Modified cavein-1 peptide formulations and methods of making and using same

Abstract

Provided herein are modified cave-1 (Cav-1) peptides and pharmaceutical formulations and methods of using the peptides or formulations for treating a subject in need thereof. In particular, provided herein are modified Cav-1 peptide formulations and methods of using the formulations for the treatment of various diseases, such as fibrosis, by extending the release of the peptide over a desired period of time.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 368,169, filed on July 12, 2022, the contents of which are incorporated by reference in their entirety. Technical Field

[0003] The present disclosure relates to extended release formulations comprising modified caveolin-1 peptides. The present disclosure also provides methods of using such formulations for treating various diseases, including but not limited to fibrosis.

[0004] Incorporation of Sequence Listing

[0005] The sequence listing associated with this application is provided in Extensible Markup Language (XML) format in lieu of a paper copy and is hereby incorporated into this specification by reference. A computer-readable format copy of the sequence listing (file name: LUTX_025_01WO_SeqList_ST26; creation date: July 6, 2023; file size: 162,438 bytes) is being submitted electronically. Background Art

[0006] Caveolin-1 (Cav-1) is an integral membrane protein that has a homeostatic function in fibrosis by participating in a series of key regulatory pathways (such as TGF-β signaling) (Shihata et al., Front. Pharmacol. 2017; 8: 567; and Gvaramia et al., Matrix Biol, 2013: 32(6): 307-315). Endogenous caveolin-1 is constitutively inhibited in bleomycin-induced IPF animal models and fibrotic lungs of IPF patients (Wang et al., J Exp Med, 2006; 203(13): 2895-2906; Sanders et al., PLoS One, 2015; 10(2), e0116995; and Sanders et al., Am J Respir Cell Mol Biol, 2017; 56(1): 50-61). Due to concerns inherent to protein drugs, including stability, delivery, cost and autoimmunogenicity, full-length caveolin-1 protein may be a less desirable drug candidate. Therefore, fragments of caveolin-1 (such as caveolin scaffold domain peptides (CSP)) have been studied and found to be effective alternatives to full-length caveolin-1 protein. Specifically, 20-mer form of CSP has been shown to prevent, limit or reverse fibrosis in animal models, and the seven amino acid fragments of CSP named CSP-7 are sufficient to effectively promote the reduction of fibrosis in vitro and in vivo (Marudamuthu et al., Sci Transl Med, 2019; 11 (522), eaat2848). Therefore, CSP is identified as a promising therapeutic agent for the treatment of fibrotic diseases (such as IPF).

[0007] Peptide therapeutics generally have a short half-life in vivo, requiring frequent administration of the therapeutic agent. Therefore, there is a need for an improved extended-release formulation of Cav-1 peptide therapeutics to reduce the burden of frequent administration and minimize adverse effects caused by peak-trough fluctuations in serum drug concentrations. Summary of the invention

[0008] The present disclosure provides formulations comprising a modified caveolin-1 peptide and at least one pharmaceutically acceptable carrier or excipient.

[0009] In one aspect, the formulation comprises a polypeptide comprising an amino acid sequence having the core sequence FTTFTVT and sucrose acetate isobutyrate (SAIB).

[0010] In some embodiments, the preparation comprising SAIB further comprises a first solvent selected from N-methyl pyrrolidone (NMP), anhydrous ethanol or ethyl acetate. In some embodiments, the first solvent is NMP or ethyl acetate. In some embodiments, SAIB is present in the first solvent with about 50% w / w to about 95% w / w or 70% w / w to about 90% w / w. In some embodiments, SAIB is present in the first solvent with about 80% w / w.

[0011] In some embodiments of the formulation comprising SAIB, the polypeptide is micronized. In some embodiments, the polypeptide has an average particle size in the range of about 0.5 μm to about 100 μm. In some embodiments, the polypeptide has an average particle size in the range of about 1 μm to about 5 μm.

[0012] In some embodiments, the formulation comprising SAIB is in the form of an emulsion, solution, microsphere, nanoparticle, nanosphere, implant, or gel. In some embodiments, the formulation is a suspension.

[0013] In some embodiments, the formulation comprising SAIB further comprises a second solvent. In some embodiments, the second solvent is sterile water, phosphate buffered saline (PBS), or any pharmaceutically acceptable carrier.

[0014] In some embodiments, the formulation comprises the polypeptide and SAIB in a weight ratio of about 1:50 to about 1: 1. In some embodiments, the weight ratio is about 1:30 to about 1:1.

[0015] In some embodiments, the preparation comprising polypeptide and SAIB releases at least about 1% to about 30% of polypeptide within four hours under physiological conditions. In some embodiments, the preparation releases at least about 5% to 25% of polypeptide within four hours under physiological conditions. In some embodiments, when measured in an aqueous buffer in the range of about pH 6 to about pH 8 at about 37 ℃, the preparation comprising polypeptide and SAIB releases at least about 1% to about 30% or at least about 5% to 25% of polypeptide within four hours. In some embodiments, when measured in an aqueous buffer of about pH 7.4 at about 37 ℃, the preparation comprising polypeptide and SAIB releases at least about 1% to about 30% or at least about 5% to about 25% of polypeptide within four hours.

[0016] In some embodiments of any of the SAIB formulations as described herein, the polypeptide comprises ASFTTFTVTK. In some embodiments, the polypeptide consists of 20 or fewer amino acids. In some embodiments, the polypeptide comprises at least one amino acid added to the N-terminus; at least one amino acid added to the C-terminus; or at least one amino acid added to the N-terminus and the C-terminus. In these embodiments, the additions made within 5 amino acids at each end have an amino acid sequence that is less than 80% identical to a continuous amino acid sequence of SEQ ID NO: 1 or 2 (human Cav-1 peptide). In some embodiments, the additions made within 5 amino acids at each end have an amino acid sequence that is less than 60% identical to a continuous amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the additions made within 5 amino acids at each end have an amino acid sequence that is less than 40% identical to a continuous amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the additions made within 5 amino acids at each end have an amino acid sequence that is less than 20% identical to a continuous amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptide sequence comprises a peptide comprising L-amino acids; the polypeptide comprises D-amino acids; or the peptide comprises both L-amino acids and D-amino acids.

[0017] In some embodiments, the polypeptide comprises at least one non-standard amino acid; or the polypeptide comprises two non-standard amino acids. In some embodiments, the non-standard amino acid is ornithine. In some embodiments, the polypeptide further comprises an N-terminal modification; a C-terminal modification; or an N-terminal and a C-terminal modification. In some embodiments, the N-terminal modification is an acylation; and / or the C-terminal modification is an amidation.

[0018] In some embodiments of any of the SAIB formulations described herein, the polypeptide comprises the amino acid sequence FTTFTVT (SEQ ID NO:3), KASFTTFTVTKGS (SEQ ID NO:4), KASFTTFTVTKGS-NH2 (SEQ ID NO:5), aaEGKASFTTFTVTKGSaa (SEQ ID NO:6), aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO:7), Ac-aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO:8), OASFTTFTVTOS (SEQ ID NO:9), or OASFTTFTVTOS-NH2 (SEQ ID NO:10). In some embodiments, the polypeptide consists of an amino acid sequence selected from the group consisting of FTTFTVT (SEQ ID NO: 3), KASFTTFTVTKGS (SEQ ID NO: 4), KASFTTFTVTKGS-NH2 (SEQ ID NO: 5), aaEGKASFTTFTVTKGSaa (SEQ ID NO: 6), aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO: 7), Ac-aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO: 8), OASFTTFTVTOS (SEQ ID NO: 9), or OASFTTFTVTOS-NH2 (SEQ ID NO: 10). In some embodiments, the polypeptide consists of the amino acid sequence Ac-aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO: 8).

[0019] In another aspect, the present disclosure provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of any of the formulations described herein. In some embodiments, the disease is fibrosis. In some embodiments, the fibrosis is interstitial lung disease, liver fibrosis, renal fibrosis, skin fibrosis, glomerulonephritis, systemic sclerosis, cardiac fibrosis, myocardial fibrosis, kidney fibrosis, liver cirrhosis, nephrosclerosis, arteriosclerosis, macular degeneration, ocular scars, cataracts, retinal and vitreoretinopathy, Graves' sophthalmopathy, neurofibromatosis, scleroderma, glioblastoma, keloid and hypertrophic scars, peritoneal fibrotic disease, chronic obstructive pulmonary disease, postoperative uterine fibroids, diabetic nephropathy, gynecological cancer, myeloproliferative syndrome, myeloid leukemia, myelodysplastic syndrome, inflammatory bowel disease, nonalcoholic fatty liver disease, fibrosarcoma, rheumatoid arthritis, nonalcoholic steatohepatitis, Alport syndrome, or chronic COVID syndrome.

[0020] In some embodiments, the interstitial lung disease is idiopathic pulmonary fibrosis, familial pulmonary fibrosis, idiopathic nonspecific interstitial pneumonia, conventional interstitial pneumonia, cryptogenic organizing pneumonia, or sarcoidosis. In some embodiments, the interstitial lung disease is idiopathic pulmonary fibrosis.

[0021] In some embodiments, the disease is nephropathy or renal condition. In some embodiments, nephropathy or renal condition is selected from the group consisting of: chronic kidney disease, end-stage renal disease, glomerulonephritis, focal segmental glomerulosclerosis, renal fibrosis, polycystic kidney disease, IgA nephropathy, lupus nephritis, nephrotic syndrome, Alport syndrome, amyloidosis, Goodpasture syndrome, granulomatosis with polyangiitis or acute kidney injury. In some embodiments, nephropathy or renal condition is renal fibrosis. In some embodiments, nephropathy or renal condition is Alport syndrome. In some embodiments, administration is intraocular, intradermal, transdermal, intramuscular or subcutaneous administration. In some embodiments, the subject is a person.

[0022] In some embodiments of any of the formulations as described herein, the formulation releases the peptide for at least 7 days after a single dose is administered. In some embodiments, the formulation releases the polypeptide for at least 21 days after a single dose is administered. In some embodiments, the formulation releases the polypeptide for at least 28 days after a single dose is administered. In some embodiments, the formulation is administered to a subject at a dose of about 0.01 mg / kg to about 250 mg / kg. In some embodiments, the formulation is administered to a subject at a dose of about 0.05 mg / kg to about 50 mg / kg.

[0023] It is contemplated that any method or formulation described herein may be implemented relative to any other method or formulation described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that although the detailed description indicates specific embodiments of the present disclosure, they are given by way of illustration only, as various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art who read this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Exemplary release (%) of modified Cav-1 peptide (APi2335, SEQ ID NO: 8) from exemplary SAIB formulations over time (h).

[0025] Figure 2 Illustrated are the mean plasma concentrations of modified Cav-1 peptide (APi2355, SEQ ID NO: 8) in rat tissues following administration of an exemplary SAIB formulation. DETAILED DESCRIPTION

[0026] The present disclosure overcomes the challenges associated with the prior art by providing a modified caveolin-1 (Cav-1) peptide in a controlled drug release dosage form. In some embodiments, a pharmaceutical formulation of a modified Cav-1 peptide is provided. In some embodiments, the peptide is formulated for subcutaneous delivery. In some embodiments, the peptide is formulated for extended release. Also provided herein is a method of treating or preventing fibrosis or chronic kidney disease by administering to a subject a therapeutically effective amount of any of the modified Cav-1 peptide formulations described herein.

[0027] I. Definitions

[0028] As used herein, the articles "a" and "an" refer to one or more than one of the grammatical objects of the article. As used in the claims herein, when used in conjunction with the word "comprising," the articles "a" and "an" refer to one or more than one of the grammatical objects of the article.

[0029] Although the present disclosure supports definitions referring to only alternatives and "and / or," the use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to only alternatives or the alternatives are mutually exclusive. As used herein, "another" may mean at least a second or more.

[0030] As used herein, the term "about" indicates a value that includes inherent error variations for the device, the method used to determine the value, variations that exist between study subjects, or values ​​that are within 10% of the stated value.

[0031] The term "peptide" or "polypeptide" refers to an amino acid sequence consisting of a single chain of amino acids connected by peptide bonds. The term can be used interchangeably with "protein" in its broad sense to refer to molecules of two or more amino acids, amino acid analogs or peptidomimetics. In some embodiments, the amino acids are connected by peptide bonds. In some embodiments, the amino acids are connected by other types of 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 optical isomers or L optical isomers) as well as amino acid analogs and peptidomimetics.

[0032] Typically, unless otherwise defined, a peptide or polypeptide contains at least two amino acid residues and is less than about 50 amino acids in length (e.g., 40 amino acids, 30 amino acids, 20 amino acids, or any number therein). In some embodiments, the peptide or polypeptide carries a counterion. In some embodiments, the peptide or polypeptide comprises an N-terminal and / or C-terminal modification, such as a blocking modification that reduces degradation.

[0033] The term "peptidomimetic" or "peptide mimetic" means that the peptide according to the invention is modified in such a way that it contains at least one non-peptide bond, such as, for example, a urea bond, a carbamate bond, a sulfonamide bond, a hydrazine bond or any other covalent bond. If the peptide chain is shorter, a peptide of three or more amino acids is generally referred to as an oligopeptide. If the peptide chain is longer, the peptide is generally referred to as a peptide or protein.

[0034] In some embodiments, a peptide or peptides have a certain percentage (e.g., 80%, 85%, 90% or 95%) of "sequence identity" or "homology" with another sequence, meaning that when aligned, the percentage of amino acids that are identical when comparing the two sequences. In some embodiments, the term "identity" or "homology" refers to the percentage of amino acid residues in the candidate sequence that are identical to the residues of the compared corresponding sequence after aligning the sequences and introducing spaces (if necessary) to achieve the maximum percentage identity of the entire sequence and not considering any conservative substitutions as part of the sequence identity. In some embodiments, N-terminal or C-terminal extensions or insertions should not be interpreted as reducing identity or homology. Alternatively, in some embodiments, N-terminal or C-terminal extensions or insertions make the newly formed sequence non-isologous to the original sequence. Alignment and percent homology or sequence identity can be determined using software programs known in the art, such as those described in Current Protocols In Molecular Biology (FM Ausubel et al., eds., 1987) 30th Suppl., Section 7.7.18, Table 7.7.1.

[0035] The term "insertion" or "deletion" is generally in the range of about 1 to 5 amino acids. The variation allowed can be determined experimentally by producing the peptide synthetically while systematically inserting, deleting or substituting nucleotides in the sequence using recombinant DNA technology.

[0036] The term "substitution" when referring to a peptide or polypeptide refers to a change in an amino acid for a different entity (e.g., another amino acid or amino acid moiety). Amino acid substitutions include changes in which 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 the amino acid residue contained in the peptide is replaced by another naturally occurring amino acid with similar characteristics in terms of polarity, side chain functionality, or size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present invention can also be "non-conservative," in which the amino acid residues present in the peptide are replaced by amino acids with different properties (such as naturally occurring amino acids from different groups) (e.g., using alanine to replace charged or hydrophobic amino acids), or alternatively wherein naturally occurring amino acids are replaced by unconventional amino acids. In some embodiments, amino acid substitutions are conservative. In some embodiments, amino acid substitutions are non-conservative.

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

[0038] The term "covalently bonded" refers to peptides or polypeptides that are linked directly or indirectly (eg, through a linker) via a covalent chemical bond. In some embodiments, the fusion peptide is covalently bonded.

[0039] As used herein, the term "fusion protein" refers to a recombinant protein of two or more proteins. Fusion proteins can be produced, for example, by linking a nucleic acid sequence encoding one protein to a nucleic acid encoding another protein so that they constitute a single open reading frame that can be translated into a single peptide with all the expected proteins in the cell. The order in which the proteins are arranged can be changed. The fusion protein can contain an epitope tag or a half-life extender. Epitope tags include biotin, FLAG tags, c-myc, hemagglutinin, His6, digoxigenin, FITC, Cy3, Cy5, green fluorescent protein, V5 epitope tag, GST, β-galactosidase, AU1, AU5 and avidin. Half-life extenders include Fc domains and serum albumin.

[0040] A "biologically active" caveolin-1 (Cav-1) peptide refers to a polypeptide that increases p53 protein levels, decreases urokinase plasminogen activator (uPA) and uPA receptor (uPAR), and / or increases plasminogen activator inhibitor 1 (PAI-1) expression in cells (such as fibrotic lung fibroblasts). In some embodiments, a biologically active peptide has at least 20% of the biological or biochemical activity (e.g., as measured by an in vitro or in vivo assay) of a native Cav-1 peptide of SEQ ID NO: 1. In some embodiments, a biologically active peptide has increased biological or biochemical activity compared to a native Cav-1 peptide.

[0041] As used herein, the term "isolated" refers to a peptide or polypeptide that has been separated from any natural environment, such as body fluids (eg, blood), and from components that naturally accompany the peptide.

[0042] As used herein, "substantially free" with respect to a specified component means that the specified component is not intentionally formulated into a preparation or composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the specified component resulting from any accidental contamination of the preparation is much less than 0.01%. Preparations or preparations in which the amount of the specified component cannot be detected using standard analytical methods are most preferred.

[0043] The term "substantially pure" refers to a peptide or polypeptide that has been separated from components that naturally accompany it and purified to at least some degree. Typically, a peptide or polypeptide is substantially pure when it is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% free by weight of the proteins and naturally occurring organic molecules with which it is naturally associated. For example, a substantially pure peptide or polypeptide can be obtained by extraction from a natural source, by expressing a recombinant nucleic acid in a cell that does not normally express the protein, or by chemical synthesis.

[0044] The terms "subject", "individual" and "patient" are used interchangeably herein and refer to an animal, such as a human or non-human animal (e.g., a mammal), to which a modified Cav-1 peptide or a pharmaceutical formulation thereof as disclosed herein is provided for treatment (including prophylactic treatment). As used herein, the term "subject" refers to humans and non-human animals. The term "non-human animal" includes all vertebrates, such as mammals such as non-human primates (particularly higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and non-mammals such as chickens, amphibians, reptiles, etc. In some embodiments, the subject is a human. In some embodiments, the subject is an experimental animal or animal substitute as a disease model. Non-human mammals include mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, and cattle. In some embodiments, the non-human animal is a companion animal such as a dog or a cat.

[0045] "Treating" a disease or disorder in a subject or "treating" a patient with a disease or disorder refers to subjecting an individual to drug therapy, such as administration of a drug, such that at least one symptom of the disease or disorder is reduced or stabilized. Typically, when the peptides of the present disclosure are administered therapeutically as a treatment, the peptides are administered to a subject who exhibits one or more symptoms of pathogen-induced lung damage.

[0046] As used herein, the term "variant" refers to a peptide or nucleic acid that differs from a peptide or nucleic acid by the deletion, addition, substitution or side chain modification of one or more amino acids or nucleic acids, but still retains one or more specific functions or biological activities of a naturally occurring molecule. Amino acid substitutions include changes in which amino acids are replaced by different naturally occurring or unconventional amino acid residues. Such substitutions can be classified as "conservative", in which case the amino acid residues contained in the peptide are replaced by another naturally occurring amino acid with similar characteristics in terms of polarity, side chain functionality or size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present invention can also be "non-conservative", in which the amino acid residues present in the peptide are replaced by amino acids with different properties (such as naturally occurring amino acids from different groups) (e.g., using alanine to replace charged or hydrophobic amino acids), or alternatively, wherein naturally occurring amino acids are replaced by unconventional amino acids. In some embodiments, amino acid substitutions are conservative. When used with reference to a polynucleotide or peptide, the term variant is also encompassed, referring to a polynucleotide or peptide that can change the primary, secondary or tertiary structure, respectively, compared to a reference polynucleotide or polypeptide (e.g., compared to a wild-type polynucleotide or polypeptide).

[0047] The term "micronized" refers to a substance having a size of several micrometers.

[0048] The phrase "effective amount" or "therapeutically effective amount" means a dose of a drug or agent sufficient to produce a desired therapeutic outcome. The desired therapeutic outcome can be subjective or objective improvement in the recipient of the dose, reduced infection, reduced inflammation, increased lung growth, increased lung repair, reduced tissue edema, increased DNA repair, reduced apoptosis, reduced tumor size, reduced cancer cell growth rate, reduced metastasis, or any combination of the above.

[0049] As used herein, "excipient" refers to a pharmaceutically acceptable carrier, which is a relatively inert substance used to facilitate the administration or delivery of an active pharmaceutical ingredient (API) into a subject, or to facilitate the processing of an API into a pharmaceutical preparation that can be used pharmaceutically for delivery to a subject's site of action. Excipients or pharmaceutically acceptable carriers include all inactive ingredients in a dosage form other than the active ingredient. Non-limiting examples of excipients include carriers, bulking agents, stabilizers, surfactants, surface modifiers, solubility enhancers, buffers, encapsulating agents, antioxidants, preservatives, nonionic wetting agents or clarifying agents, viscosity enhancers, and absorption enhancers. "Excipient-free" means that the modified Cav-1 peptide pharmaceutical formulation does not contain any excipients.

[0050] The term "physiological conditions" refers to a set of conditions that simulate those of a living subject, including temperature, salt concentration, pH. Conditions include physiologically relevant conditions for in vitro assays. Typically, a physiological buffer contains salts at physiological concentrations and is adjusted to a neutral pH, ranging from about 6 to about 8, from about 6.5 to about 7.8, or from about 7.0 to about 7.5. The physiologically relevant temperature range is from about 25°C to about 38°C or from about 30°C to about 37°C. In some embodiments, the physiological buffer is a phosphate buffer, such as about 10mM to about 100mM phosphate buffer or phosphate buffered saline or simulated body fluid. In some embodiments, the physiological buffer is isotonic (such as about 250mOsm / kg to about 350mOsm / kg) with a soluble material (including but not limited to sodium chloride or glucose). In some embodiments, the physiological buffer may also include a surfactant.

[0051] The phrases "pharmaceutical formulation", "pharmaceutically acceptable formulation" or "pharmacologically acceptable formulation" refer to molecular entities and formulations that do not produce side effects, allergic reactions or other adverse reactions when administered to animals (such as humans) as appropriate. In light of the present disclosure, one skilled in the art will know how to prepare pharmaceutical formulations comprising modified Cav-1 peptides (such as CSP7) or additional active ingredients. In addition, for animal (e.g., human) administration, it should be understood that the preparation should meet bioburden, sterility, pyrogenicity, general safety and / or purity standards as required by the FDA or other recognized regulatory agencies.

[0052] As used herein, "pharmaceutically acceptable carriers" include any and all excipients, processing aids, aqueous solvents (e.g., water, alcohol / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, disintegrants, lubricants, flavor modifiers (e.g., sweeteners, flavoring agents), and the like materials and combinations thereof known to those of ordinary skill in the art. The pH and exact concentrations of the various components in the pharmaceutical formulation are adjusted according to well-known parameters. In some embodiments, the carrier may encapsulate the therapeutic agent, but it itself cannot be consumed or administered to a subject (e.g., a shell capsule encapsulating a dry powder formulation, such as used in a dry powder inhaler). See, e.g., Remington's Pharmaceutical Sciences, 18th ed., 1990, which is incorporated herein by reference.

