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

Abstract

The present disclosure relates to modified peptide fragments of the CAV-1 protein and their use in the treatment of fibrosis. Provided herein are compositions comprising a modified caveolar protein 1 (Cav-1) peptide. Also provided are methods of treating pulmonary infection or acute or chronic lung injury, in particular pulmonary fibrosis, using the modified Cav-1 peptides.

Description

[0001] This application is a divisional application of the invention patent filed on September 10, 2019, with application number 201980059209.6 and invention title "Modified peptide fragments of CAV-1 protein and their use in the treatment of fibrosis".

[0002] 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.

[0003] This invention is the result of activities conducted within the scope of a joint research agreement that was in effect at the time of its preparation. The parties to this joint research agreement are the Board of Regents of the University of Texas System and Lung Therapeutics. Technical Field

[0004] This invention generally relates to the fields of molecular biology and medicine. More specifically, this invention relates to compositions and methods for delivering therapeutic peptide compositions to subjects (such as by delivery to the respiratory system). Background Technology

[0005] During lung injury, increased p53 expression induces plasminogen activator inhibitor 1 (PAI-1) and inhibits 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 modulating 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 peptides that can be used to prevent or treat lung injury, and particularly for formulations and methods for the therapeutic delivery of such peptides. Summary of the Invention

[0006] According to this 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 may be a standard amino acid, a non-standard amino acid, or a chemical modification. Peptide polymers of the peptides of this disclosure are provided. A pharmaceutical composition of the peptides is also provided. The peptides of this disclosure may be used to treat lung injury, infection, or disease. In other aspects, the peptides of the embodiments may be used to treat fibrotic conditions (e.g., organ fibrosis) or inflammation.

[0007] In some embodiments, this 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 lacking 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 both the N-terminus and C-terminus. In some aspects, the peptide retains the biological activity of caverin 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 an L-amino acid. In some aspects, the peptide comprises a D-amino acid. In some aspects, the peptide comprises both L-amino acids and D-amino acids.

[0009] In some aspects, the peptide contains at least one non-standard amino acid. In some aspects, the peptide contains two or more non-standard amino acids. In some aspects, the peptide contains four 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 a 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 both N-terminal and C-terminal modifications. In some aspects, the N-terminal modification is acylation. In some aspects, the C-terminal modification is 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, this 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, this disclosure provides a composition comprising the peptides disclosed herein. In some aspects, the peptides are substantially pure. In some aspects, the peptides are 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, this 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, intra-articular, parenteral, enteral, topical, subcutaneous, intramuscular, buccal, sublingual, rectal, vaginal, penile, intraocular, epidural, intracranial, or inhalation administration. In some aspects, the pharmaceutical composition is formulated for pulmonary infusion. In some aspects, the pharmaceutical composition is formulated as a nebulized solution.

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

[0017] In some aspects, the peptide composition of the embodiments can be used in methods of treating or preventing a disease in a subject. In some aspects, the disease is a fibrotic disease or an inflammatory disease. For example, the fibrotic disease can be an organ fibrosis disease, such as kidney, liver, lung, or heart fibrosis. In some aspects, the inflammatory disease is an inflammatory eye disease. The composition of the embodiments can be applied systemically or locally (e.g., at the site of diseased tissue).

[0018] In some embodiments, this 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 the peptide as described herein to the subject. In some aspects, the subject suffers from lung inflammation. In some aspects, the subject is undergoing chemotherapy or radiation therapy. 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, obliterative bronchiolitis, asthma, acute respiratory distress syndrome (ARDS), or acute lung injury induced by inhaled smoke (ISALI). In some aspects, the lung disease is a fibrotic condition of the lungs. In some aspects, the lung disease is interstitial lung disease. In some aspects, the lung disease is idiopathic pulmonary fibrosis (IPF) or pulmonary scarring. In some aspects, the administration comprises nebulizing a solution containing the peptide. In some aspects, the method further comprises administering at least one additional antifibrotic therapeutic agent. In some aspects, the at least one additional antifibrotic agent is an NSAID, steroid, DMARD, immunosuppressant, biological response modifier, or bronchodilator. In some aspects, the subject is a human.

[0019] It is anticipated that any method or composition described herein can be implemented in relation to any other method or composition described herein. Other objects, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that while the detailed description and specific examples indicate particular embodiments of this disclosure, they are given by way of illustration only, as various changes and modifications within the spirit and scope of this disclosure will become apparent to those skilled in the art upon reading this detailed description. Attached Figure Description

[0020] The following figures form part of this specification and are included to further illustrate certain aspects of the invention. The invention can be better understood by referring to one or more of these figures in conjunction with the detailed description of the specific embodiments presented herein.

[0021] Figure 1 Western blot analysis of SMA and tubulin expression in idiopathic pulmonary fibrosis cells treated with Cav-1 peptide. IPF cells were treated with the following substances: 1: untreated, 2: 10 μM LTI-03, 3: 90 μM LTI-03, 4: 10 μM APi2350, 5: 10 μM MAPi2354, 6: 10 μM APi2355, 7: 10 μM APi2356, and 8: DMSO. SMA and tubulin expression were assessed by Western blot analysis.

[0022] Figure 2Treatment with Cav-1 peptide increased SMA relative to tubulin in IPF cells. A graphical representation of the SMA / tubulin ratio in cells receiving the specified treatment. Detailed Implementation

[0023] This disclosure overcomes challenges associated with the prior art by providing a modified caveolin 1 (Cav-1) peptide and its use in the treatment and prevention of diseases, particularly pulmonary fibrosis. In some aspects, pharmaceutical formulations of the modified Cav-1 peptide are provided. For example, in some aspects, the peptide is formulated for delivery to the respiratory system. For example, the peptide can be prepared for administration to a subject's airway by reconstituted in an aqueous solution and nebulized using a nebulizer. In other aspects, the peptide can be formulated for injection. This document also provides a method for treating lung injury and disease by administering a therapeutically effective amount of the modified Cav-1 peptide to the subject, e.g., via the airway.

[0024] I. Definition

[0025] As used herein, "substantially free of" with respect to a specified component means that the specified component was not intentionally formulated into the composition and / or is present only as a contaminant or in trace amounts. Therefore, the total amount of the specified component due to any accidental contamination of the composition is well below 0.01%. Compositions in which the amount of the specified component cannot be detected using standard analytical methods are preferred.

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

[0027] While this disclosure supports only the definition of alternatives and "and / or", the term "or" is used in the claims to mean "and / or" unless it is explicitly stated that only alternatives are referred to or that 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 inherent error variations in the means or methods used to determine the value, or variations that exist between the subjects under study.

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

[0030] "Bioactive" caveolin 1 (Cav-1) peptides are peptides that increase p53 protein levels, decrease urokinase plasminogen activator (uPA) and uPA receptor (uPAR), and / or increase plasminogen activator inhibitor 1 (PAI-1) expression in cells such as fibrotic lung fibroblasts. In some aspects, the bioactive peptides possess at least 20% of the biological or biochemical activity (e.g., as measured by in vitro or in vivo assays) of the natural Cav-1 polypeptide of SEQ ID NO:1. In some aspects, the bioactive peptides have increased biological or biochemical activity compared to the natural Cav-1 polypeptide.

[0031] The terms “identity” or “homology” should be interpreted as meaning the percentage of amino acid residues in a candidate sequence that are identical to those in the corresponding sequence being compared, after sequence alignment and the introduction of vacancies (if necessary) to achieve the maximum percentage of identity across the entire sequence, without considering any conservation substitutions as part of sequence identity. N-terminal or C-terminal extensions or insertions should not be interpreted as a reduction in identity or homology. The methods and computer programs used for alignment are well known in the art. Sequence identity can be measured using sequence analysis software.

[0032] The terms "peptide" or "protein" in their broadest sense refer to compounds consisting of two or more subunits of amino acids, amino acid analogs, or peptide mimics. These subunits may be linked by peptide bonds. In another embodiment, the subunits may be linked 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), as well as amino acid analogs and peptide mimics. The term "peptide mimic" or "peptide analog" means a peptide according to the invention modified in such a way that it contains at least one non-peptide bond, such as a urea bond, carbamate bond, sulfonamide bond, hydrazine bond, or any other covalent bond. Peptides with three or more amino acids are generally called oligopeptides if the peptide chain is short. Peptides are generally called polypeptides or proteins if the peptide chain is long.

[0033] The terms “subject” and “individual” and “patient” are used interchangeably herein and refer to an animal, such as a human or a non-human animal (e.g., a mammal), to which the pharmaceutical composition disclosed herein is provided for treatment (including prophylactic treatment). As used herein, the term “subject” refers to both humans and non-human animals. The term “non-human animal” includes all vertebrates, such as mammals like 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 used 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 respects, non-human animals are companion animals such as dogs or cats.

