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

Abstract

Provided herein are compositions comprising modified caveolin-1 (Cav-1) peptides. Also provided are methods of using the modified Cav-1 peptides to treat lung infections or acute or chronic lung injury, particularly pulmonary fibrosis.

Description

[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] The present invention is the result of activities conducted within the scope of a joint research agreement in effect at the time the present invention was prepared. The parties to the joint research agreement were the Board of Regents of the University of Texas System and Lung Therapeutics. Background Art

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

[0004] 2. Related technical description

[0005] During lung injury, p53 expression increases, thereby inducing plasminogen activator inhibitor 1 (PAI-1), while inhibiting the expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR), leading to 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 regulate these interactions can be used in methods for inhibiting apoptosis of injured or damaged lung epithelial cells and in methods for treating acute lung injury and subsequent pulmonary fibrosis. Therefore, there is a need for polypeptides that can be used to prevent or treat lung injury, and in particular for preparations and methods for therapeutic delivery of such polypeptides. Summary of the invention

[0006] According to the present disclosure, a peptide comprising the amino acid sequence SEQ ID NO: 2 is provided, wherein the peptide comprises at least one N-terminal or C-terminal addition. The N-terminal or C-terminal addition can be a standard amino acid, a non-standard amino acid or a chemical modification. A peptide multimer of the peptide of the present disclosure is provided. A pharmaceutical composition of the peptide is also provided. The peptide of the present disclosure can be used to treat lung injury, infection or disease. In other aspects, the peptide of the embodiment can be used to treat fibrotic conditions (e.g., organ fibrosis) or inflammation.

[0007] 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-terminal 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-terminus and the C-terminus. In some aspects, the peptide retains the biological activity of caveolin-1 (Cav-1). In other aspects, the peptide of the embodiments may comprise one or more deuterated residues.

[0008] In some aspects, the peptide comprises L-amino acids. In some aspects, the peptide comprises D-amino acids. In some aspects, the peptide comprises both L-amino acids and D-amino acids.

[0009] In some aspects, the peptide comprises at least one non-standard amino acid. In some aspects, the peptide comprises 2 or more non-standard amino acids. In some aspects, the peptide comprises 4 or more non-standard amino acids. In some aspects, the non-standard amino acid is ornithine. In some aspects, the non-standard amino acid is D-alanine.

[0010] In some aspects, the peptide comprises an N-terminal or C-terminal modification. In some aspects, the peptide comprises an N-terminal modification. In some aspects, the peptide comprises a C-terminal modification. In some aspects, the peptide comprises an N-terminal and a C-terminal modification. In some aspects, the N-terminal modification is an acylation. In some aspects, the C-terminal modification is an amidation.

[0011] In some aspects, the peptide comprises the amino acid sequence KASFTTFTVTKGS (SEQ ID NO: 4). In some aspects, the peptide comprises the amino acid sequence aaEGKASFTTFTVTKGSaa (SEQ ID NO: 6). In other aspects, the peptide comprises the amino acid sequence OASFTTFTVTOS (SEQ ID NO: 9). In other aspects, the peptide comprises the amino acid sequence aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO: 7). In other aspects, the peptide comprises the amino acid sequence Ac-aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO: 8). In other aspects, the peptide comprises the amino acid sequence OASFTTFTVTOS-NH2 (SEQ ID NO: 10).

[0012] 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 consisting of GRKKRRQRRRPPQ (SEQ ID NO: 21), RQIKIWFQNRRMKWKK (SEQ ID NO: 22), and GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 23).

[0013] In some embodiments, the present disclosure provides a peptide polymer comprising at least two peptides as disclosed herein. In some aspects, the first peptide of the at least two peptides is substantially identical to the second peptide of the at least two peptides. In other aspects, the first peptide of the at least two peptides is not identical to the second peptide of the at least two peptides.

[0014] In some embodiments, the present disclosure provides a composition comprising a peptide disclosed herein. In some aspects, the peptide is substantially pure. In some aspects, the peptide is at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, or at least 99% pure.

[0015] In some embodiments, the disclosure provides a pharmaceutical composition comprising 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, intravaginal, intrapenis, 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 an aerosolized solution.

[0016] In some embodiments, the present disclosure provides a polynucleotide comprising a nucleic acid sequence encoding a peptide as described herein.

[0017] In some aspects, the peptide composition of the embodiment can be used for the method of treating or preventing the disease of the 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, which can be kidney, liver, lung or heart fibrosis. In some aspects, the inflammatory disease is an inflammatory eye disease. The composition of the embodiment can be administered systemically or locally (e.g., at the site of the diseased tissue).

[0018] 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 an effective amount of a peptide as described herein to the subject. In some aspects, the subject suffers from pulmonary inflammation. In some aspects, the subject is undergoing chemotherapy or radiotherapy. In some aspects, the subject suffers from acute lung injury or infection. In some aspects, the subject suffers from chemically induced lung injury. In some aspects, the subject suffers from 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 aspects, the lung disease is a fibrotic condition of the lung. In some aspects, the lung disease is interstitial lung disease. In some aspects, the lung disease is idiopathic pulmonary fibrosis (IPF) or lung scarring. In some aspects, the administration includes atomizing a solution comprising the peptide. In some aspects, the method further comprises administering at least one additional anti-fibrotic therapeutic agent. In some aspects, the at least one additional anti-fibrotic agent is an NSAID, a steroid, a DMARD, an immunosuppressant, a biological response modifier, or a bronchodilator. In some aspects, the subject is a human.

[0019] It is contemplated that any method or composition described herein may be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become clear from the following detailed description. However, it should be understood that although the detailed description and specific examples indicate specific embodiments of the present disclosure, they are given only by way of illustration, because various changes and modifications within the spirit and scope of the present disclosure will become clear to those skilled in the art who read this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The following drawings form part of this specification and are included to further illustrate 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.

[0021] Figure 1 : Western blot of SMA and tubulin idiopathic pulmonary fibrosis cells treated with Cav-1 peptide. IPF cells were treated with: 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 were assessed by western blot.

[0022] Figure 2: Treatment with Cav-1 peptide increases SMA relative to tubulin in IPF cells. Graphical representation of the SMA to tubulin ratio in cells receiving the indicated treatments. DETAILED DESCRIPTION

[0023] The present disclosure overcomes challenges associated with the prior art by providing modified caveolin-1 (Cav-1) peptides and their use for disease treatment and prevention, particularly pulmonary fibrosis. In some aspects, pharmaceutical formulations of modified Cav-1 peptides are provided. For example, in some aspects, the peptides are formulated for delivery to the respiratory system. For example, peptides can be prepared for administration to the airways of a subject by formulating in an aqueous solution and atomizing the solution using a nebulizer. In other aspects, the peptides can be formulated for injection. Also provided herein is a method for treating lung injury and disease, which is performed by administering a therapeutically effective amount of a modified Cav-1 peptide to the subject (e.g., via the airways).

[0024] I. Definitions

[0025] As used herein, "substantially free" with respect to a specified component is used herein to mean that the specified component is not intentionally formulated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the specified component resulting from any accidental contamination of the composition is much less than 0.01%. Compositions in which the amount of the specified component cannot be detected using standard analytical methods are most preferred.

[0026] As used in the specification of this invention, "a / an" may mean one or more than one. As used in one or more claims of this invention, when used in conjunction with the word "comprising", the word "a / an" may mean one or more than one.

[0027] Although the present disclosure supports definitions referring to alternatives only and "and / or", the use of the term "or" in the claims is used to mean "and / or" unless explicitly stated to refer to alternatives only or the alternatives are mutually exclusive. As used herein, "another" may mean at least a second or more.

[0028] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device or method being employed to determine the value or the variation that exists between study subjects.

[0029] As used herein, the term "peptide" generally refers to an amino acid sequence consisting of a single chain of amino acids linked by peptide bonds. Typically, unless otherwise defined, a peptide comprises at least two amino acid residues and is less than about 50 amino acids in length.

[0030] 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 aspects, the biologically active peptide has at least 20% of the biological or biochemical activity (e.g., as measured by an in vitro or in vivo assay) of the native Cav-1 polypeptide of SEQ ID NO: 1. In some aspects, the biologically active peptide has increased biological or biochemical activity compared to the native Cav-1 polypeptide.

[0031] The term "identity" or "homology" should be interpreted as meaning the percentage of amino acid residues in the candidate sequence that are identical to the residues in the compared corresponding sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent identity of the entire sequence and not considering any conservative substitutions as part of the sequence identity. N-terminal or C-terminal extensions or insertions should not be interpreted as reducing identity or homology. Methods and computer programs for comparison are well known in the art. Sequence identity can be measured using sequence analysis software.

[0032] The term "polypeptide" or "protein" is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits may be connected by peptide bonds. In another embodiment, the subunits may be connected by other bonds (e.g., esters, ethers, etc.). As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids (including glycine and D or L optical isomers) and amino acid analogs and peptidomimetics. The term "peptidomimetics" or "peptide mimetics" means that the peptide according to the present invention is modified in this way so that it includes at least one non-peptide bond, such as urea bonds, carbamate bonds, sulfonamide bonds, hydrazine bonds or any other covalent bonds. If the peptide chain is shorter, a peptide of three or more amino acids is generally referred to as an oligopeptide. If the peptide chain is longer, the peptide is generally referred to as a polypeptide or protein.

[0033] The terms "subject", "individual" and "patient" are used interchangeably herein and refer to animals, such as humans or non-human animals (e.g., mammals), to which a pharmaceutical composition as disclosed herein is provided for treatment (including preventive treatment). As used herein, the term "subject" refers to humans and non-human animals. The term "non-human animal" includes all vertebrates, such as mammals such as non-human primates (particularly higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and non-mammals such as chickens, amphibians, reptiles, etc. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal substitute as a disease model. Non-human mammals include mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits and cattle. In some aspects, non-human animals are companion animals such as dogs or cats.

