Dry powder formulations of cavein-1 peptides and methods of use thereof

Abstract

Provided herein are compositions comprising a Cav-1 (Cav-1) peptide. Also provided are methods of treating pulmonary infections or acute or chronic lung injuries, in particular pulmonary fibrosis, using the Cav-1 peptides.

Description

[0001] This application is a divisional application of Chinese patent application No. 201980073144.0.

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 729,010, filed September 10, 2018, which is incorporated herein by reference in its entirety.

[0003] The present invention is the result of activities conducted within the scope of a joint research agreement that was in effect at the time the present invention was made. The parties to the joint research agreement are The University of Texas System and the Board of Trustees of Pulmonary Therapeutics. Technical Field

[0004] The present invention relates generally to the fields of molecular biology, pharmacy and medicine. More specifically, the present invention relates to compositions and methods for delivering dry powder therapeutic polypeptide compositions to a subject, such as by delivery to the respiratory system. Background Art

[0005] During lung injury, p53 expression increases, inducing plasminogen activator inhibitor 1 (PAI-1) while inhibiting the expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR), leading to apoptosis of lung epithelial cells (LECs). The mechanism of injury involves cell surface signaling interactions between uPA, uPAR, caveolin-1 ("Cav-1"), and β1-integrin (Shetty et al., 2005). Compositions that modulate these interactions can be used in methods for inhibiting apoptosis in injured, diseased, or damaged tissues. For example, for treating inflammatory or fibrotic conditions, such as pulmonary fibrosis. Therefore, there is a need for polypeptides that can be used to prevent or treat lung injury and disease, particularly stable formulations and simple methods for therapeutic delivery of such polypeptides. Summary of the Invention

[0006] According to the present disclosure, a dry powder composition of a peptide is provided, which comprises the amino acid sequence of SEQ ID NO: 2.

[0007] In a first embodiment, a pharmaceutical composition is provided comprising a dry powder of a peptide comprising any one of the following sequences: SEQ ID NOs: 2-20. In some aspects, the peptide is 7-20 amino acids in length. In a specific aspect, the peptide comprises the amino acid sequence of SEQ ID NO: 2. In other aspects, the peptide comprises at least one amino acid added to the N-terminus of the peptide of SEQ ID NO: 2. In other aspects, the peptide comprises at least one amino acid added to the C-terminus of the peptide of SEQ ID NO: 2. In another aspect, the peptide comprises at least one amino acid added to both the N-terminus and the C-terminus of the peptide of SEQ ID NO: 2. In certain aspects, the peptide may comprise an L-amino acid or a D-amino acid or both an L-amino acid and a D-amino acid. In other aspects, the peptide may comprise at least one non-standard amino acid. In several aspects, the peptide comprises two non-standard amino acids. In a specific aspect, the non-standard amino acid is ornithine.

[0008] In other aspects, the peptide may comprise an N-terminal modification or a C-terminal modification or both. In a specific aspect, the N-terminal modification is an acylation. In another aspect, the C-terminal modification is an amidation.

[0009] In some aspects, the peptide may comprise the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 10, or SEQ ID NO: 20. In several aspects, the peptide comprises at least two repeats of the sequence of any one of SEQ ID NO: 2-20. In specific aspects, the at least two repeats have the same amino acid sequence. In other aspects, the at least two repeats have different amino acid sequences. In other aspects, the pharmaceutical composition further comprises a cell penetrating peptide (CPP). In certain aspects, the CPP comprises an amino acid sequence selected from the group consisting of GRKKRRQRRRPPQ (SEQ ID NO: 23), RQIKIWFQNRRMKWKK (SEQ ID NO: 24), and GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 25).

[0010] In other aspects, the dry powder is produced by a grinding process. In several aspects, the dry powder is produced by a spray drying process. In alternative aspects, the dry powder is produced by jet grinding, ball milling or wet grinding. In some aspects, the dry powder comprises less than 10% (by weight) water. On the other hand, the dry powder comprises less than 1% (by weight) water. In certain aspects, the pharmaceutical composition is substantially free of excipients. In a specific aspect, the pharmaceutical composition is free of excipients. In specific aspects, the pharmaceutical composition is formulated for pulmonary delivery. On the other hand, the pharmaceutical composition is formulated for dry powder inhalation. In other aspects, the pharmaceutical composition is formulated for inhalation pressurized metered dose inhalation. In some aspects, the pharmaceutical composition is formulated for oral administration, topical application or injection.