[0034] "Treating" a subject's disease or condition, or "treating" a patient suffering from a disease or condition, means administering a drug to the individual, such as by administering a drug, to reduce or stabilize at least one symptom of the disease or condition. Typically, when the peptide is administered therapeutically as a treatment, it is administered to a subject exhibiting one or more symptoms of lung injury or pulmonary fibrosis.

[0035] "Separated" means that the polypeptide has been separated from any natural environment such as bodily fluids (e.g., blood) and from the components that naturally accompany the peptide.

[0036] "Isolated" and "substantially pure" means a polypeptide that has been isolated from and purified to at least some degree from its naturally occurring accompanying components. Typically, a polypeptide is substantially pure when it contains 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, the proteins and naturally occurring organic molecules associated with it. For example, substantially pure polypeptides can be obtained by extraction from natural sources, by expression of recombinant nucleic acids in cells that do 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 naturally occurring molecule by one or more amino acid deletions, additions, substitutions, or side-chain modifications, but still retains one or more specific functions or biological activities of the molecule. Amino acid substitution includes alterations where an amino acid is replaced by a different naturally occurring or unconventional amino acid residue. Such substitutions can be classified as "conservative," in which case an amino acid residue contained in the polypeptide is replaced by another naturally occurring amino acid with similar characteristics in polarity, side-chain functionality, or size. Such conserved substitutions are well known in the art. Substitutions covered by this invention can also be "non-conservative," wherein an amino acid residue present in the peptide is replaced by an amino acid with different properties (such as naturally occurring amino acids from different groups) (e.g., using alanine to replace a charged or hydrophobic amino acid), or alternatively, wherein a naturally occurring amino acid is replaced by an unconventional amino acid. In some embodiments, amino acid substitutions are conserved. The term "variant" also encompasses, when used with reference polynucleotides or polypeptides, polynucleotides or polypeptides that can have their primary, secondary, or tertiary structures altered, respectively, compared to a reference polynucleotide or polypeptide (e.g., compared to a wild-type polynucleotide or polypeptide).

[0038] The terms "insertion" or "deletion" typically range from about 1 to 5 amino acids. Permissible variations can be experimentally determined by generating peptides synthetically while systematically inserting, deleting, or substituting nucleotides into the sequence using recombinant DNA technology.

[0039] When referring to peptides, the term "substitution" refers to a change in the amino acid composition of a different entity (e.g., another amino acid or a moiety of amino acids). Substitution can be either conservative or non-conservative.

[0040] A "molecular analog" (such as a peptide) is a molecule that functions similarly to the whole molecule or a fragment thereof. The term "analyte" is also intended to include allelic species and induced variants. Analytes often differ from naturally occurring peptides at one or more positions due to conserved substitutions. Analytes typically exhibit at least 80% or 90% sequence identity with the natural peptide. Some analogs also contain 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, and σ-N-methylarginine. Fragments and analogs may be screened in transgenic animal models for prophylactic or therapeutic efficacy as described below.

[0041] "Covalent bonding" means a connection directly or indirectly (e.g., through a linker) via a covalent chemical bond. In some aspects of all embodiments of the present 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 generated, for example, by linking a nucleic acid sequence encoding one protein to a nucleic acid sequence encoding another protein, such that they form a single open reading frame (ORF), which can be translated into a single polypeptide carrying all the intended proteins in the cell. The protein sequence can be altered. Fusion proteins may contain epitope tags or half-life extenders. Epitope tags include biotin, FLAG tags, c-myc, hemagglutinin, His6, digitoxin, 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" in this text refers to any part of the respiratory tract, including the upper respiratory tract, the respiratory airways, and the lungs. The upper respiratory tract includes the nose and nasal passages, the mouth, and the pharynx. 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 “inhaled smoke-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 presence of one or more of the following conditions 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 heart catheterization as clinically indicated; and 3) PaO2 / FiO2 <300 mmHg (40 kPa). In some implementations, 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 fragments, and alveolar fibrin deposition.

[0045] The terms "nebulizing" and other grammatical variations herein refer 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. Cavernin 1-peptide

[0047] Embodiments of this disclosure provide peptide variants of the caveolin 1 (Cav-1) protein. The caveolin 1 (Cav-1) scaffold domain or peptide interferes with the interaction of Cav-1 with Src kinase, mimicking the combined action of uPA and an anti-β1 integrin antibody. Natural 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 with SEQ ID NO:2FTTFTVT. The peptide may comprise substitutions, deletions, or insertions of 1, 2, 3, 4, or more amino acids relative to sequence SEQ ID NO:1 to obtain polypeptides with 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 residues. In certain aspects, the peptide is a truncated form of a natural 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 disclosed herein are bioactive derivatives that exhibit the activity of a natural CAV-1 polypeptide in in vitro or in vivo assays of binding or bioactivity. In a particular aspect, the peptides inhibit or prevent BLM-induced apoptosis of LECs in vitro or in vivo, with an activity of at least about 20% of the activity of the natural CAV-1 polypeptide, or at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, about 95%, 97%, 99%, and any range derived therefrom, such as 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 natural CAV-1 polypeptide. Assays for testing bioactivity (e.g., antifibrotic activity, the ability to affect the expression of uPA, uPAR, and PAI-1 mRNA, or the ability to inhibit lung fibroblast proliferation) are well known in the art.

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

[0056] Conservative amino acid substitution involves replacing one amino acid with another that has similar structure and / or chemical properties, such as replacing leucine with isoleucine or valine, replacing aspartic acid with glutamic acid, or replacing threonine with serine. Therefore, conserved substitution of a specific amino acid sequence refers to the substitution of amino acids that are not critical to peptide activity, or the substitution of amino acids with other amino acids that have similar properties (e.g., acidity, basicity, positive or negative charge, polarity or nonpolarity), such that even the substitution of critical amino acids does not reduce peptide activity. Conservative representations of functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservedly substituted 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 herein by reference in its entirety.) In some embodiments, alterations, additions, or deletions of individual amino acids or small percentages of amino acids may also be considered conserved substitutions if the changes do not reduce the activity of the peptide. The range of insertions or deletions is typically from about 1 to 5 amino acids. The selection of conserved amino acids may be based on the position of the amino acid to be substituted in the peptide, for example, when the amino acid is external to the peptide and exposed to a solvent or internal to the peptide and not exposed to a solvent.

[0057] In alternative embodiments, the amino acid to replace the existing amino acid can be selected based on its position in the solvent (i.e., its exposure to the solvent (i.e., if the amino acid is exposed to the solvent or present on the outer surface of the peptide or polypeptide compared to an internally located amino acid that is not exposed to the solvent)). Such selection of conserved amino acid substitutions is well known in the art, for example, as disclosed in Dordo et al., J. Mol Biol, 1999, 217, 721-739; Taylor et al., J. Theor. Biol. 119 (1986); 205-218; and S. French and B. Robson, J. Mol. Evol. 19 (1983) 171. Therefore, conserved amino acid substitutions suitable for the amino acids outside the protein or peptide (i.e., amino acids exposed to the solvent) can be selected, for example, but not limited to, the following substitutions: replacing Y with F, replacing T with S or K, replacing P with A, replacing E with D or Q, replacing N with D or G, replacing R with K, replacing G with N or A, replacing T with S or K, replacing D with N or E, replacing I with L or V, replacing F with Y, replacing S with T or A, replacing R with K, replacing G with N or A, replacing K with R, and replacing A with S, K, or P.

[0058] In alternative embodiments, conservative amino acid substitutions applicable to amino acids within the protein or peptide may also be selected. For example, suitable conservative substitutions of amino acids within the protein or peptide (i.e., amino acids not exposed to the solvent) may be used, such as, but not limited to, the following conservative substitutions: wherein Y is replaced by F, T by A or S, I by L or V, W by Y, M by L, N by D, G by A, T by A or S, D by N, I by L or V, F by Y or L, S by A or T, and A by S, G, T, or V. In some embodiments, non-conservative amino acid substitutions are also covered within the terminology variations.

[0059] In some respects, the polypeptide is a derivative of the natural Cav-1 polypeptide. As used herein, the term "derivative" refers to a peptide that has been chemically modified, such modification being, for example but not limited to, by techniques such as acetylation, ubiquitination, labeling, PEGylation (using polyethylene glycol derivatization), esterification, glycosylation, amidation, or the addition of other molecules. A molecule is also a "derivative" of another molecule when it contains additional chemical parts that are not normally part of the molecule. Such parts may alter pH or improve the molecule's solubility, absorption, biological half-life, etc. Such parts may alternatively reduce the toxicity of the molecule, eliminate or mitigate any undesirable side effects of the molecule, etc. Parts capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences, 18th edition, ed. ARGennaro, 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 present invention whose biological activity (functional or structural) is substantially similar to that of the entity or molecule as a functional derivative or functional variant thereof. The term "functional derivative" is intended to include fragments of the molecule, analogs, or chemical derivatives.