[0034] "Treating" a disease or condition in a subject or "treating" a patient suffering from a disease or condition refers to subjecting the individual to drug therapy, such as administration of a drug, such that at least one symptom of the disease or condition is alleviated or stabilized. Typically, when the peptide is administered therapeutically as a treatment, the peptide is administered to a subject exhibiting one or more symptoms of lung injury or pulmonary fibrosis.

[0035] By "isolated" is meant that the polypeptide has been separated from any natural environment, such as body fluids (eg, blood), and from components which naturally accompany the peptide.

[0036] Isolated and "substantially pure" means a polypeptide that has been separated from components that naturally accompany it and purified to at least some degree. Typically, a polypeptide is substantially pure when it is at least about 60% by weight, or at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, 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 can be obtained by extraction from a natural source, by expressing a recombinant nucleic acid in a cell that does not normally express the protein, or by chemical synthesis.

[0037] As used herein, the term "variant" refers to a polypeptide or nucleic acid that differs from a polypeptide or nucleic acid by the deletion, addition, substitution or side chain modification of one or more amino acids or nucleic acids, but still retains one or more specific functions or biological activities of a naturally occurring molecule. Amino acid substitutions include changes in which amino acids are replaced by different naturally occurring or unconventional amino acid residues. Such substitutions can be classified as "conservative", in which case the amino acid residues contained in the polypeptide are replaced by another naturally occurring amino acid with similar characteristics in terms of 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 amino acid residues present in the peptide are replaced by amino acids with different properties (such as naturally occurring amino acids from different groups) (e.g., using alanine to replace charged or hydrophobic amino acids), or alternatively, in which naturally occurring amino acids are replaced by unconventional amino acids. In some embodiments, amino acid substitutions are conservative. When used with reference to a polynucleotide or polypeptide, the term variant also encompasses polynucleotides or polypeptides that can change the primary, secondary or tertiary structure, respectively, compared to a reference polynucleotide or polypeptide (e.g., compared to a wild-type polynucleotide or polypeptide).

[0038] The term "insertion" or "deletion" is generally in the range of about 1 to 5 amino acids. The variation allowed can be determined experimentally by producing the peptide synthetically while systematically inserting, deleting or substituting nucleotides in the sequence using recombinant DNA technology.

[0039] The term "substitution" when referring to a peptide refers to a change of an amino acid to a different entity, such as another amino acid or amino acid moiety. A substitution may be a conservative substitution or a non-conservative substitution.

[0040] "Analogs" of molecules (such as peptides) refer to molecules whose functions are similar to those of the entire molecule or its fragments. The term "analogs" is also intended to include allele species and induced variants. Analogs are often different from naturally occurring peptides in one or several positions due to conservative substitutions. Analogs typically exhibit at least 80% or 90% sequence identity with natural peptides. Some analogs also include modifications of non-natural amino acids or N-terminal or C-terminal amino acids. Examples of non-natural amino acids are, for example, but 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, σ-N-methylarginine. Fragments and analogs can be screened for preventive or therapeutic efficacy in transgenic animal models as described below.

[0041] "Covalently bonded" means attached directly or indirectly (eg, through a linker) via a covalent chemical bond. In some aspects of all embodiments of the invention, the fusion peptide is covalently bonded.

[0042] As used herein, the term "fusion protein" refers to a recombinant protein of two or more proteins. Fusion proteins can be produced, for example, by linking a nucleic acid sequence encoding one protein to a nucleic acid encoding another protein so that they constitute a single open reading frame that can be translated into a single polypeptide with all the expected proteins in the cell. The order in which the proteins are arranged can be changed. The fusion protein may contain an epitope tag or a half-life extender. Epitope tags include biotin, FLAG tags, 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.

[0043] The term "airway" herein refers to any part of the respiratory tract, including the upper respiratory tract, respiratory 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 the respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli.

[0044] The terms "smoke inhalation-induced acute lung injury" and "ISALI" are used interchangeably herein and refer to a form of acute lung injury (ALI) caused by smoke inhalation. ALI is also known as "mild acute respiratory distress syndrome; ARDS". ARDS can be defined by the finding of one or more of the following in a subject: 1) bilateral pulmonary infiltrates on chest x-ray; 2) pulmonary capillary wedge pressure <18 mmHg (2.4 kPa) when measured by right cardiac catheterization as clinically indicated; and 3) PaO 2 / FiO 2 <300 mmHg (40 kPa). In some embodiments, treatment of ISALI includes treatment of one or more of the following conditions: decreased oxygenation, airway obstruction (including severe airway obstruction), fibrinous airway casts or debris, and alveolar fibrin deposition.

[0045] The term "nebulizing" and other grammatical variations refer herein to the process of converting a liquid into small aerosol droplets. In some embodiments, the median diameter of the aerosol droplets is about 2-10 μm. In some embodiments, the median diameter of the aerosol droplets is about 2-4 μm.

[0046] II. Caveolin-1 peptide

[0047] Embodiments of the present disclosure provide peptide variants of caveolin-1 (Cav-1) proteins. Caveolin-1 (Cav-1) scaffold domains or polypeptides interfere with the interaction of Cav-1 with Src kinase, mimicking the combined effects 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).

[0048]

[0049] In some aspects, the peptide is a scaffold domain peptide comprising an amino acid sequence having at least about 40%, 50%, 60%, 70%, 80%, 85%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2FTTFTVT. The peptide may comprise 1, 2, 3, 4 or more amino acid substitutions, deletions or insertions relative to the sequence SEQ ID NO: 1, thereby obtaining a polypeptide of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 residues. In a specific aspect, the peptide is a truncated form of a native Cav-1 polypeptide, such as the exemplary polypeptides shown in Table 1.

[0050] Table 1: Exemplary Cav-1 peptides.

[0051]

[0052]

[0053] (a = D-alanine, O = ornithine)

[0054] The peptides provided by the present disclosure are biologically active derivatives that have the activity of a native CAV-1 polypeptide in an in vitro or in vivo assay of binding or biological activity. In particular aspects, the peptides inhibit or prevent apoptosis of LEC induced by BLM in vitro or in vivo, and their activity is at least about 20% of the activity of the native CAV-1 polypeptide, or at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, about 95%, 97%, 99% and any range derivable therein, such as, for example, about 70% to 80%, and more preferably about 81% to about 90%; or even more preferably about 91% to about 99%. The peptides may have 100% or even higher activity compared to the native CAV-1 polypeptide. Assays for testing biological activity (e.g., anti-fibrotic activity, the ability to affect the expression of uPA, uPAR and PAI-1 mRNA or the ability to inhibit the proliferation of lung fibroblasts) are well known in the art.

[0055] The peptides disclosed herein are peptides of natural Cav-1 polypeptides or modified versions thereof. The peptides may be synthetic, recombinant or chemically modified peptides isolated or produced using methods well known in the art. The amino acids may be modified at the N-terminus, C-terminus or internally. The N-terminal modification may be, for example, but not limited to, acylation, acetylation or C-terminal amidation. The peptides may contain conservative or non-conservative amino acid changes as described below. Changes in the polynucleotide may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. The peptides may also contain insertions, deletions or substitutions of amino acids, including insertions and substitutions of amino acids (and other molecules) that do not normally occur in the peptide sequence that is the basis of the modified variant, such as, but not limited to, insertions of L-amino acids or non-standard amino acids such as ornithine that do not normally occur in human proteins. When describing a peptide, the term conservative substitution refers to a change in the amino acid composition of the peptide that does not substantially change the activity of the peptide. For example, conservative substitution refers to the substitution of an amino acid residue for a different amino acid residue having similar chemical properties. Conservative amino acid substitutions include replacement of leucine with isoleucine or valine, replacement of aspartic acid with glutamic acid, or replacement of threonine with serine.

[0056] Conservative amino acid substitutions are substitutions of one amino acid for another amino acid with similar structural and / or chemical properties, such as substitutions of leucine with isoleucine or valine, substitutions of aspartic acid with glutamic acid, or substitutions of threonine with serine. Thus, conservative substitutions of a particular amino acid sequence refer to substitutions of those amino acids that are not critical to the activity of the polypeptide or substitutions of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.), such that even substitutions of key amino acids do 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 each 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, WH Freeman and Company (1984), which is incorporated by reference in its entirety). In some embodiments, individual substitutions, deletions or additions that change, add or delete a single amino acid or a small percentage of amino acids may also be considered conservative substitutions if the change does not reduce the activity of the peptide. The range of insertions or deletions is generally about 1 to 5 amino acids. The selection of conservative amino acids can be selected based on the position of the amino acid to be substituted in the peptide, for example, when the amino acid is on the outside of the peptide and exposed to the solvent or on the inside and not exposed to the solvent.

[0057] In alternative embodiments, the amino acid that will replace the existing amino acid can be selected based on the position of the existing amino acid, i.e., its exposure to the solvent (i.e., if the amino acid is exposed to the solvent or is present on the outer surface of the peptide or polypeptide compared to an internally positioned amino acid that is not exposed to the solvent). 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, conservative amino acid substitutions may be selected for amino acids on the outside of the protein or peptide (i.e., those exposed to the solvent), for example, but not limited to, the following substitutions may be used: substitution of F for Y, S or K for T, A for P, D or Q for E, D or G for N, K for R, N or A for G, S or K for T, N or E for D, L or V for I, F for Y, S for T or A, K for R, G for N or A, K for R, A for S, K or P.

[0058] In alternative embodiments, conservative amino acid substitutions applicable to amino acids within the interior of a protein or peptide may also be selected, for example, suitable conservative substitutions for amino acids within a protein or peptide (i.e., where the amino acids are not exposed to a solvent) may be used, for example, but not limited to, the following conservative substitutions may be used: wherein Y is substituted with F, T is substituted with A or S, I is substituted with L or V, W is substituted with Y, M is substituted with L, N is substituted with D, G is substituted with A, T is substituted with A or S, D is substituted with N, I is substituted with L or V, F is substituted with Y or L, S is substituted with A or T, and A is substituted with S, G, T, or V. In some embodiments, non-conservative amino acid substitutions are also encompassed within the term variant.