[0011] In some aspects, the dry powder formulation of the embodiment comprises a water content of less than about 10%, 9%, 8%, 7%, 6% or 5%. In other aspects, the composition comprises a water content of about 0.01% to about 10%, 0.1% to about 10%, 1.0% to about 8% or 1% to about 5%. In other aspects, the dry powder formulation of the embodiment comprises an average particle size of less than 10 μm. In some aspects, the average particle size is about 0.01 μm to about 10 μm; about 0.1 μm to about 8 μm; about 0.5 μm to about 7 μm or about 1 μm to about 5 μm. In some aspects, at least about 50%, 55%, 60%, 65% or 70% of the dry powder composition of the embodiment includes a particle size of about 1 μm to about 5 μm. In some aspects, the dry powder formulation of the peptide of the embodiment (e.g., CSP7) is composed of at least 70% (e.g., 70%-80%) particles with a particle size of about 1 μm to about 5 μm. In preferred aspects, at least about 70%, 75%, 80%, or 85% (eg, 75%-95%) of the particles in the dry powder formulation have a particle size of less than 5 μm.

[0012] A further embodiment of the present invention provides a nebulizer device comprising the pharmaceutical composition of the above embodiments and aspects.

[0013] In another embodiment, a method for treating a subject is provided, the method comprising administering an effective amount of the pharmaceutical composition of the above embodiments and aspects to the subject, administering the subject. In certain aspects, the subject suffers from inflammatory diseases. In other aspects, the subject suffers from fibrotic conditions. In several aspects, the subject suffers from pulmonary inflammation, acute lung injury, lung infection or lung. On the other hand, the subject suffers from pulmonary inflammation. In a specific aspect, the subject suffers from chronic obstructive pulmonary disease (COPD). In other aspects, the subject may suffer from acute lung injury or infection, lung infection, chemically induced lung injury, plastic bronchitis, asthma, acute respiratory distress syndrome (ARDS), acute lung injury induced by smoke inhalation (ISALI), bronchiolitis or bronchiolitis obliterans. In specific aspects, the lung disease is fibrotic conditions, interstitial lung disease or idiopathic pulmonary fibrosis (IPF) or lung scarring of the lung. In other aspects, the administration comprises dry powder inhalation. In other aspects, the administration comprises a solution comprising atomized variant polypeptides.

[0014] In other aspects, the method further comprises administering at least one additional anti-fibrotic therapeutic agent. In certain aspects, the at least one additional anti-fibrotic therapeutic agent is an NSAID, a steroid, a DMARD, an immunosuppressant, a biological response modifier, or a bronchodilator. In several aspects, the subject is a human.

[0015] Yet another embodiment of the present invention provides a pharmaceutical composition comprising a peptide of SEQ ID No: 2-20 formulated as a ground dry powder having a respirable particle size. For example, in certain aspects, the ground dry powder comprises a mass median aerodynamic diameter (MMAD) of less than about 10 microns. Methods for determining MMAD are provided, for example, in Carvalho et al., 2011, which is incorporated herein by reference.

[0016] In yet another embodiment, a method of treating a subject is provided, the method comprising administering to the subject by inhalation an effective amount of a composition of the embodiments.

[0017] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that although preferred embodiments of the present invention are indicated, the detailed description and specific examples are given by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art through this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following drawings form part of this specification and are included to further illustrate certain aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0019] Figure 1 Scanning electron microscope image of CSP7 bulk powder. The powder sample was sputtered onto a sample tray and dispersed by blowing compressed nitrogen gas. The sample was observed using a Hitachi S5500 SEM / STEM scanning electron microscope. The scale bar is shown in the lower right corner of the image.

[0020] Figure 2 : Optical microscope images of CSP7 bulk powder. The powder samples were sputtered onto a glass slide and observed using a Leica optical microscope (Leica CTR6500). The scale bar is shown in the lower left corner of each image. Arrows point to agglomerated particles of pure CSP7 powder.

[0021] Figure 3 X-ray powder diffraction of CSP7 bulk powder particles. CSP7 bulk powder particles were evaluated using X-ray powder diffraction to determine crystallinity. The powder was measured between 2 and 40 2θ degrees using a step size of 0.025 2θ degrees and a speed of 2 degrees / min.

[0022] Figure 4 Polarized light microscopy of CSP7 bulk powder. Crystallinity was assessed using polarized light microscopy. Representative images are shown. White arrows point to crystalline regions.

[0023] Figure 5 Differential Scanning Calorimetry of CSP7 Bulk Powder. Bulk CSP7 powder was analyzed by differential scanning calorimetry using a TA Instruments Q20 Differential Scanning Calorimeter. The figure shows a modulated DSC curve with a frequency of 1°C / 60s and a rate of 2°C / min, from 25°C to 300°C.

[0024] Figure 6 Thermogravimetric analysis of CSP7 bulk powder. Thermogravimetric analysis was performed using a Mettler TGA / DSC thermogravimetric analyzer. The figure shows the TGA curve. The heating rate was set at 10°C / min from 25°C to 500°C.