[0061] In some respects, amino acid substitutions can occur at one or more sites on a polypeptide, wherein the substitutions are made against amino acids with similar hydrophilicity. The importance of the hydrophilic amino acid index in conferring biological functions of protein-protein interactions is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydrophilicity of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the protein's interactions with other molecules such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Therefore, such conserved substitutions can occur in polypeptides and may have only a minor effect on their activity. As detailed in U.S. Patent 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 guidelines, and therefore substitutions of amino acids with hydrophilicity values ​​within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. Thus, any polypeptide described herein can be modified by substituting amino acids with different but homologous amino acids having similar hydrophilicity values. Amino acids with hydrophilicity values ​​within + / -1.0 or + / -0.5 are considered homologous.

[0062] Modified Cav-1 peptides may include co-translational and post-translational (C-terminal peptide cleavage) modifications, such as disulfide bond formation, glycosylation, acetylation, phosphorylation, and proteolytic cleavage (e.g., via furin or metalloproteinases). To a certain extent, such modifications do not affect the anti-inflammatory properties of the isolated peptide or its ability to improve glycemic control.

[0063] In some respects, modified Cav-1 peptides contain non-naturally occurring amino acids. Peptides may contain a combination of naturally occurring and non-naturally occurring amino acids, or may contain only non-naturally occurring amino acids. Non-naturally occurring amino acids may include synthetic non-natural amino acids, substituted amino acids, or, in some cases, one or more D-amino acids desired in the peptide (or other components of the composition besides the protease recognition sequence). Peptides containing D-amino acids exhibit increased stability in vitro or in vivo compared to forms containing L-amino acids. 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 drugs and conjugates, improved bioavailability of membrane-permanent complexes (see further discussion below), and prolonged intravascular and interstitial lifespan when such properties are required. The use of D-isomer peptides can also enhance transdermal and oral transepithelial delivery of drugs and other carrier molecules. Furthermore, D-peptides cannot be efficiently processed for restricted presentation to T helper cells in major histocompatibility complex class II, and are therefore unlikely to induce humoral immune responses throughout the organism. Therefore, peptide conjugates can be constructed using, for example, D-isomers of cell-penetrating peptide sequences, L-isomers of cleavage sites, and D-isomers of therapeutic peptides.

[0064] In addition to the 20 “standard” L-amino acids, D-amino acids that are well-defined in the art, or non-standard, modified, or unusual amino acids, are also contemplated for use in this disclosure. Phosphorylated amino acids (Ser, Thr, Tyr), glycosylated amino acids (Ser, Thr, Asn), β-amino acids, GABA, and ω-amino acids are further contemplated for use in this disclosure. These include, for example, β-alanine (β-Ala) and other ω-amino acids such as 3-aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid, etc.; α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly); ornithine (Orn); citrulline (Cit); tert-butylalanine (t-BuA); tert-butylglycine (t-BuG); N-methylisoleucine (MeIle); phenylglycine (Phg); ortholeucine (Nle); 4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F)); 3-Fluorophenylalanine (Phe(3-F)); 4-Fluorophenylalanine (Phe(4-F)); Penicillamine (Pen); 1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (Tic); Homoarginine (hArg); N-acetyllysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,4-diaminobutyric acid (Dab); p-aminophenylalanine (Phe(pNH2)); N-methylvaline (MeVal); homocysteine ​​(hCys), homophenylalanine (hPhe), and homoserine (hSer); hydroxyproline (Hyp), homoproline (hPro), N-methylated amino acids, and peptides (N-substituted glycine).

[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 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 can be capped at its N-terminus with an acyl group (abbreviated "Ac") and at its C-terminus with an amide group (abbreviated "Am"), for example, with an acetyl group (CH3CO-) at the N-terminus and an amide group (-NH2) at the C-terminus. A wide range of N-terminal capping functional groups are desired, preferably linked to a terminal amino group, such as a formyl group.

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

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

[0069] Acyl groups having 1 to 10 carbon atoms, such as hex-5-acyl groups;

[0070] Aromatic acyl groups, such as benzoyl or 1-naphthoyl;

[0071] heteroaryl groups, such as 3-pyrrole or 4-quinolinyl;

[0072] Alkyl sulfonyl groups, such as methanesulfonyl groups;

[0073] arylsulfonyl groups, such as benzenesulfonyl or sulfanyl;

[0074] Heteroarylsulfonyl groups, such as pyridine-4-sulfonyl groups;

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

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

[0077] Substituted acetylacetyl groups having 1 to 10 carbon atoms, such as 3-hydroxy-hexane-5-acetylacetyl;

[0078] Substituted aromatic acyl groups, such as 4-chlorobenzoyl or 8-hydroxy-naphthalene-2-acyl;

[0079] Substituted heteroaryl groups, such as 2,4-dioxo-1,2,3,4-tetrahydro-3-methyl-quinazoline-6-acyl;

[0080] Substituted alkyl sulfonyl groups, such as 2-aminoethanesulfonyl groups;

[0081] Substituted aryl sulfonyl groups, such as 5-dimethylamino-1-naphthalenesulfonyl;

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

[0083] Carbamoyl or thiocarbamoyl;

[0084] A substituted carbamoyl group (R'-NH-CO) or a substituted thiocarbamoyl group (R'-NH-CS), wherein R' is an 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 an alkanoyl, enoyl, alkynyl, aromatic acyl, heteroaromatic acyl, substituted alkanoyl, substituted enoyl, substituted alkynyl, substituted aromatic acyl, or substituted heteroaromatic acyl, as defined above.

[0086] C-terminal capping functional groups can be located in amide or ester bonds with the terminal carboxyl group. Capping functional groups providing amide bonds are named NR. 1 R 2 , where R 1 and R 2 It can be obtained independently from the following group: hydrogen;

[0087] Preferably, it is an alkyl group having 1 to 10 carbon atoms, such as methyl, ethyl, or isopropyl.

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

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

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

[0091] Alkenyl groups having 1 to 10 carbon atoms, such as hydroxyalkenyl, alkoxyalkenyl, mercaptoalkenyl, alkylthioalkenyl, haloalkenyl, cyanoalkenyl, aminoalkenyl, alkylaminoalkenyl, dialkylaminoalkenyl, alkylacylalkenyl, carboxylalkenyl, carbamoylalkenyl.

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

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

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

[0095] heteroaryl groups, such as 4-quinolinyl;

[0096] An alkanoyl group having 1 to 10 carbon atoms, such as an acetyl or butyryl group;

[0097] Aromatic acyl groups, such as benzoyl groups;

[0098] heteroaryl groups, such as 3-quinolinyl;

[0099] OR' or NR'R", where R' and R" are independently hydrogen, alkyl, aryl, heteroaryl, acyl, aromatic acyl, sulfonyl, sulfinyl, or SO2-R"' or SO-R"', where R"' is a substituted or unsubstituted alkyl, aryl, heteroaryl, alkenyl, or alkynyl group.

[0100] The end-capping functional group that provides the ester bond is named OR, where R can be: alkoxy; aryloxy; heteroaryloxy; arylalkoxy; heteroarylalkoxy; substituted alkoxy; substituted aryloxy; substituted heteroaryloxy; substituted arylalkoxy; or substituted heteroarylalkoxy.

[0101] The N-terminal or C-terminal capping functional groups, or both, can have such a structure, allowing the capped molecule to act as a prodrug (a pharmacologically inactive derivative of the parent drug molecule), undergo spontaneous or enzymatic conversion in vivo to release the active drug, and possess improved delivery properties relative to the parent drug molecule (Bundgaard H ed.: Design of Prodrugs, Elsevier, Amsterdam, 1985).

[0102] Choosing the right capping group allows for the addition of additional activity to the peptide. For example, the presence of a thiol group attached to the N-terminus or C-terminus cap will allow the derived peptide to be conjugated with other molecules.