[0059] In some aspects, 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 (derivation with polyethylene glycol), lipidation, glycosylation, amidation, or addition of other molecules. When a molecule contains an additional chemical moiety that is not normally part of the molecule, the molecule is also a "derivative" of another molecule. Such a moiety can change the pH or improve the solubility, absorption, biological half-life, etc. of the molecule. The moiety can alternatively reduce the toxicity of the molecule, eliminate or reduce any undesirable side effects of the molecule, etc. The moiety capable of mediating such an effect is disclosed in Remington's Pharmaceutical Sciences, 18th edition, A.R. Gennaro, ed., Mack Publ., Easton, PA (1990), which is incorporated herein by reference in its entirety.

[0060] When used in conjunction with "derivative" or "variant", the term "functional" refers to a polypeptide of the invention having a biological activity (functional or structural) substantially similar to that of an entity or molecule of which it is a functional derivative or functional variant. The term functional derivative is intended to include fragments, analogs or chemical derivatives of a molecule.

[0061] In some aspects, amino acid substitutions can be made at one or more positions of a polypeptide, wherein the substitutions are for amino acids with similar hydrophilicity. The importance of the hydropathic amino acid index in conferring interactive biological function on proteins is generally understood in the art (Kyte and Doolittle, 1982). It is recognized that the relative hydrophilicity of amino acids contributes 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. Therefore, such conservative substitutions can be made in a polypeptide and may have only a minor effect on its 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); glutamic acid (+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, and thus substitution of amino acids with hydrophilicity values ​​within ±2 is preferred, substitution of amino acids within ±1 is particularly preferred, and substitution of amino acids within ±0.5 is even more particularly preferred. Thus, any polypeptide described herein can be modified by substituting amino acids for different but homologous amino acids with similar hydrophilicity values. Amino acids with hydrophilicity within + / -1.0 or + / -0.5 points are considered homologous.

[0062] Modified Cav-1 peptides may contain co-translational and post-translational (C-terminal peptide cleavage) modifications, such as, for example, disulfide bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., by furin or metalloproteinase cleavage), etc., to the extent that such modifications do not affect the anti-inflammatory properties of the isolated peptide or its ability to improve glycemic control.

[0063] In some aspects, the modified Cav-1 peptide comprises non-naturally occurring amino acids. The polypeptide may comprise a combination of naturally occurring amino acids and non-naturally occurring amino acids, or may comprise only non-naturally occurring amino acids. The non-naturally occurring amino acids may include synthetic non-natural amino acids, substituted amino acids, or one or more D-amino acids that are desired in certain cases to the peptide (or other components of the composition other than the protease recognition sequence). Compared to the L-amino acid-containing form, peptides containing D-amino acids exhibit increased stability in vitro or in vivo. Therefore, the construction of peptides incorporating D-amino acids can 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 further discussion below), and extended intravascular and interstitial life when such properties are required. 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 and are therefore unlikely to induce a humoral immune response in the whole organism. Thus, 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.

[0064] In addition to the 20 "standard" L-amino acids, D-amino acids or non-standard, modified or unusual amino acids that are well defined in the art are also expected to be used in the present disclosure. Phosphorylated amino acids (Ser, Thr, Tyr), glycosylated amino acids (Ser, Thr, Asn), β-amino acids, GABA, ω-amino acids are further expected to be used in the present 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-aminocaproic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly); ornithine (Orn); citrulline (Cit); tert-butylalanine (t-BuA); tert-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-acetyl lysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,4-diaminobutyric acid (Dab); p-aminophenylalanine (Phe(pNH 2 )); N-methylvaline (MeVal); homocysteine ​​(hCys), homophenylalanine (hPhe) and homoserine (hSer); hydroxyproline (Hyp), homoproline (hPro), N-methylated amino acids and peptoids (N-substituted glycines).

[0065] Carboxyl 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, benzyloxycarbonyl (Cbz); acetylation and biotinylation. Amino-terminal modifications include: (i) acylation with carboxylic acids: formic acid, acetic acid, propionic acid, fatty acids (myristic acid, palmitic acid, stearic acid, etc.), succinic acid, benzoic acid, benzyloxycarbonyl (Cbz); (ii) biotinylation; (iii) amidation; (iv) attachment of dyes such as fluorescein (FITC, FAM, etc.), 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.

[0066] The polypeptide may be terminated at its N-terminus with an acyl group (abbreviated as "Ac") and an amide group (abbreviated as "Am"), respectively, such as an acetyl group (CH 3 CO-) and the C-terminus was capped with an amide group (-NH 2 ) end-capping. A wide range of N-terminal capping functional groups are contemplated, preferably linked to the terminal amino group, for example: formyl;

[0067] Alkanoyl having 1 to 10 carbon atoms, such as acetyl, propionyl, butyryl;

[0068] an alkenoyl group having 1 to 10 carbon atoms, such as hex-3-enoyl;

[0069] an alkynoyl group having 1 to 10 carbon atoms, such as hex-5-ynoyl;

[0070] Aroyl, such as benzoyl or 1-naphthoyl;

[0071] heteroaroyl, such as 3-pyrrolyl or 4-quinolinyl;

[0072] Alkylsulfonyl, such as methylsulfonyl;

[0073] Arylsulfonyl, such as benzenesulfonyl or sulfonyl;

[0074] heteroarylsulfonyl, such as pyridine-4-sulfonyl;

[0075] Substituted alkanoyl having 1 to 10 carbon atoms, such as 4-aminobutyryl;

[0076] Substituted alkenoyl having 1 to 10 carbon atoms, such as 6-hydroxy-hex-3-enoyl;

[0077] Substituted alkynoyl having 1 to 10 carbon atoms, such as 3-hydroxy-hex-5-ynoyl;

[0078] Substituted aroyl, such as 4-chlorobenzoyl or 8-hydroxy-naphthalen-2-yl;

[0079] Substituted heteroaroyl, such as 2,4-dioxo-1,2,3,4-tetrahydro-3-methyl-quinazolin-6-yl;

[0080] Substituted alkylsulfonyl, such as 2-aminoethanesulfonyl;

[0081] Substituted arylsulfonyl, such as 5-dimethylamino-1-naphthalenesulfonyl;

[0082] Substituted heteroarylsulfonyl, such as 1-methoxy-6-isoquinolinesulfonyl;

[0083] Carbamoyl or thiocarbamoyl;

[0084] Substituted carbamoyl (R'-NH-CO) or substituted thiocarbamoyl (R'-NH-CS), wherein R' is alkyl, alkenyl, alkynyl, aryl, heteroaryl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted aryl or substituted heteroaryl;

[0085] Substituted carbamoyl (R'-NH-CO) and substituted thiocarbamoyl (R'-NH-CS), wherein R' is alkanoyl, alkenoyl, alkynyl, aroyl, heteroaroyl, substituted alkanoyl, substituted alkenoyl, substituted alkynyl, substituted aroyl or substituted heteroaroyl, all as defined above.

[0086] The C-terminal capping functional group can be in an amide bond or an ester bond with the terminal carboxyl group. The capping functional group that provides an amide bond is named NR 1 R 2 , where R 1 and R 2 may be independently derived from the following groups: hydrogen;

[0087] Alkyl groups preferably having 1 to 10 carbon atoms, such as methyl, ethyl, isopropyl;

[0088] Preferably alkenyl having 1 to 10 carbon atoms, such as prop-2-enyl;

[0089] Preferably an alkynyl group having 1 to 10 carbon atoms, such as prop-2-ynyl;

[0090] Substituted alkyl groups having 1 to 10 carbon atoms, such as hydroxyalkyl, alkoxyalkyl, mercaptoalkyl, alkylthioalkyl, haloalkyl, cyanoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkanoylalkyl, carboxyalkyl, carbamoylalkyl;

[0091] Substituted alkenyl having 1 to 10 carbon atoms, such as hydroxyalkenyl, alkoxyalkenyl, mercaptoalkenyl, alkylthioalkenyl, haloalkenyl, cyanoalkenyl, aminoalkenyl, alkylaminoalkenyl, dialkylaminoalkenyl, alkanoylalkenyl, carboxyalkenyl, carbamoylalkenyl;

[0092] Substituted alkynyl groups having 1 to 10 carbon atoms, such as hydroxyalkynyl, alkoxyalkynyl, mercaptoalkynyl, alkylthioalkynyl, haloalkynyl, cyanoalkynyl, aminoalkynyl, alkylaminoalkynyl, dialkylaminoalkynyl, alkanoylalkynyl, carboxyalkynyl, carbamoylalkynyl;

[0093] Aroylalkyl having up to 10 carbon atoms, such as benzoyl or 2-benzoylethyl;

[0094] Aryl, such as phenyl or 1-naphthyl;

[0095] heteroaryl, such as 4-quinolinyl;

[0096] Alkanoyl having 1 to 10 carbon atoms, such as acetyl or butyryl;

[0097] Aroyl groups such as benzoyl;

[0098] heteroaroyl, such as 3-quinolinyl;

[0099] OR' or NR'R", wherein R' and R" are independently hydrogen, alkyl, aryl, heteroaryl, acyl, aroyl, sulfonyl, sulfinyl, or SO 2 -R"' or SO-R"', wherein R"' is substituted or unsubstituted alkyl, aryl, heteroaryl, alkenyl or alkynyl.

[0100] The end-capping functional group providing the ester bond is designated as OR, where R can be: alkoxy; aryloxy; heteroaryloxy; aralkyloxy; heteroaralkyloxy; substituted alkoxy; substituted aryloxy; substituted heteroaryloxy; substituted aralkyloxy; or substituted heteroaralkyloxy.

[0101] The N-terminal or C-terminal capping functional group, or both, may have such a structure that the capped molecule acts as a prodrug (a pharmacologically inactive derivative of a parent drug molecule), undergoes spontaneous or enzymatic conversion in vivo to release the active drug, and has improved delivery characteristics relative to the parent drug molecule (Bundgaard H, ed.: Design of Prodrugs, Elsevier, Amsterdam, 1985).