[0025] Figure 7 Dynamic vapor sorption of CSP7 bulk powder. Bulk CSP7 powder was run on a Surface Measurement Systems DVS instrument, performing a complete sorption / desorption cycle from 0% to 90% relative humidity in 10% steps at 25°C. Water desorption at 0% humidity and mass change at 90% humidity are shown.

[0026] Figure 8 Figure 2: Particle size distribution of spray-dried CSP7. CSP7 was mixed with leucine, trehalose, sodium citrate, or leucine and trehalose and spray-dried. Particle size was assessed using a Malvern Mastersizer 2000 (laser diffraction, Fraunhofer approximation; dispersion pressure: 3.0 bar). The graph shows the curves for each spray-dried mixture.

[0027] Figure 9 Visual observation of a homogenized CSP7 suspension. A. Ethanol treatment at maximum rotor-stator power for 1 minute; B. Untreated CSP7-ethanol suspension; red arrows indicate large particles / aggregates; C. CSP7-ethanol suspension treated at maximum rotor-stator power for 1 minute, resulting in a dark gray color; D. CSP7-ethanol suspension treated at minimum rotor-stator power for 1 minute, resulting in a light gray color.

[0028] Figure 10 : Optical microscopy of jet-milled CSP7 powder. Powder samples collected from designated locations of the jet mill were imaged using an optical microscope. Representative images are shown.

[0029] Figure 11 : Scanning electron microscopy of jet-milled CSP7 powder. The milled CSP7 powder (lot 171027) was imaged by scanning electron microscopy (SEM) under the same conditions as bulk CSP7 powder. A representative SEM image of the milled CSP7 powder is shown.

[0030] Figure 12 : CSP7 optical microscopy of the film after freezing. The powder sample was sputtered onto a glass slide and observed using a Leica optical microscope (Leica CTR6500). The scale bar is shown in the lower left corner of each image.

[0031] Figure 13 : Scanning electron microscopy of spray-dried CSP7. The particle morphology of the spray-dried CSP7 mixture (A = 100% LTI, B = leucine, C = trehalose, d = sodium citrate, e = leucine and trehalose) was examined using SEM. Representative images are shown in the figure.

[0032] Figure 14 : X-ray powder diffraction of jet-milled CSP7 powder. Shows X-ray powder diffraction patterns of milled (batch 171027) and untreated bulk CSP7 powder. The diffraction curves show that the crystallinity of milled CSP7 decreases.

[0033] Figure 15: Physical state of spray-dried CSP7. The spray-dried CSP7 mixtures were examined by X-ray diffraction. Curves indicating the crystallinity or lack of crystallinity of each spray-dried CSP7 mixture are shown.

[0034] Figure 16 : HPLC analysis of spray-dried CSP7 The purity of the spray-dried CSP7 mixture was checked by determining its chemical potency using HPLC.

[0035] Figure 17 HPLC analysis of the stability of jet-milled CSP7. The stability of untreated bulk CSP7 powder and jet-milled CSP7 (lot 171027) was examined by measuring their chemical potency using HPLC. Samples of each were stored under three different conditions (4°C, 25°C / 60RH, and 40°C / 75RH) with the vial cap open or closed, and the chemical potency was measured after 5, 15, and 32 days of storage.

[0036] Figure 18 Figure 2: Particle deposition of aerosolized CSP7. After aerosolization using a Next Generation Impactor (NGI), all collection surfaces were rinsed with a known volume of 20 mM Tris buffer (pH 10.3). Powder deposited in the throat, pre-separator, and 1-MOC stage was extracted and measured. Indicated is the percentage of untreated or air-jet-milled (collected from the collection vessel) (Batch 171013) CSP7 powder that deposited at a specific location.

[0037] Figure 19 Figure 2: Aerodynamic particle size distribution of jet-milled CSP7. After atomization using a Next Generation Impactor (NGI), all collection surfaces were rinsed with a known volume of 20 mM Tris buffer (pH 10.3). Powder deposited in the capsule, device, adapter, throat, pre-separator, and 1-MOC stage was extracted and measured. The position of untreated or jet-milled (Batch 171027) CSP7 powder (collected from all fractions of ground powder) in the mill was determined. The percentage of ground powder present at each position is shown.

[0038] Figure 20 Aerosol performance of a spray-dried CSP7 mixture. After atomization by NGI, all collection surfaces were rinsed with a known volume of 20 mM Tris buffer (pH 10.3). Powder deposited in the capsule, device, adapter, throat, pre-separator, and 1-MOC stage was extracted and measured. The percentage of powder deposited at a specific location is indicated.

[0039] Figure 21Dynamic Vapor Sorption of Jet-Milled CSP7 Powder. Milled CSP7 powder (Batch 171027) was run on a Surface Measurement Systems DVS instrument, performing a complete sorption / desorption cycle from 0% to 90% relative humidity in 10% steps at 25°C. Water desorption at 0% humidity and mass change at 90% humidity are shown.