[0103] On the other hand, the peptide or its fragments or derivatives can be "retro-inversopeptides." A "retro-inversopeptide" is a peptide in which the direction of the peptide bonds at at least one position is reversed, i.e., the amino and carboxyl terms are reversed relative to the amino acid side chains. Therefore, retro-inversopeptides have reversed ends and reversed peptide bonds while maintaining a topological structure of side chains that is substantially the same as in the native peptide sequence. Retro-inversopeptides may contain L-amino acids or D-amino acids, or a mixture of L-amino and D-amino acids, with at most all amino acids being D-isomers. Partially retro-inversopeptide analogs are polypeptides in which only a portion of the sequence is reversed and replaced by enantiomeric amino acid residues. Because the retro-inversopeptide portion of such analogs has reversed amino and carboxyl terms, the amino acid residues side-attached to the retro-inversopeptide portion are replaced by α-substituted gem-diaminomethane and malonic acid esters with similar side chains. Retro-inversopeptide forms of cell-penetrating peptides have been found to be as efficient as the native form in transmembrane translocation. The synthesis of reverse-inverted 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. Patent No. 6,261,569, all of which are incorporated herein by reference in their entirety. A partial solid-phase synthesis method for reverse-inverted peptide analogs (EP 97994-B) has also been described and is incorporated herein by reference in its entirety.

[0104] A percentage (e.g., 80%, 85%, 90%, or 95%) of “sequence identity” or “homology” between a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) and another sequence refers to the percentage of bases (or amino acids) that are identical when comparing the two sequences during alignment. This alignment and percentage of homology or sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology (FMAusubel et al., ed., 1987), Supplement 30, Section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, which uses default parameters. Specifically, the preferred procedures are BLASTN and BLASTP, which use the following default parameters: genetic code = standard; filter = none; strands = two; cutoff value = 60; expected value = 10; matrix = BLOSUM62; description = 50 sequences; sorting method = high score; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+SwissProtein+SPupdate+PIR.

[0105] A. Polymeric polypeptides

[0106] Embodiments of this 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 can 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 linkers or spacer regions).

[0107] B. Peptide mimicry

[0108] Modified Cav-1 peptides can be peptide-mimicking compounds that mimic the biological effects of natural Cav-1 polypeptides. Peptidomics can be non-natural peptides or non-peptide agents that recreate the stereochemical properties of the binding elements of natural Cav-1 polypeptides, thereby acquiring the binding and biological activities of natural Cav-1 polypeptides. Similar to natural Cav-1 polypeptides or polypeptide polymers, peptide mimics will have both a binding surface (which interacts with any ligand that binds to natural Cav-1) and a non-binding surface.

[0109] In some aspects, this disclosure also includes compounds that retain some peptide characteristics. For example, any protein hydrolysis-unstable bonds within the peptides of the present 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] Peptide-mimicking compounds (whether agonists, substrates, or inhibitors) of various bioactive peptides / peptides such as opioid peptides, VIP, thrombin, and HIV protease have been described. Methods for designing and preparing peptide-mimicking 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). Some mimics of 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 peptide-mimicking compounds that at least have binding affinity and specificity to natural Cav-1 peptides and preferably also have biological activity. In view of this 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 peptide mimics can be identified by examining the three-dimensional structure of the free peptides of the present invention or those complexed with ligands (e.g., soluble uPAR or fragments thereof). Alternatively, the structure of the peptides of the present invention bound to their ligands can be obtained by nuclear magnetic resonance spectroscopy. Further knowledge of the stereochemistry of the interaction between the peptides and their ligands or receptors will allow for the rational design of such peptide mimics. In the absence of ligands, the structures of the peptides or polypeptides of the present invention can also provide a scaffold for designing mimic molecules.

[0112] C. PEGylation

[0113] Modified Cav-1 peptides can be conjugated to heterologous polypeptide segments or polymers such as polyethylene glycol. The polypeptide can be linked to PEG to increase the enzyme's hydrodynamic radius and thus serum persistence. The polypeptide can be conjugated to any targeting agent, such as a ligand with the ability to specifically and stably bind to an external receptor (US Patent Publication 2009 / 0304666).

[0114] In some aspects, the methods and compositions of the embodiments involve the PEGylation of disclosed peptides. PEGylation is a process of covalently attaching a poly(ethylene glycol) polymer chain to another molecule (typically 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 effects of the host's immune system (reducing immunogenicity and antigenicity) or increase the hydrodynamic size (size in solution) of the agent, thereby prolonging 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 ends. PEG with the same reactive moiety activated at each end is called "homobifunctional," while PEG derivatives with different functional groups are called "heterobifunctional" or "heterofunctional." Chemically active or activated derivatives of the PEG polymer are prepared to attach PEG to the desired molecule.

[0116] The selection of suitable functional groups for PEG derivatives is based on the type of reactive groups available on the molecule to which the PEG will be 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 can also be used.

[0117] The techniques used to form first-generation PEG derivatives typically involve reacting the PEG polymer with groups that can react with hydroxyl groups (usually acid anhydrides, acyl chlorides, chloroformates, and carbonates). In second-generation PEGylation chemistries, more effective functional groups such as aldehydes, esters, and amides can be used for conjugation.

[0118] As PEGylation applications become increasingly sophisticated and complex, the demand for heterobifunctional PEGs for conjugation is also growing. These heterobifunctional PEGs are highly useful for joining two entities where hydrophilic, flexible, and biocompatible spacers are required. Preferred end groups for 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-based PEG (mPEG) molecules. Their activity depends on the addition of protein-modifying groups to the alcohol terminus. In some cases, polyethylene glycol (PEG diol) is used as a precursor molecule. The diol is then modified at both ends to prepare heterodimer or homodimer PEG-linked molecules.

[0120] Proteins are typically PEGylated at nucleophilic sites such as unprotonated thiols (cysteyl residues) or amino groups. Examples of cysteyl-specific modifiers include PEG-maleimide, PEG-iodoacetate, PEG-thiol, and PEG-vinyl sulfone. All four are strongly cysteyl-specific under mild conditions and at neutral to weakly alkaline pH, but each has some drawbacks. The thioether formed with maleimide may be somewhat unstable under alkaline conditions, thus limiting formulation options using this linker. The thiocarbamate bond formed with iodide-PEG is more stable, but free iodine can modify tyrosine residues under certain conditions. PEG-thiol forms disulfide bonds with protein thiols, but this bond can also be unstable under alkaline conditions. Compared to maleimide and iodide-PEG, PEG-vinyl sulfone is relatively slower to react; however, the resulting thioether bond is quite stable. Its slower reaction rate also makes the PEG-vinyl sulfone reaction easier to control.

[0121] Site-specific PEGylation at native cysteine ​​residues is rarely performed because these residues are typically disulfide bonds or essential for biological activity. On the other hand, site-directed mutagenesis can be used to incorporate cysteine ​​PEGylation sites with thiol-specific linkers. Cysteine ​​mutations must be engineered to make them accessible to the PEGylating agent and retain biological activity after PEGylation.

[0122] Amine-specific modifiers include PEG NHS esters, PEG tresylates, PEG aldehydes, PEG isothiocyanates, and several other modifiers. All reactions are carried out under mild conditions and are highly specific to amino groups. PEG NHS esters are among the most reactive; however, their high reactivity makes PEGylation difficult to control on a large scale. PEG aldehydes form imines with amino groups, which are then reduced to secondary amines using sodium cyanoborohydride. Unlike sodium borohydride, sodium cyanoborohydride does not reduce disulfide bonds. However, this chemical is highly toxic and must be handled with care, especially at lower pH levels where it can become volatile.

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

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

[0125] For both amine-specific and thiol-specific modifications, reaction conditions can affect protein stability. This may limit temperature, protein concentration, and pH. Furthermore, the reactivity of the PEG linker should be known before initiating the PEGylation reaction. For example, if the PEGylating agent has only 70% activity, 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] Some embodiments of the present invention relate to fusion proteins of modified Cav-1 peptides. These molecules may be polypeptides having a heterologous domain linked at the N-terminus or C-terminus. For example, fusions may also use a leader sequence from another species to allow recombinant expression of the protein in a heterologous host. The fusion protein may contain a half-life extender. Another useful fusion involves adding protein affinity tags, such as serum albumin affinity tags or six histidine residues, or adding immunologically active domains, such as preferably cleavable antibody epitopes, to facilitate purification of the fusion protein. Non-restrictive affinity tags include multihistidine, chitin-binding protein (CBP), maltose-binding protein (MBP), and glutathione S-transferase (GST).

[0128] In a specific implementation scheme, the peptide of the scheme may be linked to a peptide that increases the in vivo half-life, such as... Peptides (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 de novo synthesis of a complete fusion protein, or by attaching a DNA sequence encoding a heterologous domain and then expressing the complete fusion protein.

[0130] The generation of fusion proteins that restore the functional activity of the parent protein can be facilitated by linking a gene to a bridging DNA segment of a peptide linker that splices between tandemly linked polypeptides. The linker will have sufficient length to allow the resulting fusion protein to fold properly.

[0131] 1. Connector

[0132] In some embodiments, the peptides of the embodiments may be chemically conjugated using bifunctional cross-linking agents or fused at the protein level using peptide linkers.