[0102] Judicious choice of capping groups allows the addition of other activities to the peptide. For example, the presence of a sulfhydryl group attached to the N-terminal or C-terminal cap will allow the derivatized peptide to be conjugated to other molecules.

[0103] On the other hand, the peptide or fragment or derivative thereof may be a "retro-inversopeptide". A "retro-inversopeptide" refers to a peptide in which the direction of the peptide bonds at at least one position is reversed, i.e., the amino and carboxyl termini are reversed relative to the side chains of the amino acids. Thus, a retro-inversopeptide analog has reversed termini and reversed direction of peptide bonds, while retaining substantially the same side chain topology as in the native peptide sequence. A retro-inversopeptide may comprise L-amino acids or D-amino acids, or a mixture of L-amino acids and D-amino acids, with all amino acids being D-isomers at most. A partial retro-inversopeptide analog is a polypeptide in which only a portion of the sequence is reversed and replaced with enantiomeric amino acid residues. Because the retro-inversopeptide portion of such an analog has reversed amino and carboxyl termini, the amino acid residues flanking the retro-inversopeptide portion are replaced with a-substituted gem-diaminomethane and malonate, respectively, with similar side chains. Retro-inversopeptide forms of cell penetrating peptides have been found to be as effective as the native form in translocation across membranes. The synthesis of retro-inverse peptide analogs is described in Bonelli, F. et al., Int J Pept Protein Res. 24 (6): 553-6 (1984); Verdini, A and Viscomi, GC, J. Chem. Soc. Perkin Trans. 1: 697-701 (1985); and U.S. Pat. No. 6,261,569, which are incorporated herein by reference in their entirety. A solid phase synthesis method for partial retro-inverse peptide analogs has been described (EP 97994-B), which is also incorporated herein by reference in its entirety.

[0104] 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" with another sequence, meaning that when the comparison is made, the percentage of bases (or amino acids) that are identical when the two sequences are compared. This comparison and homology or sequence identity percentage can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology (FM Ausubel et al., 1987) 30th Supplement, Section 7.7.18, Table 7.7.1. Preferably, default parameters are used for comparison. A preferred comparison program is BLAST, which uses default parameters. In particular, the preferred programs are BLASTN and BLASTP, which use the following default parameters: genetic code = standard; filter = none; chain = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sorting mode = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR.

[0105] A. Polymer polypeptides

[0106] Embodiments of the present disclosure also include longer polypeptides constructed from repeating units of modified Cav-1 variant polypeptides. Polypeptide multimers may comprise different combinations of polypeptides. Such multimeric polypeptides may be prepared by chemical synthesis or by recombinant DNA techniques as discussed herein. When produced by chemical synthesis, the oligomer preferably has 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 not more than 100 residues (or its equivalent, when including a linker or spacer).

[0107] B. Peptidomimetics

[0108] The modified Cav-1 peptide can be a peptidomimetic compound that mimics the biological effects of a natural Cav-1 polypeptide. A peptidomimetic agent can be a non-natural peptide or non-peptide agent that recreates the three-dimensional spatial characteristics of the binding element of a natural Cav-1 polypeptide, thereby giving it the binding activity and biological activity of a natural Cav-1 polypeptide. Similar to a natural Cav-1 polypeptide or polypeptide multimer, a peptidomimetic will have a binding surface (which interacts with any ligand bound by natural Cav-1) and a non-binding surface.

[0109] In some aspects, the disclosure also includes compounds that retain some of the peptide characteristics. For example, any proteolytically unstable bonds within the peptides of the invention can be selectively replaced by non-peptide elements such as isosteres (N-methylated; D-amino acids) or reduced peptide bonds, while the remainder of the molecule retains its peptide properties.

[0110] A variety of peptidomimetic compounds (whether agonists, substrates or inhibitors) of biologically active peptides / polypeptides such as opioid peptides, VIP, thrombin, HIV protease, etc. have been described. 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). Certain mimetics that mimic secondary structures are described in Johnson et al., In: Biotechnology and Pharmacy, Pezzuto et al., Chapman and Hall (eds.), NY, 1993. These methods are used to prepare peptidomimetics that have at least the binding ability and specificity of native Cav-1 polypeptides and preferably also have biological activity. In view of the present disclosure, the knowledge of peptide chemistry and general organic chemistry available to those skilled in the art is sufficient for the design and synthesis of such compounds.

[0111] For example, such peptidomimetics can be identified by checking the three-dimensional structure of the polypeptide of the present invention free or compound binding with 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. More knowledge of the interactive stereochemistry of the peptide and its ligand or receptor will allow rational design of such peptidomimetics. In the absence of a ligand, the structure of the peptide of the present invention or polypeptide can also provide a scaffold for designing a simulation molecule.

[0112] C. PEGylation

[0113] The modified Cav-1 peptide can be conjugated to 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 and thus increase serum persistence. The polypeptide can be conjugated to any targeting agent, such as a ligand having the ability to specifically and stably bind to an external receptor (US Patent Publication No. 2009 / 0304666).

[0114] In certain aspects, the methods and compositions of the embodiments relate to the PEGylation of the disclosed 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. The covalent attachment of PEG to a drug or therapeutic protein can "mask" the agent from the host's immune system (reducing immunogenicity and antigenicity), or increase the hydrodynamic size (size in solution) of the agent, thereby extending its circulation time by reducing renal clearance. PEGylation can also provide water solubility for hydrophobic drugs and proteins.

[0115] The first step in PEGylation is to appropriately functionalize the PEG polymer at one or both termini. PEGs activated with the same reactive moiety at each terminus are referred to as "homobifunctional," while PEG derivatives are referred to as "heterobifunctional" or "heterofunctional" if the functional groups present are different. Chemically active or activated derivatives of PEG polymers are prepared to attach PEG to desired molecules.

[0116] The selection of suitable functional groups for PEG derivatives is based on the type of available reactive groups on the molecule to which the PEG is coupled. For 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.

[0117] The technique used to form the first generation of PEG derivatives is generally to react the PEG polymer with a group that can react with a hydroxyl group (usually anhydrides, acid chlorides, chloroformates and carbonates). In the second generation of PEGylation chemistry, more effective functional groups such as aldehydes, esters, amides, etc. can be used for conjugation.

[0118] As the applications of PEGylation become more advanced and complex, the demand for heterobifunctional PEGs for conjugation is also increasing. These heterobifunctional PEGs are very useful in connecting two entities where a hydrophilic, flexible and biocompatible spacer is required. The preferred end groups of heterobifunctional PEGs are maleimide, vinyl sulfone, pyridyl disulfide, amine, carboxylic acid and NHS ester.

[0119] The most common modifiers or linkers are based on methoxy PEG (mPEG) molecules. Their activity depends on the addition of a protein modifying group at the alcohol end. In some cases, polyethylene glycol (PEG diol) is used as a precursor molecule. The diol is then modified at both ends to prepare heterodimeric or homodimeric PEG-linked molecules.

[0120] Proteins are usually PEGylated at nucleophilic sites such as unprotonated thiols (cysteinyl residues) or amino groups. Examples of cysteinyl-specific modification agents include PEG maleimide, PEG iodoacetate, PEG thiol and PEG vinyl sulfone. Under mild conditions and at neutral to weakly alkaline pH, all four examples are strongly cysteinyl-specific, but each has some disadvantages. The thioether formed with maleimide may be somewhat unstable under alkaline conditions, so the formulation options using this joint may have some limitations. The thiocarbamate linkage formed with iodine PEG is more stable, but free iodine can modify tyrosine residues under some conditions. PEG thiol forms a disulfide bond with protein thiol, but this linkage may also be unstable under alkaline conditions. Compared with maleimide and iodine PEG, the reactivity of PEG-vinyl sulfone is relatively slow; however, the thioether linkage formed is quite stable. Its slower reaction rate can also make the PEG-vinyl sulfone reaction easier to control.

[0121] Site-specific PEGylation is rarely performed at native cysteinyl residues because these residues are usually in the form of disulfide bonds or are required for biological activity. On the other hand, site-directed mutagenesis can be used to incorporate cysteinyl PEGylation sites for thiol-specific linkers. Cysteine ​​mutations must be designed to make them accessible to PEGylation reagents and still have biological activity after PEGylation.

[0122] Amine-specific modifiers include PEG NHS esters, PEG tresylate, PEG aldehydes, PEG isothiocyanates, and several others. All reactions are performed under mild conditions and are very specific for amino groups. PEG NHS esters may be one of the more reactive agents; however, their high reactivity can make PEGylation reactions difficult to control on a large scale. PEG aldehydes form imines with amino groups and are then reduced to secondary amines with sodium cyanoborohydride. Unlike sodium borohydride, sodium cyanoborohydride will not reduce disulfide bonds. However, this chemical is highly toxic and must be handled with caution, especially at lower pH where it becomes volatile.

[0123] Site-specific PEGylation can be a challenge due to multiple lysine residues on most proteins. Fortunately, because these reagents react with unprotonated amino groups, PEGylation can be directed to amino groups with lower pK by reacting at lower pH. Typically, the pK of α-amino groups is 1-2 pH units lower than the ε-amino groups of lysine residues. By PEGylating the molecule at pH 7 or lower pH, high selectivity to the N-terminus can often be achieved. However, this is feasible only when the N-terminal portion of the protein is not required for biological activity. Nevertheless, the pharmacokinetic benefits from PEGylation often exceed the significant loss of in vitro biological activity, resulting in a product having higher in vivo biological activity regardless of the chemical method of PEGylation.