[0040] Figure 22 Thermal analysis of jet-milled CSP7 powder. Differential scanning calorimetry (DSC) was performed on ground CSP7 (lot 171027) using the calorimeter described above. The figure shows the DSC curve with a frequency of 1°C / 60s and a rate of 2°C / min, from 25 to 300°C.

[0041] Figure 23 Thermal properties of spray-dried 100% CSP7. Excipient-free spray-dried CSP7 was analyzed by modulated differential scanning calorimetry. The figure shows the mDSC curve.

[0042] Figure 24 Thermal properties of spray-dried CSP7 with leucine. A spray-dried CSP7 mixture containing 25% leucine was analyzed by modulated differential scanning calorimetry. The figure shows the mDSC curve.

[0043] Figure 25 Thermal properties of spray-dried CSP7 with trehalose. A spray-dried CSP7 mixture containing 25% trehalose was analyzed by modulated differential scanning calorimetry. The figure shows the mDSC curve.

[0044] Figure 26 Thermal properties of spray-dried CSP7 with sodium citrate. A spray-dried CSP7 mixture containing 25% sodium citrate was analyzed by modulated differential scanning calorimetry. The figure shows the mDSC curve.

[0045] Figure 27 Thermal properties of spray-dried CSP7 with leucine and trehalose. A spray-dried CSP7 mixture containing 15% leucine and 10% trehalose was analyzed by modulated differential scanning calorimetry. The figure shows the mDSC curve.

[0046] Figure 28 : Thermal properties of all spray-dried CSP7 mixtures. Each manufactured spray-dried CSP7 mixture was analyzed by mDSC. The figure shows all mDSC curves for these powders.

[0047] Figure 29Wet weight of mouse lung tissue after autopsy. Euthanized mice were dissected, and their lungs were removed and weighed. Mice were treated with saline, bleomycin to induce pulmonary fibrosis, or bleomycin and CSP7 peptide for 12 or 60 minutes.

[0048] Figure 30 : Collagen content of mouse lung tissue. Untreated, bleomycin-treated, or bleomycin and CSP7-treated lung tissue was homogenized and analyzed for collagen content using the Quickzyme collagen assay. The graph depicts total collagen content in the lung.

[0049] Figure 31 Figure 2: Ashcroft score of mouse lung tissue. Untreated, bleomycin-treated, or bleomycin and CSP7-treated lung tissue was homogenized and analyzed for collagen content using the Quickzyme collagen assay. The Ashcroft score was determined as described in Hubner et al. 2008, incorporated herein by reference. This figure depicts the total collagen content of the lung.

[0050] Figure 32 : Collagen content of mouse lung tissue. Untreated, bleomycin-treated, or bleomycin and CSP7-treated lung tissue was homogenized and analyzed for collagen content using the Quickzyme collagen assay. The graph depicts total collagen content in the lung.

[0051] Figure 33 : Stability of CSP7 (ammonium counterion) after up to 5 freeze-thaw cycles.

[0052] Figure 34 : Specific surface area of ground pure CSP7 (ammonium counterion) powder.

[0053] Figure 35 : Thermogravimetric analysis of ground pure CSP7 (ammonium counterion) powder.

[0054] Figure 36 : SEM image of milled pure CSP7 (ammonium counterion) powder.

[0055] Figure 37 : Appearance of milled CSP7 (ammonium counterion) powder in stability study.

[0056] Figure 38 : Crystallinity of milled CSP7 (ammonium counterion) in stability studies. DETAILED DESCRIPTION

[0057] I. Definition

[0058] As used herein, "substantially free" with respect to a particular component is used herein to mean that the particular component is not intentionally formulated into the composition and / or that the particular component is present only as a contaminant or in trace amounts. Thus, the total amount of the particular component resulting from any accidental contamination of the composition is well below 0.01%. Most preferred are compositions in which the amount of the particular component cannot be detected using standard analytical methods.

[0059] As used in this specification, "a" or "an" may refer to one or more than one. As used herein in the claims, when used in conjunction with the word "comprising," the word "a" or "an" may refer to one or more than one.

[0060] Unless explicitly stated to refer to only alternatives or the alternatives are mutually exclusive, the use of the term "or" in the claims means "and / or," although the present disclosure supports a definition involving only alternatives and "and / or." As used herein, "another" can mean at least a second or more.

[0061] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, method being employed to determine the value, or variation that exists between study subjects. Unless otherwise indicated, "about" means + / - 10%.

[0062] As used herein, the term "peptide" generally refers to an amino acid sequence consisting of a single chain of amino acids connected by peptide bonds. Typically, unless otherwise defined, a peptide comprises at least two amino acid residues and is less than about 50 amino acids in length. In some aspects, a counterion can be provided to the peptide. Similarly, in some cases, the peptide can include N and / or C-terminal modifications, such as closed modifications that reduce degradation.