[0133] Bifunctional crosslinking agents have been widely used for various purposes, including the 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 link peptides in implementation schemes, such as the Gly-Ser linker.

[0134] Bifunctional reagents, carrying two identical functional groups, have proven highly effective in inducing cross-linking between identical and different macromolecules or macromolecular subunits, as well as the linking of peptide ligands to their specific binding sites. Heterofunctional reagents contain two distinct functional groups. By utilizing the differential reactivity of the two functional groups, cross-linking can be selectively and sequentially controlled. Bifunctional cross-linking reagents can be classified according to the specificity of their functional groups (e.g., amino, mercapto, guanidino, indole, carboxyl specific groups). Among these cross-linking reagents, those targeting free amino groups are particularly popular due to their commercial availability, ease of synthesis, and the mild reaction conditions they can be applied.

[0135] Most heterobifunctional crosslinking agents contain both primary amine reactive groups and thiol reactive groups. In another embodiment, a heterobifunctional crosslinking agent and a method of using said crosslinking agent are described (U.S. Patent No. 5,889,155, which is specifically incorporated herein by reference in its entirety). The crosslinking agent combines a nucleophilic hydrazide residue with an electrophilic maleimide residue, allowing, in one instance, the coupling of an aldehyde to a free thiol. The crosslinking agent can be modified to crosslink various functional groups.

[0136] In addition, any other linker / coupling agent and / or mechanism known to those skilled in the art can be used in combination with peptides for implementation purposes, such as antibody-antigen interactions, avidin-biotin binding, amide binding, ester binding, thioester binding, ether binding, thioether binding, phosphate binding, phosphoramide binding, anhydride binding, disulfide binding, ionic and hydrophobic interactions, bispecific antibodies and antibody fragments or combinations thereof.

[0137] Preferably, a crosslinking agent with reasonable stability in blood will be used. Various types of disulfide bond-containing linkers are known, which have been successfully used to conjugate targeting agents and therapeutic / prophylactic agents. Linkers containing disulfide bonds as steric hindrance have demonstrated greater stability in vivo. Therefore, these linkers constitute a group of connecting agents.

[0138] In addition to sterically hindered crosslinking agents, non-sterically hindered joints may also be used in this case. Other useful crosslinking agents considered not to contain or generate protected disulfides include SATA, SPDP, and 2-iminothionane (Wawrzynczak and Thorpe, 1987). The uses of such crosslinking agents are well known in the art. Another embodiment involves the use of flexible joints.

[0139] Once chemical conjugation occurs, the peptide is typically purified to separate the conjugate from the non-conjugating agent and other contaminants. Numerous purification techniques are available to provide conjugates of sufficient purity for clinical use.

[0140] Size-based purification methods (such as gel filtration, gel permeation, or high-performance liquid chromatography) are generally the most commonly used. Other chromatographic techniques, such as blue-sepharose separation, can also be used. Conventional methods for purifying fusion proteins from inclusion bodies can be useful, such as using a mild detergent such as sodium N-lauroyl-sarcosinate (SLS).

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

[0142] In addition, in some respects, the modified Cav-1 peptide may also include a cell-binding domain or a cell-penetrating peptide (CPP). As used herein, the terms “cell-penetrating peptide” and “membrane translocation domain” are used interchangeably and refer to a segment of a polypeptide sequence that allows the polypeptide to cross the cell membrane (e.g., the plasma membrane in the case of eukaryotic cells). Examples of CPP segments include, but are not limited to, segments derived from HIV Tat (e.g., GRKKRRQRRRPPQ (SEQ ID NO:21)), herpesvirus VP22, Drosophila antennal foot homeobox gene products, protegrin I, penetrating peptide (RQIKIWFQNRRMKWKK (SEQ ID NO:22)), or melives venom peptide (GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO:23)). In some respects, the CPP contains the 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 the polypeptides described herein and their mutants, variants, analogs, or derivatives. Specifically, these methods involve administering to a subject any of the polypeptides described herein, or pharmaceutically acceptable modifications thereof, as a pharmaceutically acceptable carrier, in a composition for the treatment or prevention of lung diseases, injuries, or infections (e.g., pulmonary fibrosis), said composition comprising the polypeptide of the embodiment in the pharmaceutically acceptable carrier.

[0145] A. Pharmaceutical Composition

[0146] The modified Cav-1 peptide is intended to be administered systemically or locally to inhibit apoptosis and to treat and prevent damage to lung tissue. The peptide can be administered intravenously, intrathecally, and / or intraperitoneally. In certain aspects, the peptide is delivered locally to the airways, such as by administering a nebulized or dry powder formulation for inhalation. The peptide can be administered alone or in combination with antifibrotic compounds.

[0147] The modified Cav-1 peptide can be combined with, concurrently with, or sequentially with at least one additional therapeutic agent for pulmonary fibrosis. The additional therapeutic agent can be an NSAID, steroid, DMARD, immunosuppressant, biological response modifier, bronchodilator, or antifibrotic agent such as pirfenedone (whose antifibrotic mechanism of action is not fully understood but may involve TGF-β blockade), the broad-spectrum tyrosine kinase blocker nintedanib, or any other antifibrotic agent.Suitable NSAIDs are selected from non-selective COX inhibitors such as acetylsalicylic acid, mesalazine, ibuprofen, naproxen, flurbiprofen, fenoprofen, fenbufen, ketoprofen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprafen, amiloride, and tiaprofenicol. acid), fluprofen, indomethacin, sulindac, tolmetin, zomepirac, nabumetone, diclofenac, fenclofenac, alclofenac, bromfenac, ibufenac, aceclofenac, acemetacin, fentiazac, clidanac, etodolac, oxpinac, mefenamic acid, meclofenamic acid, flufenamic acid, nifluminicacid, tolfenamic acid diflunisal, flufenisal, piroxicam, tenoxicam, lornoxicam, and nimesulide, and their pharmaceutically acceptable salts; selective COX 2 inhibitors meloxicam, celecoxib, and rofecoxib, and their pharmaceutically acceptable salts.Suitable steroids include prednisone, prednisolone, methylprednisolone, dexamethasone, budenoside, fluocortolone, and triamcinolone. Suitable DMARDs include sulfasalazine, olsalazine, chloroquine, gold derivatives (Auranofin), D-penicillamine, and cell inhibitors such as methotrexate and cyclophosphamide. Suitable immunosuppressants include cyclosporine A and its derivatives, mycophenolate mofetil, FK 506, OKT-3, ATG, desoxyspergualin, mizoribine, misoprostol, rapamycin, reflunomide, and azathioprine. Suitable bioresponse modifiers include interferon β, anti-TNF-α (etanercept), IL-10, and anti-CD3 or anti-CD25. Suitable bronchodilators include ipratropium bromide, oxytropium bromide, tiotropium bromide, epinephrine hydrochloride, salbutamol, terbutalin sulfate, fenoterol hydrobromide, salmeterol, and formoterol. In such combinations, each active ingredient may be administered at a dose within its usual dose range or below. The dose of the combined NSAID, steroid, DMARD, immunosuppressant, and bioresponse modifier is suitably 1 / 50 to 1 / 1 of the normally recommended minimum dose, preferably 1 / 20 to 1 / 2, and more preferably 1 / 10 to 1 / 5. The normally recommended dose of the combined drug should be understood, for example, in Rote… Dosage disclosed in the 2002 Edition, Cantor Verlag Aulendorf, Germany, or Physician's Desk Reference.

[0148] When considering clinical applications, it may be necessary to prepare pharmaceutical compositions comprising proteins, antibodies, and drugs in a form suitable for the intended use. Typically, pharmaceutical compositions may comprise effective amounts of one or more embodiments of a polypeptide or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase “pharmaceutically or pharmacologically acceptable” means a molecular entity and composition that, when administered to animals (e.g., humans) where appropriate, does not produce side effects, allergic reactions, or other adverse reactions. Based on this disclosure, those skilled in the art will recognize the preparation of pharmaceutical compositions comprising at least one polypeptide or additional active ingredient isolated by the methods disclosed herein, as illustrated in Remington's Pharmaceutical Sciences, 18th edition, 1990, which is incorporated herein by reference. Furthermore, for animal (e.g., human) administration, it should be understood that the product should meet the standards of sterility, pyrogenicity, general safety, and purity as required by the FDA Office of Biological Standards.

[0149] As used herein, “pharmaceuticalally acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, pharmaceuticals, pharmaceutical stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and other materials and combinations thereof, as known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th edition, 1990, which is incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, its use in pharmaceutical compositions should be considered.

