the lungs. When inflamed, the bronchi secrete mucus, causing chronic coughing.

[0129] In emphysema, the alveolar sacs become overinflated as a result of damage to the lung's elastin framework. Inflammatory cells in emphysematous lungs release the enzyme elastase, which breaks down or damages elastin fibers within the lung matrix. Emphysema has many causes, including smoking, exposure to environmental pollutants, alpha-1 antitrypsin deficiency, and aging.

[0130] Bronchiolitis is most commonly caused by viral lower respiratory tract infections and is primarily characterized by acute inflammation, edema, necrosis of epithelial cells lining the small airways, and increased mucus production (Ralston et al., 2014). Signs and symptoms usually begin with rhinitis and cough and may progress to tachypnea, wheezing, rales, accessory muscle use, and / or nasal flaring.

[0131] Bronchiolitis obliterans is a progressive reduction in airflow as a result of abnormal remodeling of the small airways of the lung (Meyer et al., 2014). Bronchiolitis obliterans syndrome is a major complication of lung transplantation and is often used to explain delayed allograft dysfunction, resulting in persistent declines in forced expiratory volume and power not caused by other known causes (Meyer et al., 2014).

[0132] The term "asthma" can refer to acute asthma, chronic asthma, intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma, chronic persistent asthma, mild to moderate asthma, mild to moderate persistent asthma, mild to moderate chronic persistent asthma, allergic (extrinsic) asthma, non-allergic (intrinsic) asthma, nocturnal asthma, bronchial asthma, exercise-induced asthma, occupational asthma, seasonal asthma, asymptomatic asthma, gastroesophageal asthma, idiopathic asthma, and cough variant asthma. During asthma, the airways are persistently inflamed and may sometimes spasm. [Example]

[0133] V. Working Examples The following examples are included to support preferred embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and as such can be considered to constitute preferred modes of practice thereof. However, those of skill in the art should, in light of the present disclosure, understand that many changes can be made in the specific embodiments disclosed and still obtain like or similar results without departing from the spirit and scope of the invention. Example 1 - Cav-1 peptide solubility

[0134] To determine which peptides were most soluble in the liquid formulation, 50 mg of each Cav-1 peptide was dissolved in 5 mL of Tris-buffered saline, pH 7.51. Each sample was vortexed to ensure complete dissolution. Absorbance at 600 nm was measured immediately after dissolution for insoluble peptides or after 10 minutes for soluble peptides. Absorbance was measured again after 10 minutes for insoluble peptides, except for samples APi2348, APi2352, and APi2353, which were measured a second time at 15 minutes, 5 minutes, or 15 minutes after dissolution, respectively. Sample APi2345 was only measured 20 minutes after dissolution due to incomplete dissolution (Table 2). pH was also tested after 24 hours.

[0135] Samples APi2350, APi2354, APi2355, and APi2356 had increased solubility at pH 7.51 compared to the other peptides tested (Table 2). After 24 hours, the pH remained stable at approximately pH 7.5 for all samples.

[0136] [Table 2]

[0137] Example 2 - Cav-1 peptides increase smooth muscle actin production Cav-1 peptides were dissolved in DMSO to prepare a 10 mM stock solution. The 10 mM stock solution of each peptide was then diluted with HBSS to prepare a 900 μM working stock solution. The DMSO-resuspended polypeptides and working stocks were stored at -20°C. For culture media, the working stocks were added to DMEM culture medium to a final concentration of 10 μM Cav-1 peptide.

[0138] Idiopathic pulmonary fibrosis (IPF) cell line 2051 was purchased, and IPF cells from passage 4 were seeded onto 100 mm plates containing DMEM, 10% FBS, and 1% P / S. IPF cells were washed with 4 mL of DMEM + 1% P / S and serum-starved overnight. Next, cells were treated with either 44 μL of HBSS (negative control), 10 μM LTI-03 (SEQ ID NO: 2), 90 μM LTI-03 (positive control), 10 μM APi2350, 10 μM APi2354, 10 μM APi2355, 10 μM APi2356, or 20 μL of DMSO (negative control) for 2 days.