[0063] A "biologically active" caveolin-1 (Cav-1) peptide is a peptide that increases p53 protein levels, decreases urokinase plasminogen activator (uPA) and uPA receptor (uPAR), and / or increases plasminogen activator inhibitor 1 (PAI-1) expression in cells such as fibrotic lung fibroblasts. In some aspects, the biologically active peptide has at least 20% of the biological or biochemical activity of a native Cav-1 polypeptide having the sequence: SEQ ID NO: 1 (e.g., as measured by an in vitro or in vivo assay). In some aspects, the biologically active peptide has the same or increased biological or biochemical activity as a native Cav-1 polypeptide.

[0064] The term "identity" or "homology" should be interpreted as meaning the percentage of amino acid residues in a candidate sequence that are identical to the residues in the corresponding sequence being compared, after aligning the sequences and introducing gaps (if necessary) to obtain the maximum percent identity over the entire sequence, and not taking into account any conservative substitutions as part of the sequence identity. N- or C-terminal extensions or insertions should not be construed as reducing identity or homology. Methods and computer programs for comparison are well known in the art. Sequence identity can be measured using sequence analysis software.

[0065] In a broad sense, the term "polypeptide" or "protein" refers to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits can be connected by amide bonds. In another embodiment, the subunits can be connected by other bonds, such as by ester bonds, ether bonds. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine and both D or L optical isomers, as well as amino acid analogs and peptidomimetics. The term "peptidomimetic" refers to a peptide according to the present invention that is modified in such a way that it includes 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 short, a peptide of three or more amino acids is generally referred to as an oligopeptide. If the peptide chain is long (for example, longer than 50 amino acids), the peptide is generally referred to as a polypeptide or protein.

[0066] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to animals, such as humans or non-human animals (e.g., mammals), for which the pharmaceutical compositions disclosed herein are used 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, for example, mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, and non-mammals, such as chickens, amphibians, reptiles, and the like. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal substitute used as a disease model. Non-human mammals include mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, and cows. In some aspects, the non-human animal is a companion animal, such as a dog or cat.

[0067] "Treating" a disease or condition in a subject or "treating" a patient suffering from a disease or condition means subjecting the individual to a drug, e.g., administering a drug, such as to reduce or stabilize at least one symptom of the disease or condition. Typically, when the peptide is administered therapeutically as a therapeutic agent, it is administered to a subject exhibiting one or more symptoms of lung injury or pulmonary fibrosis.

[0068] By "isolated" is meant that the polypeptide has been separated from any of its natural environment, such as body fluids (eg, blood), and from components that naturally accompany the polypeptide.

[0069] Isolated and "substantially pure" refer to polypeptides that have been separated and purified to at least some degree from naturally associated components. Generally, a polypeptide is substantially pure when it is at least about 60%, or at least about 70%, at least about 80%, at least about 90%, at least about 95%, or even at least about 99%, by weight, free from proteins and naturally occurring organic molecules with which they are naturally associated. For example, a substantially pure polypeptide can be obtained by extraction from a natural source, by expressing a recombinant nucleic acid in a cell that does not normally express the protein, or by chemical synthesis.

[0070] The term "variant" as used herein refers to a polypeptide that is different from the polypeptide described, which differs in that one or more amino acids are deleted, added, substituted or modified in the side chain, but retains one or more specific functions or biological activities of the native molecule. 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 polypeptide is replaced by another naturally occurring amino acid with similar characteristics in terms of polarity, side chain function 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., replacing a charged or hydrophobic amino acid with alanine); alternatively, wherein a naturally occurring amino acid is replaced by an unconventional amino acid. In some embodiments, the amino acid substitution is conservative. When used in relation to a polynucleotide or polypeptide, the term variant also encompasses a polynucleotide or polypeptide that can change the primary, secondary or tertiary structure compared to a reference polynucleotide or polypeptide (e.g., compared to a wild-type polynucleotide or polypeptide).

[0071] The term "insertion" or "deletion" generally ranges from about 1 to 5 amino acids. Allowable variants can be determined experimentally by synthetically producing peptides and systematically inserting, deleting or substituting nucleotides in the sequence using recombinant DNA technology.

[0072] The term "substitution" when referring to peptides refers to the change of an amino acid to a different entity, such as another amino acid or amino acid moiety. Substitutions can be conservative or non-conservative substitutions.

[0073] An "analog" of a molecule, such as a peptide, refers to a molecule that functions similarly to the entire molecule or a fragment thereof. The term "analog" is also intended to include allelic species and induced variants. Analogs typically differ from naturally occurring peptides at one or several positions, typically due to conservative substitutions. Analogs typically exhibit at least 80% or 90% sequence identity with the native peptide. Some analogs also include non-natural amino acids or modifications of the N- or C-terminal amino acids. Examples of non-natural amino acids include, but are not limited to, unsubstituted amino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, and σ-N-methylarginine. Fragments and analogs can be screened for prophylactic or therapeutic efficacy in transgenic animal models as described below.