[0053] II. Caveolin-1 peptide

[0054] Embodiments of the present disclosure provide modified versions of native caveolin-1 (Cav-1) proteins, including but not limited to fragments, derivatives and variants of native Cav-1 proteins. In some embodiments, the modified Cav-1 peptide is a truncation of a native Cav-1 peptide, such as the exemplary peptides shown in Table 2 and / or Table 3.

[0055] Native human Cav-1 is 178 amino acids in length (see SEQ ID NO: 1 in Table 1 below) and has a molecular weight of 22 kDa. Caveolin-1 is an integral membrane protein associated with endocytosis, extracellular matrix organization, cholesterol distribution, cell migration and signal transduction. See, Boscher and Nabi, Adv Exp Med Biol, 2012; 729-29-50.

[0056] Table 1. Amino acid sequences of native human Cav-1 and Cav-1 scaffold domains

[0057]

[0058] In some embodiments, the modified Cav-1 peptide is a Cav-1 scaffold domain (CSD). The CSD consists of amino acids 82-101 of caveolin-1 (see SEQ ID NO: 2 in Table 1 above). The CSD of caveolin-1 plays a key role in caveolin-1 dimerization and regulation of various signaling intermediates (Shetty et al., Am J Respir Cell Mol Biol 2012; 47: 474-83; Fridolfsson et al., FASEB J 2014; 28: 3823-31; Degryse et al., Am J Physiol Cell Mol Physiol 2010; 299: L442-L452; and Egger et al., PLos One, 2013; 8: e63432). The CSD domain of caveolin-1 has been shown to inhibit Wnt signaling, β-catenin-mediated transcription, activation of SRC, EGFR, MEK1, and ERK-2, as well as various other factors (see, Shetty et al., Am J Respir Cell Mol Biol 2012; 47:474-83; Bhandary et al., Am J Physiol Cell Mol Physiol 2012; 302:L463-L473; Bhandary et al., Am J Pathol 2013; 183:131-143; Fridolfsson et al., FASE B J 2014; 28:3823-31; Degryse et al., Am J Physiol Cell Mol Physiol 2010; 299:L442-L452; and Fiddler et al., Ann Am Thorac Soc, 2016; 13:1430-2). For example, the CSD of Cav-1 interferes with the interaction of Cav-1 with SRC kinase and mimics the combined effects of uPA and anti-β1 integrin antibodies. The endogenous CSD domain can form homodimers with other Cav-1 proteins and interact with proteins with the caveolin binding domain sequence (CBD) motif. It is estimated that up to 30% of all endogenous proteins have a CBD motif and it is speculated that the caveolin-1 CSD domain can provide stability to these proteins (see, Marudamuthu et al., Am J Pathol 2015; 185: 55-68).Treatment with CSP20-mer (full-length CSD of caveolin-1) results in reduced lung αSMA and lung epithelial cell apoptosis, reduced collagen deposition, and downregulated expression of pro-fibrotic signaling molecules (see, Bhandary et al., Am J Phys Lung Cell Mol Phys, 2012, 302(5), L463-L473; Razani et al., JBC, 2001, 276(9), 6727-6738; and Lee et al., Biochem Biophys Res Commun, 2007, 359(2): 385-390).

[0059] In some embodiments, the modified Cav-1 peptide is CSP-7. CSP-7 is a seven amino acid fragment of the CSD of human caveolin-1 (see SEQ ID NO: 3 in Table 3 below).

[0060] Exemplary amino acid sequences of modified Cav-1 peptides are shown in Tables 2 and 3 below. Capital letters represent L-amino acids and lowercase letters represent D-amino acids (e.g., lowercase "a" represents D-alanine). The term "Ac" refers to an acetyl group and the term "NH2" refers to an amide group. "O" represents ornithine.

[0061] Table 2. Illustrative modified Cav-1 peptides

[0062]

[0063] Table 3. Additional illustrative modified Cav-1 peptides

[0064]

[0065]

[0066] In some embodiments, the Cav-1 peptide or modified Cav-1 peptide:

[0067] a) consisting of any one of the amino acid sequences of SEQ ID NOs: 2-111;

[0068] b) a core sequence comprising any one of the amino acid sequences of SEQ ID NOs: 2-111; or

[0069] c) a core sequence comprising any one of the amino acid sequences of SEQ ID NOs: 2-111, wherein the core sequence includes one or more amino acid substitutions, insertions, deletions or chemical modifications.

[0070] In some embodiments, the Cav-1 peptide comprises or consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the Cav-1 peptide comprises an amino acid sequence having at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the Cav-1 peptide comprises an amino acid sequence of SEQ ID NO: 1 having one or more mutations relative to SEQ ID NO: 1. For example, in some embodiments, the Cav-1 peptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations relative to SEQ ID NO: 1. In some embodiments, the Cav-1 peptide comprises an amino acid sequence of SEQ ID NO: 1 having 1-5, 5-10, 11-5, 15-20, 10-25, 25-30, or more than 30 mutations. In some embodiments, the modified Cav-1 peptide comprises 1, 2, 3, 4, 5, or more amino acid substitutions, deletions, or insertions relative to the sequence of SEQ ID NO: 1, resulting in a peptide of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 residues.

[0071] In some embodiments, the modified Cav-1 peptide comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 2-111. In some embodiments, the modified Cav-1 peptide comprises an amino acid sequence having at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 2-111. In some embodiments, the modified Cav-1 peptide comprises an amino acid sequence of any one of SEQ ID NOs: 2-111 having one or more mutations relative to any one of SEQ ID NOs: 2-111. For example, in some embodiments, the modified Cav-1 peptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations relative to any one of SEQ ID NOs: 2-111. In some embodiments, the modified Cav-1 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 2-111 with 1-5, 5-10, or 11-15 or more mutations. In some embodiments, the modified Cav-1 peptide comprises an additional 1-5 amino acids at the N-terminus or C-terminus or both termini of any one of SEQ ID NOs: 2-111.

[0072] In some embodiments, the modified Cav-1 peptide comprises or consists of the amino acid sequence of SEQ ID NO:2-10. In some embodiments, the modified Cav-1 peptide comprises an amino acid sequence having at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 85% sequence identity with SEQ ID NO:2-10. In some embodiments, the modified Cav-1 peptide comprises an amino acid sequence of SEQ ID NO:3 having one or more mutations relative to SEQ ID NO:3. For example, in some embodiments, the modified Cav-1 peptide comprises 1, 2, 3, 4, or 5 mutations relative to SEQ ID NO:2-10. In some embodiments, the modified Cav-1 peptide comprises an additional 1-5 amino acids at the N-terminus or C-terminus or at both ends of SEQ ID NO:2-10. In some embodiments, the modified Cav-1 peptide of SEQ ID NO:2-10 comprises an N-terminal and / or C-terminal modification. In some embodiments, the N-terminal modification is acylation. In some embodiments, the C-terminal modification is amidation.

[0073] In some embodiments, the polypeptide of the present disclosure consists of 20 or less amino acids. In some embodiments, the polypeptide comprising the amino acid sequence of SEQ ID NO:3 further comprises at least one amino acid added to the N-terminus; at least one amino acid added to the C-terminus; or at least one amino acid added to the N-terminus and the C-terminus. In these embodiments, the addition within 5 amino acids at each end has an amino acid sequence that is less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 0% (or any value or sub-range therebetween) identical to the continuous amino acid sequence of SEQ ID NO:1. In these embodiments, the addition within 5 amino acids at each end has an amino acid sequence that is less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 0% (or any value or sub-range therebetween) identical to the continuous amino acid sequence of SEQ ID NO:2. In these embodiments, the additions made within 5 amino acids of each terminus have an amino acid sequence that is less than 80% identical to the contiguous amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the additions made within 5 amino acids of each terminus have an amino acid sequence that is less than 60% identical to the contiguous amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the additions made within 5 amino acids of each terminus have an amino acid sequence that is less than 40% identical to the contiguous amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the additions made within 5 amino acids of each terminus have an amino acid sequence that is less than 20% identical to the contiguous amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the polypeptide sequence comprises a peptide containing L-amino acids; the peptide comprises D-amino acids; or the peptide comprises both L-amino acids and D-amino acids.

[0074] In some embodiments, the polypeptides of the present disclosure comprise at least one non-standard amino acid; or the polypeptide comprises two non-standard amino acids. In some embodiments, the non-standard amino acid is ornithine. In some embodiments, the polypeptide further comprises an N-terminal modification; a C-terminal modification; or an N-terminal and a C-terminal modification. In some embodiments, the N-terminal modification is an acylation and / or the C-terminal modification is an amidation.

[0075] In some embodiments, the polypeptide comprises the core sequence ASFTTFTVT.

[0076] In some embodiments, the polypeptide comprises the amino acid sequence FTTFTVT (SEQ ID NO:3), KASFTTFTVTKGS (SEQ ID NO:4), KASFTTFTVTKGS-NH2 (SEQ ID NO:5), aaEGKASFTTFTVTKGSaa (SEQ ID NO:6), aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO:7), Ac-aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO:8), OASFTTFTVTOS (SEQ ID NO:9), or OASFTTFTVTOS-NH2 (SEQ ID NO:10). In some embodiments, the polypeptide consists of an amino acid sequence selected from the group consisting of FTTFTVT (SEQ ID NO: 3), KASFTTFTVTKGS (SEQ ID NO: 4), KASFTTFTVTKGS-NH2 (SEQ ID NO: 5), aaEGKASFTTFTVTKGSaa (SEQ ID NO: 6), aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO: 7), Ac-aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO: 8), OASFTTFTVTOS (SEQ ID NO: 9), or OASFTTFTVTOS-NH2 (SEQ ID NO: 10). In some embodiments, the polypeptide consists of the amino acid sequence Ac-aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO: 8).

[0077] In some embodiments, the modified Cav-1 peptide comprises 1, 2, 3, 4 or more amino acid substitutions, deletions or insertions relative to the sequence of SEQ ID NO: 1, resulting in a polypeptide of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 residues.

[0078] The modified Cav-1 peptides provided in the present disclosure exhibit similar or identical biological activity to the natural Cav-1 peptide in an in vitro or in vivo assay. In some embodiments, the modified Cav-1 peptide inhibits or prevents apoptosis of lung epithelial cells induced by bleomycin in vitro or in vivo, and its activity is at least about 20% of the activity of the natural Cav-1 peptide, or at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, about 95%, 97%, 99% and any range derivable therein, such as, for example, about 70% to about 80%, about 81% to about 90%; or about 91% to about 99%. The modified Cav-1 peptide may have 100% of the activity of the natural Cav-1 peptide or even greater activity than the natural Cav-1 peptide. Assays for testing biological activity (e.g., anti-fibrotic activity, the ability to affect the expression of uPA, uPAR and PAI-1 mRNA or the ability to inhibit the proliferation of lung fibroblasts) are well known in the art.

[0079] The modified Cav-1 peptides disclosed herein are fragments, derivatives or variants of natural Cav-1 peptides. The peptides may be synthetic, recombinant or chemically modified peptides isolated or produced using methods well known in the art. The amino acids at the N-terminus, C-terminus or internally may be modified. The peptides may contain conservative or non-conservative amino acid changes as described below. Changes in the polynucleotides may result in amino acid substitutions, additions, deletions, fusions and truncations in the Cav-1 peptides encoded by the reference sequence. The peptides may also contain insertions, deletions or substitutions of amino acids, including insertions and substitutions of amino acids (and other molecules) that do not normally appear in the peptide sequence that is the basis of the modified variant, such as, but not limited to, insertions of L-amino acids or non-standard amino acids (such as ornithine) that do not normally appear in human proteins.

[0080] A. Replace

[0081] In some embodiments, the modified Cav-1 peptide comprises one or more conservative amino acid substitutions. Conservative amino acid substitutions are produced by replacing one amino acid with another amino acid having similar structural and / or chemical properties, such as replacing leucine with isoleucine or valine, replacing aspartic acid with glutamic acid, or replacing threonine with serine. Thus, conservative substitutions of a particular amino acid sequence refer to substitutions of those amino acids that are not critical to the activity of the peptide or substitutions of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.), such that even substitutions of key amino acids do not reduce the activity of the peptide. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). In some embodiments, individual substitutions, deletions or additions that change, add or delete a single amino acid or a small percentage of amino acids may also be considered conservative substitutions if the change does not reduce the activity of the peptide. Insertions or deletions typically range from about 1 to 6 amino acids.

[0082] In some embodiments, the amino acid that will replace the existing amino acid can be selected based on the position of the existing amino acid, i.e., its exposure to the solvent (i.e., if the amino acid is exposed to the solvent or is present on the outer surface of the peptide or peptide compared to an internally positioned amino acid that is not exposed to the solvent). The selection of such conservative amino acid substitutions is well known in the art, for example, as disclosed in Dordo et al., J. Mol Biol, 1999, 217, 721-739 and Taylor et al., J. Theor. Biol. 119 (1986); 205-218 and S. French and B. Robson, J. Mol. Evol. 19 (1983) 171. Thus, conservative amino acid substitutions may be selected for amino acids on the outside of the protein or peptide (i.e., those exposed to the solvent), for example, but not limited to, the following substitutions may be used: substitution of F for Y, S or K for T, A for P, D or Q for E, D or G for N, K for R, N or A for G, S or K for T, N or E for D, L or V for I, F for Y, S for T or A, K for R, G for N or A, K for R, A for S, K or P.

[0083] In some embodiments, conservative amino acid substitutions suitable for amino acids within the interior of a protein or peptide may be selected, for example, suitable conservative substitutions for amino acids within a protein or peptide (i.e., where the amino acids are not exposed to a solvent) may be used, for example, but not limited to, the following conservative substitutions may be used: wherein Y is substituted with F, T is substituted with A or S, I is substituted with L or V, W is substituted with Y, M is substituted with L, N is substituted with D, G is substituted with A, T is substituted with A or S, D is substituted with N, I is substituted with L or V, F is substituted with Y or L, S is substituted with A or T, and A is substituted with S, G, T, or V. In some embodiments, non-conservative amino acid substitutions are also encompassed within the term variant.

[0084] In some embodiments, amino acid substitutions can be made at one or more positions of a peptide, wherein the substitution is for an amino acid with a similar hydrophilicity. The importance of the hydrophilic amino acid index in conferring biological functions on protein interactions is generally understood in the art. It is recognized that the relative hydrophilicity of the amino acid contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Therefore, such conservative substitutions can be made in a peptide and may only have a minor effect on its activity. As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values ​​have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). These values ​​can be used as a guide, and thus substitution of amino acids with hydrophilicity values ​​within ±2 is preferred, substitution of amino acids within ±1 is particularly preferred, and substitution of amino acids within ±0.5 is even more particularly preferred. Thus, any of the Cav-1 peptides described herein can be modified by substituting amino acids for different but homologous amino acids with similar hydrophilicity values. Amino acids with hydrophilicity within + / -1.0 points or + / -0.5 points are considered homologous.

[0085] In some embodiments, the modified Cav-1 peptide comprises non-naturally occurring amino acids. In some embodiments, the modified Cav-1 peptide comprises a combination of naturally occurring amino acids and non-naturally occurring amino acids, or comprises 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 (or other components of the formulation other than the protease recognition sequence) that are desired. 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 linked drugs and conjugates, improved bioavailability of membrane-permanent complexes, and extended intravascular and interstitial life when such properties are desired. In addition, D-peptides cannot be effectively processed for major histocompatibility complex class II restricted presentation to T helper cells, and are therefore less likely to induce humoral immune responses in the whole organism.

[0086] In addition to the 20 "standard" L-amino acids, D-amino acids or non-standard, modified or unusual amino acids well defined in the art are also contemplated for use in the present disclosure. Phosphorylated amino acids (Ser, Thr, Tyr), glycosylated amino acids (Ser, Thr, Asn), β-amino acids, GABA, ω-amino acids are further contemplated for use in the present disclosure. These include, for example, β-alanine (β-Ala) and other ω-amino acids, such as 3-aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid, etc.; α-aminoisobutyric acid (Aib); ε-aminocaproic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly); ornithine (Orn); citrulline (Cit); tert-butylalanine (t-BuA); tert-butylglycine (t-BuG); N-methylisoleucine (MeIle); phenylglycine (Phg); norleucine (Nle); 4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe( 2-F)); 3-fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); homoarginine (hArg); N-acetyllysine (AcLys); 2,4-diaminobutyric acid (Dbu; Dab); p-aminophenylalanine (Phe(pNH2)); N-methylvaline (MeVal); homocysteine ​​(hCys), homophenylalanine (hPhe) and homoserine (hSer); hydroxyproline (Hyp), homoproline (hPro), N-methylated amino acids and peptoids (N-substituted glycines).

[0087] A peptide or peptide region has a certain percentage (e.g., 80%, 85%, 90% or 95%) of "sequence identity" or "homology" with another sequence, meaning that when aligned, the percentage of bases (or amino acids) that are identical when comparing the two sequences. Such alignments and homology or sequence identity percentages can be determined using software programs known in the art, such as those described in Current Protocols In Molecular Biology (FM Ausubel et al., 1987) 30th Supplement, Section 7.7.18, Table 7.7.1. Default parameters can be used for alignment.

[0088] B. Derivatives

[0089] In some embodiments, the modified Cav-1 peptide is a derivative of a native Cav-1 peptide. As used herein, the term "derivative" refers to a Cav-1 peptide that has been chemically modified by using techniques including, but not limited to, acetylation, ubiquitination, labeling, pegylation (derivatization with polyethylene glycol), lipidation, glycosylation, amidation, cyclization, or addition of other molecules. In some embodiments, the peptide is provided in a cyclic form, for example as a cyclic peptide or as a lactam. Alternatively or additionally, in some embodiments, the peptide is provided as a branched peptide. When a molecule contains an additional chemical moiety that is not normally part of the molecule, the molecule is also a "derivative" of another molecule. Such moieties can change the pH or improve the solubility, absorption, biological half-life, etc. of the molecule. The moiety can alternatively reduce the toxicity of the molecule, eliminate or attenuate any undesirable side effects of the molecule, etc. The moiety capable of mediating such effects is disclosed in Remington's Pharmaceutical Sciences, 18th edition, edited by AR Gennaro, Mack Publ., Easton, PA (1990), which is incorporated herein by reference in its entirety.

[0090] When used in conjunction with "derivative" or "variant", the term "functional" refers to a peptide of the invention having a biological activity (functional or structural) substantially similar to the biological activity of an entity or molecule of which it is a functional derivative or functional variant. The term functional derivative is intended to include fragments, analogs or chemical derivatives of a molecule.

[0091] In some embodiments, the modified Cav-1 peptide may comprise co-translational and post-translational (e.g., C-terminal peptide cleavage) modifications, such as, for example, disulfide bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., by furin or metalloproteinase cleavage), etc., to the extent that such modifications do not affect the function of the modified Cav-1 peptide.

[0092] In some embodiments, the modified Cav-1 peptide may be a "retro-inversopeptide". A "retro-inversopeptide" refers to a peptide in which the direction of the peptide bonds at at least one position is reversed, i.e., the amino and carboxyl termini are reversed relative to the side chains of the amino acids. Thus, a retro-inversopeptide analog has reversed termini and reversed direction of peptide bonds, while maintaining substantially the same side chain topology as in the native peptide sequence. A retro-inversopeptide may contain L-amino acids or D-amino acids, or a mixture of L-amino acids and D-amino acids, with all amino acids being D-isomers at most. A partial retro-inversopeptide analog is a peptide in which only a portion of the sequence is reversed and replaced with enantiomeric amino acid residues. Because the retro-inversopeptide portion of such an analog has reversed amino and carboxyl termini, the amino acid residues flanking the retro-inversopeptide portion are replaced with a-substituted gem-diaminomethane and malonate, respectively, with similar side chains. Retro-inversopeptide forms of cell penetrating peptides have been found to be as effective as the native form in translocation across membranes. The synthesis of retro-inverse peptide analogs is described in Bonelli, F. et al., Int J Pept Protein Res. 24 (6): 553-6 (1984); Verdini, A and Viscomi, GC, J. Chem. Soc. Perkin Trans. 1: 697-701 (1985); and U.S. Pat. No. 6,261,569, which are incorporated herein by reference in their entirety. A solid phase synthesis method for partial retro-inverse peptide analogs has been described (EP97994-B), which is also incorporated herein by reference in its entirety.

[0093] C.Terminal modification

[0094] In some embodiments, the Cav-1 peptides of the present disclosure are modified at their amino or carboxyl termini (when linear). Examples of amino terminal modifications include, for example, N-glycosylation, N-alkylation, N-acetylation, or N-acylation amino acids. Terminal modifications may include pegylation. Examples of carboxyl terminal modifications are C-terminal amidated amino acids. In some embodiments, the peptides are cross-linked or have cross-linking sites (e.g., the modified Cav-1 peptides have cysteinyl residues and thus form cross-linked dimers in vitro or in vivo). In some embodiments, one or more peptidyl bonds are replaced by non-peptidyl bonds; the N-terminus or C-terminus is replaced, and the individual amino acid moieties are modified by treatment with an agent capable of reacting with a selected side chain or terminal residue, etc. The C-terminus or N-terminus or both of the amino acid sequence may be connected to a carboxylic acid functional group or an amine functional group, respectively. In some embodiments, the modified Cav-1 peptides include N-terminal modifications. In some embodiments, the modified Cav-1 peptides include C-terminal modifications. In some embodiments, the modified Cav-1 peptide comprises N-terminal and C-terminal modifications.

[0095] Non-limiting illustrative examples of N-terminal protecting groups include acyl (-CO-R1) and alkoxycarbonyl or aryloxycarbonyl (-CO-O-R1), wherein R1 is an aliphatic group, a substituted aliphatic group, a benzyl group, a substituted benzyl group, an aromatic group or a substituted aromatic group. Specific examples of acyl groups include, but are not limited to, acetyl, (ethyl)-CO-, n-propyl-CO-, isopropyl-CO-, n-butyl-CO-, sec-butyl-CO-, tert-butyl-CO-, hexyl, lauroyl, palmitoyl, myristoyl, stearoyl, oleoyl, phenyl-CO-, substituted phenyl-CO-, benzyl-CO- and (substituted benzyl)-CO-. Examples of alkoxycarbonyl groups and aryloxycarbonyl groups include, but are not limited to, CH3-O-CO-, (ethyl)-O-CO-, n-propyl-O-CO-, isopropyl-O-CO-, n-butyl-O-CO-, sec-butyl-O-CO-, tert-butyl-O-CO-, phenyl-O-CO-, (substituted phenyl)-O-CO-, benzyl-O-CO-, and (substituted benzyl)-O-CO-. To facilitate N-acylation, one to four glycine residues may be present at the N-terminus of the molecule.