[0124] When developing a PEGylation procedure, several parameters should be considered. Fortunately, there are usually no more than four or five key parameters. The "experimental design" approach to optimizing PEGylation conditions can be very useful. For thiol-specific PEGylation reactions, the parameters that should be considered include: protein concentration, PEG to protein ratio (on a molar basis), temperature, pH, reaction time, and in some cases, excluding oxygen. (Oxygen can cause proteins to form intermolecular disulfides, which will reduce the yield of PEGylated products). For amine-specific modifications, the same factors (except oxygen) should be considered, except that pH may be more critical, especially when targeting the N-terminal amino group.

[0125] For both amine-specific and thiol-specific modifications, the reaction conditions can affect the stability of the protein. This may limit temperature, protein concentration, and pH. In addition, the reactivity of the PEG linker should be known before starting the PEGylation reaction. For example, if the PEGylation agent is only 70% active, the amount of PEG used should ensure that only active PEG molecules are counted in the stoichiometry of the protein-PEG reaction.

[0126] D. Fusion protein

[0127] Certain embodiments of the present invention relate to fusion proteins of modified Cav-1 peptides. These molecules may have a polypeptide of an embodiment connected to a heterologous domain at the N-terminus or C-terminus. For example, the fusion may also use a leader sequence from another species to allow the protein to be recombinantly expressed in a heterologous host. The fusion protein may include a half-life extender. Another useful fusion includes adding a protein affinity tag, such as a serum albumin affinity tag or six histidine residues, or adding an immunologically active domain, such as an antibody epitope that is preferably cleavable, 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).

[0128] In specific embodiments, the peptides of the embodiments may be linked to peptides that increase in vivo half-life, such as polypeptides (Schellenberger et al., 2009), IgG Fc domains, albumin or albumin binding peptides.

[0129] Methods for producing fusion proteins are well known to those skilled in the art. Such proteins can be produced, for example, by synthesizing the complete fusion protein de novo, or by attaching a DNA sequence encoding a heterologous domain and then expressing the complete fusion protein.

[0130] The production of fusion proteins that restore the functional activity of the parent proteins can be facilitated by connecting the genes to a bridging DNA segment encoding a peptide linker that is spliced ​​between the tandemly linked polypeptides. The linker will be of sufficient length to allow the resulting fusion protein to fold properly.

[0131] 1. Connector

[0132] In certain embodiments, the polypeptides of the embodiments can be chemically conjugated using bifunctional cross-linking reagents or fused at the protein level using peptide linkers.

[0133] Bifunctional cross-linking reagents have been widely used for a variety of purposes, including preparation of affinity matrices, modification and stabilization of various structures, identification of ligand and receptor binding sites, and structural studies. Suitable peptide linkers can also be used to connect the polypeptides of the embodiments, such as Gly-Ser linkers.

[0134] Homobifunctional reagents carrying two identical functional groups have been shown to be very effective in inducing crosslinking between identical and different macromolecules or macromolecular subunits and the connection of polypeptide ligands to their specific binding sites. Heterobifunctional reagents contain two different functional groups. By utilizing the differential reactivity of the two different functional groups, crosslinking can be selectively and sequentially controlled. Bifunctional crosslinking reagents can be divided according to the specificity of their functional groups (e.g., amino, sulfhydryl, guanidinyl, indolyl, carboxyl specific groups). Among these crosslinking reagents, reagents for free amino groups have become particularly popular because of their commercial availability, ease of synthesis, and the mild reaction conditions that can be applied.

[0135] Most heterobifunctional cross-linking reagents contain a primary amine reactive group and a thiol reactive group. In another embodiment, heterobifunctional cross-linking reagents and methods of using the same are described (U.S. Patent No. 5,889,155, which is specifically incorporated herein by reference in its entirety). The cross-linking reagent combines a nucleophilic hydrazide residue with an electrophilic maleimide residue, in one example, allowing the coupling of an aldehyde to a free thiol. The cross-linking reagent can be modified to allow various functional groups to be cross-linked.

[0136] Furthermore, any other linking / coupling agents and / or mechanisms known to those skilled in the art can be used to combine the polypeptides of the embodiments, such as, for example, antibody-antigen interactions, avidin-biotin linkages, amide linkages, ester linkages, thioester linkages, ether linkages, thioether linkages, phosphate linkages, phosphoramide linkages, anhydride linkages, disulfide linkages, ionic and hydrophobic interactions, bispecific antibodies and antibody fragments, or combinations thereof.

[0137] Preferably, a cross-linking agent with reasonable stability in blood will be used. Various types of disulfide-containing linkers are known that can be successfully used to conjugate targeting agents and therapeutic / prophylactic agents. Linkers containing disulfide bonds that act as steric hindrances may demonstrate greater stability in vivo. Therefore, these linkers are a group of linking agents.

[0138] In addition to sterically hindered crosslinkers, non-sterically hindered linkers can also be used based on the circumstances. Other useful crosslinkers that are believed not to contain or generate protected disulfides include SATA, SPDP, and 2-iminothiolane (Wawrzynczak and Thorpe, 1987). The use of such crosslinkers is well known in the art. Another embodiment involves the use of flexible linkers.

[0139] Once chemical conjugation occurs, the peptide is typically purified to separate the conjugate from non-conjugated agents and other contaminants. A number of purification techniques are available to provide the conjugate of sufficient purity to make it clinically useful.

[0140] Purification methods based on size separation (such as gel filtration, gel permeation or high performance liquid chromatography) will generally be the most commonly used. Other chromatographic techniques, such as blue-agarose separation, may also be used. Conventional methods for purifying fusion proteins from inclusion bodies may be useful, such as using weak detergents such as sodium N-lauroyl-sarcosinate (SLS).

[0141] 2. Cell-penetrating peptides and membrane-translocating peptides

[0142] In addition, in certain aspects, the modified Cav-1 peptide may also 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 a polypeptide to pass through a cell membrane (e.g., a plasma membrane in the case of a eukaryotic cell). 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 products, protegrin I, penetrating peptide (RQIKIWFQNRRMKWKK (SEQ ID NO: 22)), or bee venom peptide (GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 23)). In certain aspects, the CPP comprises a T1 (TKIESLKEHG (SEQ ID NO:24)), T2 (TQIENLKEKG (SEQ ID NO:25)), 26 (AALEALAEALEALAEALEALAEAAAA (SEQ ID NO:26)), or INF7 (GLFEAIEGFIENGWEGMIEGWYGCG (SEQ ID NO:27)) CPP sequence.

[0143] III. How to use

[0144] One aspect of the invention relates to the use of polypeptides described herein and mutants, variants, analogs or derivatives thereof. Specifically, these methods relate to administering to a subject any polypeptide described herein or a pharmaceutically acceptable modification thereof in a pharmaceutically acceptable carrier, i.e., a composition for treating or preventing a disease, injury or infection of the lung (e.g., a fibrotic condition of the lung), the composition comprising a polypeptide of the embodiment in a pharmaceutically acceptable carrier.

[0145] A. Pharmaceutical Composition

[0146] It is contemplated that the modified Cav-1 peptides can be administered systemically or locally to inhibit apoptosis and for the treatment and prevention of damage to lung tissue. The peptides can be administered intravenously, intrathecally and / or intraperitoneally. In particular aspects, the polypeptides are delivered locally to the airways, such as by administering an aerosolized formulation or a dry powder formulation for inhalation. The polypeptides can be administered alone or in combination with an anti-fibrotic compound.

[0147] The modified Cav-1 peptide can be combined, administered simultaneously or sequentially with at least one additional therapeutic agent for pulmonary fibrosis. The additional therapeutic agent can be an NSAID, a steroid, a DMARD, an immunosuppressant, a biological response modifier, a bronchodilator, or an anti-fibrotic agent such as pirfenedone (whose anti-fibrotic mechanism of action is not fully understood, but may involve the blockade of TGF-β), the generalized tyrosine kinase blocker nintedanib, or any other anti-fibrotic agent.Suitable NSAIDs are selected from the group consisting of 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, and tiaprofenic acid. acid), fluprofen, indomethacin, sulindac, tolmetin, zomepirac, nabumetone, diclofenac, fenclofenac, alclofenac, bromfenac, ibufenac, aceclofenac, acemetacin, fentiazac, clidanac, etodolac, oxpinac, mefenamic acid, meclofenamic acid, flufenamic acid, nifluminic acid, tolfenamic acid acid, diflunisal, flufenisal, piroxicam, tenoxicam, lornoxicam and nimesulide and pharmaceutically acceptable salts thereof; selective COX 2 inhibitors meloxicam, celecoxib and rofecoxib and pharmaceutically acceptable salts thereof.Suitable steroids are prednisone, prednisolone, methylprednisolone, dexamethasone, budenoside, fluocortolone and triamcinolone. Suitable DMARDs are sulfasalazine, olsalazine, chloroquin, gold derivatives (Auranofin), D-penicillamine and cytostatics such as methotrexate and cyclophosphamide. Suitable immunosuppressants are cyclosporin A and its derivatives, mycophenolate mofetil, FK 506, OKT-3, ATG, 15-deoxyspergualin, mizoribine, misoprostol, rapamycin, reflunomide and azathioprine. Suitable biological response modifiers are interferon beta, anti-TNF-alpha (Etanercept), IL-10, anti-CD3 or anti-CD25. Suitable bronchodilators are ipratropium bromide, oxytropium bromide, tiotropium bromide, epinephrine hydrochloride, salbutamole, terbutalin sulfate, fenoterol hydrobromide, salmeterole and formoterole. In such combinations, each active ingredient can be administered according to its usual dosage range or a dosage lower than its usual dosage range. The dosage of the combined NSAID, steroid, DMARD, immunosuppressant and biological response modifier is appropriately 1 / 50 of the usually recommended minimum dosage to 1 / 1 of the usually recommended dosage, preferably 1 / 20 to 1 / 2, and more preferably 1 / 10 to 1 / 5. The usually recommended dosage of the combined drug should be understood as, for example, in Rote. 2002, Editio Cantor Verlag Aulendorf, Germany or the dosages disclosed in the Physician's Desk Reference.