[0074] "Covalently bonded" refers to attachment directly or indirectly (eg, via a linker) via a covalent chemical bond. In some aspects of all embodiments of the invention, the fusion peptide is covalently bonded.

[0075] 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 connecting a nucleic acid sequence encoding one protein to a nucleic acid encoding another protein so that they constitute a single open reading frame that can be translated into a single polypeptide having all the desired proteins in the cell. The order in which the proteins are arranged can vary. The fusion protein can include 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.

[0076] The term "airway" as used herein refers to any part of the respiratory tract, including the upper respiratory tract, respiratory tract, and lungs. The upper respiratory tract includes the nose and nasal cavity, mouth, and throat. The respiratory airways include the larynx, trachea, bronchi, and bronchioles. The lungs include the respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli.

[0077] The terms "acute lung injury from smoke inhalation" and "ISALI" are used interchangeably herein to refer to a form of acute lung injury (ALI) caused by smoke inhalation. ALI is also known as "mild acute respiratory distress syndrome; ARDS." ARDS can be defined by the finding of one or more of the following conditions in a subject: 1) bilateral pulmonary infiltrates on chest x-ray; 2) pulmonary capillary wedge pressure <18 mmHg (2.4 kPa) when measured by right cardiac catheterization as clinically indicated; and 3) PaO2 / FiO2 <300 mmHg (40 kPa). In some embodiments, treatment of ISALI includes treatment of one or more of the following conditions: decreased oxygenation, airway obstruction (including severe airway obstruction), fibrous airway casts or debris, and alveolar fibrin deposition.

[0078] The term "jet mill" refers to a device or method that reduces particle size by using a jet of compressed gas to cause particles to collide with each other, thereby crushing the particles. A jet mill can be used to reduce the size of peptide particles. Other mechanical grinding devices that perform the same function can also be used interchangeably with a jet mill. Jet milling can be performed under various environmental parameters, such as temperature, pressure, relative / absolute humidity, oxygen content, etc.

[0079] The term "ball mill" refers to a device or method for reducing particle size by adding the target particles and grinding media to the interior of a cylinder and rotating the cylinder. As the grinding media rotates, the target particles break down as they rise and fall along the exterior of the cylinder. A ball mill can be used to reduce the size of peptide particles. Other mechanical milling devices that perform the same function can also be used interchangeably with a jet mill.

[0080] The term "wet grinder" or "media grinder" refers to a device or method for reducing particle size by adding the target particles to a device with an agitator, which contains a medium comprising a liquid and grinding media. As the target particles are added, the energy of the agitator's rotation causes the grinding media and target particles to come into contact and break down the target particles. Other mechanical grinding devices that perform the same function may also be used interchangeably with jet mill.

[0081] The term "high-pressure homogenization" refers to a method of reducing particle size by subjecting the target particles to a device that combines pressure and mechanical forces to break them down. Mechanical forces used in high-pressure homogenization may include impact, shear, and cavitation, among others. Other mechanical milling devices that perform the same function may also be used interchangeably with a jet mill.

[0082] The term "cryogenic mill" refers to a device or method for reducing particle size by first cooling the target particles with dry ice, liquid nitrogen, or other cryogenic liquids and then grinding the target particles to reduce their size. Other mechanical grinding devices that perform the same function may also be used interchangeably with jet mill.

[0083] The phrase "effective amount" or "therapeutically effective" refers to a dose of a drug or pharmaceutical agent sufficient to produce the desired therapeutic outcome. The desired therapeutic outcome can be subjective or objective improvement in the recipient of the dose, reduction in infection, reduction in 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 thereof.

[0084] As used herein, "excipient" refers to a pharmaceutical carrier, which is a relatively inert substance used to facilitate the administration or delivery of an active pharmaceutical ingredient (API) to a subject, or to facilitate the processing of an API into a pharmaceutically acceptable pharmaceutical formulation for delivery to a subject's site of action. Excipients or pharmaceutical carriers include all inactive components of a dosage form other than the active ingredient. Non-limiting examples of excipients include carriers, fillers, stabilizers, surfactants, surface modifiers, solubility enhancers, buffers, encapsulating agents, antioxidants, preservatives, nonionic wetting or clarifying agents, viscosity enhancers, and absorption enhancers. "Excipient-free" refers to a pharmaceutical composition of interest in a formulation that does not contain any excipients.

[0085] The term "pharmaceutical composition" or "pharmaceutical composition" refers to molecular entities and compositions that, when properly administered to an animal (such as a human), do not produce adverse, allergic, or other untoward reactions. In light of the present disclosure, the preparation of pharmaceutical compositions comprising a Cav-1 peptide such as CSP7 or additional active ingredients is known to those of ordinary skill in the art. In addition, for administration to an animal (e.g., a human), it will be understood that the formulation should meet the bioburden, sterility, pyrogenicity, general safety, and / or purity standards required by the FDA or other regulatory bodies.