[0096] Carboxyl terminal modifications include acylation with carboxylic acids: formic acid, acetic acid, propionic acid, fatty acids (myristic acid, palmitic acid, stearic acid), succinic acid and benzoic acid; carbonylation such as benzyloxycarbonylation (Cbz); acetylation and biotinylation. Amino-terminal modifications include, but are not limited to: (i) acylation with carboxylic acids: formic acid, acetic acid, propionic acid, fatty acids (myristic acid, palmitic acid, stearic acid, etc.), succinic acid, benzoic acid; (ii) carbonylation (such as benzyloxycarbonylation (Cbz)); (iii) biotinylation; (iv) amidation; (v) attachment of dyes such as fluorescein (FITC, FAM, etc.), 7-hydroxy-4-methylcoumarin-3-acetic acid, 7-hydroxycoumarin-3-acetic acid, 7-methoxycoumarin-3-acetic acid and other coumarins; rhodamine (5-carboxyrhodamine 110 or 6G, 5(6)-TAMRA, ROX); N-[4-(4-dimethylamino)phenylazo]benzoic acid (Dabcyl), 2,4-dinitrobenzene (Dnp), 5-dimethylaminonaphthalene-1-sulfonic acid (Dansyl) and other dyes; and (vi) pegylation.

[0097] The carboxyl group at the C-terminus of the peptide can be protected, for example, by including but not limited to amides (i.e., the hydroxyl group at the C-terminus is replaced by -NH2, -NHR2 and -NR2R3) or esters (i.e., the hydroxyl group at the C-terminus is replaced by -OR2) groups. R2 and R3 are optionally independently aliphatic groups, substituted aliphatic groups, benzyl groups, substituted benzyl groups, aryl groups or substituted aryl groups. In addition, R2 and R3 together with nitrogen-atoms can optionally form a C4 to C8 heterocycle with about 0-2 additional heteroatoms (such as nitrogen, oxygen or sulfur). Non-limiting examples of heterocycles include but are not limited to piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl or piperazinyl. Examples of C-terminal protecting groups include, but are not limited to, -NH2, -NHCH3, -N(CH3)2, -NH(ethyl), -N(ethyl)2, -N(methyl)(ethyl), -NH(benzyl), -N(C1-C4 alkyl)(benzyl), -NH(phenyl), -N(C1-C4 alkyl)(phenyl), -OCH3, -O-(ethyl), -O-(n-propyl), -O-(n-butyl), -O-(isopropyl), -O-(sec-butyl), -O-(tert-butyl), -O-benzyl and -O-phenyl.

[0098] D. Side chain modification

[0099] In some embodiments, the modified Cav-1 peptides of the present disclosure comprise modified amino acid side chains. Non-limiting examples of modification include carboxymethylation, acylation, phosphorylation, glycosylation, or fatty acylation. An ether bond may optionally be used to connect a serine or threonine hydroxyl group to a hydroxyl group of a sugar. An amide bond may optionally be used to connect a glutamic acid or aspartic acid carboxyl group to an amino group on a sugar (Gang and Jeanloz, Advances in Carbohydrate Chemistry and Biochemistry, Vol. 43, Academic Press (1985); Kunz, Ang. Chem. Int. Ed. English 26: 294-308 (1987)). Acetal bonds and ketal bonds may also optionally be formed between amino acids and carbohydrates. Fatty acid acyl derivatives can optionally be prepared, for example, by acylation of free amino groups (eg, lysine) (Toth et al., Peptides: Chemistry, Structure and Biology, Rivier and Marshal, eds., ESCOMPubl., Leiden, 1078-1079 (1990)).

[0100] As used herein, the term "chemically modified" when referring to the modified Cav-1 peptides of the present disclosure refers to peptides in which at least one amino acid residue is modified by natural processes (such as processing or other post-translational modifications) or by chemical modification techniques well known in the art. Examples of many known modifications generally include, but are not limited to: acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of a lipid or lipid derivative, methylation, myristylation, pegylation, prenylation, phosphorylation, ubiquitination, or any similar process.

[0101] Other types of modifications optionally include the addition of cycloalkane moieties to biomolecules such as proteins as described in PCT Application No. WO 2006 / 050262, which is hereby incorporated by reference in its entirety. These moieties are designed for biomolecules and can optionally be used to impart various properties to proteins.

[0102] In addition, any point on the protein can be optionally modified. For example, PEGylation of glycosylated moieties on the protein can be optionally performed as described in PCT Application No. WO 2006 / 050247, which is hereby incorporated by reference in its entirety. Optionally, one or more polyethylene glycol (PEG) groups can be added to the O-linked and / or N-linked glycosylation. The PEG group can optionally be branched or linear. Optionally, any type of water-soluble polymer can be attached to the glycosylation site on the protein via a glycosyl linker.

[0103] Covalent modifications of the modified Cav-1 peptides disclosed herein are included within the scope of the invention. Other types of peptide covalent modifications are introduced into the molecule by reacting targeted amino acid residues with an organic derivatizing agent capable of reacting with selected side chains or the N- or C-terminal residues.

[0104] Cysteinyl residues are most commonly reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxamidomethyl derivatives. Cysteinyl residues can also be derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidazolyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyl disulfide, p-chloromercurybenzoate, 2-chloromercury-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0105] Histidyl residues are derivatized by reaction with diethyl pyrocarbonate at pH 5.5-7.0, as this agent is relatively specific for the histidyl side chain. P-Bromophenacyl bromide is also useful; in some embodiments, the reaction is carried out in 0.1 M sodium cacodylate at pH 6.0.

[0106] Lysyl and amino terminal residues react with succinic acid or other carboxylic anhydrides. Derivatization with these agents has the effect of reversing the charge of lysine residues. Other suitable reagents for derivatizing α-amino residues include imidoesters such as pyridine imide acid methyl ester, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactions with glyoxylic acid.

[0107] Arginyl residues are modified by reaction with one or several conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin.

[0108] Due to the high pKa of the guanidine functional group, derivatization of arginine residues requires that the reaction be performed under alkaline conditions. In addition, these reagents react with lysine groups as well as with the arginine epsilon-amino group.

[0109] Tyrosyl residues can be specifically modified, and of particular interest is the introduction of spectral labels into tyrosyl residues by reaction with aromatic diazo compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using 125I or 131I to prepare labeled peptides for radioimmunoassay.

[0110] The carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimide (RN=C=N-R'), wherein R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-aza-4,4-dimethylpentyl)carbodiimide. In addition, aspartyl and glutamyl residues are converted into asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0111] Derivatization using bifunctional agents can be used for crosslinking to a water-insoluble support matrix or surface for use in methods of purifying anti-CHF antibodies, and vice versa. Commonly used crosslinking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters (e.g., esters with 4-azidosalicylic acid), homobifunctional imidoesters (including disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate)), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propionimidate can generate photoactivated intermediates that are capable of forming crosslinks in the presence of light. Alternatively, protein immobilization is performed using reactive water-insoluble matrices such as cyanogen bromide activated carbohydrates and reactive substrates as described in US Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440.

[0112] Glutaminyl residues and asparaginyl residues are usually deamidated to the corresponding glutamyl residues and aspartyl residues, respectively. These residues are deamidated under neutral or alkaline conditions. The deamidated forms of these residues fall within the scope of the present invention.

[0113] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86

[1983] ), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0114] E. End Capping

[0115] In some embodiments, the modified Cav-1 peptide is terminated at its N-terminus and C-terminus with an acyl group (abbreviated as "Ac") and an amide group (abbreviated as "Am"), respectively, such as an acetyl group (CH3CO-) at the N-terminus and an amide group (-NH2) at the C-terminus. In some embodiments, the modified Cav-1 peptide is terminated at its N-terminus with an acyl group, such as an acetyl group (CH3CO-) at the N-terminus. In some embodiments, the modified Cav-1 peptide is terminated at its C-terminus with an amide group, such as an amide (-NH2) at the C-terminus.

[0116] In some embodiments, the modified Cav-1 peptide is capped at its N-terminus. A wide range of N-terminal capping functionalities are contemplated, such as linkages to the terminal amino group, for example:

[0117] Formyl;

[0118] Alkanoyl having 1 to 10 carbon atoms, such as acetyl, propionyl, butyryl;

[0119] an alkenoyl group having 1 to 10 carbon atoms, such as hex-3-enoyl;

[0120] an alkynoyl group having 1 to 10 carbon atoms, such as hex-5-ynoyl;

[0121] Aroyl, such as benzoyl or 1-naphthoyl;

[0122] heteroaroyl, such as 3-pyrrolyl or 4-quinolinyl;

[0123] Alkylsulfonyl, such as methylsulfonyl;

[0124] Arylsulfonyl, such as benzenesulfonyl or sulfonyl;

[0125] heteroarylsulfonyl, such as pyridine-4-sulfonyl;

[0126] Substituted alkanoyl having 1 to 10 carbon atoms, such as 4-aminobutyryl;

[0127] Substituted alkenoyl having 1 to 10 carbon atoms, such as 6-hydroxy-hex-3-enoyl;

[0128] Substituted alkynoyl having 1 to 10 carbon atoms, such as 3-hydroxy-hex-5-ynoyl;

[0129] Substituted aroyl, such as 4-chlorobenzoyl or 8-hydroxy-naphthalen-2-yl;

[0130] Substituted heteroaroyl, such as 2,4-dioxo-1,2,3,4-tetrahydro-3-methyl-quinazolin-6-yl;

[0131] Substituted alkylsulfonyl, such as 2-aminoethanesulfonyl;

[0132] Substituted arylsulfonyl, such as 5-dimethylamino-1-naphthalenesulfonyl;

[0133] Substituted heteroarylsulfonyl, such as 1-methoxy-6-isoquinolinesulfonyl;

[0134] Carbamoyl or thiocarbamoyl;

[0135] Substituted carbamoyl (R'-NH-CO) or substituted thiocarbamoyl (R'-NH-CS), wherein R' is alkyl, alkenyl, alkynyl, aryl, heteroaryl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted aryl or substituted heteroaryl;

[0136] Substituted carbamoyl (R'-NH-CO) and substituted thiocarbamoyl (R'-NH-CS), wherein R' is alkanoyl, alkenoyl, alkynyl, aroyl, heteroaroyl, substituted alkanoyl, substituted alkenoyl, substituted alkynyl, substituted aroyl or substituted heteroaroyl, all of which are as defined above.

[0137] In some embodiments, the modified Cav-1 peptide is capped at its C-terminus. The C-terminal capping functional group may be in an amide bond or an ester bond with the terminal carboxyl group. The capping functional group providing the amide bond is designated as NR 1 R 2 , where R 1 and R 2 may be independently derived from the following groups: hydrogen;

[0138] Such as alkyl having 1 to 10 carbon atoms, such as methyl, ethyl, isopropyl;

[0139] Such as alkenyl having 1 to 10 carbon atoms, such as prop-2-enyl;

[0140] Such as alkynyl having 1 to 10 carbon atoms, such as prop-2-ynyl;

[0141] Substituted alkyl groups having 1 to 10 carbon atoms, such as hydroxyalkyl, alkoxyalkyl, mercaptoalkyl, alkylthioalkyl, haloalkyl, cyanoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkanoylalkyl, carboxyalkyl, carbamoylalkyl;

[0142] Substituted alkenyl having 1 to 10 carbon atoms, such as hydroxyalkenyl, alkoxyalkenyl, mercaptoalkenyl, alkylthioalkenyl, haloalkenyl, cyanoalkenyl, aminoalkenyl, alkylaminoalkenyl, dialkylaminoalkenyl, alkanoylalkenyl, carboxyalkenyl, carbamoylalkenyl;

[0143] Substituted alkynyl groups having 1 to 10 carbon atoms, such as hydroxyalkynyl, alkoxyalkynyl, mercaptoalkynyl, alkylthioalkynyl, haloalkynyl, cyanoalkynyl, aminoalkynyl, alkylaminoalkynyl, dialkylaminoalkynyl, alkanoylalkynyl, carboxyalkynyl, carbamoylalkynyl;

[0144] Aroylalkyl having up to 10 carbon atoms, such as benzoyl or 2-benzoylethyl;

[0145] Aryl, such as phenyl or 1-naphthyl;

[0146] heteroaryl, such as 4-quinolinyl;

[0147] Alkanoyl having 1 to 10 carbon atoms, such as acetyl or butyryl;

[0148] Aroyl groups such as benzoyl;

[0149] heteroaroyl, such as 3-quinolinyl;

[0150] OR' or NR'R", wherein R' and R" are independently hydrogen, alkyl, aryl, heteroaryl, acyl, aroyl, sulfonyl, sulfinyl, or SO2-R"' or SO-R"', wherein R"' is substituted or unsubstituted alkyl, aryl, heteroaryl, alkenyl or alkynyl.

[0151] The end-capping functional group providing the ester bond is designated as OR, where R can be: alkoxy; aryloxy; heteroaryloxy; aralkyloxy; heteroaralkyloxy; substituted alkoxy; substituted aryloxy; substituted heteroaryloxy; substituted aralkyloxy; or substituted heteroaralkyloxy.

[0152] In some embodiments, the N-terminal or C-terminal capping functional group or both have such a structure that the capped molecule acts as a prodrug (a pharmacologically inactive derivative of a parent drug molecule), undergoes spontaneous or enzymatic conversion in vivo to release the active drug, and has improved delivery characteristics relative to the parent drug molecule (Bundgaard H, ed.: Design of Prodrugs, Elsevier, Amsterdam, 1985).

[0153] Judicious choice of capping groups allows the addition of other activities to the peptide. For example, the presence of a thiol group attached to an N-terminal cap or a C-terminal cap will allow the derivatized peptide to be conjugated to other molecules.

[0154] F. Polymerization

[0155] Embodiments of the present disclosure also include longer peptides constructed from repeating units of modified Cav-1 peptides. In some embodiments, the peptide multimers contain different peptide combinations. In some embodiments, the multimeric peptides are prepared by chemical synthesis or by recombinant DNA technology as discussed herein. When produced by chemical synthesis, in some embodiments, the oligomers have 2-5 repeats of the core peptide sequence, and the total number of amino acids in the multimer should not exceed about 160 residues or not more than 100 residues (or its equivalent, when including a linker or spacer).

[0156] In some embodiments, the modified Cav-1 peptide is a multimer comprising at least two peptides of the present disclosure. In some embodiments, the first peptide of at least two peptides is substantially identical to the second peptide of at least two peptides. In some embodiments, the first peptide of at least two peptides is not identical to the second peptide of at least two peptides.

[0157] G. Peptidomimetics

[0158] In some embodiments, the modified Cav-1 peptide is a peptidomimetic compound that mimics the biological effects of the natural Cav-1 peptide. In some embodiments, the peptidomimetic agent is a non-natural peptide or non-peptide agent that reconstructs the steric properties of the binding element of the natural Cav-1 peptide so that it has the binding activity and biological activity of the natural Cav-1 peptide. Similar to the natural Cav-1 peptide or peptide multimer, the peptidomimetic will have a binding surface (which interacts with any ligand bound by the natural Cav-1 peptide) and a non-binding surface.

[0159] In some embodiments, the present disclosure also includes modified Cav-1 peptides that retain some of the peptide characteristics. For example, any proteolytically unstable bonds within the Cav-1 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 rest of the molecule retains its peptide properties.

[0160] Many bioactive peptides / peptides such as opioid peptides, VIP, thrombin, HIV protease, etc. have been described as peptidomimetic compounds (whether agonists, substrates or inhibitors). Methods for designing and preparing peptidomimetic compounds are known in the art (Hruby, VJ, Biopolymers 33: 1073-1082 (1993); Wiley, RA et al., Med. Res. Rev. 13: 327-384 (1993); Moore et al., Adv. in Pharmacol 33: 91-141 (1995); Giannis et al., Adv. in Drug Res. 29: 1-78 (1997)). Some mimics of secondary structure are described in Johnson et al., In: Biotechnology and Pharmacy, Pezzuto et al., Chapman and Hall (eds.), NY, 1993. These methods are used to prepare peptidomimetics that have at least the binding capacity and specificity of native Cav-1 peptides and in some embodiments 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.

[0161] For example, such peptidomimetics can be identified by checking the three-dimensional structure of the peptide of the present invention free or compound binding with ligand (e.g., soluble uPAR or its fragment). Alternatively, the structure of the peptide of the present invention bound to its ligand can be obtained by nuclear magnetic resonance spectroscopy. More knowledge of the interactive stereochemistry of the peptide and its ligand or receptor will allow the rational design of such peptidomimetics. In the absence of a ligand, the peptide of the present invention or the structure of the peptide can also provide a scaffold for designing a simulated molecule.

[0162] H.PEGylation

[0163] In some embodiments, the modified Cav-1 peptides of the present disclosure are conjugated to heterologous peptide segments or polymers such as polyethylene glycol. In some embodiments, the modified Cav-1 peptides are linked to PEG to increase the hydrodynamic radius of the enzyme and thus increase serum persistence. In some embodiments, the modified Cav-1 peptides are conjugated to any targeting agent, such as a ligand having the ability to specifically and stably bind to an external receptor (see, e.g., U.S. Patent Publication No. 2009 / 0304666).

[0164] In some embodiments, the present disclosure provides methods and formulations related to PEGylation of Cav-1 peptides. PEGylation is the process of covalently attaching a poly(ethylene glycol) polymer chain to another molecule (usually a drug or therapeutic protein). PEGylation is conventionally achieved by incubating a reactive derivative of PEG with a target macromolecule. Covalent attachment of PEG to a drug or therapeutic protein can "mask" the agent from the host's immune system (reducing immunogenicity and antigenicity), or increase the hydrodynamic size (size in solution) of the agent, thereby extending its circulation time by reducing renal clearance. PEGylation can also provide water solubility to hydrophobic drugs and proteins.

[0165] The first step in PEGylation is to appropriately functionalize the PEG polymer at one or both termini. PEGs activated with the same reactive moiety at each terminus are referred to as "homobifunctional," while PEG derivatives are referred to as "heterobifunctional" or "heterofunctional" if the functional groups present are different. Chemically active or activated derivatives of PEG polymers are prepared to attach PEG to the desired molecule.

[0166] The selection of suitable functional groups for the PEG derivative is based on the types of available reactive groups on the modified Cav-1 peptide to which the PEG is to be coupled. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. N-terminal amino and C-terminal carboxylic acid groups may also be used.

[0167] The technique used to form the first generation of PEG derivatives is generally to react the PEG polymer with a group that can react with a hydroxyl group (usually anhydrides, acid chlorides, chloroformates and carbonates). In the second generation of PEGylation chemistry, more effective functional groups such as aldehydes, esters, amides, etc. can be used for conjugation.

[0168] As the applications of PEGylation become more advanced and complex, the demand for heterobifunctional PEGs for conjugation is also increasing. These heterobifunctional PEGs are very useful in connecting two entities where a hydrophilic, flexible and biocompatible spacer is required. The preferred end groups of heterobifunctional PEGs are maleimide, vinyl sulfone, pyridyl disulfide, amine, carboxylic acid and N-hydroxysuccinimide (NHS) ester.

[0169] The most common modifiers or linkers are based on methoxy PEG (mPEG) molecules. Their activity depends on the addition of protein modifying groups at the alcohol end. In some embodiments, 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.

[0170] Proteins are usually PEGylated at nucleophilic sites such as unprotonated thiols (cysteinyl residues) or amino groups. Examples of cysteinyl-specific modification agents include PEG maleimide, PEG iodoacetate, PEG thiol and PEG vinyl sulfone. Under mild conditions and at neutral to weakly alkaline pH, all four examples are strongly cysteinyl-specific, but each has some disadvantages. The thioether formed with maleimide may be somewhat unstable under alkaline conditions, so the formulation options using this joint may have some limitations. The thiocarbamate linkage formed with iodine PEG is more stable, but free iodine can modify tyrosine residues under some conditions. PEG thiol forms a disulfide bond with protein thiol, but this linkage may also be unstable under alkaline conditions. Compared with maleimide and iodine PEG, the reactivity of PEG-vinyl sulfone is relatively slow; however, the thioether linkage formed is quite stable. Its slower reaction rate can also make the PEG-vinyl sulfone reaction easier to control.

[0171] Site-specific PEGylation is rarely performed at native cysteinyl residues because these residues are usually in the form of disulfide bonds or are required for biological activity. On the other hand, site-directed mutagenesis can be used to incorporate cysteinyl PEGylation sites for thiol-specific linkers. Cysteine ​​mutations must be designed to make them accessible to PEGylation reagents and still have biological activity after PEGylation.

[0172] Amine-specific modifiers include PEG NHS esters, PEG tresylate, PEG aldehydes, PEG isothiocyanates, and several others. All reactions are performed under mild conditions and are very specific for amino groups. PEG NHS esters may be one of the more reactive agents; however, their high reactivity can make PEGylation reactions difficult to control on a large scale. PEG aldehydes form imines with amino groups and are then reduced to secondary amines with sodium cyanoborohydride. Unlike sodium borohydride, sodium cyanoborohydride will not reduce disulfide bonds. However, this chemical is highly toxic and must be handled with caution, especially at lower pH where it becomes volatile.

[0173] Site-specific PEGylation can be a challenge due to the multiple lysine residues on most proteins. Because these reagents react with unprotonated amino groups, it is possible to direct PEGylation to amino groups with lower pK by reacting at lower pH. Typically, the pK of the α-amino group is 1-2 pH units lower than the ε-amino group of lysine residues. By PEGylating the molecule at pH 7 or lower pH, high selectivity for the N-terminus can usually be achieved. 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 generally outweigh the significant loss of in vitro biological activity, resulting in a product with greater in vivo biological activity regardless of the chemical method of PEGylation.

[0174] When developing a PEGylation procedure, several parameters should be considered. Fortunately, there are usually no more than four or five key parameters. The "experimental design" approach to optimizing PEGylation conditions can be very useful. For thiol-specific PEGylation reactions, the parameters that should be considered include: protein concentration, PEG to protein ratio (on a molar basis), temperature, pH, reaction time, and in some cases, excluding oxygen. (Oxygen can contribute to the formation of intermolecular disulfides in proteins, which will reduce the yield of PEGylated products). For amine-specific modifications, the same factors (except oxygen) should be considered, except that pH can be more critical, especially when targeting the N-terminal amino group.

[0175] For both amine-specific and thiol-specific modifications, the reaction conditions can affect the stability of the protein. This can limit temperature, protein concentration, and pH. In addition, the reactivity of the PEG linker should be known before starting the PEGylation reaction. For example, if the PEGylating agent is only 70% active, the amount of PEG used should ensure that only active PEG molecules are counted in the stoichiometry of the protein-PEG reaction.