[0150] Some embodiments of the invention may include different types of carriers, depending on whether it is administered in solid, liquid, or aerosol form, and whether sterility is required for the route of administration (such as injection). The composition may be administered intravenously, intrathecally, intradermally, percutaneously, intrathecally, intra-arterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly, subcutaneously, orally, topically, locally, by inhalation (e.g., inhaled nebulizers or dry powder formulations), by injection, by infusion, by continuous infusion, by direct local perfusion of target cells with a lipid composition (e.g., liposomes) (via catheter, via irrigation), or by other methods as known to those skilled in the art, or any combination thereof (see, for example, Remington's Pharmaceutical Sciences, 18th edition, 1990, which is incorporated herein by reference).

[0151] Modified polypeptides can be formulated into compositions in the form of free bases, neutral compounds, or salts. Pharmaceutically acceptable salts include acid addition salts, such as acid addition salts formed with the free amino group of a 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. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine, or procaine. After formulation, the solution is administered in a dosage form compatible with the dosage formulation and in a therapeutically effective amount. The formulation is readily applicable in various dosage forms, such as for parenteral administration (e.g., injectable solutions), for delivery to the lungs (e.g., aerosols), or for dietary administration (e.g., drug-release capsules).

[0152] Further according to certain aspects of the invention, the applicable composition may be provided in a pharmaceutically acceptable carrier, with or without an inert diluent. The carrier shall be absorbable and comprise liquid, semi-solid (i.e., paste), or solid carriers. Its use in an applicable composition for carrying out the method is appropriate unless any conventional medium, agent, diluent, or carrier is detrimental to the recipient or the therapeutic efficacy of the composition contained therein. Examples of carriers or diluents include fats, oils, water, salt solutions, lipids, liposomes, resins, binders, fillers, etc., or combinations thereof. The composition may also contain various antioxidants that prevent oxidation of one or more components. Furthermore, antimicrobial action may be achieved by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0153] According to certain aspects of the invention, the composition is combined with the carrier in any convenient and practical manner (i.e., by dissolution, suspension, emulsification, mixing, encapsulation, absorption, etc.). Such procedures are conventional to those skilled in the art.

[0154] In a specific embodiment of the invention, the composition is thoroughly combined or mixed with a semi-solid or solid carrier. Mixing can be performed by any convenient method, such as grinding. Stabilizers may also be added during mixing to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers used for the composition include buffers, amino acids (such as glycine and lysine), carbohydrates, or lyophilization protectants (such as glucose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.).

[0155] In some respects, pharmaceutical formulations contain one or more surfactants. Surfactants used according to the disclosed methods include ionic and nonionic surfactants. Representative nonionic surfactants include polysorbates, such as... -20 and Surfactants (Bridgewater, ICIAmericas, NJ); poloxamer (e.g., poloxamer 188); Surfactants (Sigma-Aldrich, St. Louis, Mo.); Sodium dodecyl sulfate (SDS); Sodium lauryl sulfate; Sodium octyl glycoside; Lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; Lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, or stearyl sarcosine; Linoleyl betaine, myristyl betaine, or hexadecyl betaine; Lauramidopropyl dimethylamine, cocamidopropyl dimethylamine, linoleidopropyl dimethylamine, myristamidopropyl dimethylamine, palmitamidopropyl dimethylamine, or (e.g., lauramidopropyl); myristamidopropyl dimethylamine, palmitamidopropyl dimethylamine, or isostearamidopropyl dimethylamine; Sodium methylcocoyl taurate or disodium methyloleoyl taurate; MONAQUAT TM Surfactants (Paterson, Mona Industries, NJ); polyethylene glycol; polypropylene glycol; block copolymers of ethylene glycol and propylene glycol, such as Surfactants (BASF, Mt. Olive, NJ); oligo(ethylene oxide) alkyl ethers; alkyl (thio)glucosides, alkyl maltodextrins; and phospholipids. For example, the surfactant may be present in the formulation in amounts of about 0.01% to about 0.5% (w / w, the weight of the surfactant relative to the total weight of other solid components in the formulation), 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 respects, the pharmaceutical formulations of the embodiments are substantially free of nonionic surfactants or substantially free of all surfactants.

[0156] Regarding the treatment methods of this invention, it is contemplated that the administration of one or more peptides as disclosed herein, or their mutants, variants, analogs, or derivatives thereof, is not limited to a specific mode of administration, dosage, or frequency of administration; the invention considers all modes of administration, including intramuscular, intravenous, intraperitoneal, intravascular, intra-articular, intralesional, subcutaneous, or any other route sufficient to provide a dose adequate for treating inflammation-related conditions. The therapeutic agent may be administered to the patient in a single dose or multiple doses. When multiple doses are administered, the doses may be spaced apart, for example, one hour, three hours, six hours, eight hours, one day, two days, one week, two weeks, or one month. For example, the therapeutic agent may be administered for, for example, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more weeks. It should be understood that, for any particular subject, the specific dosing regimen should be adjusted over time based on 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 may be increased.

[0157] While the attending physician will ultimately determine the appropriate dosage and dosing regimen, therapeutically effective doses of one or more peptides, or mutants, variants, analogs, or derivatives thereof, as disclosed herein, may be provided at doses 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 testing bioassays or systems.

[0158] The dosage for a particular patient or subject can be determined by a person skilled in the art using conventional considerations, such as appropriate conventional pharmacological protocols. For example, a physician may initially prescribe a relatively low dose and then increase it 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 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 or mutants, variants, analogs, or derivatives thereof disclosed herein, and the patient's condition, as well as the weight or surface area of ​​the patient to be treated.

[0159] In some respects, a single dose is administered once daily to treat subjects (preferably mammals, more preferably individuals with or susceptible to pulmonary fibrosis resulting from this), with a single dose between about 0.2 mg / kg and about 250 mg / kg, such as between about 10 mg / kg and about 50 mg / kg, for example, via infusion (by inhalation). This dose may be administered daily anywhere for about 3 days to one or more weeks. Long-term administration is also possible, although the dose may need to be adjusted downwards as is well known in the art. However, the foregoing ranges are suggestive because the number of variables in a single treatment regimen is large, and considerable deviations from these preferred values ​​are expected.

[0160] For continuous administration, such as via a pump system (such as the osmotic pump used in some experiments described below), the total dose over a period of about 1-2 weeks is preferably in the range of 1 mg / kg to 1 g / kg, preferably 20-300 mg / kg, and more preferably 50-200 mg / kg. Following 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] The effective concentration of the active compound used to inhibit or prevent the inhibition of apoptosis in vitro is in the range of about 0.5 nM to about 100 nM, more preferably about 2 nM to about 20 nM. The effective dose and the optimal dose range can be determined in vitro using the methods described herein.

[0162] B. Aerosol dispersion and atomization device

[0163] The formulation can be atomized using any suitable device, including but not limited to jet nebulizers, ultrasonic nebulizers, metered-dose inhalers (MDIs), and devices that atomize liquids by forcibly passing them through a jet or nozzle (e.g., Aradigm from Hayward, Calif.). (Drug delivery device). Furthermore, 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 combined with a mask, mouthpiece, mist inhalation device, and / or a platform that guides the user to inhale correctly and automatically delivers the drug during respiration at the appropriate time. Representative atomizing devices that can be used with the method according to the present invention include, but are not limited to, the atomizing 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, as well as U.S. Patent Application Publication Nos. 20020020412 and 20020020409.

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

[0165] Metered-dose inhalers (MDIs) can be used to deliver the compositions of the invention in a more concentrated form than that typically delivered using nebulizers. For optimal efficacy, MDI delivery systems require appropriate administration techniques, including coordinated aerosol delivery and inhalation, slow inhalation of approximately 0.5–0.75 liters per second, deep breathing with a near-deep inspiratory volume, and breath-holding for at least 4 seconds. Lung delivery using MDIs is convenient and suitable when treatment benefits from relatively short treatment times and low costs. Optionally, the formulation may be heated to approximately 25°C to approximately 90°C during nebulization to promote effective droplet formation and subsequent delivery. See, for example, U.S. Patent No. 5,299,566.

[0166] The aerosol composition of the described embodiment comprises microdroplets of the composition, the size of which is suitable for effective delivery within the lungs. In some cases, the surfactant formulation is delivered to the bronchioles, more preferably to the bronchioles, even more preferably to the alveolar ducts, and even more preferably to the alveoli. The diameter of the aerosol microdroplets is typically less than about 15 μm, less than about 10 μm, less than about 5 μm, or less than about 2 μm. For effective delivery to the alveolar bronchi of a human subject, the aerosol composition may preferably comprise microdroplets with a diameter of about 1 μm to about 5 μm.