[0139] After two days of treatment, cells were washed once with cold sterile HBSS. HBSS was removed, and 150 μL of lysis buffer containing a protease inhibitor cocktail was added to the cells. The cells were incubated with the lysis buffer for 10 minutes. The cell lysate was scraped off the plate and collected. The cell lysate was then sonicated twice. After sonication, the lysate was centrifuged at 13,000 RPM for 20 minutes. The lysate was then flash-frozen in liquid nitrogen, thawed, vortexed, and centrifuged again at 13,000 RPM for 30 minutes. The supernatant was then collected and the pellet discarded. The concentration of the cell lysate was then determined by BCA assay.

[0140] Western blots were performed to assess the effects of treatment. Briefly, 12 μg of each lysate was run on a 10% polyacrylamide gel. The gel was then transferred to a membrane and washed. The results of the Western blots using primary antibodies against smooth muscle actin (SMA) and tubulin can be seen in Figure 1. The treatments of each lysate in the lanes shown are as follows: 1: untreated, 2: 10 μM LTI-03, 3: 90 μM LTI-03, 4: 10 μM APi2350, 5: 10 μM APi2354, 6: 10 μM APi2355, 7: 10 μM APi2356, and 8: DMSO.

[0141] Western blots were photographed and analyzed with ImageJ to determine the ratio of smooth muscle actin to tubulin (Figure 2). As expected, LTI-03 induced an increase in SMA production relative to tubulin. Treatment with the Cav-1 peptides APi2350, APi2354, APi2355, and APi2356 all also increased SMA expression relative to tubulin (Figure 2). Example 3 - Cav-1 peptides preserve AEC2 cells in fibrotic lung biopsies

[0142] To evaluate the effect of the Cav-1 peptide APi2355 (SEQ ID NO: 8) on AEC2 cell viability, surgical biopsies were obtained for the preparation of nonspecific interstitial pneumonia (NSIP) precision-cut lung slices (PCLS). One individual was treated with nonspecific interstitial pneumonia (NSIP) and another with end-stage IPF. Lysotracker staining, which stains the acidic compartment of viable cells and selectively accumulates in the lamellar bodies of pulmonary AEC2 cells, was performed (Van der Velden et al., 2013). Cav-1 peptide was suspended in DMEM / 5% FBS, and PCLS slices (n = 5 replicates / treatment group) were treated with 10, 100, or 500 μM LTI-03 or APi2355 (Var 55). Lysotracker staining (Green DND-26, Promega) was performed on NSIP PCLS 48 h after a single treatment. A strong dose-dependent increase in AEC2 cell viability was observed. Additionally, Lysotracker staining (Red DND-99, Promega) was performed in end-stage IPF on days 1, 2, 3, 5, and 7 after daily treatment with LTI-03 or APi2355. A dose-dependent increase in AEC2 cell viability was observed in end-stage IPF biopsies treated with LTI-03 for 7 consecutive days. The therapeutic effect of APi2355 (Var 55) was observed up to day 3.