[0086] As used herein, "pharmaceutically acceptable carrier" includes any and all excipients, processing aids, aqueous solvents (e.g., water, alcohol / water 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), isotonicity agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, disintegrants, lubricants, taste modifiers (e.g., sweeteners, flavorings), and the like, and combinations thereof, as known to those of ordinary skill in the art. The pH and exact concentration of the various ingredients in a pharmaceutical composition are adjusted according to known parameters. In some aspects, a carrier can encapsulate a therapeutic agent but is not itself consumed or administered to a subject (e.g., a shell capsule enclosing a dry powder composition, such as used in a dry powder inhaler).

[0087] II. Caveolin-1 peptide

[0088] Embodiments of the present disclosure provide dry powder formulations of caveolin-1 (Cav-1) peptides. Caveolin-1 (Cav-1) scaffold domains or peptides interfere with the interaction of Cav-1 with Src kinase, mimicking the combined effects of uPA and anti-β1 integrin antibodies. Native human Cav-1 is 178 amino acids long and has a molecular weight of 22 kDa. The amino acid sequence of Cav-1 is shown below (SEQ ID NO: 1).

[0089] 1MSGGKYVDSE GHLYTVPIRE QGNIYKPNNK AMADELSEKQ VYDAHTKEID LVNRDPKHLN

[0090] 61DDVVKIDFED VIAEPEGTHS FDGIWKAS FT TFTVT KYWFY RLLSALFGIP MALIWGIYFA

[0091] 121lLSFLHIWAVVPCIKSFLIEIQCISRVYSI YVHTVCDPLF EAVGKIFSNVRINLQKEI

[0092] In some aspects, the peptide is a scaffold domain peptide comprising an amino acid sequence having at least about 40%, 50%, 60%, 70%, 80%, 85%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the following sequence: SEQ ID NO: 2, FTTFTVT. The peptide can comprise 1, 2, 3, 4, or more amino acid substitutions, deletions, or insertions relative to the sequence of SEQ ID NO: 1, such as resulting in a polypeptide of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 residues. In particular aspects, the peptide is a truncated native Cav-1 polypeptide, such as the exemplary peptides shown in Table 1.

[0093] Table 1: Exemplary Cav-1 peptides.

[0094]

[0095]

[0096] (a = D-alanine, O = ornithine)

[0097] The peptides provided herein are biologically active derivatives that have the activity of a native Cav-1 polypeptide in an in vitro or in vivo binding or biological activity assay. In particular aspects, the peptides inhibit or prevent bleomycin-induced apoptosis of lung epithelial cells (LECs) in vitro or in vivo with an activity of at least about 20%, or at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, about 95%, 97%, 99%, and any range derivable therein, such as, for example, from about 70% to about 80%, and more preferably from about 81% to about 90%; or even more preferably from about 91% to about 99%. The peptides may have 100% of the activity of a native Cav-1 polypeptide or even higher activity. Assays for testing biological activities such as anti-fibrotic activity, the ability to affect the expression of uPA, uPAR, and PAI-1 mRNA, or the ability to inhibit lung fibroblast proliferation are well known in the art.

[0098] The peptides disclosed herein are peptides of natural Cav-1 polypeptides or modified forms thereof. The peptides may be synthetic, recombinant, or chemically modified peptides isolated or produced using methods well known in the art. Modifications may be made to the N-terminus, C-terminus, or internal amino acids. N-terminal modifications may be, for example, but not limited to, acylation, acetylation, or C-terminal amidation. The peptides may include conservative or non-conservative amino acid changes, as described below. Changes in the polynucleotide may result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence. The peptides may also include insertions, deletions, or substitutions of amino acids, including insertions and substitutions of amino acids (and other molecules) that do not normally occur in the amino acid sequence underlying the peptide, such as, but not limited to, insertions of L-amino acids, or non-standard amino acids (such as ornithine) that do not normally occur in human proteins. When describing peptides, the term conservative substitution refers to changes in the amino acid composition of the peptide that do not substantially alter the activity of the peptide. For example, conservative substitutions refer to the replacement of different amino acid residues with similar chemical properties with amino acid residues. Conservative amino acid substitutions include replacement of leucine with isoleucine or valine, replacement of aspartic acid with glutamic acid, or replacement of threonine with serine.