[0176] I. Fusion Protein

[0177] In some embodiments, the present disclosure provides a fusion protein of a modified Cav-1 peptide. For example, the fusion may employ a leader sequence from another species to allow the protein to be recombinantly expressed in a heterologous host. The fusion protein may comprise a half-life extender. Another useful fusion includes adding a protein affinity tag, such as a serum albumin affinity tag or six histidine residues, or adding an immunologically active domain, such as an antibody epitope (including those that are cleavable), to facilitate purification of the fusion protein. Non-limiting affinity tags include polyhistidine, chitin binding protein (CBP), maltose binding protein (MBP), and glutathione-S-transferase (GST). In some embodiments, the modified Cav-1 peptide comprises a heterologous peptide or protein connected at the N-terminus and / or C-terminus. In some embodiments, the heterologous peptide or protein is a leader sequence, a half-life extender, a protein affinity tag, or an immunologically active domain.

[0178] In some embodiments, the modified Cav-1 peptide is linked to a peptide that increases in vivo half-life, such as peptides (Schellenberger et al., 2009), IgG Fc domain, albumin or albumin binding peptides.

[0179] Methods for producing fusion proteins are well known to those skilled in the art.Such proteins can be produced, for example, by synthesizing the complete fusion protein de novo, or by attaching a DNA sequence encoding a heterologous domain followed by expression of the complete fusion protein.

[0180] The production of fusion proteins that restore the functional activity of the parent proteins can be facilitated by connecting the genes to a bridging DNA segment encoding a peptide linker that is spliced ​​between the tandemly linked peptides. The linker will be of sufficient length to allow the resulting fusion protein to fold properly.

[0181] i) Connectors

[0182] In some embodiments, the modified Cav-1 peptides are chemically conjugated using bifunctional cross-linking reagents or fused at the protein level using peptide linkers.

[0183] Bifunctional cross-linking reagents have been widely used for various purposes, including preparation of affinity matrices, modification and stabilization of various structures, identification of ligand and receptor binding sites, and structural studies. In some embodiments, a peptide linker such as a Gly-Ser linker is used to link the modified Cav-1 peptides of the present disclosure.

[0184] Homobifunctional reagents carrying two identical functional groups have been shown to be very effective in inducing crosslinking between identical and different macromolecules or macromolecular subunits and the connection of peptide ligands to their specific binding sites. Heterobifunctional reagents contain two different functional groups. By utilizing the differential reactivity of two different functional groups, crosslinking can be selectively and sequentially controlled. Bifunctional crosslinking reagents can be divided according to the specificity of their functional groups (e.g., amino, sulfhydryl, guanidinyl, indolyl, carboxyl specific groups). Among these crosslinking reagents, reagents for free amino groups have become particularly popular because of their commercial availability, ease of synthesis, and the mild reaction conditions that can be applied.

[0185] Most heterobifunctional cross-linking reagents contain a primary amine reactive group and a thiol reactive group. In another example, heterobifunctional cross-linking reagents and methods of using the same are described (U.S. Pat. No. 5,889,155, which is incorporated herein by reference in its entirety). The cross-linking reagent combines a nucleophilic hydrazide residue with an electrophilic maleimide residue, in one example, allowing the coupling of an aldehyde to a free thiol. The cross-linking reagent can be modified to allow the cross-linking of various functional groups.

[0186] Additionally, any other linking / coupling agents and / or mechanisms known to those skilled in the art can be used to combine the modified Cav-1 peptides of the present disclosure, such as, for example, antibody-antigen interactions, avidin-biotin linkages, amide linkages, ester linkages, thioester linkages, ether linkages, thioether linkages, phosphate linkages, phosphoramide linkages, anhydride linkages, disulfide linkages, ionic and hydrophobic interactions, bispecific antibodies and antibody fragments, or combinations thereof.

[0187] In some embodiments, the modified Cav-1 peptide comprises a cross-linker that has reasonable stability in blood. Many types of disulfide-containing linkers are known that can be successfully used to conjugate targeting agents and therapeutic / prophylactic agents. Linkers containing disulfide bonds that are sterically hindered may demonstrate greater stability in vivo. Therefore, in some embodiments, the modified Cav-1 peptide comprises a sterically hindered cross-linker.

[0188] In addition to sterically hindered cross-linkers, non-sterically hindered linkers may also be used, depending on the circumstances. In some embodiments, the modified Cav-1 peptide comprises a non-sterically hindered cross-linker. Other useful cross-linkers that are believed not to contain or generate protected disulfides include SATA, SPDP, and 2-iminothiolane (Wawrzynczak and Thorpe, 1987). The use of such cross-linkers is well known in the art.

[0189] In some embodiments, the modified Cav-1 peptide comprises a flexible linker.

[0190] Once chemical conjugation occurs, the modified Cav-1 peptide is typically purified to separate the conjugate from non-conjugated agents and other contaminants. A number of purification techniques are available to provide the conjugate of sufficient purity to be clinically useful.

[0191] Purification methods based on size separation (such as gel filtration, gel permeation or high performance liquid chromatography) will usually be the most commonly used. Other chromatographic techniques, such as blue-agarose separation (Blue-Sepharose separation) can also be used. Conventional methods for purifying fusion proteins from inclusion bodies can be useful, such as using weak detergents such as sodium N-lauroyl-sarcosinate (SLS).

[0192] ii) Cell penetrating peptides and membrane translocation peptides

[0193] In some embodiments, the modified Cav-1 peptide comprises a cell binding domain or a cell penetrating peptide (CPP). As used herein, the terms "cell penetrating peptide," "membrane translocation domain," and "protein transduction domain" are used interchangeably and refer to a segment of a peptide sequence that allows the peptide to pass through a cell membrane (e.g., the plasma membrane in the case of eukaryotic cells). Examples of CPPs include, but are not limited to, segments derived from HIV binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22-derived or similar peptides, HSV VP22 (herpes simplex), protegrin I, MAP, KALA or protein transduction domain (PTD), PpT620, proline-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptides, Pep-1, L-oligomers, calcitonin peptides, Antennapedia-derived peptides (particularly from Drosophila Antennapedia), pAntp, T1 (TKIESLKEHG, SEQ ID NO: 115), T2 (TQIENLKEKG, SEQ ID NO: 116), 26 (AALEALAEALEALAEALEALAEAAAA, SEQ ID NO: 117), INF7 (GLFEAIEGFIENGWEGMIEGWYGCG, SEQ ID NO: 118), NO:118) pIsl, FGF, lactoferrin, transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptide, SAP or histone.

[0194] CPP generally has the following amino acid composition: either containing relatively high positively charged amino acids, such as lysine or arginine, or having a sequence containing polar / charged amino acids and non-polar hydrophobic amino acids alternating patterns. These two types of structures are respectively referred to as polycationic or amphipathic. Typically, CPP is a peptide of 8 to 50 residues that can pass through the cell membrane and enter most cell types. Frankel and Pabo described the ability of the transactivating transcription activator from human immunodeficiency virus type 1 (HIV-TAT) to penetrate into cells (Frankel, AD and COPabo, Cellular uptake of thetat protein from human immunodeficiency virus. Cell, 1988. 55 (6): 1189-93 pages). In 1991, the transduction of the homeodomain (DNA binding domain) from Drosophila antennapedia into neural cells was also described (Joliot, A. et al., Antennapediahomeoboxpeptideregulatesneural morphogenesis. Proc Natl Acad Sci USA, 1991. 88(5): p. 1864-8). In 1994, the first 16-mer peptide CPP, called penetratin (RQIKIWFQNRRMKWKK, SEQ ID NO: 113), was characterized from the third helix of the homeodomain of the Drosophila Antennapedia homeobox gene product (Derossi, D. et al., The third helix of the Antennapedia homeodomain translocates through biological membranes. J Biol Chem, 1994. 269(14): p. 10444-50), followed by the identification of the minimal TAT domain required for protein transduction in 1998 (e.g., GRKKRRQRRRPPQ, SEQ ID NO: 112) (Vives, E., P. Brodin, and B. Lebleu, Atruncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem, 1994. 269(14): p. 10444-50). Chem, 1997. 272(25): p. 16010-7).In the past two decades, dozens of peptides from different sources have been described, including viral proteins, such as herpes virus VP22 (Elliott, G. and P. O'Hare, Intercellular trafficking and protein delivery by a herpes virus structural protein. Cell, 1997. 88 (2): pp. 223-33), or from venom, such as melittin (GIGAVLKVLTTGLPALISWIKRKRQQ, SEQ ID NO: 114) (Dempsey, CE, The actions of melittin on membranes. Biochim Biophys Acta, 1990. 1031 (2): pp. 143-61), mast cell degranulating peptide (mastoporan) (Konno, K. et al., Structure and biological activities of eumenine mastoparan-AF (EMP-AF), a new mast cell degranulating peptide in the venom of the solitary wasp (Anterhynchium flavomarginatummicado). Toxicon, 2000. 38(11): 1505-15), maurocalcine (Esteve, E. et al., Transduction of the scorpiontoxin maurocalcine into cells. Evidence that the toxin crosses the plasma membrane. J Biol Chem, 2005. 280(13): p. 12833-9), crotamine (Nascimento, FD et al., Crotamine mediates gene delivery into cells through the binding to heparan sulfate proteoglycans. J Biol Chem, 2007. 282(29): p. 21349-60) or buforin (Kobayashi, S. et al., Membrane translocation mechanism of the antimicrobial peptide buforin 2. Biochemistry, 2004. 43(49): p. 15610-6).Synthetic CPPs have also been designed, including polyarginine (R8, R9, R10 and R12) (Futaki, S. et al., Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J Biol Chem, 2001. 276 (8): p. 5836-40) or transport peptides (Pooga, M. et al., Cell penetration by transportan. FASEB J, 1998. 12 (1): p. 67-77). Any of the above CPPs can be used in the modified Cav-1 peptides disclosed herein. Many other CPPs described in Milletti F. (Drug Discov Today 17 (15-16): 850-60, 2012) can also be used in the modified Cav-1 peptides disclosed herein.

[0195] III. Pharmaceutical Preparations

[0196] In some embodiments, the present disclosure relates to pharmaceutical formulations comprising a modified Cav-1 peptide. In some embodiments, the pharmaceutical formulations as described herein can be used to treat or prevent a disease, injury, or infection as described herein.

[0197] In some embodiments, the pharmaceutical formulations comprising the modified Cav-1 peptides are formulated for administration intravenously, intrathecally, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intravesically, intraarticularly, intralesionally, intrarectally, intramuscularly, subcutaneously, mucosally, orally, topically, locally, by inhalation (e.g., inhalation of aerosolized or dry powder formulations), by injection, by infusion, by continuous infusion, by direct local perfusion of target cells with lipid compositions (e.g., liposomes) (via catheter, via lavage), or by other methods known to those of ordinary skill in the art, or any combination of the foregoing (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, 1990, which is incorporated herein by reference). In some embodiments, the pharmaceutical formulations are formulated for intravenous, intrathecal, subcutaneous, and / or intraperitoneal administration.

[0198] In one aspect, the present disclosure provides a pharmaceutical formulation comprising a modified Cav-1 peptide containing a core sequence FTTFTVT and sucrose acetate isobutyrate (SAIB). In some embodiments, the pharmaceutical formulation comprises a modified Cav-1 peptide comprising a core sequence ASFTTFTVT and SAIB. In some embodiments, SAIB is a mixed ester of sucrose esterified with two acetate and six isobutyrate groups, wherein the ester is completely non-crystalline and has a viscosity of more than 100,000 cP at 30°C.

[0199] In some embodiments, the formulation comprising a modified Cav-1 peptide is selected from any of the polypeptides disclosed herein. In some embodiments, the formulation comprises SAIB.

[0200] In some embodiments, SAIB is mixed with a modified Cav-1 peptide to prepare a suspension. In some embodiments, SAIB and a modified Cav-1 peptide are mixed at a temperature between room temperature and about 37°C. In some embodiments, SAIB and a modified Cav-1 peptide are mixed at a temperature of about 37°C or below. In some embodiments, SAIB and a modified Cav-1 peptide may be mixed with one or more biocompatible solvents. In some embodiments, SAIB and a modified Cav-1 peptide may be mixed with one or more biocompatible solvents selected from water, ethanol, isopropanol, saline or phosphate buffered saline (PBS). In some embodiments, the use of different solvents can change the viscosity of the SAIB and Cav-1 peptide solutions. In some embodiments, the modified Cav-1 peptide formulation is an injectable formulation.

[0201] In some embodiments, SAIB preparation further comprises the first solvent, and the first solvent is selected from acetone, benzyl alcohol, butanediol, caprolactam, caprolactone, dimethyl sulfoxide, ethanol, ethyl acetate, ethyl lactate, glycerine, glycerine formal, tetrahydrofuran polyethylene glycol ether, tetraethylene glycol, N-methyl-2-pyrrolidone, polyethylene glycol, methoxy polyethylene glycol, alkoxy polyethylene glycol, propylene carbonate, 2-pyrrolidone, triacetin, triethyl citrate or its combination.In some embodiments, the first solvent is N-Methyl pyrrolidone (NMP), dehydrated alcohol and / or ethyl acetate.In some embodiments, the first solvent is NMP and / or ethyl acetate.

[0202] In some embodiments, SAIB is present in the first solvent at about 50% w / w to about 95% w / w, including any value or subrange therebetween. In some embodiments, SAIB is present in the first solvent at about 50% w / w, about 51% w / w, about 52% w / w, about 53% w / w, about 54% w / w, about 55% w / w, about 56% w / w, about 57% w / w, about 58% w / w, about 59% w / w, about 60% w / w, about 61% w / w, about 62% w / w, about 63% w / w, about 64% w / w, about 65% w / w, about 66% w / w, about 67% w / w, about 68% w / w, about 69% w / w, about 70% w / w, about 71% w / w, about 72% w / w, about 73% w / w, about 74% w / w, about 75% w / w, about 76% w / w, about 77% w / w, about 78% w / w, about 79% w / w, about 80% w / w, about 81% w / w, about 82% w / w, about 83% w / w, about 84% w / w, about 85% w / w, about 86% w / w, about 87% w / w, about 88% w / w, about 89% w / w, about 90% w / w, about 91% w / w, about 92% w / w, about 93% w / w, about 94% w / w In some embodiments, SAIB is present in the first solvent with about 70% w / w to about 90% w / w, including any value or sub-range therebetween. In some embodiments, SAIB is present in the first solvent with about 80% w / w.

[0203] In some embodiments, the modified Cav-1 peptides of the present disclosure are micronized. In some embodiments, the modified Cav-1 peptides are micronized before mixing with SAIB. In some embodiments, the modified Cav-1 peptides are micronized during the formulation process. In some embodiments, the size of the modified Cav-1 peptide particles is reduced by using techniques including, but not limited to, grinding, milling (e.g., air milling (jet milling), ball milling), coacervation, complex coacervation, high pressure homogenization, spray drying, and / or supercritical fluid crystallization. In some embodiments, the size of the modified Cav-1 peptide particles is reduced by mechanical impact (e.g., by a hammer mill, a ball mill, and / or a pin mill). In some embodiments, the modified Cav-1 peptides are micronized via fluid energy (e.g., by a spiral jet mill, a loop jet mill, and / or a fluidized bed jet mill). In some embodiments, the size of the modified Cav-1 peptide particles is reduced by spray drying. In some embodiments, the modified Cav-1 peptides are micronized by spray drying.

[0204] In some embodiments, the formulations described herein comprise one or more multi-particle modified Cav-1 peptides. In some embodiments, the formulations described herein comprise micronized modified Cav-1 peptides. In some embodiments, the average particle size of the micronized polypeptide is in the range of about 0.5 μm to about 500 μm, including any value or sub-range therebetween. In some embodiments, the average particle size of the micronized polypeptide is in the range of about 0.5 μm to about 200 μm, including any value or sub-range therebetween. In some embodiments, the average particle size of the micronized polypeptide is in the range of about 0.5 μm to about 100 μm, including any value or sub-range therebetween. In some embodiments, the average particle size of the micronized polypeptide is in the range of about 1 μm to about 50 μm, including any value or sub-range therebetween. In some embodiments, the average particle size of the micronized polypeptide is in the range of about 1 μm to about 5 μm, including any value or sub-range therebetween. In some embodiments, the micronized polypeptide has an average particle size of less than about 10 μm, less than about 5 μm, or less than about 3 μm. In some embodiments, the micronized peptide has a size of about 1.0 μm, about 1.1 μm, about 1.2 μm, about 1.3 μm, about 1.4 μm, about 1.5 μm, about 1.6 μm, about 1.7 μm, about 1.8 μm, about 1.9 μm, about 2.0 μm, about 2.1 μm, about 2.2 μm, about 2.3 μm, about 2.4 μm, about 2.5 μm, about 2.6 μm, about 2.7, about 2.8 μm, about 2.9 μm. , about 3.0μm, about 3.1μm, about 3.2μm, about 3.3μm, about 3.4μm, about 3.5μm, about 3.6μm, about 3.7μm, about 3.8μm, about 3.9μm, about 4.0μm, about 4.1μm, about 4.2μm, about 4.3μm, about 4.4μm, about 4.5μm, about 4.6μm, about 4.7μm, about 4.8μm, about 4.9μm or about 5.0μm in average particle size.

[0205] In some embodiments, SAIB and modified Cav-1 peptides can be combined by any suitable means known in the art, such as direct mixing, extrusion coating, spray drying, blending and encapsulation. In some embodiments, SAIB and modified Cav-1 peptides are combined and formulated into a pharmaceutical formulation as described in Example 1.

[0206] In some embodiments, the preparation is in the form of emulsion, solution, microsphere, nanoparticle, nanosphere, implant or gel. In some embodiments, the preparation is a suspension. In some embodiments, the preparation further comprises a second solvent. In some embodiments, the second solvent is sterile water, phosphate buffered saline (PBS) or any pharmaceutically acceptable diluent known in the art. In some embodiments, the second solvent is PBS. In some embodiments, the SAIB preparation is in the form of emulsion, solution, microsphere, nanoparticle, nanosphere, implant or gel.

[0207] In one aspect, the present disclosure provides a pharmaceutical formulation comprising a modified Cav-1 peptide comprising the core sequence FTTFTVT and a biodegradable polymer.

[0208] In some embodiments, the formulation comprising the modified Cav-1 peptide described herein is an extra-intestinal depot formulation or an in situ forming extra-intestinal system (ISFI). In some embodiments, the formulation comprises a mixture of a modified Cav-1 peptide and a biodegradable polymer dissolved or suspended in a pharmaceutically acceptable water-miscible organic solvent. ISFI formulations and methods are further described in Musmade et al., J. Biol. Chem. Chron., 2019, 5(1), 14-21; Ko et al., Progress in Polymer Science, Vol. 38, 2013; and Kanwar and Sinha, Crit. Rev. Ther. Drug Carrier Systems, Vol. 36, 2019, which are incorporated herein by reference in their entirety.

[0209] In some embodiments, the formulation comprising a modified Cav-1 peptide described herein comprises a biodegradable polymer. In some embodiments, the biodegradable polymer is poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), poly(ethylene glycol), poly(vinyl alcohol), polysiloxane, poly(ethylene-vinyl acetate), polyurethane, polyalkyl cyanoacrylate, poly-ε-caprolactone, or a hydrogel polymer.

[0210] In some embodiments, the formulation comprises a modified Cav-1 peptide and SAIB in a weight ratio of about 1:50 to about 1:1, about 1:50 to about 1:5, about 1:50 to about 1:10, or any value or sub-range therebetween. In some embodiments, the weight ratio of the modified Cav-1 peptide and SAIB is about 1:40 to about 1:1 or about 1:40 to about 1:5, including any value or sub-range therebetween. In some embodiments, the weight ratio of the modified Cav-1 peptide and SAIB is about 1:30 to about 1:1 or 1:30 to about 1:5, including any value or sub-range therebetween. In some embodiments, the weight ratio of the modified Cav-1 peptide and SAIB is about 1:20 to about 1:1 or about 1:20 to about 1:5, including any value or sub-range therebetween. In some embodiments, the weight ratio of the modified Cav-1 peptide to SAIB is about 1:30, about 1:29, about 1:28, about 1:27, about 1:26, about 1:25, about 1:24, about 1:23, about 1:22, about 1:21, about 1:20, about 1:19, about 1:18, about 1:17, about 1:16, about 1:15, about 1:14, about 1:13, about 1:12, about 1:11, about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, or any value or subrange therebetween. In some embodiments, the weight ratio of the modified Cav-1 peptide to SAIB is about 1:50. In some embodiments, the weight ratio of the modified Cav-1 peptide to SAIB is about 1:40. In some embodiments, the weight ratio of the modified Cav-1 peptide to SAIB is about 1:30. In some embodiments, the weight ratio of the modified Cav-1 peptide to SAIB is about 1:17. In some embodiments, the weight ratio of the modified Cav-1 peptide to SAIB is about 1:13. In some embodiments, the weight ratio of the modified Cav-1 peptide to SAIB is about 1:10. In some embodiments, the weight ratio of the modified Cav-1 peptide to SAIB is about 1:8. In some embodiments, the weight ratio of the modified Cav-1 peptide to SAIB is about 1:5. In some embodiments, the weight ratio of the modified Cav-1 peptide to SAIB is about 1:1.

[0211] In some embodiments, the formulation further comprises any molecule or material for minimizing chemical degradation of the modified Cav-1 peptide and / or maintaining the physicochemical stability of the formulation. Exemplary molecules or materials include amphiphilic molecules such as mono-C 12-18sodium salts of alkyl sulfates, dialkyl ester sulfosuccinic acid derivatives having 3 to 16 carbon atoms, dioctyl sulfosuccinic acid, benzenesulfonic acid, naphthalene-1,5-disulfonic acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, dodecylsulfuric acid, p-toluenesulfonic acid, naphthalene-2-sulfonic acid, cholesterol sulfate, heptanesulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, oleic acid, palmitic acid, pamoic acid, benzoic acid, stearic acid, undecylenic acid and phospholipids; other polymers such as polylactic acid, polyglycolide, polycaprolactone, polyanhydride, polyamine, polyurethane, polyesteramide, polyorthoester, polydioxanone, polyacetal, polyketal, polycarbonate, polyphosphate, polyoxaester, polyorthocarbonate, Polyphosphazene, succinate, poly(malic acid), poly(amino acid), polyvinyl pyrrolidone, polyethylene glycol, polyhydroxycellulose, chitin, chitosan, hyaluronic acid and copolymers, terpolymers and mixtures thereof; and free radical scavengers and stabilizers, such as cysteine ​​or methionine, d-alpha tocopheryl acetate, dl-alpha tocopherol, ascorbyl palmitate, butylated hydroxyaniline, butylated hydroxyanisole, butylated hydroxyquinone, butylated hydroxyanisole, hydroxycoumarin, butylated hydroxytoluene, cephalosporin, ethyl gallate, propyl gallate, octyl gallate, lauryl gallate, propyl hydroxybenzoate, trihydroxybutyrophenone, dimethylphenol, di-tert-butylphenol, vitamin E and lecithin. In some embodiments, the preparation comprises SAIB.