[0148] In the case of considering clinical applications, it may be necessary to prepare a pharmaceutical composition comprising proteins, antibodies and drugs in a form suitable for the intended application. Generally, the pharmaceutical composition may include an effective amount of one or more polypeptides or additional agents of the embodiments dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce side effects, allergic reactions or other adverse reactions when applied to animals (such as humans) when appropriate. According to the present disclosure, those skilled in the art will know the preparation of pharmaceutical compositions comprising at least one polypeptide or additional active ingredients of the embodiments separated by the methods disclosed herein, as exemplified by Remington's Pharmaceutical Sciences, 18th edition, 1990, which is incorporated herein by reference. In addition, for animal (e.g., human) administration, it should be understood that the product should meet the sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards.

[0149] As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes and the like materials and combinations thereof, as known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, 1990, which is incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, it is contemplated for use in the pharmaceutical composition.

[0150] Certain embodiments of the present invention may include different types of carriers, depending on whether it is used in solid, liquid or aerosol form, and whether sterility is required for route of administration (such as injection). The composition can be intravenous, intrathecal, intradermal, percutaneous, intrathecal, intraarterial, intraperitoneal, intranasal, intravaginal, intrarectal, intramuscular, subcutaneous, mucosal, oral, local (topically), local (locally), by inhalation (e.g., inhalation atomization or dry powder formulation), by injection, by infusion, by continuous infusion, by directly soaking target cells (via catheter, via lavage) with lipid composition (e.g., liposome) local perfusion, or by other methods as known to those of ordinary skill in the art or any combination of the foregoing to use (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, 1990, the document is incorporated herein by reference).

[0151] The modified polypeptide can be formulated into a composition in the form of a free base, neutral or salt. Pharmaceutically acceptable salts include acid addition salts, such as acid addition salts formed with the free amino groups of the protein composition, or acid addition 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. The salt formed with the free carboxyl group can also be derived from an inorganic base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide; or an organic base such as isopropylamine, trimethylamine, histidine or procaine. After preparation, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The preparation is easy to be administered in a variety of dosage forms, such as formulated for parenteral administration such as injectable solutions, or for delivery to the aerosol of the lung, or formulated for dietary administration such as drug release capsules.

[0152] According to some aspects of the present invention further, the composition suitable for use can be provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be absorbable and include liquid, semisolid (i.e. paste) or solid carrier. Unless any conventional medium, agent, diluent or carrier is harmful to the therapeutic effectiveness of the recipient or the composition contained therein, it is appropriate to use it in the composition for implementing the method. The example of carrier or diluent includes fat, oil, water, saline solution, lipid, liposome, resin, binder, filler, etc. or its combination. The composition may also include various antioxidants that prevent one or more components from being oxidized. In addition, the effect of preventing microorganisms can be achieved by preservatives, such as various antibacterial agents and antifungal agents, including but not limited to parabens (such as methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal or its combination.

[0153] According to certain aspects of the invention, the composition is combined with the carrier in any convenient and practical manner, ie, by dissolving, suspending, emulsifying, mixing, encapsulating, absorbing, etc. Such procedures are routine to those skilled in the art.

[0154] In a specific embodiment of the invention, the composition is thoroughly combined or mixed with a semisolid or solid carrier. Mixing can be carried out in any convenient way such as grinding. Stabilizers can also be added during the mixing process to protect the composition from losing its therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for compositions include buffers, amino acids (such as glycine and lysine), carbohydrates, or lyoprotectants (such as glucose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.).

[0155] In some aspects, the pharmaceutical formulation comprises one or more surfactants. Surfactants used according to the disclosed methods include ionic surfactants and nonionic surfactants. Representative nonionic surfactants include polysorbates, such as and Surfactant (ICI Americas, Bridgewater, NJ); Poloxamer (e.g., Poloxamer 188); Surfactants (Sigma, St. Louis, Mo.); sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, or stearyl sarcosine; linoleyl betaine, myristyl betaine, or cetyl betaine; lauroamidopropyl dimethylamine, cocamidopropyl dimethylamine, linoleamidopropyl dimethylamine, myristamidopropyl dimethylamine, palmitamidopropyl dimethylamine, or (e.g., lauroamidopropyl); myristamidopropyl dimethylamine, palmitamidopropyl dimethylamine, or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleoyl taurate; MONAQUAT TM Surfactants (Mona Industries, Paterson, NJ); polyethylene glycol; polypropylene glycol; block copolymers of ethylene glycol and propylene glycol, such as Surfactants (BASF of Mt. Olive, NJ); oligo(ethylene oxide) alkyl ethers; alkyl(thio)glucosides, alkyl maltosides; and phospholipids. For example, the surfactant may be present in the formulation in an amount of about 0.01% to about 0.5% (the weight of the surfactant relative to the total weight of other solid components in 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 other aspects, the pharmaceutical formulations of the embodiments are substantially free of nonionic surfactants or substantially free of all surfactants.

[0156] With regard to the treatment methods of the present invention, it is contemplated that the administration of one or more peptides as disclosed herein or mutants, variants, analogs or derivatives thereof is not limited to a specific mode of administration, dosage or frequency of administration; the present invention contemplates all modes of administration, including intramuscular, intravenous, intraperitoneal, intravascular, intraarticular, intralesional, subcutaneous or any other route sufficient to provide a dose sufficient to treat inflammation-related conditions. The therapeutic agent can be administered to the patient in a single dose or multiple doses. When multiple doses are administered, the doses can be separated from each other, for example, by one hour, three hours, six hours, eight hours, one day, two days, one week, two weeks or one month. For example, the therapeutic agent can be administered for, for example, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20 or more weeks. It should be understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition. For example, if a lower dose does not provide sufficient therapeutic activity, the therapeutic dose can be increased.

[0157] Although the attending physician will ultimately decide the appropriate amount and dosing regimen, a therapeutically effective amount of one or more polypeptides as disclosed herein, or mutants, variants, analogs or derivatives thereof, can be provided at 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.

[0158] 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 regimens). For example, a physician may first prescribe a relatively low dose, then increase the dose until an appropriate response is obtained. Depending on the application, the dose administered to the patient is sufficient to produce a beneficial therapeutic response in the patient over time, or, for example, to reduce symptoms, or to produce other appropriate activity. The dosage is determined by the efficacy of the particular formulation, and the activity, stability or serum half-life of one or more polypeptides as disclosed herein, or their mutants, variants, analogs or derivatives, and the patient's condition, as well as the body weight or surface area of ​​the patient to be treated.

[0159] In some aspects, a single dose is given to a subject, and a single dose of about 0.2 mg / kg to about 250 mg / kg is given once a day to treat a subject (preferably a mammal, more preferably a person suffering from or susceptible to the pulmonary fibrosis caused thereby), such as about 10 mg / kg to about 50 mg / kg, for example, via instillation (by inhalation). This dosage can be applied anywhere every day for about 3 days to one or more weeks. Long-term administration is also possible, although as is well known in the art, the dosage may need to be adjusted downward. However, the aforementioned range is suggestive, because the number of variables in a single treatment regimen is large, and it is expected that there is a considerable deviation from these preferred values.

[0160] For continuous administration, for example, by a pump system (such as the osmotic pump used in some experiments described below), the total dose over a course of about 1-2 weeks is preferably in the range of 1 mg / kg to 1 g / kg, preferably 20-300 mg / kg, more preferably 50-200 mg / kg. After this continuous dosing regimen, the total concentration of the active compound is preferably in the range of about 0.5 μM to about 50 μM, preferably about 1 μM to about 10 μM.

[0161] Effective concentrations of active compounds for inhibiting or preventing apoptosis in vitro range from about 0.5 nM to about 100 nM, more preferably from about 2 nM to about 20 nM. Effective doses and optimal dose ranges can be determined in vitro using the methods described herein.

[0162] B. Aerosol Dispersion and Atomization Devices

[0163] The formulation may be aerosolized using any suitable device, including, but not limited to, a jet nebulizer, an ultrasonic nebulizer, a metered dose inhaler (MDI), and a device that aerosolizes a liquid by forcing it through a jet or nozzle (e.g., the NEBU-1000 from Aradigm, Inc. of Hayward, Calif.). Drug delivery device). In addition, the compound can be formulated as a dry powder for delivery using a dry powder inhaler device. To deliver the formulation to a subject, as further described below, the pulmonary delivery device may also include a ventilator, optionally in combination with a mask, a mouthpiece, a mist inhalation device, and / or a platform that guides the user to inhale correctly and automatically delivers the drug in the breath at the appropriate time. Representative aerosolization devices that can be used in accordance with the methods of the present invention include, but are not limited to, the aerosolization devices 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. Published Patent Application Nos. 20020020412 and 20020020409.

[0164] With a jet nebulizer, compressed gas from a compressor or hospital air line passes through a narrow constriction called an ejector. This creates a low-pressure area, and liquid medication from a reservoir is drawn in through a feed tube and broken into droplets by the airflow. Only the smallest droplets leave the nebulizer directly, while most droplets hit baffles and walls and return to the reservoir. Therefore, the time required to perform jet nebulization varies depending on factors such as the volume of the composition to be nebulized, and such time can be easily adjusted by those skilled in the art.

[0165] Metered dose inhaler (MDI) can be used to deliver the compositions of the present invention in a more concentrated form than that usually delivered by a nebulizer. For optimal effect, the MDI delivery system requires appropriate application techniques, which include the coordinated drive of aerosol delivery and suction, the slow suction of about 0.5-0.75 liter per second, the deep breathing close to the deep inspiration volume suction and the breath holding of at least 4 seconds. When treatment benefits from relatively short treatment time and lower cost, it is convenient and suitable to use MDI to carry out pulmonary delivery. Optionally, the preparation can be heated to about 25 ℃ to about 90 ℃ during atomization to promote effective droplet formation and subsequent delivery. See, for example, U.S. Patent number 5,299,566.