[0167] Droplet size can be evaluated using techniques known in the art, such as cascading, collision, laser diffraction, and optical patterning. See McLean et al. (2000) Anal Chem 72:4796-804, Fults et al. (1991) J PharmPharmacol 43:726-8, and Vecellio None et al. (2001) J AerosolMed 14:107-14.

[0168] Protein stability after aerosolization can be assessed using techniques known in the art, including size exclusion chromatography; electrophoresis; spectroscopic techniques (such as UV spectroscopy and circular dichroism spectroscopy); and protein activity (measured in vitro or in vivo). For 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 administration of the composition to the lungs of a subject, the aerosol device is adapted 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 atomizer" herein refers to any atomizer that operates according to the general principle of using a vibrating mesh or vibrating plate (perforated plate) with multiple holes to produce a low-velocity aerosol of fine particles. Some atomizers may comprise a mesh / membrane with between 1,000 and 7,000 holes, which vibrates on top of a liquid reservoir (see, for example, U.S. Patent Publication 20090134235 and Waldrep and Dhand 2008, each of which is incorporated herein by reference). In some embodiments, the vibrating mesh atomizer is Professional atomizer, Omron Pari Or EZ inhalation nebulizers. In some cases, vibrating mesh nebulizers have vibration frequencies between approximately 50-250 kHz, 75-200 kHz, 100-150 kHz, or approximately 120 kHz. These devices offer high efficiency in delivering aerosols to the lungs and minimize the volume of liquid remaining in the device, which is advantageous for expensive and potent compounds such as plasminogen activators.

[0170] In some aspects, the atomizing composition of the embodiments described above is produced using a vibrating mesh atomizer. For example, the composition may be produced using an active vibrating mesh atomizer (e.g., (Professional atomizer systems) are used to manufacture them. Descriptions of such systems and their operation can be found, for example, in U.S. Patent Nos. 6,921,020; 6,926,208; 6,968,840; 6,978,941; 7,040,549; 7,083,112; 7,104,463; and 7,360,536, each of which is incorporated herein by reference in its entirety. In other aspects, the compositions of the described embodiments can use passive vibrating mesh atomizers such as Omron. Or it can be produced using EZ breathing nebulizers.

[0171] IV. Treatment of lung symptoms

[0172] The modified peptides of this invention can be used to treat a variety of lung conditions. The treated lung conditions 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 injury

[0174] In some respects, the subject suffered from acute lung injury (ALI) or infection- or chemically induced lung injury. Specifically, the subject suffered from acute respiratory distress syndrome (ARDS), acute lung injury induced by inhaled smoke (ISALI), bronchiectasis, airway disease induced by inhaled toxins (e.g., chlorine or other induced airway diseases), 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 following acute lung injury, including ARDS). Acute lung injury (ALI) is a serious medical problem among U.S. military personnel. During combat, ALI can be caused by a very wide range of etiologies.

[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 outcomes. No effective preventative measures are available other than a barrier with a breathing mask. Significant progress has been made in the management of ARDS, but it remains largely supportive, requiring careful attention to await the activation of intrinsic healing mechanisms; and in-hospital mortality remains above 40% (Matthay et al., 2012). Survivors of ALI often suffer from chronic respiratory dysfunction and a declining quality of life. Any model that can accelerate recovery and / or prevent subsequent complications such as chronic respiratory failure and pulmonary fibrosis would be highly desirable. There is an urgent need to improve the early diagnosis of ALI, and more importantly, to improve its prevention and treatment. The pathophysiology of ALI caused by ARDS resulting from direct inhalation lung injury or systemic disease is extremely complex and heterogeneous, encompassing systemic and local cardiopulmonary factors such as increased membrane permeability, influx of inflammatory cytokines, oxidative cell damage, atrioventricular fluid shift, ion channel disruption, and many other factors (Matthay et al., 2012). Clearly, novel therapies are needed to treat and prevent lung conditions such as ALI.

[0176] In some embodiments, a method is provided for treating or preventing acute lung injury, lung infection, or lung disease in a subject, comprising administering to the subject an effective amount of a 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, a method of administering the pharmaceutical formulation of the embodiments comprises nebulizing a solution containing the variant polypeptide. In a specific aspect, 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 (such as those arising from lupus, scleroderma, or mixed connective tissue disease), interstitial lung diseases (such as idiopathic pulmonary fibrosis or sarcoidosis), and acute and chronic lung injuries that lead 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 and exocrine sweat glands of the digestive and respiratory systems. This disease is typically characterized by chronic respiratory infections, pancreatic insufficiency, abnormally thick and sticky secretions, and premature death. Cystic fibrosis (CF) is characterized by progressive airflow obstruction. Individual subgroups with CF also exhibit airway hyperresponsiveness to inhaled cholinergic agonists (Weinberger, 2002 and Mitchell et al., 1978) and reversible airflow limitation in response to bronchodilators (van Haren et al., 1991 and van Haren et al., 1992). The presence of bronchial hyperresponsiveness and airway obstruction suggests a possible shared etiology between CF and other airway stenosis diseases, such as asthma or chronic obstructive pulmonary disease (COPD), where airway smooth muscle dysfunction is thought to contribute to disease progression.

[0180] Lung infections can be caused by bacteria. Infectious bacteria can include *Pseudomonas aeruginosa*, *Bacillus anthracis*, *Listeria monocytogenes*, *Staphylococcus aureus*, *Salmonella*, *Yersina pestis*, *Mycobacterium leprae*, *Mycobacterium africanum*, *Mycobacterium asiaticum*, *Mycobacterium avium*, and *Mycobacterium chelonei*. Mycobacterium abscessus, Mycobacterium fallax, Mycobacterium fortuitum, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium malmoense, Mycobacterium shimoidei, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium xenopi, Mycobacterium tuberculosis, Brucella melitensis, Brucella suis, Brucella abortus, Brucella canis, Legionella pneumonophilia, Francisella tularensis, Pneumocystis carinii, Mycoplasma or Burkholderia (cepacia). Bacterial infections can lead to pneumonia.

[0181] Chronic obstructive pulmonary disease (COPD) is a term used to classify two main airflow obstruction conditions: chronic bronchitis and emphysema. Approximately 16 million Americans have COPD, and 80-90% of them are smokers for most of their lives. COPD is the leading cause of death in the United States, accounting for 122,283 deaths in 2003. Direct healthcare costs for COPD in the United States in 2003 were approximately $20.9 billion. 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 overinflated due to damage to the elastin backbone of the lungs. Inflammatory cells in emphysematous lungs release elastase, an enzyme that degrades or damages 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 viral lower respiratory tract infections and is characterized by acute inflammation, edema, epithelial cell necrosis in the small airways, and increased mucus production (Ralston et al., 2014). Signs and symptoms usually begin with rhinitis and cough, which may progress to tachypnea, wheezing, rales, use of accessory muscles, and / or nasal flaring.

[0184] Bronchiolitis obliterans is a progressive reduction in airflow caused by abnormal remodeling of the small airways in the lungs (Meyer et al., 2014). Bronchiolitis obliterans syndrome is a major complication of lung transplantation and is often used to describe delayed allogeneic graft dysfunction, which results in a persistent decrease in forced expiratory volume and strength not caused by other known reasons (Meyer et al., 2014).

[0185] The term "asthma" can refer to acute asthma, chronic asthma, intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma, chronic persistent asthma, mild to moderate asthma, mild to moderate persistent asthma, mild to moderate chronic persistent asthma, allergic (exogenous) asthma, non-allergic (endogenous) asthma, nocturnal asthma, bronchial asthma, exercise-induced asthma, occupational asthma, seasonal asthma, asymptomatic asthma, gastroesophageal asthma, idiopathic asthma, and cough-variant asthma. During asthma attacks, the airways are persistently inflamed and may occasionally experience spasms.

[0186] V. Example

[0187] The following embodiments are included to demonstrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques discovered by the inventors that work well in the practice of the invention and can therefore be considered as constituting preferred modes of practice. However, based on this disclosure, those skilled in the art will understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention and still obtaining similar or analogous results.

[0188] Example 1 - Cav-1 peptide solubility

[0189] To determine which peptide was most soluble in the liquid formulation, 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 aid complete dissolution. For insoluble peptides, absorbance was measured at 600 nm immediately after dissolution, or for soluble peptides, absorbance was measured after 10 minutes. Except for samples APi2348, APi2352, and APi2353, which were measured a second time at 15 minutes, 5 minutes, or 15 minutes after dissolution, the absorbance of other insoluble peptides was measured again after 10 minutes. Sample APi2345 was measured only after 20 minutes of dissolution due to incomplete dissolution (Table 2). pH was also tested after 24 hours.