[0212] In some embodiments, the formulation further comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, the formulation comprises SAIB.

[0213] In some embodiments, the formulation releases at least about 1% to about 20% of the modified Cav-1 peptide within four hours under physiological conditions. In some embodiments, when tested in a dissolution apparatus at neutral pH, the formulation releases at least about 1% to about 20% of the modified Cav-1 peptide within four hours. In some embodiments, when tested in a dissolution apparatus at about 37° C., the formulation releases at least about 1% to about 20% of the modified Cav-1 peptide within four hours. In some embodiments, the formulation releases at least about 2% to about 15% within four hours under physiological conditions, including any value or sub-range therebetween. In some embodiments, the formulation releases at least about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% of the peptide within four hours under physiological conditions. In some embodiments, physiological conditions are in an aqueous buffer ranging from about pH 6 to about pH 8 at about 37° C. In some embodiments, physiological conditions are in an aqueous buffer at about pH 7.4 at about 37° C. In some embodiments, the formulation comprises SAIB.

[0214] In some embodiments, the formulation releases the modified Cav-1 peptide under physiological conditions for at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days or 42 days or any value therein. In some embodiments, the formulation releases the modified Cav-1 peptide under physiological conditions for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks or any value or range therebetween. In some embodiments, the physiological condition is an aqueous buffer in the range of about pH 6 to about pH 8 at about 37°C. In some embodiments, physiological conditions are at about 37°C in an aqueous buffer at about pH 7.4.

[0215] In some embodiments, the formulations as described herein release the modified Cav-1 peptides under physiological conditions for at least eight hours. In some embodiments, the formulations as described herein release the modified Cav-1 peptides under physiological conditions for at least twelve hours. In some embodiments, the formulations as described herein release the modified Cav-1 peptides under physiological conditions for at least one day. In some embodiments, the formulations as described herein release the modified Cav-1 peptides under physiological conditions for at least 2 days. In some embodiments, the formulations release the modified Cav-1 peptides under physiological conditions for at least 5 days. In some embodiments, the formulations release the modified Cav-1 peptides under physiological conditions for at least 7 days. In some embodiments, the formulations release the modified Cav-1 peptides under physiological conditions for at least 14 days. In some embodiments, the formulations release the modified Cav-1 peptides under physiological conditions for at least 21 days. In some embodiments, the formulations release the modified Cav-1 peptides under physiological conditions for at least 28 days. In some embodiments, the formulations release the modified Cav-1 peptides under physiological conditions for at least 35 days. In some embodiments, the formulations release the modified Cav-1 peptides under physiological conditions for at least 42 days. In some embodiments, the release of the modified Cav-1 peptide under physiological conditions is measured by HPLC after the formulation is placed in a medium simulating physiological conditions (such as PBS). In some embodiments, the formulation is placed in a medium simulating physiological conditions while stirring or shaking. In some embodiments, the physiological condition is an aqueous buffer in the range of about pH 6 to about pH 8 at about 37°C. In some embodiments, the physiological condition is an aqueous buffer at about pH 7.4 at about 37°C.

[0216] In some embodiments, a single dose of a formulation described herein releases a modified Cav-1 peptide at an average of about mg peptide / 1 kg of subject body weight (1 mg / kg) to about 3 mg / kg per day under physiological conditions for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or any value therein. In some embodiments, a single dose of the formulation releases the modified Cav-1 peptide at an average of about 1 mg / kg to about 3 mg / kg per day under physiological conditions for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or any value therein. In some embodiments, the physiological conditions are in an aqueous buffer ranging from about pH 6 to about pH 8 at about 37°C. In some embodiments, the physiological conditions are in an aqueous buffer at about pH 7.4 at about 37°C. In some embodiments, the formulation comprises SAIB.

[0217] In some embodiments, a single dose of a formulation as described herein releases a modified Cav-1 peptide at an average of about 1 mg peptide / 1 kg of subject body weight per day (1 mg / kg per day) to about 3 mg / kg for at least 2 days under physiological conditions. In some embodiments, a single dose of a formulation releases a modified Cav-1 peptide at an average of about 1 mg / kg to about 3 mg / kg per day for at least 5 days under physiological conditions. In some embodiments, a single dose of a formulation releases a modified Cav-1 peptide at an average of about 1 mg / kg to about 3 mg / kg per day for at least 7 days under physiological conditions. In some embodiments, a single dose of a formulation releases a modified Cav-1 peptide at an average of about 1 mg / kg to about 3 mg / kg per day for at least 14 days under physiological conditions. In some embodiments, a single dose of a formulation releases a modified Cav-1 peptide at an average of about 1 mg / kg to about 3 mg / kg per day for at least 21 days under physiological conditions. In some embodiments, a single dose of a formulation releases a modified Cav-1 peptide at an average of about 1 mg / kg to about 3 mg / kg per day for at least 28 days under physiological conditions. In some embodiments, a single dose of the formulation releases the modified Cav-1 peptide at an average of about 1 mg / kg to about 3 mg / kg per day for at least 35 days under physiological conditions. In some embodiments, a single dose of the formulation releases the modified Cav-1 peptide at an average of about 1 mg / kg to about 3 mg / kg per day for at least 42 days under physiological conditions. In some embodiments, the physiological conditions are in an aqueous buffer in the range of about pH 6 to about pH 8 at about 37°C. In some embodiments, the physiological conditions are in an aqueous buffer at about pH 7.4 at about 37°C.

[0218] In some embodiments, a single dose of a formulation as described herein releases a modified Cav-1 peptide at an average of about 1 mg peptide / 1 kg subject body weight (1 mg / kg), about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, or about 3 mg / kg per day for at least 2 days under physiological conditions. In some embodiments, a single dose of a formulation as described herein releases a modified Cav-1 peptide at an average of about 1 mg peptide / 1 kg of subject body weight (1 mg / kg), about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, or about 3 mg / kg per day for at least 7 days, at least 14 days, at least 21 days, at least 28 days, at least 35 days, or at least 42 days under physiological conditions. In some embodiments, physiological conditions are at about 37°C in an aqueous buffer in the range of about pH 6 to about pH 8. In some embodiments, physiological conditions are at about 37°C in an aqueous buffer at about pH 7.4.

[0219] IV. Methods of Using the Formulations for Therapeutic Use

[0220] In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is used to treat or prevent a fibrotic disease or condition. In some embodiments, the modified Cav-1 peptide is used to treat or prevent a fibrotic disease or condition, such as interstitial lung disease, liver fibrosis, renal fibrosis, skin fibrosis, glomerulonephritis, systemic sclerosis, cardiac fibrosis, myocardial fibrosis, kidney fibrosis, liver cirrhosis, nephrosclerosis, arteriosclerosis, macular degeneration, ocular scars, cataracts, retinal and vitreoretinopathy, Graves' ophthalmopathy, neurofibromatosis, scleroderma, glioblastoma, keloid and hypertrophic scars, peritoneal fibrosis, chronic obstructive pulmonary disease, postoperative uterine fibroids, diabetic nephropathy, gynecological cancer, myeloproliferative syndrome, myeloid leukemia, myelodysplastic syndrome, inflammatory bowel disease, non-alcoholic fatty liver disease, fibrosarcoma, rheumatoid arthritis, non-alcoholic steatohepatitis, Alport syndrome, or chronic COVID syndrome.

[0221] In some embodiments, the present disclosure provides a method for treating or preventing a fibrotic disease or condition in a subject, wherein the method comprises administering an effective amount of a modified Cav-1 peptide or a pharmaceutical preparation thereof to the subject. In some embodiments, the modified Cav-1 peptide or a pharmaceutical preparation thereof is used to treat or prevent a lung disease or lung condition in a subject. In some embodiments, the modified Cav-1 peptide is used to treat or prevent a lung disease or lung condition, such as, for example, acute lung injury (ALI), chronic lung injury, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), asthma, interstitial lung disease, pulmonary fibrosis, pneumonia, hypersensitivity pneumonitis, bronchiolitis, sarcoidosis, scleroderma, or lung infection. In some embodiments, the modified Cav-1 peptide is used to treat or prevent a lung disease or lung condition in an elderly or advanced age subject. In some embodiments, an elderly subject suffers from an interstitial lung disease, such as idiopathic pulmonary fibrosis.

[0222] In some embodiments, the modified Cav-1 peptides or formulations disclosed herein are used to treat or prevent a lung infection, such as a bacterial, viral or fungal infection, in a subject. In some embodiments, the lung infection causes one or more lung diseases or lung conditions in the subject, including but not limited to ALI, ARDS, COPD, asthma, interstitial lung disease, pulmonary fibrosis, pneumonia, hypersensitivity pneumonitis, bronchiolitis, sarcoidosis and scleroderma.

[0223] In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation of the present disclosure is used to treat or prevent bacterial infection in a subject. Examples of bacteria that cause lung infection include, but are not limited to, Pseudomonas aeruginosa, Bacillus anthracis, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Enterobacteriaceae, Nocardia, Actinomyces, Moraxella catarrhalis, Klebsiella pneumoniae, Chlamydia trachomatis, and Streptococcus pneumoniae. trachomatis, Chlamydophilia pneumoniae, Chlamydophilia psittaci, Coxiella burnetti, Salmenellosis, Yersinia pestis, Mycobacterium leprae, Mycobacterium africanum, Mycobacterium asiaticum, Mycobacterium aviuin-intracellulaire, Mycobacterium chelonei, Mycobacterium abscessus, Mycobacterium fallax, Mycobacterium fortuitum, Mycobacterium kansasii, Mycobacterium leprae leprae), Mycobacterium malmoense, Mycobacterium shimoidei, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium xenopi, Mycobacterium tuberculosisIn some embodiments, the bacterial infection causes pneumonia in the subject.

[0224] In some embodiments, the modified Cav-1 peptide or pharmaceutical preparation of the present disclosure is used to treat or prevent viral infection in a subject. In some embodiments, the modified Cav-1 peptide is used to treat or prevent infection in a subject caused by a double-stranded DNA (dsDNA) virus, a single-stranded DNA (ssDNA) virus, a single-stranded RNA (ssRNA) virus, or a double-stranded RNA (dsRNA) virus. In some embodiments, the ssRNA virus is a positive sense ssRNA virus (+ssRNA). In some embodiments, the ssRNA virus is a negative sense ssRNA virus (-ssRNA). Examples of viruses causing lung infections include, but are not limited to, coronaviruses (e.g., SARS-CoV-1, SARS-CoV-2, or MERS-CoV), influenza, respiratory syncytial virus, metapneumovirus, bocavirus, parainfluenza virus, rhinovirus, enterovirus, norovirus, adenovirus, varicella-zoster virus, hantavirus, parechovirus, Epstein-Barr virus, herpes simplex virus, mimivirus, cytomegalovirus, torquetenovirus, and Middle East Respiratory Syndrome coronavirus. In some embodiments, viral infection causes pneumonia in a subject. In some embodiments, viral infection causes pulmonary fibrosis in a subject. In some embodiments, viral infection causes bronchiolitis in a subject. In some embodiments, viral infection causes ALI or ARDS in a subject. In some embodiments, viral infection causes interstitial lung disease in a subject. In some embodiments, viral infection causes asthma in a subject. In some embodiments, viral infection causes sarcoidosis in a subject. In some embodiments, the viral infection causes scleroderma in the subject.

[0225] In some embodiments, SARS-CoV-1 causes severe acute respiratory syndrome (SARS) in a subject. In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation is used to treat or prevent SARS in a subject. SARS is initially characterized by systemic symptoms of muscle pain, headache, and fever, followed by respiratory symptoms within 2-14 days, mainly cough, dyspnea, and pneumonia.

[0226] In some embodiments, MERS-CoV causes Middle East respiratory syndrome (MERS) in a subject. In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation is used to treat or prevent MERS in a subject. Clinical features of MERS include asymptomatic or mild illness, acute respiratory distress syndrome, and multiple organ failure leading to death, especially in individuals with underlying comorbidities. There is currently no specific drug treatment for MERS, and infection prevention and control measures are essential to prevent its spread in healthcare facilities. See Zumla et al. Lancet 2015; 386(9997): 995-1007.

[0227] In some embodiments, SARS-CoV-2 causes COVID-19 in a subject. In some embodiments, a modified Cav-1 peptide or pharmaceutical formulation is used to treat or prevent an infection caused by SARS-CoV-2. In some embodiments, a variant of SARS-CoV-2 causes COVID-19 in a subject. In some embodiments, a modified Cav-1 peptide or pharmaceutical formulation is used to treat or prevent an infection caused by a variant of SARS-CoV-2. In some embodiments, a modified Cav-1 peptide or pharmaceutical formulation is used to treat or prevent an infection caused by a SARS-CoV-2 alpha variant. In some embodiments, a modified Cav-1 peptide or pharmaceutical formulation is used to treat or prevent an infection caused by a SARS-CoV-2 beta variant. In some embodiments, a modified Cav-1 peptide or pharmaceutical formulation is used to treat or prevent an infection caused by a SARS-CoV-2 gamma variant. In some embodiments, a modified Cav-1 peptide or pharmaceutical formulation is used to treat or prevent an infection caused by a SARS-CoV-2 delta variant. In some embodiments, a modified Cav-1 peptide or pharmaceutical formulation is used to treat or prevent an infection caused by a SARS-CoV-2 epsilon variant. In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation is used to treat or prevent an infection caused by a SARS-CoV-2ζ variant. In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation is used to treat or prevent an infection caused by a SARS-CoV-2η variant. In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation is used to treat or prevent an infection caused by a SARS-CoV-2θ variant. In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation is used to treat or prevent an infection caused by a SARS-CoV-2ι variant. In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation is used to treat or prevent an infection caused by a SARS-CoV-2κ variant. In some embodiments, the modified Cav-1 peptide is used to treat or prevent acute post-COVID-19 caused by a SARS-CoV-2λ variant. In some embodiments, the modified Cav-1 peptide is used to treat or prevent acute post-COVID-19 caused by a SARS-CoV-2μ variant. In some embodiments, the modified Cav-1 peptide is used to treat or prevent acute post-COVID-19 caused by a SARS-CoV-2ο variant.In some embodiments, the SARS-CoV-2 variant is B.1.1.7 (also known as 501Y.V1 or VOC-202012 / 01), B.1.1.317, B.1.1.318, B.1.1.529, B.1.351 (also known as 501Y.V2), B.1.429, B1.427, B1.1.207, A.23.1, COH.20G / 501Y, B.1.525, B.1.526, B.1.617, B.1.618, B.1.621, C.37, P.1, P.2 or P.3 or a subvariant thereof or a recombinant form thereof. See Konings et al., Variants of Interest and Concern naming scheme conducive for global discourse. Nature Microbiology (2021). In some embodiments, the subvariant of SARS-CoV-2 variant B.1.1.529 is BA.1 (B1.1.529.1), BA1.1 (B1.1.529.1.1), BA.2 (B1.1.529.2), BA.3 (B1.1.529.3), BA.4 (B1.1.529.4), or BA.5 (B1.1.529.5). In some embodiments, the subvariant of SARS-CoV-2 variant B.1.1.7 is Q.1, Q.2, Q.3, Q.4, Q.5, Q.6, Q.7, or Q.8. In some embodiments, the subvariant of SARS-CoV-2 variant B.1.351 is B.1.351.1, B.1.351.2, B.1.351.3, B.1.351.4, or B.1.351.5. In some embodiments, the subvariant of SARS-CoV-2 variant P.1 is P.1.1, P.1.2, P.1.3, P.1.4, P.1.5, P.1.6, P.1.7, P.1.7.1, P.1.8, P.1.9, P.1.10, P.1.10.1, P.1.10.2, P.1.11, P.1.12, P.1.12.1, P.1.13, P.1.14, P.1.15, P.1.16, P.1.17, or P.1.17.1. In some embodiments, the subvariant of SARS-CoV-2 variant B.1.617 is B.1.617.1, B.1.617.2, or B.1.617.3. In some embodiments, the subvariant of SARS-CoV-2 variant B.1.526 is B.1.526.1. In some embodiments, the subvariant of SARS-CoV-2 variant B.1.621 is B.1.621.1, B.1.621.2, BB.1 or BB.2.In some embodiments, the subvariant of SARS-CoV-2 variant C.37 is C.37.1.

[0228] In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation disclosed herein is used to treat or prevent fungal infection in a subject. Examples of fungi causing lung infection include, but are not limited to, Candida (e.g., Candida albicans, Candida glabrata, Candida akrusei), Aspergillus, Pneumocystis, Coccidioides (e.g., Coccidioides immitis, Coccidioides posadasii), Blastomyces (e.g., Blastomyces dermatitidis), Histoplasma (e.g., Histoplasma capsule capsulatum), Cryptococcus (e.g., Cryptococcus neoformans, Cryptococcus gattii), Sporothrix (e.g., Sporothrix schenckii), Mucor, and Paracoccidioides. In some embodiments, the fungal infection causes pneumonia in the subject. In some embodiments, the fungal infection causes invasive pulmonary aspergillosis in the subject. In some embodiments, the fungal infection causes allergic asthma, allergic bronchopulmonary aspergillosis, or hypersensitivity pneumonitis in the subject. In some embodiments, the fungal infection causes ARDS. In some embodiments, the fungal infection causes pulmonary fibrosis in the subject. In some embodiments, the fungal infection causes pulmonary edema in the subject.

[0229] In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation of the present disclosure is used to treat or prevent interstitial lung disease in a subject. Interstitial lung disease is a group of conditions that cause interstitial fibrosis and inflammation. In some embodiments, the interstitial lung disease is idiopathic pulmonary fibrosis, lymphangioleiomyomatosis, nonspecific interstitial pneumonia, idiopathic interstitial pneumonia, cryptogenic organizing pneumonia, acute interstitial pneumonia, respiratory bronchiolitis-associated interstitial lung disease, desquamative interstitial pneumonia, lymphocytic interstitial pneumonia, pulmonary sarcoidosis, diffuse alveolar damage, systemic sclerosis, polymyositis, systemic lupus erythematosus, rheumatoid arthritis, drug-induced interstitial lung disease or occupational interstitial lung disease. In some embodiments, the interstitial lung disease is idiopathic pulmonary fibrosis.

[0230] In some embodiments, the modified Cav-1 peptides or pharmaceutical formulations disclosed herein are used to treat or prevent acute lung injury (ALI) in a subject. ALI is an acute inflammatory condition that causes destruction of the pulmonary endothelium and epithelial barrier. ALI may be the result of inhalation injury or the result of systemic disease (such as sepsis or severe hypovolemic shock). In some embodiments, ALI is chemical-induced ALI. In some embodiments, ALI is smoke inhalation-induced acute lung injury (ISALI). In some embodiments, ALI is ARDS.

[0231] In some embodiments, the modified Cav-1 peptides or pharmaceutical formulations disclosed herein are used to treat or prevent ARDS in a subject. ARDS is the most severe form of ALI and is distinguished by the severity of the oxygenation defect. ARDS is a life-threatening lung injury that occurs when fluid accumulates in the tiny elastic air sacs (alveoli) of the lungs. The fluid in the alveoli prevents the lungs from filling with oxygen, resulting in less oxygen reaching the blood and causing difficulty breathing.

[0232] In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation is used to treat or prevent cystic fibrosis (CF) in a subject. CF is a genetic disease that primarily affects the exocrine glands and exocrine sweat glands of the digestive and respiratory systems. The disease is typically characterized by chronic respiratory infections, pancreatic insufficiency, abnormally thick, viscous secretions, and premature death. CF is characterized by progressive airflow obstruction. A subgroup of individuals with CF also develop airway hyperresponsiveness to inhaled cholinergic agonists (Weinberger, 2002 and Mitchell et al., 1978), and reversibility of 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 that there may be a common disease etiology between CF and other airway narrowing diseases (such as asthma or COPD), in which airway smooth muscle dysfunction is thought to lead to disease progression.

[0233] In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation is used to treat or prevent COPD in a subject. COPD is a term used to classify the two main airflow obstruction conditions: chronic bronchitis and emphysema. 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. In emphysema, the alveolar sacs over-expand due to damage to the elastin skeleton of the lungs. Inflammatory cells in the emphysematous lung release elastase, which degrades or damages the elastin fibers within the lung matrix. Emphysema has many causes, including smoking, exposure to environmental pollutants, alpha-1 antitrypsin deficiency, and aging.

[0234] In some embodiments, the modified Cav-1 peptides or pharmaceutical formulations disclosed herein are used to treat or prevent bronchiolitis in a subject. Bronchiolitis is most commonly caused by viral lower respiratory tract infections and is primarily characterized by acute inflammation, edema, epithelial cell necrosis within the small airways, and increased mucus production (Ralston et al., 2014). Signs and symptoms typically begin with rhinitis and cough, which may progress to shortness of breath, wheezing, rales, use of accessory muscles, and / or nasal flaring.

[0235] In some embodiments, the modified Cav-1 peptides or pharmaceutical formulations disclosed herein are used to treat or prevent bronchiolitis obliterans in a subject. 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 is a major complication of lung transplantation and is often used to describe delayed allograft dysfunction, which results in a persistent decrease in forced expiratory volume and force that is not caused by other known causes (Meyer et al., 2014).

[0236] In some embodiments, the modified Cav-1 peptides or pharmaceutical preparations disclosed herein are used to treat or prevent asthma in a subject. The term "asthma" may refer to acute asthma, chronic asthma, intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma, chronic persistent asthma, mild to moderate asthma, mild to moderate persistent asthma, mild to moderate chronic persistent asthma, allergic (extrinsic) asthma, non-allergic (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, the airways remain inflamed and spasms may occur occasionally.

[0237] In some embodiments, the modified Cav-1 peptides or pharmaceutical formulations disclosed herein are used to treat or prevent hypersensitivity pneumonitis in a subject. Hypersensitivity pneumonitis is a complex syndrome caused by the inhalation of various antigens by susceptible and sensitized individuals. These antigens are present in the environment and are primarily derived from avian proteins and fungi. Hypersensitivity pneumonitis is characterized by excessive humoral and cellular immune responses affecting the small airways and lung parenchyma. Hypersensitivity pneumonitis can be classified into acute, chronic non-fibrotic, and chronic fibrotic forms. Acute hypersensitivity pneumonitis is caused by intermittent, high-level exposure to inducing antigens, usually occurring within a few hours after exposure, while chronic hypersensitivity pneumonitis is mostly caused by long-term, low-level exposure (usually to birds or mold in the home), which is not easily defined in time and may occur weeks, months, or even years after exposure. Even if exposure is completely avoided, some patients with fibrosing hypersensitivity pneumonitis may develop a progressive phenotype. See Costabel et al., Nature Reviews Disease Primers 2020; 6 (65).