[0166] The aerosol composition of the embodiment comprises droplets of the composition, the size of the droplets being suitable for effective delivery in the lung. In some cases, the surfactant preparation is delivered to the pulmonary bronchi, more preferably to the bronchioles, still more preferably to the alveolar ducts, and still more preferably to the alveoli. The diameter of the aerosol droplets is generally less than about 15 μm, less than about 10 μm in diameter, less than about 5 μm in diameter, or less than about 2 μm in diameter. In order to effectively deliver to the alveolar bronchi of a human subject, the aerosol composition may preferably comprise droplets having a diameter of about 1 μm to about 5 μm.

[0167] 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.

[0168] Protein stability after aerosolization can be assessed using techniques known in the art, including size exclusion chromatography; electrophoresis techniques; spectroscopic techniques (such as UV spectroscopy and circular dichroism) and protein activity (measured in vitro or in vivo). In order to perform in vitro assays of protein stability, the aerosol composition can be collected and then distilled or absorbed onto a filter. For in vivo assays or for administering the composition to the lungs of a subject, the device for aerosolization is suitable for inhalation by the subject. For example, protein stability can be assessed by determining the level of protein aggregates. Preferably, the aerosol composition of the present invention is substantially free of protein aggregates. The presence of soluble aggregates can be qualitatively determined using DLS (DynaPro-801TC, ProteinSolutions, Charlottesville, Va.) and / or by UV spectrophotometry.

[0169] The term "vibrating mesh nebulizer" refers herein to any nebulizer that operates according to the general principle of using a vibrating mesh or vibrating plate (orifice plate) with a plurality of holes to produce a low-speed aerosol of fine particles. Some nebulizers may include a mesh / membrane with between 1000 and 7000 holes that vibrates on top of a liquid reservoir (see, e.g., U.S. Patent Publication 20090134235 and Waldrep and Dhand 2008, each of which is incorporated herein by reference). In some embodiments, the vibrating mesh nebulizer is Professional atomizer, Omron Pari Or EZ Breath Nebulizer. In some aspects, the vibration frequency of the vibrating mesh nebulizer is between about 50-250 kHz, 75-200 kHz, 100-150 kHz, or about 120 kHz. These devices have high efficiency in delivering aerosols to the lungs, and the volume of liquid remaining in these devices is minimal, which is advantageous for expensive and effective compounds such as plasminogen activators.

[0170] In certain aspects, a vibrating mesh nebulizer is used to produce the aerosolized composition of the embodiments. For example, the composition can be produced using an active vibrating mesh nebulizer (e.g. The compositions of the embodiments can be produced using a passive vibrating mesh nebulizer such as an Omron nebulizer. Or EZ Breathe Nebulizer to produce.

[0171] IV. Lung Conditions Treated

[0172] The modified peptides of the present invention can be used to treat a variety of lung conditions. The lung conditions treated can be acute or chronic. Acute lung conditions can be acute lung injury, infection, or chemically induced conditions. Chronic lung conditions can be the result of injury, infection, or disease.

[0173] A. Lung damage

[0174] In some aspects, the subject suffers from acute lung injury (ALI) or infection or chemical-induced lung injury. In specific aspects, the subject suffers from 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., fibrosis occurring after acute lung injury, including ARDS).

[0175] ALI caused by inhalation injury has been treated with inhaled anticoagulants, steroids, beta-agonists, high-frequency ventilation, and extracorporeal membrane oxygenation, with varying and generally suboptimal results. No effective preventive measures are available except for barriers with respiratory masks. Significant progress has been made in the management of ARDS, but it remains largely supportive and requires careful waiting for endogenous healing mechanisms to take effect; and the in-hospital mortality rate remains over 40% (Matthay et al., 2012). Survivors of ALI often suffer from chronic respiratory dysfunction and decreased quality of life. Any model that can accelerate recovery and / or prevent subsequent complications such as chronic respiratory insufficiency and pulmonary fibrosis would be highly desirable. There is an urgent need to improve the early diagnosis of ALI, and more importantly, the prevention and treatment of ALI. The pathophysiology of ALI caused by direct inhalation lung injury or ARDS caused by systemic disease is extremely complex and heterogeneous, covering systemic as well as local cardiopulmonary factors, such as increased membrane permeability, influx of inflammatory cytokines, oxidative cell damage, chamber fluid shifts, ion channel derangements, and many other factors (Matthay et al., 2012). Clearly, new therapies are needed to treat and prevent lung disorders such as ALI.

[0176] In some embodiments, a method for treating or preventing acute lung injury, lung infection or lung disease in a subject is provided, 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 FTTFTVT (SEQ ID NO: 2), wherein the variant polypeptide retains the biological activity of caveolin 1 (Cav-1). In some aspects, the method of administering the pharmaceutical preparation of the embodiment comprises atomizing a solution comprising the variant polypeptide. In specific aspects, the subject is a human.

[0177] B. Lung disease

[0178] Lung diseases include cystic fibrosis, chronic obstructive pulmonary disease (COPD), asthma, bronchiolitis obliterans, plastic bronchitis and lung 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 injury leading to fibrosis (Murray et al., 1997; Rabe et al., 2007; Tsushima et al., 2009). These diseases constitute the third leading cause of death worldwide.

[0179] Cystic fibrosis is a hereditary disease that primarily affects the exocrine glands and exocrine sweat glands of the digestive and respiratory systems. The characteristics of this disease are usually chronic respiratory infections, pancreatic insufficiency, abnormally thick viscous viscous secretions and premature death. Cystic fibrosis (CF) is characterized by progressive airflow obstruction. A subgroup of individuals with CF also produces airway hyperresponsiveness to inhaled cholinergic agonists (Weinberger, 2002 and Mitchell et al., 1978), and reversibility of airflow limitation is produced in response to bronchodilators (van Haren et al., 1991 and van Haren et al., 1992). The presence of bronchial hyperresponsiveness and airway obstruction suggests that there may be a common cause of disease between CF and other airway narrowing diseases (such as asthma or chronic obstructive pulmonary disease (COPD)), in which airway smooth muscle dysfunction is considered to lead to disease progression.

[0180] Lung infection may be a bacterial infection. The infectious bacteria may be Pseudomonas aeruginosa, Bacillus anthracis, Listeria monocytogenes, Staphylococcus aureus, Salmenellosis, Yersinia 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 cause pneumonia.

[0181] Chronic obstructive pulmonary disease (COPD) is a term used to classify two major airflow obstruction conditions: chronic bronchitis and emphysema. Approximately 16 million Americans have COPD, of whom 80%-90% have been smokers for most of their lives. COPD is the leading cause of death in the United States, accounting for 122,283 deaths in 2003. The direct health care expenditure cost for COPD in the United States was approximately $20.9 billion in 2003. Chronic bronchitis is an inflammation of the bronchial airways. The bronchial airways connect the trachea to the lungs. When inflamed, the bronchi secrete mucus, causing a chronic cough.

[0182] In emphysema, the alveolar sacs become over-inflated due to damage to the lung's elastin framework. Inflammatory cells in the emphysematous lung release elastase, an enzyme that degrades or damages the elastin fibers within the lung matrix. Emphysema has a variety of causes, including smoking, exposure to environmental pollutants, alpha-1 antitrypsin deficiency, and aging.

[0183] Bronchiolitis is most commonly caused by a viral lower respiratory tract infection and is primarily characterized by acute inflammation, edema, epithelial cell necrosis within the small airways, and increased mucus production (Ralston et al., 2014). Signs and symptoms typically begin with rhinitis and cough, which may progress to shortness of breath, wheezing, rales, use of the accessory muscles, and / or nasal flaring.

[0184] Bronchiolitis obliterans is a progressive decrease in airflow caused by abnormal remodeling of the small airways in the lung (Meyer et al., 2014). Bronchiolitis obliterans syndrome is a major complication of lung transplantation and is often used to describe delayed allograft dysfunction that results in a persistent decrease in forced expiratory volume and force that is not due to other known causes (Meyer et al., 2014).

[0185] The term "asthma" may 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 remain inflamed and spasms may occur occasionally.

[0186] V. Examples

[0187] The following examples are included to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques found by the inventors to work well in the practice of the present invention, and therefore can be considered to constitute preferred modes for its practice. However, based on this disclosure, it will be appreciated by those skilled in the art that many changes can be made in the disclosed specific embodiments without departing from the spirit and scope of the present invention and still obtaining similar or similar results.

[0188] Example 1 - Cav-1 Peptide Solubility

[0189] In order to determine which peptide is most soluble in liquid formulations, 50 mg of each Cav-1 peptide was dissolved in 5 mL of Tris buffered saline at pH 7.51. Each sample was vortexed to help the sample dissolve completely. For insoluble peptides, the absorbance at 600 nm was measured immediately after the peptide was dissolved, or for soluble peptides, the absorbance was measured after 10 minutes. Except for samples APi2348, APi2352, and APi2353, which were measured for the second time at 15 minutes, 5 minutes, or 15 minutes after dissolution, other insoluble peptides measured absorbance again after 10 minutes. Sample APi2345 was measured only after 20 minutes after dissolution because the dissolution was incomplete (Table 2). pH was also tested after 24 hours.

[0190] Samples APi2350, APi2354, APi2355 and APi2356 showed increased solubility at pH 7.51 compared to the other peptides tested (Table 2). After 24 hours, the pH of all samples remained stable at approximately pH 7.5.

[0191]

[0192] Example 2 - Cav-1 peptide increases smooth muscle actin production

[0193] Cav-1 peptides were dissolved in DMSO to prepare 10 mM stock solutions. The 10 mM stock solutions of each peptide were then diluted with HBSS to prepare 900 μM working stock solutions. The DMSO-resuspended peptides and working stock solutions were stored at -20°C. For the culture medium, the working stock solution was added to DMEM medium to make the final concentration of Cav-1 peptide 10 μM.