[0190] Compared with other peptides tested, samples APi2350, APi2354, APi2355, and APi2356 showed increased solubility at pH 7.51 (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 the production of smooth muscle actin

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

[0194] Idiopathic pulmonary fibrosis (IPF) cell line 2051 was purchased, and fourth-generation IPF cells were seeded into 100 mm plates containing DMEM, 10% FBS, and 1% P / S. IPF cells were washed with 4 mL of DMEM + 1% P / S and allowed to starve overnight with serum. Cells were then treated for 2 days with 44 μL HBSS (negative control), 10 μM LTI-03 (SEQ ID NO:2), 90 μM LTI-03 (positive control), 10 μM APi2350, 10 μM APi2354, 10 μM APi2355, 10 μM APi2356, or 20 μL DMSO (negative control).

[0195] Two days after treatment, the cells were washed once in cold, sterile HBSS. The HBSS was removed, and 150 μL of lysis buffer containing a mixture of protease inhibitors was added to the cells. The cells were incubated with the lysis buffer for 10 minutes. Cell lysates were scraped from the plates and collected. The cell lysates were then sonicated twice. After sonication, the lysates were centrifuged at 13,000 RPM for 20 minutes. The lysates were then flash-frozen in liquid nitrogen, thawed, vortexed, and centrifuged again at 13,000 RPM for 30 minutes. The supernatant was then collected, and the precipitate was discarded. The concentration of the cell lysates was then determined by BCA assay.

[0196] Western blotting was performed to assess the presence of therapeutic effect. Briefly, 12 μg of each lysate was run on a 10% polyacrylamide gel. The gel was then transferred to a membrane and washed. Results of the first antibody Western blotting against smooth muscle actin (SMA) and tubulin are available in [link to relevant documentation]. Figure 1 The treatment of each lysate in the lanes shown in the figure is as follows: 1: Untreated, 2: 10 μM LTI-03, 3: 90 μM LTI-03, 4: 10 μM APi2350, 5: 10 μMAPi2354, 6: 10 μM APi 2355, 7: 10 μMAPi2356, and 8: DMSO.

[0197] The protein blots were photographed and analyzed using ImageJ to determine the ratio of smooth muscle actin to tubulin. Figure 2 As expected, LTI-03 induced an increase in SMA production relative to tubulin. Treatment with 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 in fibrotic lung biopsies

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

[0200] ***

[0201] According to this disclosure, all methods disclosed and claimed herein can be prepared and performed without excessive experimentation. While the compositions and methods of the invention have been described by way of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the methods described herein and to the steps or sequence of steps of the methods described herein without departing from the spirit and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant agents can replace the agents described herein while achieving the same or similar results. All such similar substitutions and modifications that will be apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0202] V. References

[0203] The following references, which are incorporated herein by reference, supplement the exemplary procedures or other details listed herein.

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

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

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Claims

1. A peptide comprising the amino acid sequence ASFTTFTVT (SEQ ID NO:3), wherein the peptide comprises at least one N-terminal and / or C-terminal addition that lacks identity with SEQ ID NO:

1.

2. The peptide of claim 1, wherein the peptide comprises: a) At least one amino acid added to the N-terminus; b) At least one amino acid added to the C-terminus; or c) At least one amino acid added to the N-terminus and C-terminus.

3. The peptide according to claim 1 or 2, wherein: a) The peptide contains L-amino acids; b) The peptide contains D-amino acids; or c) The peptide contains both L-amino acids and D-amino acids.

4. The peptide according to any one of claims 1-3, wherein the peptide comprises at least one non-standard amino acid.

5. The peptide of claim 4, wherein the non-standard amino acid is ornithine.

6. The peptide of any one of claims 1-5, wherein the peptide comprises: a) N-terminal modification; b) C-terminal modification; or c) N-terminal modification and C-terminal modification.

7. The peptide of claim 6, wherein the N-terminal modification is acylation; and / or the C-terminal modification is amidation.

8. The peptide according to any one of claims 1-7, wherein the peptide comprises the amino acid sequence KASFTTFTVTKGS (SEQ ID NO:4), KASFTTFTVTKGS-NH2 (SEQ ID NO:5), aaEGKASFTTFTVTKGSaa (SEQ ID NO:6), aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO:7), OASFTTFTVTOS (SEQ ID NO:9) or OASFTTFTVTOS-NH2 (SEQ ID NO:10).

9. The peptide of claim 1, wherein the peptide comprises the amino acid sequence Ac-aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO:8).

10. The peptide of claim 1, wherein the peptide comprises the amino acid sequence KASFTTFTVTKGS (SEQ ID NO:4).

11. The peptide according to any one of claims 1-9, further comprising a cell-penetrating peptide (CPP).

12. The peptide of claim 11, wherein the CPP comprises an amino acid sequence selected from the following: GRKKRRQRRRPPQ (SEQ ID NO:21), RQIKIWFQNRRMKWKK (SEQ ID NO:22), and GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO:23).

13. A peptide polymer comprising at least two peptides according to any one of claims 1-12.

14. A composition comprising a peptide as described in any one of claims 1-12 or a peptide polymer as described in claim 13.

15. The composition of claim 14, wherein the peptide or peptide polymer is at least 95% pure.

16. The composition of claim 14 or 15, wherein the peptide or peptide polymer is soluble in an aqueous solution.

17. A pharmaceutical composition comprising a peptide as described in any one of claims 1-12 or a peptide polymer as described in claim 13.

18. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient.

19. The pharmaceutical composition of claim 17 or 18, wherein the pharmaceutical composition is formulated for oral, intravenous, intra-articular, parenteral, enteral, topical, subcutaneous, intramuscular, buccal, sublingual, rectal, vaginal, penile, intraocular, epidural, intracranial, or inhalation administration.

20. The pharmaceutical composition of claim 17 or 18, wherein the pharmaceutical composition is formulated for pulmonary infusion.

21. The pharmaceutical composition of claim 17 or 18, wherein the pharmaceutical composition is formulated as an atomized solution.

22. The pharmaceutical composition of claim 17 or 18, wherein the pharmaceutical composition is formulated as a dry powder.

23. A polynucleotide comprising a nucleic acid sequence encoding a peptide as described in any one of claims 1-12 or a peptide multimer as described in claim 13.

24. Use of the peptide of any one of claims 1-12 or the peptide polymer of claim 13 in the preparation of a medicament for treating or preventing a disease in a subject, wherein an effective amount of the peptide or peptide polymer is administered to the subject, wherein the disease is fibrosis, inflammatory disease, lung disease, or lung infection.

25. The use as claimed in claim 24, wherein the fibrosis is organ fibrosis.

26. The use as claimed in claim 25, wherein the organ fibrosis is renal fibrosis, liver fibrosis, pulmonary fibrosis, or cardiac fibrosis.

27. The use as claimed in claim 24, wherein the inflammatory disease is an inflammatory eye disease.

28. The use as claimed in claim 24, wherein the lung disease is pulmonary inflammation, chronic obstructive pulmonary disease (COPD), acute lung injury, lung infection, chemically induced lung injury, plastic bronchitis, asthma, acute respiratory distress syndrome (ARDS), acute lung injury induced by inhaled smoke (ISALI), bronchiolitis, obliterative bronchiolitis, fibrotic symptoms of the lungs, interstitial lung disease, nonspecific interstitial pneumonia, idiopathic pulmonary fibrosis (IPF), or lung scarring.

29. The use as described in any one of claims 24-28, wherein the subject is undergoing chemotherapy or radiation therapy.

30. The use as described in any one of claims 24-29, wherein the peptide or peptide polymer is administered systemically or locally to the diseased tissue of the subject.

31. The use as described in any one of claims 24-30, wherein the peptide or peptide polymer is administered to the subject via a dry powder inhaler.

32. The use as described in any one of claims 24-30, wherein the peptide or peptide polymer is administered to the subject using a nebulizer.

33. The use as described in any one of claims 24-30, wherein the peptide or peptide polymer is administered to the subject intravenously or subcutaneously.

34. The use as described in any one of claims 24-33, wherein at least one additional therapeutic agent is also applied.

35. The use as described in claim 34, wherein the at least one additional therapeutic agent is a nonsteroidal anti-inflammatory drug (NSAID), a steroid, a disease-modifying antirheumatic drug (DMARD), an immunosuppressant, a biological response modifier, or a bronchodilator.

36. Use of the peptide of any one of claims 1-12 or the peptide multimer of claim 13 in the preparation of a medicament for increasing the viability of lung epithelial cells in a subject, wherein an effective amount of the peptide or peptide multimer is administered to the subject.

37. The use of claim 36, wherein the peptide or peptide polymer increases the activity of type 2 alveolar epithelial cells.

38. The use of claim 36 or 37, wherein the subject suffers from lung disease, lung infection, lung inflammation, or acute lung injury.

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