[0143] * * * All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods, and in the steps or in the sequence of method steps, described herein without departing from the concept, spirit, or scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0144] V. References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. Bonelli, F. et al., Int J Pept Protein Res.24(6):553-6(1984). Bundgaard H,Ed:Design of Prodrugs,Elsevier,Amsterdam,1985 Current Protocols In Molecular Biology (FMAusubel et al., eds., 1987) Dordo et al, J. Mol Biol, 1999, 217, 721-739 Fults et al. (1991) J Pharm Pharmacol 43:726-8 Giannis et al.,Adv.in Drug Res.29:1-78(1997). Hruby, VJ, Biopolymers 33:1073-1082 (1993) Johnson et al., In: Biotechnology and Pharmacy, in Pezzuto et al., Chapman and Hall (Eds.), NY, 1993 Kyte and Doolittle, J. Mol. Biol., 157(1):105-32, 1982. McLean et al. (2000) Anal Chem 72:4796-804. Meyer et al.,European Respiratory Journal,44:1479-1503,2014. Moore et al.,Adv.in Pharmacol 33:91-141(1995) Ralston et al., Pediatrics, 134(5):e1474-e1502, 2014. Remington's Pharmaceutical Sciences, 18th edition, ARGennaro, Ed., Mack Publ., Easton, PA (1990). S. French and B. Robson, J. Mol. Evol. 19 (1983) 171 Taylor et al,J.Theor.Biol.119(1986);205-218 U.S. Patent No. 4,554,101 U.S. Patent No. 5,277,175 U.S. Patent No. 5,284,133 U.S. Patent No. 5,355,872 U.S. Patent No. 5,660,166 U.S. Patent No. 5,797,389 U.S. Patent No. 5,823,179 U.S. Patent No. 5,889,155 U.S. Patent No. 6,016,974 U.S. Patent No. 6,041,776 U.S. Patent No. 6,044,841 U.S. Patent No. 6,241,159 U.S. Patent No. 6,261,569 U.S. Patent No. 6,261,569 U.S. Patent No. 6,354,516 U.S. Patent No. 6,357,671 U.S. Patent No. 6,921,020 U.S. Patent No. 6,926,208 U.S. Patent No. 6,968,840 U.S. Patent No. 6,978,941 U.S. Patent No. 7,040,549 U.S. Patent No. 7,083,112 U.S. Patent No. 7,104,463 U.S. Patent No. 7,360,536 U.S. Patent Application Publication No. 2002 / 0020409 U.S. Patent Application Publication No. 2002 / 0020412 U.S. Patent Application Publication No. 2009 / 0134235 U.S. Patent Application Publication No. 2009 / 0304666 Vecellio None et al. (2001) J Aerosol Med 14:107-1 Verdini, A and Viscomi, GC, J. Chem. Soc. Perkin Trans. 1 :697-701, 1985. Waldrep and Dhand, Curr. Drug Deliv., 5(2):114-9, 2008. Wawrzynczak and Thorpe, Cancer Treat Res., 37:239-51, 1988. Wiley, RA et al.,Med.Res.Rev.13:327-384,1993.

Claims

1. 1. A dry powder pharmaceutical composition comprising a peptide consisting of the amino acid sequence Ac-aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO: 8), or a pharmaceutically acceptable salt thereof, wherein the dry powder pharmaceutical composition is formulated for use in a dry powder inhaler.

2. 10. The dry powder pharmaceutical composition of claim 1, wherein the pharmaceutically acceptable salt is an ammonium salt.

3. 10. The dry powder pharmaceutical composition of claim 1, wherein the pharmaceutically acceptable salt is an acetate salt.

4. 4. The dry powder pharmaceutical composition of any one of claims 1 to 3, wherein the peptide is at least 95% (w / v) pure.

5. 4. The dry powder pharmaceutical composition of any one of claims 1 to 3, wherein the peptide is at least 99% (w / v) pure.

6. 6. The dry powder pharmaceutical composition of any one of claims 1 to 5, wherein the composition is for use in treating or preventing a disease in a subject, the disease being lung inflammation, chronic obstructive pulmonary disorder (COPD), acute lung injury, lung infection, chemical-induced lung injury, plastic bronchitis, asthma, acute respiratory distress syndrome (ARDS), inhalant smoke-induced acute lung injury (ISALI), bronchiolitis, bronchiolitis obliterans, a pulmonary fibrotic condition, interstitial lung disease, idiopathic pulmonary fibrosis (IPF), or lung scarring.

7. 6. The dry powder pharmaceutical composition of any one of claims 1 to 5 for use in treating or preventing IPF in a subject.

8. 6. The dry powder pharmaceutical composition of any one of claims 1 to 5 for use in treating or preventing fibrosis in a subject.

9. 1. A pharmaceutical composition for treating or preventing fibrosis in a subject, the pharmaceutical composition comprising a peptide consisting of the amino acid sequence KASFTTFTVTKGS (SEQ ID NO: 4), aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO: 7), Ac-aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO: 8), or OASFTTFTVTOS (SEQ ID NO: 9), or a pharmaceutically acceptable salt thereof.

10. 10. The pharmaceutical composition of claim 9, wherein the peptide consists of the amino acid sequence Ac-aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO: 8), or a pharmaceutically acceptable salt thereof.

11. 11. The pharmaceutical composition of claim 9 or 10, further comprising a pharmaceutically acceptable carrier.

12. 11. The pharmaceutical composition of claim 9 or 10, wherein the pharmaceutical composition is formulated for oral, intravenous, intraarticular, parenteral, enteral, subcutaneous, intramuscular, topical, buccal, sublingual, intraocular, epidural, intracranial, or inhalation administration.

13. 11. The pharmaceutical composition of claim 9 or 10, wherein the pharmaceutical composition is formulated as an aqueous solution.