[0238] In some embodiments, the modified Cav-1 peptide or pharmaceutical preparation is used to treat or prevent systemic sclerosis or scleroderma in a subject. Systemic sclerosis is a systemic autoimmune disease characterized by endothelial dysfunction leading to small vessel lesions, fibroblast dysfunction leading to excessive collagen production and fibrosis, and immune abnormalities. The classification of systemic sclerosis is subdivided into diffuse cutaneous sclerosis, limited cutaneous sclerosis, or systemic sclerosis without scleroderma according to the degree of skin involvement. Although almost any organ system can be involved in the disease process, the fibrotic and vascular pulmonary manifestations of systemic sclerosis, including interstitial lung disease and pulmonary hypertension, are the cause of death. Although some pulmonary manifestations may be more common in subgroups of systemic sclerosis (i.e., ILD is more common in diffuse cutaneous sclerosis, and pulmonary hypertension is more common in limited cutaneous sclerosis), all known pulmonary manifestations reported have been described in each disease subgroup. Lung disease may even occur in systemic sclerosis without skin involvement (called non-scleroderma). See Solomon et al., Eur Respir Rev 2013; 22(127): 6-19.

[0239] In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation is used to treat or prevent sarcoidosis in a subject. Sarcoidosis is a multisystem disorder characterized by non-caseating epithelioid cell granulomas that may affect almost any organ. Chest involvement is common and accounts for the majority of morbidity and mortality associated with the disease. Chest abnormalities are observed in approximately 90% of patients with sarcoidosis, and an estimated 20% of patients develop chronic lung disease leading to pulmonary fibrosis. Pulmonary sarcoidosis can present in a variety of forms: the most common is bilateral hilar lymphadenopathy, followed by interstitial lung disease. The most typical findings of pulmonary involvement are micronodules distributed around lymphatic vessels, fibrotic changes, and bilateral perihilar shadows. Atypical manifestations such as mass-like or alveolar shadows, honeycomb cysts, miliary shadows, mosaic attenuation, tracheobronchial involvement, and pleural disease and complications such as aspergilloma may also be seen. See Criado et al., Chest Imaging 2010; 30(6).

[0240] In some embodiments, the present disclosure provides a method of treating or preventing a lung disease or condition in an elderly subject, wherein the method comprises administering to the elderly subject an effective amount of a modified Cav-1 peptide or a pharmaceutical formulation thereof.

[0241] In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is used to treat or prevent a renal disease or renal disorder (e.g., chronic kidney disease) in a subject. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is used to treat or prevent a pulmonary disease or pulmonary disorder (e.g., idiopathic pulmonary fibrosis) in a subject. In some embodiments, the subject is an elderly or advanced age subject.

[0242] In some embodiments, the modified Cav-1 peptide formulation is used to treat or prevent renal disease or renal disorder, such as, for example, chronic kidney disease, end-stage renal disease, glomerulonephritis, focal segmental glomerulosclerosis, renal fibrosis, polycystic kidney disease, IgA nephropathy, lupus nephritis, nephrotic syndrome, Alport syndrome, amyloidosis, Goodbastier syndrome, Wegener's granulomatosis, or acute kidney injury. In some embodiments, the renal disease is characterized by fibrosis. In some embodiments, the renal disease or renal disorder is acute. In some embodiments, the renal disease or renal disorder is chronic. In some embodiments, the subject is an elderly or elderly subject. In some embodiments, the renal disease or renal disorder of the subject is caused by infection, such as, for example, viral, bacterial, fungal or parasitic infection. In some embodiments, the renal disease or renal disorder of the subject is caused by high blood pressure / hypertension. In some embodiments, the renal disease or renal disorder of the subject is caused by diabetes. In some embodiments, the subject's kidney disease or kidney disorder is caused by excessive use of drugs, such as over-the-counter painkillers and heroin. In some embodiments, the subject suffers from hypertension or diabetes.

[0243] In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is used to delay the progression of renal disease or renal disorder in a subject. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is used to improve progression-free survival. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is used to prolong the survival of a subject.

[0244] In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation of the present disclosure is used to treat or prevent chronic kidney disease in a subject. Chronic kidney disease is a gradual and progressive loss of the kidney's ability to excrete waste, concentrate urine, and preserve electrolytes. The progressive loss of renal function is caused by the deposition of fibrous tissue between renal functional units or nephrons (interstitial fibrosis) and the continuous replacement of the filtration surface by fibrous tissue (glomerulosclerosis). Renal fibrosis is a pathological hallmark of chronic kidney disease and a major contributor to progression to end-stage renal disease. In one embodiment, chronic kidney disease is chronic renal fibrosis.

[0245] In some embodiments, the modified Cav-1 peptides or pharmaceutical formulations disclosed herein are used to treat or prevent end-stage renal disease in a subject. End-stage renal disease is the final stage of chronic kidney disease, in which the kidneys have ceased to function and the individual requires long-term dialysis or a kidney transplant to survive.

[0246] In some embodiments, the modified Cav-1 peptides or pharmaceutical formulations disclosed herein are used to treat or prevent focal segmental glomerulosclerosis in a subject. Focal segmental glomerulosclerosis is a kidney disease characterized by scarring of the glomeruli, resulting in loss of protein into the urine.

[0247] In some embodiments, the modified Cav-1 peptides or pharmaceutical formulations disclosed herein are used to treat or prevent glomerulonephritis in a subject. Glomerulonephritis, also known as glomerular disease, is a kidney disease in which the glomeruli are damaged and cannot normally remove waste and fluid from the body. In some embodiments, the glomerulonephritis is acute glomerulonephritis. In some embodiments, the glomerulonephritis is chronic glomerulonephritis.

[0248] In some embodiments, the modified Cav-1 peptides or pharmaceutical formulations of the present disclosure are used to treat or prevent polycystic kidney disease in a subject. Polycystic kidney disease is a genetic disorder in which clusters of cysts develop primarily within the kidneys, causing the kidneys to enlarge and lose function over time.

[0249] In some embodiments, the modified Cav-1 peptides or pharmaceutical formulations disclosed herein are used to treat or prevent IgA nephropathy in a subject. IgA nephropathy, also known as Berger's disease, is a kidney disease that occurs when immunoglobulin IgA accumulates in the kidneys, leading to local inflammation that prevents the kidneys from filtering waste products from the blood.

[0250] In some embodiments, the modified Cav-1 peptides or pharmaceutical formulations disclosed herein are used to treat or prevent lupus nephritis in a subject. Lupus nephritis is a type of glomerulonephritis that constitutes one of the most serious organ manifestations of systemic lupus erythematosus and occurs when the immune system attacks the kidneys.

[0251] In some embodiments, the modified Cav-1 peptides or pharmaceutical formulations of the present disclosure are used to treat or prevent nephrotic syndrome in a subject. Nephrotic syndrome is a kidney disorder that causes the body to excrete too much protein into the urine. Nephrotic syndrome is usually caused by damage to the small blood vessels in the kidneys that filter waste and excess water from the blood.

[0252] In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation of the present disclosure is used to treat or prevent Alport syndrome in a subject. Alport syndrome is a genetic disorder characterized by progressive kidney disease and abnormalities of the inner ear and eyes. There are three genetic types: X-linked Alport syndrome (XLAS), autosomal recessive Alport syndrome (ARAS), and autosomal dominant Alport syndrome (ADAS). XLAS is caused by mutations in the COL4A5 gene, ARAS is caused by mutations in two copies of the COL4A3 or COL4A4 gene, and ADAS is caused by mutations in one copy of the COL4A3 or COL4A4 gene. Individuals with Alport syndrome exhibit chronic glomerular dysfunction, renal inflammation, and fibrosis, which are hallmarks of chronic kidney disease, and progress to end-stage renal disease. Those with Alport syndrome also develop progressive hearing loss and eye abnormalities of varying severity, but usually do not cause visual impairment.

[0253] In some embodiments, the modified Cav-1 peptides or pharmaceutical formulations disclosed herein are used to treat or prevent amyloidosis in a subject. Amyloidosis occurs when amyloid accumulates in tissues and organs and interferes with normal function. Amyloid deposition damages the kidneys, affecting the kidneys' ability to filter waste and break down proteins. In some embodiments, the amyloidosis is primary amyloidosis. In some embodiments, the amyloidosis is dialysis-related amyloidosis.

[0254] In some embodiments, the modified Cav-1 peptides or pharmaceutical formulations of the present disclosure are used to treat or prevent Goodpasture's syndrome in a subject. Goodpasture's syndrome, also known as anti-glomerular basement membrane disease, is an autoimmune disease in which antibodies attack the basement membranes of the lungs and kidneys, leading to pulmonary hemorrhage, glomerulonephritis, and renal failure.

[0255] In some embodiments, the modified Cav-1 peptides or pharmaceutical formulations disclosed herein are used to treat or prevent granulomatosis with polyangiitis in a subject. Granulomatosis with polyangiitis, also known as Wegener's granulomatosis, is an autoimmune disease that most often targets the lungs and kidneys, involving granulomatous inflammation, necrosis, and vasculitis.

[0256] In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation of the present disclosure is used to treat or prevent acute kidney injury in a subject. Acute kidney injury, also known as acute renal failure, is a sudden loss of renal excretory function. Acute kidney injury is defined as serum creatine and urine output levels that persist for less than one week.

[0257] In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation is used to treat or prevent kidney infection. In some embodiments, the modified Cav-1 peptide is used to treat or prevent pyelonephritis. Pyelonephritis is a urinary tract infection in which one or both kidneys are infected. In some embodiments, pyelonephritis is caused by bacteria or viruses, such as, for example, Escherichia coli, Klebsiella, Proteus, Pseudomonas, Enterococcus, or Taphylococcus saprophyticus.

[0258] In some embodiments, the modified Cav-1 peptide or pharmaceutical preparation is used to treat or prevent renal disease or renal disorder caused by microbial infection in a subject. In some embodiments, the microbial infection is a bacterial, viral, fungal or parasitic infection. In some embodiments, the renal disease or renal disorder is caused by Streptococcus pyogenes, Staphylococcus aureus, Staphylococcus epidermidis, Salmonella (typhoid, paratyphoid), Escherichia coli, Leptospira, Mycobacterium tuberculosis, Mycobacterium leprae, Legionella spp., Yersinia enterocolitica, Brucella, Campylobacter jejuni, Corynebacterium diphtheriae, diphtheriae), Klebsiella, Proteus, Pseudomonas, Enterococcus, Staphylococcus saprophyticus, SARS-CoV-1, SARS-CoV-2, dengue virus, hantavirus, varicella-zoster virus, parvovirus, hepatitis A virus, hepatitis B virus, hepatitis E virus, cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, and / or hepatitis C virus.

[0259] In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation is used to treat or prevent renal disease or renal disorder caused by SARS-CoV-2 infection. In some embodiments, SARS-CoV-2 causes acute kidney injury. In some embodiments, SARS-CoV-2 causes chronic kidney injury. In some embodiments, SARS-CoV-2 causes chronic kidney disease. In some embodiments, SARS-CoV-2 causes renal fibrosis. In some embodiments, SARS-CoV-2 causes renal failure.

[0260] In some embodiments, the kidney disease or kidney disorder is chronic kidney disease. In some embodiments, the kidney disease or kidney disorder is Alport syndrome. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is used to improve renal function in a subject with a kidney disease or kidney disorder. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation improves renal function by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to the subject before treatment with the modified Cav-1 peptide or its pharmaceutical preparation. In some embodiments, the improvement of renal function represents a reduction in fibrotic glomeruli, a reduction in blood urea nitrogen, a reduction in blood creatinine, an increase in blood albumin, a reduction in the urine albumin to creatinine ratio, and / or an increase in glomerular filtration rate.

[0261] In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation thereof reduces the number of fibrotic glomeruli in a subject with a kidney disease or a kidney disorder. In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation thereof reduces the number of fibrotic glomeruli by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to the subject before treatment with the modified Cav-1 peptide or pharmaceutical formulation thereof.

[0262] In some embodiments, compared with a subject (e.g., a normal, healthy subject) not suffering from renal disease or renal disorder, the expression of caveolin-1 in the kidney of a subject suffering from renal disease or renal disorder is reduced. In some embodiments, compared with a subject not suffering from renal disease or renal disorder, the expression of caveolin-1 in the glomeruli of a subject suffering from renal disease or renal disorder is reduced. In some embodiments, compared with a subject not suffering from renal disease or renal disorder, the expression of caveolin-1 in the renal endothelial cells of a subject suffering from renal disease or renal disorder is reduced. In some embodiments, compared with a subject not suffering from renal disease or renal disorder, the expression of caveolin-1 in the renal epithelial cells of a subject suffering from renal disease or renal disorder is reduced. In some embodiments, compared with a subject not suffering from renal disease or renal disorder, the expression of caveolin-1 in the renal podocytes of a subject suffering from renal disease or renal disorder is reduced. In some embodiments, caveolin-1 expression is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% in the kidney of a subject with a renal disease or disorder compared to a subject without the renal disease or disorder. In some embodiments, the epithelial cell is a parietal epithelial cell lining Bowman's capsule.

[0263] In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation reduces endothelial cell death in a subject with a renal disease or renal disorder. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation reduces epithelial cell death in a subject with a renal disease or renal disorder. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation reduces podocyte death in a subject with a renal disease or renal disorder. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation reduces cell death by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, compared to the subject before treatment with the modified Cav-1 peptide or its pharmaceutical preparation.

[0264] In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation increases endothelial cell survival in a subject with a renal disease or renal disorder. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation increases epithelial cell survival in a subject with a renal disease or renal disorder. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation increases podocyte survival in a subject with a renal disease or renal disorder. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation increases cell survival by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, compared to the subject before treatment with the modified Cav-1 peptide or its pharmaceutical preparation.

[0265] In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation promotes renal regeneration in a subject with a renal disease or renal disorder. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation promotes regeneration of renal blood vessels, glomeruli and / or tubules in the kidney of a subject. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation promotes regeneration of epithelial cells, endothelial cells, tubular cells and / or podocytes in the kidney of a subject.

[0266] In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation increases endothelial cell proliferation in a subject with a renal disease or renal disorder. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation increases epithelial cell proliferation in a subject with a renal disease or renal disorder. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation increases podocyte proliferation in a subject with a renal disease or renal disorder. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation increases cell proliferation by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, compared to the subject before treatment with the modified Cav-1 peptide or its pharmaceutical preparation.

[0267] In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation reduces blood urea nitrogen in a subject with renal disease or renal disorder. In some embodiments, high blood urea nitrogen values ​​indicate that the subject suffers from renal damage or renal disease. Generally, human blood urea nitrogen levels are considered normal in the range of 6 mg / dl to 24 mg / dl. In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation reduces blood urea nitrogen by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to the subject before treatment with the modified Cav-1 peptide or its pharmaceutical formulation. In some embodiments, the modified Cav-1 peptide or a pharmaceutical formulation thereof reduces the blood urea nitrogen of a subject to less than about 50 mg / dl, less than about 45 mg / dl, less than about 40 mg / dl, less than about 35 mg / dl, less than about 30 mg / dl, less than about 25 mg / dl, or less than about 20 mg / dl. In some embodiments, the modified Cav-1 peptide or a pharmaceutical formulation thereof reduces blood urea nitrogen to less than about 20 mg / dl.

[0268] In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation reduces blood creatinine in a subject with kidney disease or kidney disorder. In some embodiments, a high blood creatinine value indicates that the subject suffers from kidney damage or kidney disease. Generally, a blood creatinine value greater than 1.2 mg / dl for women and greater than 1.4 mg / dl for men indicates abnormal kidney function. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation reduces blood creatinine by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to the subject before treatment with the modified Cav-1 peptide or its pharmaceutical preparation. In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation thereof reduces the subject's blood creatinine to less than about 4 mg / dl, less than about 3.5 mg / dl, less than about 3.25 mg / dl, less than about 3 mg / dl, less than about 2.75 mg / dl, less than about 2.5 mg / dl, less than about 2.25 mg / dl, less than about 2 mg / dl, less than about 1.75 mg / dl, less than about 1.5 mg / dl, or less than about 1.25 mg / dl. In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation thereof reduces blood urea nitrogen to less than about 1.5 mg / dl.

[0269] In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation increases blood albumin in a subject with kidney disease or kidney disorder. In some embodiments, low blood albumin values ​​may indicate that the subject suffers from kidney damage or kidney disease. The normal level of albumin in the blood is 3.5 g / dL to 5 g / dL. In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation increases blood albumin by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to the subject before treatment with the modified Cav-1 peptide or its pharmaceutical formulation. In some embodiments, the modified Cav-1 peptide or a pharmaceutical formulation thereof increases the blood albumin of a subject to greater than about 1.5 g / dl, greater than about 1.75 g / dl, greater than about 2 g / dl, greater than about 2.25 g / dl, greater than about 2.5 g / dl, greater than about 2.75 g / dl, greater than about 3 g / dl, or greater than about 3.5 g / dl. In some embodiments, the modified Cav-1 peptide or a pharmaceutical formulation thereof increases blood albumin to greater than about 3.5 g / dl.

[0270] In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation reduces the urine albumin to creatinine ratio in a subject with kidney disease or kidney disorder. The urine albumin to creatinine ratio helps identify kidney damage or kidney disease in a subject. In humans, an albumin to creatinine ratio of less than 30 mg / g is considered normal; a ratio of 30-300 mg / g indicates microalbuminuria, while a ratio above 300 mg / g indicates macroalbuminuria. In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation reduces the urine albumin to creatinine ratio by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to the subject before treatment with the modified Cav-1 peptide or its pharmaceutical formulation. In some embodiments, the modified Cav-1 peptide or a pharmaceutical formulation thereof reduces the urine albumin to creatinine ratio of a subject to less than about 300 mg / g, less than about 250 mg / g, less than about 200 mg / g, less than about 150 mg / g, less than about 100 mg / g, less than about 75 mg / g, less than about 50 mg / g, less than about 40 mg / g, less than about 30 mg / g, or less than about 25 mg / g. In some embodiments, the modified Cav-1 peptide or a pharmaceutical formulation thereof reduces the urine albumin to creatinine ratio to less than about 30 mg / g.

[0271] In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation thereof increases the glomerular filtration rate in a subject with a kidney disease or disorder. The glomerular filtration rate measures the ability of the kidney to filter blood to remove waste products and excess water to form urine. A glomerular filtration rate above 90 mL / min / 1.73 m 2 is considered normal, while a glomerular filtration rate of less than 60 mL / min / 1.73 m 2 Can indicate kidney damage or kidney disease. Glomerular filtration rate less than 15 mL / min / 1.73 m 2 Can indicate renal failure. In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation thereof increases the glomerular filtration rate by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to the subject before treatment with the modified Cav-1 peptide or pharmaceutical formulation thereof. In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation thereof increases the glomerular filtration rate of the subject to greater than about 30 mL / min / 1.73 m 2 , greater than about 40mL / min / 1.73m 2 , greater than about 50mL / min / 1.73m 2 , greater than about 60mL / min / 1.73m 2, greater than about 70mL / min / 1.73m 2 , greater than about 80mL / min / 1.73m 2 or greater than about 90 mL / min / 1.73 m 2 In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation thereof increases the glomerular filtration rate of a subject to greater than about 60 mL / min / 1.73 m 2 .

[0272] In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is used to preserve renal function in a subject with a renal disease or renal disorder. As used herein, the term "preserve" refers to maintaining renal function in a subject with a renal disease or renal disorder or preventing further decline in renal function. In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation preserves renal function as measured by blood urine nitrogen, blood creatinine, blood albumin, urine albumin to creatinine ratio and / or glomerular filtration rate, wherein these measurements remain stable after treatment with the modified Cav-1 peptide or its pharmaceutical formulation.

[0273] In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is used to treat or prevent a disease or condition in an elderly subject. As used herein, the term "elderly" or "advanced age" refers to a subject who is 55 years old or older. In some embodiments, the elderly subject is about 55 years old, about 60 years old, about 65 years old, about 70 years old, about 75 years old, about 80 years old, about 90 years old, about 95 years old, or about 100 years old. In some embodiments, the elderly subject has an increased susceptibility to the diseases or conditions described herein compared to younger subjects. In some embodiments, the elderly subject suffers from a fibrotic disease or condition, such as idiopathic pulmonary fibrosis.

[0274] In some embodiments, elderly subjects have reduced caveolin-1 expression compared to younger subjects (e.g., young adults or middle-aged subjects). In some embodiments, elderly subjects have reduced caveolin-1 expression compared to younger subjects by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

[0275] In some embodiments, the present disclosure provides a method of treating or preventing a fibrotic disease or condition in an elderly subject, wherein the method comprises administering to the elderly subject an effective amount of a modified Cav-1 peptide or a pharmaceutical formulation thereof.

[0276] In some embodiments of any of the methods disclosed herein, the formulation comprising a modified Cav-1 peptide as disclosed herein comprises a modified Cav-1 peptide comprising an amino acid sequence of any one of SEQ ID NOs: 2-111. In some embodiments of any of the methods disclosed herein, the formulation comprising a modified Cav-1 peptide as disclosed herein comprises a modified Cav-1 peptide comprising an amino acid sequence of any one of SEQ ID NOs: 2-10. In some embodiments of any of the methods disclosed herein, the formulation comprising a modified Cav-1 peptide as disclosed herein comprises a modified Cav-1 peptide comprising an amino acid sequence of SEQ ID NO: 3. In some embodiments of any of the methods disclosed herein, the formulation comprising a modified Cav-1 peptide as disclosed herein comprises a modified Cav-1 peptide comprising an amino acid sequence of SEQ ID NO: 8. In some embodiments of any of the methods disclosed herein, a formulation comprising a modified Cav-1 peptide as disclosed herein comprises a modified Cav-1 peptide comprising at least one amino acid substitution, deletion, or insertion relative to the amino acid sequence of FTTFTVT (SEQ ID NO: 3), wherein the modified Cav-1 peptide maintains the biological activity of Cav-1.

[0277] In some embodiments of any of the methods disclosed herein, the formulation comprising a modified Cav-1 peptide as disclosed herein comprises an effective amount of a modified Cav-1 peptide comprising an amino acid sequence of any one of SEQ ID NOs: 2-111. In some embodiments of any of the methods disclosed herein, the formulation comprising a modified Cav-1 peptide as disclosed herein comprises an effective amount of a modified Cav-1 peptide comprising an amino acid sequence of any one of SEQ ID NOs: 2-10. In some embodiments of any of the methods disclosed herein, the formulation comprising a modified Cav-1 peptide as disclosed herein comprises an effective amount of a modified Cav-1 peptide comprising an amino acid sequence of SEQ ID NO: 3. In some embodiments of any of the methods disclosed herein, the formulation comprising a modified Cav-1 peptide as disclosed herein comprises an effective amount of a modified Cav-1 peptide comprising an amino acid sequence of SEQ ID NO: 8. In some embodiments of any of the methods disclosed herein, a formulation comprising a modified Cav-1 peptide as disclosed herein comprises an effective amount of a modified Cav-1 peptide comprising at least one amino acid substitution, deletion, or insertion relative to the amino acid sequence of FTTFTVT (SEQ ID NO: 3), wherein the modified Cav-1 peptide maintains the biological activity of Cav-1.