[0194] Idiopathic pulmonary fibrosis (IPF) cell line 2051 is purchased, and the IPF cells of the fourth generation are inoculated into the 100mm flat board containing DMEM, 10%FBS and 1%P / S.IPF cells are washed with 4mL DMEM+1%P / S, and their serum starvation is spent the night.Then 44uL HBSS (negative control), 10uM LTI-03 (SEQ ID NO:2), 90uM LTI-03 (positive control), 10uM APi2350, 10uM APi2354, 10uM APi2355, 10uM APi2356 or 20uL DMSO (negative control) are used to process cells for 2 days.

[0195] After 2 days of treatment, the cells were washed once in cold sterile HBSS. The HBSS was removed and 150uL of lysis buffer with a protease inhibitor cocktail was added to the cells. The cells were incubated with 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 13,000RPM for 20 minutes. The lysate was then snap frozen in liquid nitrogen, thawed, vortexed, and centrifuged again at 13,000RPM for 30 minutes. The supernatant was then collected and the pellet discarded. The concentration of the cell lysate was then determined by BCA assay.

[0196] Western blots were performed to assess the presence of treatment effects. Briefly, 12ug of each lysate was run on a 10% polyacrylamide gel. The gel was then transferred to a membrane and washed. Western blot results with primary antibodies against smooth muscle actin (SMA) and tubulin can be found at Figure 1 The treatments for each lysate in the lanes shown are: 1: untreated, 2: 10 μM LTI-03, 3: 90 μM LTI-03, 4: 10 μM APi2350, 5: 10 μM APi2354, 6: 10 μM APi 2355, 7: 10 μM APi2356, and 8: DMSO.

[0197] Western blots were photographed and analyzed using ImageJ to determine the ratio of smooth muscle actin to tubulin ( Figure 2 As expected, LTI-03 caused an increase in SMA production relative to tubulin. Treatment with the Cav-1 peptides APi2350, APi2354, APi2355, and APi2356 also increased SMA expression relative to tubulin ( Figure 2 ).

[0198] Example 3 - Cav-1 peptide protects AEC2 cells from fibrotic lung biopsies

[0199] To evaluate the effect of Cav-1 peptide APi2355 (SEQ ID NO: 8) on AEC2 cell viability, surgical biopsies were obtained for the preparation of nonspecific interstitial pneumonia precision cut lung sections (PCLS). One individual with nonspecific interstitial pneumonia (NSIP) and another with advanced IPF were treated. Lysotracker staining was performed, which stains acidic compartments in live cells and selectively accumulates in the lamellar bodies of lung AEC2 cells (Van der Velden et al., 2013). Cav-1 peptides were suspended in DMEM / 5% FBS and PCLS sections were treated with 10, 100 or 500 μM LTI-03 or APi2355 (Var55) (n=5 replicates / treatment group). Lysotracker staining (green DND-26, Promega) was performed on NSIP PCLS 48 hours after a single treatment. A strong dose-dependent increase in AEC2 cell viability was observed. In addition, lysotracker staining (red DND-99, Promega) was performed on terminal IPF at 1, 2, 3, 5, and 7 days after daily treatment with LTI-03 or APi2355. A dose-dependent increase in AEC2 cell viability was observed in IPF terminal biopsies treated with LTI-03 for 7 consecutive days. The therapeutic effect of APi2355 (Var55) was observed on day 3.

[0200] ***

[0201] In light of the present disclosure, all methods disclosed and claimed herein can be prepared and performed without undue experimentation. Although the compositions and methods of the present invention have been described in the form of preferred embodiments, it is clear to those skilled in the art that changes can be applied to the methods described herein and in the steps or step sequences of the methods described herein without departing from the concept, spirit and scope of the present invention. More specifically, it will be clear that certain agents related to chemistry and physiology can replace the agents described herein while achieving the same or similar results. All such similar substitutions and modifications clear to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0202] V. References

[0203] The following references, to the extent they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0204] Bonelli, F. et al., Iht J Pept Protein Res. 24(6): 553-6 (1984).

[0205] Bundgaard H,Ed:Design of Prodrugs,Elsevier,Amsterdam,1985

[0206] Current Protocols In Molecular Biology(F.M.Ausubel et al.,eds.,1987)

[0207] Dordo et al.J.Mol Biol,1999,217,721-739

[0208] Fults et al.(1991)J Pharm Pharmacol 43:726-8

[0209] Giannis et al.,Adv.in Drug Res.29:1-78(1997).

[0210] Hruby,VJ,Biopolymers 33:1073-1082(1993)

[0211] Johnson et al.,In:Biotechnology and Pharmacy,in Pezzuto et al.,Chapman and Hall(Eds.),NY,1993

[0212] Kyte and Doolittle,J.Mol.Biol.,157(1):105-32,1982.

[0213] McLean et al.(2000)Anal Chem72:4796-804.

[0214] Meyer et al.,European Respiratory Journal,44:1479-1503,2014.

[0215] Moore et al.,Adv.in Pharmacol 33:91-141(1995)

[0216] Ralston et al.,Pediatrics,134(5):e1474-e1502,2014.

[0217] Remington′s Pharmaceutical Sciences,18th edition,A.R.Gennaro,Ed.,MackPubl.,Easton,PA(1990).

[0218] S.French and B.Robson,J.Mol.Evol.19(1983)171

[0219] Taylor et al,J.Theor.Biol.119(1986);205-218

[0220] U.S.Patent No.4,554,101

[0221] U.S.Patent No.5,277,175

[0222] U.S.Patent No.5,284,133

[0223] U.S.Patent No.5,355,872

[0224] U.S.Patent No.5,660,166

[0225] U.S.Patent No.5,797,389

[0226] U.S.Patent No.5,823,179

[0227] U.S.Patent No.5,889,155

[0228] U.S.Patent No.6,016,974

[0229] U.S.Patent No.6,041,776

[0230] U.S.Patent No.6,044,841

[0231] U.S.Patent No.6,241,159

[0232] U.S.Patent No.6,261,569

[0233] U.S.Patent No.6,261,569

[0234] U.S.Patent No.6,354,516

[0235] U.S.Patent No.6,357,671

[0236] U.S.Patent No.6,921,020

[0237] U.S.Patent No.6,926,208

[0238] U.S.Patent No.6,968,840

[0239] U.S.Patent No.6,978,941

[0240] U.S.Patent No.7,040,549

[0241] U.S.Patent No.7,083,112

[0242] U.S.Patent No.7,104,463

[0243] U.S.Patent No.7,360,536

[0244] U.S.Patent Publication No.2002 / 0020409.

[0245] U.S.Patent Publication No.2002 / 0020412

[0246] U.S.Patent Publication No.2009 / 0134235

[0247] U.S.Patent Publication No.2009 / 0304666

[0248] Vecellio None et al.(2001)JAerosol Med 14:107-1

[0249] Verdini,A and Viscomi,G.C,J.Chem.Soc.Perkin Trans.1:697-701,1985.

[0250] Waldrep and Dhand,Curr.Drug Deliv.,5(2):114-9,2008.

[0251] Wawrzynczak and Thorpe,Cancer Treat Res.,37:239-51,1988.

[0252] Wiley,RA et al.,Med.Res.Rev.13:327-384,1993.

Claims

1. A peptide consisting of the amino acid sequence KASFTTFTVTKGS, aaEGKASFTTFTVTKGSaa-NH2 or Ac-aaEGKASFTTFTVTKGSaa-NH2.

2. The peptide of claim 1, wherein the peptide consists of the amino acid sequence KASFTTFTVTKGS or aaEGKASFTTFTVTKGSaa-NH2.

3. A peptide consisting of the amino acid sequence Ac-aaEGKASFTTFTVTKGSaa-NH2.

4. A composition comprising the peptide according to any one of claims 1 to 3.

5. The composition of claim 4, wherein the peptide is at least 95% pure.

6. A composition as claimed in claim 4 or 5, wherein the peptide is at least 98% pure.

7. A composition as claimed in claim 4 or 5, wherein the peptide is pure.

8. A pharmaceutical composition comprising the peptide according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is formulated as a nebulized solution.

10. A polynucleotide comprising a nucleic acid sequence encoding the peptide according to any one of claims 1 to 3.

11. Use of the peptide according to claim 3 in the preparation of a medicament for treating or preventing a disease in a subject, wherein an effective amount of the peptide is administered to the subject, wherein the subject suffers from nonspecific interstitial pneumonia or idiopathic pulmonary fibrosis.

12. The method of claim 11, wherein the subject suffers from nonspecific interstitial pneumonia.

13. The use of claim 11, wherein the subject suffers from idiopathic pulmonary fibrosis.

14. The use according to any one of claims 11 to 13, wherein the peptide is administered by nebulization.

15. The use according to any one of claims 11 to 13, wherein the peptide is administered systemically.

16. The use according to any one of claims 11 to 13, wherein the peptide is administered topically to the diseased tissue.

17. The use according to any one of claims 11 to 13, wherein at least one additional anti-fibrotic therapeutic agent is administered.

18. Use according to claim 17, wherein the at least one additional anti-fibrotic therapeutic agent is a NSAID.

19. The use of any one of claims 11-13, wherein a steroid, a DMARD, an immunosuppressant, a biological response modifier or a bronchodilator is also administered.

20. The use of any one of claims 11-13, wherein the subject is a human.

21. A dry powder composition comprising the peptide of any one of claims 1-3.

22. The dry powder composition of claim 21, further comprising a pharmaceutically acceptable carrier.

23. The dry powder composition of claim 21, wherein the peptide is administered by inhalation.

24. The use of a dry powder composition as claimed in claim 21 in the preparation of a medicament for treating or preventing a disease in a subject, wherein an effective amount of the peptide is administered to the subject, and wherein the subject suffers from nonspecific interstitial pneumonia or idiopathic pulmonary fibrosis, wherein the peptide consists of the amino acid sequence Ac-aaEGKASFTTFTVTKGSaa-NH2.

25. The use of claim 24, wherein the subject suffers from nonspecific interstitial pneumonia.

26. The use of claim 24, wherein the subject suffers from idiopathic pulmonary fibrosis.

27. The peptide of any one of claims 1-3, wherein the peptide is soluble in an aqueous solution.

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