14. 11. The pharmaceutical composition of claim 9 or 10, wherein the pharmaceutical composition is formulated as a dry powder.

15. 11. The pharmaceutical composition of claim 9 or 10, wherein the pharmaceutical composition is administered systemically or locally to the diseased tissue.

16. 11. The dry powder pharmaceutical composition of claim 8, or the pharmaceutical composition of claim 9 or 10, wherein the composition is administered by inhalation.

17. 17. The dry powder pharmaceutical composition or pharmaceutical composition of claim 16, wherein the composition is administered by a nebulizer.

18. 18. The dry powder pharmaceutical composition or pharmaceutical composition of claim 17, wherein the composition is administered by a vibrating mesh nebulizer, a jet nebulizer, or an ultrasonic nebulizer.

19. 17. The dry powder pharmaceutical composition or pharmaceutical composition of claim 16, wherein the composition is administered by inhaler.

20. 17. The dry powder pharmaceutical composition or pharmaceutical composition of claim 16, wherein the composition is administered by a metered dose inhaler.

21. 11. The dry powder pharmaceutical composition of claim 8, or the pharmaceutical composition of claim 9 or 10, wherein the composition is administered in a single dose.

22. 11. The dry powder pharmaceutical composition of claim 8, or the pharmaceutical composition of claim 9 or 10, wherein the composition is administered in multiple doses.

23. 23. The dry powder pharmaceutical composition or pharmaceutical composition of claim 22, wherein the composition is administered multiple times and the doses are administered once daily, once every two days, once weekly, once every two weeks, or once monthly.

24. 11. The dry powder pharmaceutical composition of claim 8, or the pharmaceutical composition of claim 9 or 10, wherein the composition is administered at a dose of 0.1 mg / kg to 500 mg / kg.

25. 11. The dry powder pharmaceutical composition of claim 8, or the pharmaceutical composition of claim 9 or 10, wherein the composition is administered at a dose of 1 mg / kg to 50 mg / kg.

26. 11. The dry powder pharmaceutical composition of claim 8, or the pharmaceutical composition of claim 9 or 10, wherein the composition is administered at a dose of 1 mg / kg to 10 mg / kg.

27. 11. The dry powder pharmaceutical composition of claim 8, or the pharmaceutical composition of claim 9 or 10, further comprising at least one additional therapeutic agent.

28. 28. The dry powder pharmaceutical composition or pharmaceutical composition of claim 27, wherein the at least one additional therapeutic agent is a nonsteroidal anti-inflammatory drug, a steroid, a disease-modifying antirheumatic drug, an immunosuppressant, a biological response modulator, a bronchodilator, or an antifibrotic agent.

29. 29. The dry powder pharmaceutical composition or pharmaceutical composition of claim 28, wherein the anti-fibrotic agent is pirfenedone.

30. 11. The dry powder pharmaceutical composition of claim 8, or the pharmaceutical composition of claim 9 or 10, wherein the fibrosis is renal fibrosis, hepatic fibrosis, cardiac fibrosis, or pulmonary fibrosis.

31. 11. The dry powder pharmaceutical composition of claim 8, or the pharmaceutical composition of claim 9 or 10, wherein the fibrosis is pulmonary fibrosis.

32. 11. The dry powder pharmaceutical composition of claim 8, or the pharmaceutical composition of claim 9 or 10, wherein the fibrosis is idiopathic pulmonary fibrosis (IPF).

33. 11. The dry powder pharmaceutical composition of claim 8, or the pharmaceutical composition of claim 9 or 10, wherein the fibrosis results from acute or chronic lung injury.

34. 11. The dry powder pharmaceutical composition of claim 8, or the pharmaceutical composition of claim 9 or 10, wherein the fibrosis is cystic fibrosis.

35. 11. The dry powder pharmaceutical composition of claim 8, or the pharmaceutical composition of claim 9 or 10, wherein administration of the composition increases the viability of epithelial cells in the subject.

36. 36. The dry powder pharmaceutical composition or pharmaceutical composition of claim 35, wherein administration of the composition increases the viability of lung epithelial cells in the subject.

37. 36. The dry powder pharmaceutical composition or pharmaceutical composition of claim 35, wherein administration of the composition modulates smooth muscle actin expression in the subject.

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