[0278] The present disclosure contemplates all modes of administration, dosing, or dosing frequencies sufficient to treat or prevent a disease or condition in a subject.Effective doses may also be extrapolated from dose-response curves derived from in vitro or animal model test bioassays or systems.

[0279] In some embodiments, the modified Cav-1 peptide or a pharmaceutical formulation thereof is administered systemically. In some embodiments, the modified Cav-1 peptide or a pharmaceutical formulation thereof is administered intravenously, intrathecally, subcutaneously, and / or intraperitoneally.

[0280] In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is delivered topically to the airways of the subject, such as by administering an aerosolized formulation using a nebulizer or administering a dry powder formulation using a dry powder inhaler. In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is administered to the lungs of the subject using a nebulizer. In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is administered to the lungs of the subject using a dry powder inhaler. In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is administered to the subject intranasally, intrabronchially, intrapleurally, intratracheally, or via inhalation.

[0281] In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is administered to the subject in a single dose or multiple doses. When multiple doses are administered, the doses may be spaced apart from each other, for example, one hour, three hours, six hours, eight hours, twelve hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, one year, or any value or range therebetween. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is administered, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 10 weeks, once every 15 weeks, once every 20 weeks, or longer. It should be understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the preparation. For example, if a lower dose does not provide sufficient therapeutic activity, the dose of the modified Cav-1 peptide or its pharmaceutical preparation may be increased.

[0282] In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is administered to a subject at a dose of about 0.0001 mg / kg to about 1,000 mg / kg. In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is administered to a subject at a dose of about 0.0001 mg / kg to about 0.01 mg / kg. In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is administered to a subject at a dose of about 0.01 mg / kg to about 1 mg / kg. In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is administered to a subject at a dose of about 1 mg / kg to about 100 mg / kg. In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is administered to a subject at a dose of about 1 mg / kg to about 50 mg / kg. In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is administered to a subject at a dose of about 1 mg / kg to about 25 mg / kg. In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is administered to a subject at a dose of about 1 mg / kg to about 10 mg / kg. In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is administered to the subject at a dose of about 10 mg / kg to about 25 mg / kg. In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is administered to the subject at a dose of about 25 mg / kg to about 50 mg / kg. In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is administered to the subject at a dose of about 50 mg / kg to about 75 mg / kg. In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is administered to the subject at a dose of about 75 mg / kg to about 100 mg / kg. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is administered to the subject at a dose of about 0.0001 mg / kg, about 0.01 mg / kg, about 0.01 mg / kg, about 0.1 mg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 25 mg / kg, about 50 mg / kg, about 100 mg / kg, about 500 mg / kg, or about 1,000 mg / kg. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is administered to the subject at a dose of about 1 mg / kg to about 10 mg / kg. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is administered to the subject at a dose of about 2 mg / kg to 5 mg / kg. In some embodiments, the modified Cav-1 peptide or a pharmaceutical formulation thereof is administered to a subject at a dose of about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, or any value or range therebetween.In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is administered to a subject at a dose of about 1 mg / kg to about 5 mg / kg. In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is administered to a subject at a dose of about 1.0 mg / kg, about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, about 1.9 mg / kg, about 2.0 mg / kg, about 2.1 mg / kg, about 2.2 mg / kg, about 2.3 mg / kg, about 2.4 mg / kg, about 2.5 mg / kg, about 2.6 mg / kg, about 2.7 mg / kg, about 2.8 mg / kg, about 2.9 mg / kg, about 3 In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is administered to a subject at a dose of about 2.2 mg / kg.

[0283] In some embodiments, the total or complete dose of the modified Cav-1 peptide or pharmaceutical formulation thereof administered to a subject is between about 1 mg and about 100 mg, such as between about 20 mg and about 100 mg, between about 50 mg and about 100 mg, between about 10 mg and about 20 mg, between about 20 mg and about 40 mg, between about 50 mg and about 70 mg, or between about 80 mg and about 90 mg. In some embodiments, the total or complete dose of the modified Cav-1 peptide or its pharmaceutical formulation administered to a subject is about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 12 mg, about 14 mg, about 16 mg, about 18 mg, about 20 mg, about 22 mg, about 24 mg, about 26 mg, about 28 mg, about 30 mg, about 32 mg, about 34 mg, about 36 mg, about 38 mg, about 40 mg, about 42 mg, about 44 mg, about about 46 mg, about 48 mg, about 50 mg, about 52 mg, about 54 mg, about 56 mg, about 58 mg, about 60 mg, about 62 mg, about 64 mg, about 66 mg, about 68 mg, about 70 mg, about 72 mg, about 74 mg, about 76 mg, about 78 mg, about 80 mg, about 82 mg, about 84 mg, about 86 mg, about 88 mg, about 90 mg, about 92 mg, about 94 mg, about 96 mg, about 98 mg, about 100 mg, about 110 mg, about 120 mg, or any value or subrange therebetween.

[0284] In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is prepared as an extended release formulation. The term "extended release" herein refers to the ability to release a component (i.e., a modified Cav-1 peptide) over a specific period of time. Without wishing to be bound by any theory, it is conceivable that the formulations disclosed herein provide a modified Cav-1 peptide over a specific period of time, such as but not limited to about one week or more, about two weeks or more, about three weeks or more, about four weeks or more, about five weeks or more, about six weeks or more, about seven weeks or more, about eight weeks or more, about 12 weeks or more, about 16 weeks or more, about 20 weeks or more, about 24 weeks or more, about 28 weeks or more, about one month or more, about two months or more, about three months or more, about four months or more, about five months or more, about six months or more. Extended release.

[0285] In some embodiments, the dosage of a modified Cav-1 peptide or pharmaceutical formulation thereof for a particular subject is determined by one of ordinary skill in the art using conventional considerations (e.g., by means of an appropriate conventional pharmacological regimen). For example, a physician may prescribe a relatively low dose initially, and subsequently increase the dose until an appropriate response is obtained. Depending on the application, the dose administered to the subject is sufficient to provide a beneficial therapeutic response in the subject over time, or, for example, to reduce symptoms or produce other appropriate activity. The dosage is determined by the efficacy of the particular formulation, as well as the activity, stability, and / or serum half-life of the modified Cav-1 peptide disclosed herein and the subject's condition, as well as the body weight or surface area of ​​the subject to be treated.

[0286] In some embodiments, a dose of a modified Cav-1 peptide or a pharmaceutical formulation thereof is administered to a subject once daily for the treatment or prevention of any of the diseases or disorders described herein. In some embodiments, the subject is an elderly or advanced age subject. In some embodiments, a single dose is between about 0.2 mg / kg and about 250 mg / kg, such as between about 1 mg / kg and about 10 mg / kg, between about 10 mg / kg and about 25 mg / kg, between about 25 mg / kg and about 50 mg / kg, between about 50 mg / kg and about 75 mg / kg, between about 75 mg / kg and about 100 mg / kg, for example, via pulmonary instillation (e.g., inhalation). Such a dose may be administered daily anywhere from about 3 days to one or more weeks or at any frequency as disclosed herein. Long-term administration of a modified Cav-1 peptide or a pharmaceutical formulation thereof is also possible, although, as is well known in the art, the dose may need to be adjusted downward. However, the foregoing ranges are suggestive because the number of variables in individual treatment regimens is large and considerable deviations from these preferred values ​​are expected.

[0287] In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is continuously administered to the subject. For example, for continuous administration by a pump system such as an osmotic pump, the total dose over a time course of about 1-2 weeks is in the range of about 1 mg / kg to about 1 g / kg, about 20 mg / kg to about 300 mg / kg, or about 50 mg / kg to about 200 mg / kg. After such a continuous dosing regimen, the total concentration of the active compound can be in the range of about 0.5 μM to about 50 μM or about 1 μM to about 10 μM.

[0288] In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is administered on a regular schedule. As used herein, a regular schedule refers to a predetermined specified time period. A regular schedule may cover time periods of the same or different lengths, as long as the schedule is predetermined. For example, a regular schedule may involve administration once a day, twice a day, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, once every six months, or any set number of days, weeks, or months in between. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is administered twice a day in the first week, followed by daily administration for several months. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is administered once a day. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is administered less than once a day, such as every other day, every three days, or once a week.

[0289] In some embodiments, the modified Cav-1 peptide formulation is administered to a subject for at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 1 year, or any set number of weeks or months in between. In some embodiments, the modified Cav-1 peptide formulation is administered to a subject with fibrosis for at least about 2 weeks. In some embodiments, the modified Cav-1 peptide formulation is administered to a subject with fibrosis for at least about 4 weeks.

[0290] In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is provided in a unit dosage form (e.g., pre-divided doses), such as capsules, blisters, or cartridges. In some embodiments, the unit dose contains at least 1 mg of the modified Cav-1 peptide formulation, such as at least about 5 mg, at least about 10 mg, at least about 15 mg, or at least about 20 mg of the modified Cav-1 peptide formulation per dose. In some embodiments, the unit dose is about 1 mg to about 10 mg (e.g., about 5 mg) of the modified Cav-1 peptide formulation. In some embodiments, the unit dosage form does not include the administration or addition of any excipients and is only used to contain the powder for inhalation (i.e., no capsules, blisters, or cartridges are administered). In some embodiments, more than one unit dosage form is administered to a subject. For example, in the case of a dry powder inhaler, the modified Cav-1 peptide formulation is provided in a unit dose capsule, and more than one unit dose capsule (e.g., 3-4) can be administered to a subject by inhalation. In some embodiments, the modified Cav-1 peptide formulation is administered at a high emission dose, such as at least about 10 mg, at least about 15 mg, or at least about 20 mg. In some embodiments, administration of a milled modified Cav-1 peptide formulation results in a higher fine particle dose entering the deep lung, such as greater than about 5 mg. In some embodiments, the fine particle dose entering the deep lung is at least about 10 mg or at least about 15 mg. In some embodiments, the particle dose is generated from 1, 2, 3, 4, or 5 or more capsules containing the peptide dose of the embodiment. In some embodiments, the fine particle dose is at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the emission dose.

[0291] In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation thereof is administered in combination with at least one additional therapeutic agent, either simultaneously or sequentially, for treating or preventing a renal disease or renal disorder in a subject. In some embodiments, the disease is chronic kidney disease. Additional therapeutic agents include, but are not limited to, angiotensin converting enzyme (ACE) inhibitors, such as (captopril), (enalapril), (fosinopril), or (lisinopril) or (ramipril); angiotensin II receptor (ARB) inhibitors such as (azilsartan), (eprosartan), (irbesartan), (losartan), (olmesartan) or (valsartan); (dapagliflozin); (darbepoetin alpha); and / or or (erythropoietin).

[0292] In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is administered in combination with dialysis for treating a renal disease or renal disorder in a subject. In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is administered in combination with dialysis for treating chronic kidney disease. In some embodiments, the dialysis is hemodialysis. In some embodiments, the dialysis is peritoneal dialysis.

[0293] In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation thereof is administered in combination with at least one additional therapeutic agent, either simultaneously or sequentially, for pulmonary fibrosis. Additional therapeutic agents include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, disease-modifying antirheumatic drugs (DMARDs), immunosuppressants, biological response modifiers, bronchodilators, or antifibrotic agents such as pirfenedone (whose antifibrotic mechanism of action is not fully understood, but may involve TGF-β blockade), the generalized tyrosine kinase blocker nintedanib, or any other antifibrotic agent.Suitable NSAIDs are selected from the group consisting of the non-selective cyclooxygenase (COX) inhibitors acetylsalicylic acid, mesalazine, ibuprofen, naproxen, flurbiprofen, fenoprofen, fenbufen, ketoprofen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, profen, miroprofen, tioxaprofen, suprofen, alminoprofen, tiaprofenic acid, fluprofen, indomethacin, sulindac, tolmetin, zomepirac, nabumetone, diclofenac, fenclofenac lofenac), alclofenac, bromfenac, ibufenac, aceclofenac, acemetacin, fentiazac, clidanac, etodolac, oxpinac, mefenamic acid, meclofenamic acid, flufenamic acid, niflumic acid The invention relates to an anti-inflammatory drug comprising the following: nifluminic acid, tolfenamic acid, diflunisal, flufenisal, piroxicam, tenoxicam, lornoxicam and nimesulide and pharmaceutically acceptable salts thereof; selective COX2 inhibitors meloxicam, celecoxib and rofecoxib and pharmaceutically acceptable salts thereof.Suitable steroids are prednisone, prednisolone, methylprednisolone, dexamethasone, budenoside, fluocortolone and triamcinolone. Suitable DMARDs are sulfasalazine, olsalazine, chloroquin, gold derivatives (auranofin), D-penicillamine and cytostatics such as methotrexate and cyclophosphamide. Suitable immunosuppressants are cyclosporin A and its derivatives, mycophenolate mofetil, FK506 (also known as tacrolimus and fugimycin), muromonab-CD3 (Orthoclone. ), antithymocyte globulin (ATG), 15-deoxyspergualin, mizoribine, misoprostol, rapamycin, reflunomide and azathioprine. Suitable biological response modifiers are interferon beta, anti-TNF-alpha antibodies (etanercept), IL-10, anti-CD3 antibodies or anti-CD25 antibodies. Suitable bronchodilators are ipratropium bromide, oxytropium bromide, tiotropium bromide, epinephrine hydrochloride, salbutamole, terbutalin sulfate, fenoterol hydrobromide, salmeterole and formoterole. In such combinations, each active ingredient may be administered according to its usual dosage range or at a dosage below its usual dosage range. The dosage of the combined NSAID, steroid, DMARD, immunosuppressant and biological response modifier is appropriately 1 / 50 of the usually recommended minimum dosage to 1 / 1, 1 / 20 to 1 / 2 or 1 / 10 to 1 / 5 of the usually recommended dosage. The usually recommended dosage of the combined drug should be understood as, for example, in Rote 2002, Editio Cantor Verlag Aulendorf, Germany or the doses disclosed in Physician's Desk Reference.

[0294] In some embodiments, the modified Cav-1 peptide or pharmaceutical formulation thereof is administered in combination with at least one additional therapeutic agent simultaneously or sequentially to treat a pathogen or pathogen-induced lung injury. Additional therapeutic agents include, but are not limited to, chloroquine, hydroxychloroquine, type I interferons, antivirals, antibiotics, remdesivir, favipiravir, lopinavir, ritonavir, nirmatrelvir, baricitinib, and molnupiravir.

[0295] Hydroxychloroquine is a chemical derivative of chloroquine that features a hydroxyethyl group instead of an ethyl group. Hydroxychloroquine is classified as an effective antimalarial drug and has shown efficacy in the treatment of systemic lupus erythematosus as well as rheumatoid arthritis and Sjögren's syndrome. Although hydroxychloroquine has long been known to increase lysosomal pH in antigen-presenting cells, its mechanism of action under inflammatory conditions has only recently been elucidated and involves blocking the activation of plasmacytoid dendritic cells by toll-like receptors. Hydroxychloroquine has demonstrated efficacy in the treatment of RNA viruses, including hepatitis C. Hydroxychloroquine can be administered in doses of 600 mg per day.

[0296] Human type I interferon (IFN) is a large subgroup of interferon proteins, which helps regulate the activity of the immune system. Mammalian types are designated as IFN-α (alpha), IFN-β (beta), IFN-κ (kappa), IFN-δ (delta), IFN-ε (epsilon), IFN-τ (tau), IFN-ω (omega) and IFN-ζ (zeta, also known as limitin). Type I interferon has been shown to resist various viruses, including the replication of Zika virus (Zikavirus), chikungunya virus (chikungunyavirus), flavivirus (flaviviruses) and hepatitis C virus. "Interferon compounds" include interferon-α, interferon-α analogs, interferon-α derivatives, interferon-α conjugates, interferon-β, interferon-β analogs, interferon-β derivatives, interferon-β conjugates and mixtures thereof. The whole protein or its fragments can be fused with other peptides and proteins (such as immunoglobulins and other cytokines). Interferon-α and interferon-β conjugates may represent, for example, formulations comprising interferon-β coupled to a non-naturally occurring polymer comprising a polyalkylene glycol moiety. Preferred interferon compounds include (interferon α-2a), (interferon α-2b), (interferon α-n3), (interferon alfacon-1), (interferon alpha), interferon alfacon-1, interferon alpha, interferon alpha analog, pegylated interferon alpha, polymeric interferon alpha, dimerized interferon alpha, interferon alpha conjugated to a carrier, interferon alpha as an oral inhaler, interferon alpha as an injectable formulation, interferon alpha as a topical formulation, (Interferon α-2a) analogs, (Interferon α-2b) analogs, (Interferon α-n3) analogs and (Interferon alfacon-1) analogs, (Interferon alpha) analogs, interferon alfacon-1 analogs, interferon beta, Avonex TM (Interferon β-1a), Betaseron TM (interferon beta-1b), Betaferon TM (interferon beta-1b), Rebif TM (interferon beta-1a), interferon beta analogs, pegylated interferon beta, polymeric interferon beta, dimerized interferon beta, interferon beta conjugated to a carrier, interferon beta as an oral inhaler, interferon beta as an injectable formulation, interferon beta as a topical formulation, Avonex TM (Interferon β-1a) analogs, Betaseron TM (Interferon beta-1b) analogs, Betaferon TM (interferon beta-1b) analogs and Rebif TM (Interferon β-1a) analogs. Alternatively, agents that induce the production of interferon α or interferon β or that mimic the effects of interferon α or interferon β may also be used. Interferon inducers include tilorone, poly(I)-poly(C), imiquimod, cridanimod, and bropirimine.

[0297] In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation comprises different types of carriers, depending on whether the formulation is administered in solid, liquid or aerosol form, and whether its route of administration (such as injection) requires sterility.

[0298] In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is administered intravenously, intrathecally, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly, subcutaneously, mucosally, orally, topically, locally, by inhalation (e.g., inhalation of an aerosolized or dry powder formulation), by injection, by infusion, by continuous infusion, by direct local perfusion of the target cells with a lipid composition (e.g., liposomes) (via a catheter, via lavage), or by other methods known to those of ordinary skill in the art, or any combination of the foregoing (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, 1990, which is incorporated herein by reference). The choice of injection volume and needle size can be selected by one of ordinary skill in the art based on the injection site, syringeability, and injectability, including consideration of the viscosity of the solution or suspension to be injected, as well as the drug concentration, pH, and osmotic pressure. In some cases, the particle size of the active agent may be selected to provide a desired dissolution rate following administration (eg, by subcutaneous injection).

[0299] In some embodiments, the formulations as disclosed herein are administered intravenously, intramuscularly or subcutaneously. In some embodiments, the formulations as disclosed herein are administered by injection. In some embodiments, the formulations as disclosed herein are sustained release formulations, controlled release formulations or extended release formulations.

[0300] In some embodiments, the formulation is an inhalable modified Cav-1 peptide formulation. Administration via inhalation includes, but is not limited to, use of an inhaler or a nebulizer.

[0301] In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is formulated as a free base, neutral or salt form. Pharmaceutically acceptable salts include acid addition salts, such as acid addition salts formed with free amino groups of the protein preparation, or acid addition salts formed with inorganic acids such as, for example, 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, for example, 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 will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulation is easy to administer in various dosage forms, such as formulated for parenteral administration such as injectable solutions, or for aerosols for delivery to the lungs, or formulated for dietary administration such as drug release capsules, etc.

[0302] In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation comprises a pharmaceutically acceptable carrier (with or without an inert diluent). The carrier should be assimilable and include a liquid, semisolid (i.e., paste) or solid carrier. Unless any conventional medium, agent, diluent or carrier is harmful to the therapeutic effectiveness of the recipient or the preparation contained therein, it is appropriate to use it in an administrable formulation for implementing the method. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, adhesives, fillers, etc. or combinations thereof. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation comprises one or more antioxidants to delay oxidation of one or more components in the formulation. In addition, prevention of the effects of microorganisms can 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 a combination thereof.

[0303] In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is combined or thoroughly mixed with a semisolid or solid carrier. Mixing can be performed in any convenient manner, such as grinding. Stabilizers can also be added during the mixing process to protect the formulation from losing its therapeutic activity, such as denaturation in the stomach. Examples of stabilizers include, but are not limited to, buffers, amino acids (such as glycine and lysine), carbohydrates, or lyoprotectants (such as glucose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.).

[0304] In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation comprises one or more surfactants. Surfactants used according to the disclosed methods include ionic surfactants and non-ionic surfactants. Representative non-ionic surfactants include polysorbates, such as and Surfactants (ICI Americas Inc. of Bridgewater, NJ); poloxamers (e.g., poloxamer 188); anionic and nonionic surfactants such as Surfactants (Sigma, St. Louis, Mo.); sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, or stearyl sarcosine; linoleyl betaine, myristyl betaine, or hexadecyl betaine; lauramidopropyl dimethylamine, Cocamidopropyl dimethylamine, linoleamidopropyl dimethylamine, myristamidopropyl dimethylamine, palmitamidopropyl dimethylamine, or isostearamidopropyl betaine (e.g. lauroyl propyl); myristamidopropyl dimethylamine, palmitamidopropyl dimethylamine, or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleoyl taurate; cationic surfactants or quaternary phospholipid surfactants such as MONAQUAT TM Surfactants (Mona Industries Inc. of Paterson, NJ); polyethylene glycol; polypropylene glycol; block copolymers of ethylene glycol and propylene glycol, such as Surfactants (BASF of Mt. Olive, NJ); oligo(ethylene oxide) alkyl ethers; alkyl(thio)glucosides, alkyl maltosides; and phospholipids. In some embodiments, one or more surfactants are present in the pharmaceutical formulation in an amount of about 0.01% to about 0.5% (the weight of the surfactant relative to the total weight of other solid components in the formulation; "w / w"), about 0.03% to about 0.5% (w / w), about 0.05% to about 0.5% (w / w), or about 0.1% to about 0.5% (w / w). In some embodiments, the modified Cav-1 peptide or its pharmaceutical formulation is substantially free of nonionic surfactants or substantially free of all surfactants.

[0305] In terms of the treatment methods of the present invention, the administration of the modified Cav-1 peptide or its pharmaceutical preparation disclosed herein is not intended to be limited to a specific mode of administration, dosage or frequency of administration. The present invention contemplates all modes of administration, including intramuscular, intravenous, intraperitoneal, intracapsular, intraarticular, intralesional, subcutaneous or any other route sufficient to provide an appropriate dose for treating a disease or condition. The modified Cav-1 peptide or its pharmaceutical preparation can be administered to the patient in a single dose or multiple doses. When multiple doses are administered, the doses may be spaced apart from each other, for example, one hour, three hours, six hours, eight hours, twelve hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, one year or any value or range therebetween. In some embodiments, the modified Cav-1 peptide or its pharmaceutical preparation is administered, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 10 weeks, once every 15 weeks, once every 20 weeks or longer. It is understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulation. For example, the dosage of the modified Cav-1 peptide or its pharmaceutical formulation may be increased if a lower dosage does not provide adequate therapeutic activity.

[0306] Example

[0307] The present disclosure is further described in detail by reference to the following examples. Unless otherwise indicated, these examples are provided for illustrative purposes only and are not intended to be limiting. Therefore, the present disclosure should never be construed as being limited to the following examples, but rather should be construed as covering any and all variations that become apparent due to the teachings provided herein.

[0308] Example 1. SAIB-based modified Cav-1 peptide formulation

[0309] A modified Cav-1 peptide (APi2355, SEQ ID NO: 8) formulation based on SAIB was prepared. Approximately 34.0 mg of micronized APi2355 was weighed into a 1 mL syringe. Approximately 567 mg of an 80% w / w SAIB solution in NMP was weighed separately into a separate 1 mL syringe. A Luer-to-Luer connector was placed between the 2 syringes and mixed until a uniform white suspension (30 mg APi2355 / 400 mg SAIB) was formed. Similarly, 10 mg APi2355 / 500 mg SAIB, 30 mg APi2355 / 500 mg SAIB, and 50 mg APi2355 / 500 mg SAIB suspensions were formed.

[0310] Approximately 150 mg of 30 mg APi2355 / 400 mg SAIB suspension was added to approximately 35 mL of phosphate buffered saline (PBS) and placed on a shaking shaker at 37°C, 150 rpm. 1 mL samples were taken out for testing at different time points and replaced with fresh preheated PBS. The samples were filtered and analyzed by HPLC for the percent release of APi2355. The in vitro release kinetic parameters of the 30 mg APi2355 / 5400 mg SAIB formulation were determined, as shown in Table 1. Figure 1 shown.

[0311] Example 2. Pharmacokinetics of Modified Cav-1 Peptide SAIB Formulations

[0312] The plasma pharmacokinetics of the Cav-1 peptide (APi2355) SAIB formulations (5.66% w / w APi235575.47% w / w SAIB and 18.87% w / w NMP, to provide 7% w / w APi2355 to 93% w / w SAIB) prepared according to Example 1 were evaluated within 42 days after a single dose of subcutaneous administration to male Sprague Dawley rats. Rats (n = 6) received one of three batches of APi2355 SAIB formulations prepared according to Example 1, with a dose of 0.033 mL / kg of 2.0 mg / kg APi2355 (concentration of 60.0 mg / mL). Control group rats (n = 2) received injections of vehicle formulations without APi2355.

[0313] Experimental Procedure

[0314] Animals were assigned to each group by a stratified randomization scheme to achieve similar group average body weights. Animals were adapted to their designated housing at least 5 days before administration on the same day. All preparations were administered with a single dose by subcutaneous (SC) injection in the back on the 0th day. Whole blood samples (40-50 μl) were collected from all animals in the APi2355 treatment group at 1h, 2h, 4h, 8h, 1 day, 2 days, 4 days, 7 days, 10 days, 14 days, 21 days, 29 days and 35 days before administration and after treatment. Whole blood samples (40-50 μl, by tail incision) were collected from all animals in the control group at 1h and 1 day before administration and after treatment. Blood was collected in K2EDTA tubes. After blood collection, the tubes were gently mixed, cooled within 30 minutes after collection and centrifuged at 3000rpm and 4°C. The resulting plasma was separated, transferred to a uniquely labeled polypropylene tube and frozen at -20°C. APi2355 concentrations in plasma and tissue samples were analyzed using a qualified LC / MS / MS analytical procedure.

[0315] On day 42 after treatment, terminal whole blood samples (maximum capacity by cardiac puncture) and tissues were collected from all animals. Tissues included tissues from the heart, liver, kidney, lung, and skin patch at the injection site. In addition, the lungs were processed to obtain bronchial lavage samples. APi2355 in tissue samples was analyzed by LC / MS / MS.

[0316] Pharmacokinetic results

[0317] All concentration values ​​of APi2355 in the control group and before administration were below the lower limit of quantification (<0.3 ng / mL). The concentration-time curve of APi2355 in the plasma of rats in the APi2355 treatment group was determined, as shown in Table 4. Figure 2 The mean concentration profiles of APi2355 in various tissues were also determined as shown in Table 5. Table 6 summarizes the mean pharmacokinetic exposure profiles of APi2355 in rat plasma measured after administration of SAIB formulations.

[0318]

[0319]

[0320] Table 7 summarizes the pharmacokinetic clearance and volume of distribution measured after administration of SAIB formulations.

[0321] Table 7. Pharmacokinetic clearance and distribution volume parameters

[0322]

[0323] T 1 / 2 : Terminal half-life

[0324] MRT last : The average residence time from time zero to the time of the last quantifiable concentration

[0325] V z / F: Apparent volume of distribution during the terminal phase

[0326] CL / F: Apparent plasma clearance of the drug

[0327] λz: terminal rate constant

[0328] Rsq:adj:Adjusted coefficient of determination

[0329] Pharmacokinetic profile

[0330] After a single subcutaneous administration, APi2355 was absorbed from the SAIB formulation, with a median T max The value is 0.083 days, and the average C maxThe value was 477 ng / mL. After administration, APi2355 was detectable in the plasma of 4 of 6 rats. The average concentration at the last quantifiable time point was 0.942 ng / mL. As shown in Table 6, AUC 0-14 The average value is 126 days*ng / mL, AUC 0-22 The average value was 146 days*ng / mL, and AUC inf The average value was 183 days*ng / mL.

[0331] AUC inf The extrapolated AUC of APi2355 was <20%, so the time points used to evaluate APi2355 concentrations fully described the systemic exposure profile, and certain PK parameters (such as AUC inf ) are considered accurate. Rsq adjusted values ​​are typically < 0.8 due to span variation, which is typically > 3 times the half-life. APi2355 concentrations vary significantly within the span, resulting in a lower Rsqadj. Half-life values ​​are presented as an average of 9.24 days. Average MRT for SAIB formulations last The value was 7.82 days. Approximately 2 / 3 of the total exposure to APi2355 occurred within the first 3 weeks after SC administration.

[0332] Numbered implementation plan

[0333] Embodiment 1. A formulation comprising: a) a polypeptide comprising an amino acid sequence having a core sequence of FTTFTVT; and b) sucrose acetate isobutyrate (SAIB).

[0334] Embodiment 2. The formulation of embodiment 1, further comprising a first solvent.

[0335] Embodiment 3. The formulation of embodiment 2, wherein the first solvent is selected from N-methylpyrrolidone (NMP), anhydrous ethanol or ethyl acetate.

[0336] Embodiment 4. The formulation of any one of embodiments 1-3, wherein SAIB is present in the first solvent at about 50% w / w to about 95% w / w.

[0337] Embodiment 5. The formulation of any one of embodiments 1-4, wherein SAIB is present in the first solvent at about 70% w / w to about 90% w / w.

[0338] Embodiment 6. The formulation of any one of embodiments 1-5, wherein SAIB is present in the first solvent at about 80% w / w.

[0339] Embodiment 7. The formulation of any one of embodiments 1-6, wherein the first solvent is NMP or ethyl acetate.

[0340] Embodiment 8. The formulation of any one of embodiments 1-7, wherein the polypeptide is micronized.

[0341] Embodiment 9. The formulation of any one of embodiments 1-8, wherein the polypeptide has an average particle size in the range of about 0.5 μm to about 100 μm.

[0342] Embodiment 10. The formulation of any one of embodiments 1-9, wherein the polypeptide has an average particle size in the range of about 1 μm to about 5 μm.

[0343] Embodiment 11. The formulation of any one of embodiments 1-10, wherein the formulation is in the form of an emulsion, a solution, a microsphere, a nanoparticle, a nanosphere, an implant, or a gel.

[0344] Embodiment 12. The formulation of any one of embodiments 1-10, wherein the formulation is a suspension.

[0345] Embodiment 13. The formulation of any one of embodiments 1-12, further comprising a second solvent.

[0346] Embodiment 14. The formulation of embodiment 13, wherein the second solvent is sterile water, phosphate buffered saline (PBS), or any pharmaceutically acceptable carrier.

[0347] Embodiment 15. The formulation of any one of embodiments 1-14, wherein the formulation comprises the polypeptide and SAIB in a weight ratio of about 1:50 to about 1:1.

[0348] Embodiment 16. The formulation of embodiment 15, wherein the weight ratio is about 1:30 to about 1:1.

[0349] Embodiment 17. The formulation of any one of embodiments 1-16, wherein the formulation releases at least about 1% to about 30% of the polypeptide within four hours under physiological conditions.

[0350] Embodiment 18. The formulation of any one of embodiments 1-16, wherein the formulation releases at least about 5% to about 25% of the polypeptide within four hours under physiological conditions.

[0351] Embodiment 19. The formulation of any one of embodiments 1-18, wherein the polypeptide comprises ASFTTFTVTK.

[0352] Embodiment 20. A formulation as described in any of embodiments 1-19, wherein the polypeptide 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 the C-terminus.

[0353] Embodiment 21. The formulation of any one of embodiments 1-20, wherein the polypeptide consists of 20 amino acids or less.

[0354] Embodiment 22. The formulation of any one of embodiments 1-21, wherein the additions made within 5 amino acids of each terminus have an amino acid sequence that is less than 80% identical to the contiguous amino acid sequence of SEQ ID NO: 1.

[0355] Embodiment 23. The formulation of embodiment 22, wherein the additions made within 5 amino acids at each end have an amino acid sequence that is less than 60% identical to the contiguous amino acid sequence of SEQ ID NO: 1.

[0356] Embodiment 24. The formulation of embodiment 22, wherein the additions made within 5 amino acids at each end have an amino acid sequence that is less than 40% identical to the contiguous amino acid sequence of SEQ ID NO: 1.

[0357] Embodiment 25. The formulation of embodiment 22, wherein the additions made within 5 amino acids at each end have an amino acid sequence that is less than 20% identical to the contiguous amino acid sequence of SEQ ID NO: 1.

[0358] Embodiment 26. A formulation as described in any of embodiments 1-25, wherein the peptide sequence comprises: a) the polypeptide comprises L-amino acids; b) the polypeptide comprises D-amino acids; or c) the polypeptide comprises both L-amino acids and D-amino acids.

[0359] Embodiment 27. The formulation of any one of embodiments 1-26, wherein: a) the polypeptide comprises at least one non-standard amino acid; or b) the polypeptide comprises two non-standard amino acids.

[0360] Embodiment 28. The formulation of embodiment 27, wherein the non-standard amino acid is ornithine.

[0361] Embodiment 29. The formulation of any one of embodiments 1-28, wherein the polypeptide further comprises: a) an N-terminal modification; b) a C-terminal modification; or c) both an N-terminal and a C-terminal modification.

[0362] Embodiment 30. A formulation as described in Embodiment 29, wherein: the N-terminal modification is acylation; and / or the C-terminal modification is amidation.

[0363] Embodiment 31. The formulation of any one of embodiments 1-30, wherein the polypeptide 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), Ac-aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO: 8), OASFTTFTVTOS (SEQ ID NO: 9), or OASFTTFTVTOS-NH2 (SEQ ID NO: 10).

[0364] Embodiment 32. A formulation as described in any of embodiments 1-31, wherein the polypeptide consists of an amino acid sequence selected from the following: KASFTTFTVTKGS (SEQ ID NO:4), KASFTTFTVTKGS-NH2 (SEQ ID NO:5), aaEGKASFTTFTVTKGSaa (SEQ ID NO:6), aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO:7), Ac-aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO:8), OASFTTFTVTOS (SEQ ID NO:9) or OASFTTFTVTOS-NH2 (SEQ ID NO:10).

[0365] Embodiment 33. The formulation of any one of embodiments 1-32, wherein the polypeptide consists of the amino acid sequence Ac-aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO: 8).

[0366] Embodiment 34. A method of treating a disease in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of the formulation of any one of embodiments 1-33.

[0367] Embodiment 35. The method of embodiment 34, wherein the disease is fibrosis.

[0368] Embodiment 36. The method of embodiment 35, wherein the fibrosis is interstitial lung disease, liver fibrosis, renal fibrosis, skin fibrosis, glomerulonephritis, systemic sclerosis, cardiac fibrosis, myocardial fibrosis, kidney fibrosis, liver cirrhosis, nephrosclerosis, arteriosclerosis, macular degeneration, ocular scars, cataracts, retinal and vitreoretinal diseases, Graves' ophthalmopathy, neurofibromatosis, scleroderma, glioblastoma, keloid and hypertrophic scars, peritoneal fibrotic disease, chronic obstructive pulmonary disease, postoperative uterine fibroids, diabetic nephropathy, gynecological cancer, myeloproliferative syndrome, myeloid leukemia, myelodysplastic syndrome, inflammatory bowel disease, nonalcoholic fatty liver disease, fibrosarcoma, rheumatoid arthritis, nonalcoholic steatohepatitis, Alport syndrome, or chronic COVID syndrome.

[0369] Embodiment 37. The method of embodiment 36, wherein the interstitial lung disease is idiopathic pulmonary fibrosis, familial pulmonary fibrosis, idiopathic nonspecific interstitial pneumonia, conventional interstitial pneumonia, cryptogenic organizing pneumonia, or sarcoidosis.

[0370] Embodiment 38. The method of embodiment 37, wherein the interstitial lung disease is idiopathic pulmonary fibrosis.

[0371] Embodiment 39. The method of embodiment 34, wherein the disease is a kidney disease or a kidney disorder.

[0372] Embodiment 40. The method of embodiment 39, wherein the renal disease or renal disorder is selected from the group consisting of chronic kidney disease, end-stage renal disease, glomerulonephritis, focal segmental glomerulosclerosis, renal fibrosis, polycystic kidney disease, IgA nephropathy, lupus nephritis, nephrotic syndrome, Alport syndrome, amyloidosis, Goodpasture's syndrome, granulomatosis with polyangiitis, or acute kidney injury.

[0373] Embodiment 41. The method of embodiment 40, wherein the renal disease or renal disorder is renal fibrosis.

[0374] Embodiment 42. The method of embodiment 40, wherein the renal disease or renal disorder is Alport's syndrome.

[0375] Embodiment 43. The method of any one of embodiments 34-42, wherein the administration is intraocular, intradermal, transdermal, intramuscular, or subcutaneous.

[0376] Embodiment 44. The method of any one of embodiments 34-43, wherein the subject is a human.

[0377] Embodiment 45. The method of any one of embodiments 34-44, wherein the formulation releases the polypeptide for at least 7 days after administration of a single dose.

[0378] Embodiment 46. The method of any one of embodiments 34-44, wherein the formulation releases the polypeptide for at least 21 days after administration of a single dose.

[0379] Embodiment 47. The method of any one of embodiments 34-44, wherein the formulation releases the polypeptide for at least 28 days after administration of a single dose.

[0380] Embodiment 48. The method of any one of embodiments 34-47, wherein the formulation is administered to the subject at a dose of about 0.01 mg / kg to about 250 mg / kg.

[0381] Embodiment 49. The method of embodiment 48, wherein the formulation is administered to the subject at a dose of about 0.05 mg / kg to about 50 mg / kg.

Claims

1. A preparation comprising: a) contains an amino acid sequence having the core sequence FTTFTVT; and b) Sucrose acetate isobutyrate (SAIB).

2. The formulation of claim 1, further comprising a first solvent.

3. The formulation of claim 2, wherein the first solvent is selected from N-methylpyrrolidone (NMP), anhydrous ethanol or ethyl acetate.

4. The formulation of any one of claims 1-3, wherein the SAIB is present in the first solvent at about 50% w / w to about 95% w / w.

5. The formulation of any one of claims 1-4, wherein the SAIB is present in the first solvent at about 70% w / w to about 90% w / w.

6. The formulation of any one of claims 1-5, wherein the SAIB is present in the first solvent at about 80% w / w.

7. The formulation of any one of claims 1-6, wherein the first solvent is NMP or ethyl acetate.

8. The formulation of any one of claims 1-7, wherein the polypeptide is micronized.

9. The formulation of any one of claims 1-8, wherein the polypeptide has an average particle size in the range of about 0.5 μm to about 100 μm.

10. The formulation of any one of claims 1-9, wherein the polypeptide has an average particle size in the range of about 1 μm to about 5 μm.

11. The formulation of any one of claims 1-10, wherein the formulation is in the form of an emulsion, a solution, a microsphere, a nanoparticle, a nanosphere, an implant or a gel.

12. The formulation of any one of claims 1-10, wherein the formulation is a suspension.

13. The formulation of any one of claims 1-12, further comprising a second solvent.

14. The formulation of claim 13, wherein the second solvent is sterile water, phosphate buffered saline (PBS), or any pharmaceutically acceptable carrier.

15. The formulation of any one of claims 1-14, wherein the formulation comprises the polypeptide and the SAIB in a weight ratio of about 1:50 to about 1:

1.

16. The formulation of claim 15, wherein the weight ratio is from about 1:30 to about 1:

1.

17. The formulation of any one of claims 1-16, wherein the formulation releases at least about 1% to about 30% of the polypeptide within four hours under physiological conditions.

18. The formulation of any one of claims 1-16, wherein the formulation releases at least about 5% to about 25% of the polypeptide within four hours under physiological conditions.

19. The formulation of any one of claims 1-18, wherein the polypeptide comprises ASFTTFTVTK.

20. The formulation of any one of claims 1-19, wherein the polypeptide 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 the C-terminus.

21. The formulation of any one of claims 1-20, wherein the polypeptide consists of 20 amino acids or less.

22. The formulation of any one of claims 1-21, wherein the additions made within 5 amino acids at each end have an amino acid sequence that is less than 80% identical to the contiguous amino acid sequence of SEQ ID NO:

1.

23. The formulation of claim 22, wherein the additions made within 5 amino acids at each end have an amino acid sequence that is less than 60% identical to the contiguous amino acid sequence of SEQ ID NO:

1.

24. The formulation of claim 22, wherein the additions made within 5 amino acids at each end have an amino acid sequence that is less than 40% identical to the contiguous amino acid sequence of SEQ ID NO:

1.

25. The formulation of claim 22, wherein the additions made within 5 amino acids at each end have an amino acid sequence that is less than 20% identical to the contiguous amino acid sequence of SEQ ID NO:

1.

26. The formulation of any one of claims 1-25, wherein the peptide sequence comprises: a) the polypeptide comprises L-amino acids; b) the polypeptide comprises D-amino acids; or c) The polypeptide comprises both L-amino acids and D-amino acids.

27. The formulation of any one of claims 1-26, wherein: a) the polypeptide comprises at least one non-standard amino acid; or b) The polypeptide comprises two non-standard amino acids.

28. The formulation of claim 27, wherein the non-standard amino acid is ornithine.

29. The formulation of any one of claims 1-28, wherein the polypeptide further comprises: a) N-terminal modification; b) C-terminal modification; or c) N-terminal and C-terminal modifications.

30. The formulation of claim 29, wherein: The N-terminal modification is acylation; and / or The C-terminal modification is amidation.

31. The formulation of any one of claims 1-30, wherein the polypeptide 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), Ac-aaEGKA SFTTFTVTKGSaa-NH2 (SEQ ID NO:8), OASFTTFTVTOS (SEQ ID NO:9), or OASFTTFTVTOS-NH2 (SEQ ID NO:10).

32. The formulation of any one of claims 1-31, wherein the polypeptide consists of an amino acid sequence selected from the group consisting of: KASFTTFTVTKGS (SEQ ID NO: 4), KA SFTTFTVTKGS-NH2 (SEQ ID NO: 5), aaEGKASFTTFTVTKGSaa (SEQ ID NO: 6), aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO: 7), Ac-aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO: 8), OASFTTFTV TOS (SEQ ID NO:9) or OASFTFTVTOS-NH2 (SEQ ID NO:10).

33. The formulation of any one of claims 1-32, wherein the polypeptide consists of the amino acid sequence Ac-aaEGKASFTTFTVTKGSaa-NH2 (SEQ ID NO: 8).

34. A method of treating a disease in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of the formulation of any one of claims 1-33.

35. The method of claim 34, wherein the disease is fibrosis.

36. The method of claim 35, wherein the fibrosis is interstitial lung disease, liver fibrosis, renal fibrosis, skin fibrosis, glomerulonephritis, systemic sclerosis, cardiac fibrosis, myocardial fibrosis, kidney fibrosis, liver cirrhosis, nephrosclerosis, arteriosclerosis, macular degeneration, ocular scars, cataracts, retinal and vitreoretinal diseases, Graves' ophthalmopathy, neurofibromatosis, scleroderma, glioblastoma, keloid and hypertrophic scars, peritoneal fibrotic disease, chronic obstructive pulmonary disease, postoperative uterine fibroids, diabetic nephropathy, gynecological cancer, myeloproliferative syndrome, myeloid leukemia, myelodysplastic syndrome, inflammatory bowel disease, nonalcoholic fatty liver disease, fibrosarcoma, rheumatoid arthritis, nonalcoholic steatohepatitis, Alport syndrome, or chronic COVID syndrome.

37. The method of claim 36, wherein the interstitial lung disease is idiopathic pulmonary fibrosis, familial pulmonary fibrosis, idiopathic nonspecific interstitial pneumonia, conventional interstitial pneumonia, cryptogenic organizing pneumonia, or sarcoidosis.

38. The method of claim 37, wherein the interstitial lung disease is idiopathic pulmonary fibrosis.

39. The method of claim 34, wherein the disease is a kidney disease or a kidney disorder.

40. The method of claim 39, wherein the renal disease or renal disorder is selected from the group consisting of chronic kidney disease, end-stage renal disease, glomerulonephritis, focal segmental glomerulosclerosis, renal fibrosis, polycystic kidney disease, IgA nephropathy, lupus nephritis, nephrotic syndrome, Alport syndrome, amyloidosis, Goodpasture's syndrome, granulomatosis with polyangiitis, or acute kidney injury.

41. The method of claim 40, wherein the renal disease or disorder is renal fibrosis.

42. The method of claim 40, wherein the renal disease or disorder is Alport's syndrome.

43. The method of any one of claims 34-42, wherein the administration is intraocular, intradermal, transdermal, intramuscular, or subcutaneous.

44. The method of any one of claims 34-43, wherein the subject is a human.

45. The method of any one of claims 34-44, wherein the formulation releases the polypeptide for at least 7 days after administration of a single dose.

46. ​​The method of any one of claims 34-44, wherein the formulation releases the polypeptide for at least 21 days after administration of a single dose.

47. The method of any one of claims 34-44, wherein the formulation releases the polypeptide for at least 28 days after administration of a single dose.

48. The method of any one of claims 34-47, wherein the formulation is administered to the subject at a dose of about 0.01 mg / kg to about 250 mg / kg.

49. The method of claim 48, wherein the formulation is administered to the subject at a dose of about 0.05 mg / kg to about 50 mg / kg.

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