[0099] Conservative amino acid substitutions are generated by replacing an amino acid with another amino acid having similar structure and / or chemical properties, such as replacing leucine with isoleucine or valine, replacing aspartic acid with glutamic acid, or replacing threonine with serine. Thus, conservative substitutions of a particular amino acid sequence refer to substitutions of amino acids that are not critical for the activity of the polypeptide, or substitutions of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar), such that even substitutions of critical 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 one another: 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); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins, WH Freeman and Company (1984), which is incorporated by reference in its entirety.) In some embodiments, individual substitutions, deletions, or additions that alter, add, or delete a single amino acid or a small percentage of amino acids can also be considered conservative substitutions if the alteration does not reduce the activity of the peptide. Insertions or deletions typically range from about 1 to 5 amino acids. The choice of conservative amino acids can be based on the position of the amino acid to be substituted in the peptide, for example, if the amino acid is on the outside of the peptide and exposed to the solvent, or on the inside and not exposed to the solvent.

[0100] In alternative embodiments, the amino acid to be substituted for an existing amino acid can be selected based on its position, i.e., its exposure to the solvent (i.e., if the amino acid is exposed to the solvent or is present on the outer surface of the peptide or polypeptide compared to an amino acid that is not exposed to the solvent and is on the interior). The selection of such conservative amino acid substitutions is well known in the art, e.g., 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 can be selected that are appropriate for amino acids on the outside of the protein or peptide (i.e., amino acids exposed to the solvent). For example, but not limited to, the following substitutions can 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, Y for F, T or A for S, K for R, N or A for G, K for R, and S, K or P for A.

[0101] In alternative embodiments, conservative amino acid substitutions suitable for amino acids within proteins or peptides can also be selected. For example, appropriate conservative substitutions can be used for amino acids within proteins or peptides (i.e., amino acids that are not exposed to solvents). For example, but not limited to, the following conservative substitutions can be used: substitution of Y with F, substitution of T with A or S, substitution of I with L or V, substitution of W with Y, substitution of M with L, substitution of N with D, substitution of G with A, substitution of T with A or S, substitution of D with N, substitution of I with L or V, substitution of F with Y or L, substitution of S with A or T, substitution of A with S, G, T or V. In some embodiments, the term "variant" also encompasses non-conservative amino acid substitutions.

[0102] In some aspects, the polypeptide is a derivative of a native Cav-1 polypeptide. As used herein, the term "derivative" refers to a peptide that has been chemically modified, for example, but not limited to, by modifications such as acetylation, ubiquitination, labeling, pegylation (derivation with polyethylene glycol), lipidation, glycosylation, amidation, or the addition of other molecules. A molecule is also a "derivative" of another molecule when it contains other chemical moieties that are not normally part of the other molecule. Such moieties can alter the pH or improve the stability, solubility, absorbability, biological half-life, etc. of the molecule. These moieties can alternatively reduce the toxicity of the molecule, eliminate or mitigate any adverse side effects of the molecule, etc. Methods for mediating such effects are disclosed in Remington's Pharmaceutical Sciences, 18th edition, A.R. Gennaro, Ed., Mack Publ., Easton, PA (1990), which is incorporated herein by reference in its entirety.

[0103] The term "functional" when used in conjunction with "derivative" or "variant" refers to a polypeptide of the present invention having a biological activity (functional or structural) substantially similar to the biological activity of the entity or molecule of which it is a functional derivative or a functional variant. The term functional derivative is intended to include fragments, analogs or chemical derivatives of a molecule.

[0104] In some aspects, amino acid substitutions can be made at one or more positions of a polypeptide, wherein the substitution is directed to an amino acid with similar hydrophilicity. The importance of the hydrophilic amino acid index in conferring a biological function on protein interactions is well known in the art (Kyte and Doolittle, 1982). It is generally acknowledged that the relative hydrophilicity of amino acids contributes to the secondary structure of the resulting protein, which then defines the interaction of the protein with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Therefore, such conservative substitutions can be made in a polypeptide and may only have a minor impact on its activity. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). These values can be used as a guide, whereby substitution of amino acids having hydrophilicity values within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. Thus, any polypeptide described herein can be modified by substituting amino acids with different but homologous amino acids having similar hydrophilicity values. Amino acids having hydrophilicity values within + / - 1.0 or + / - 0.5 points are considered homologous.

[0105] Cav-1 peptides may contain co-translational and post-translational (C-terminal peptide cleavage) modifications, such as disulfide bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., by furin or metalloproteinases), etc., to the extent that such modifications do not affect the anti-inflammatory properties of the isolated peptide or its ability to improve glycemic control.

[0106] In some aspects, the Cav-1 peptide comprises non-naturally occurring amino acids. The peptide may comprise a combination of naturally occurring amino acids and non-naturally occurring amino acids, or may comprise only non-naturally occurring amino acids. The non-naturally occurring amino acids in the peptide (or other components of the composition other than the protease recognition sequence) may include synthetic non-natural amino acids, substituted amino acids, or one or more D-amino acids, which may be desirable in certain circumstances. 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 may be particularly useful when greater in vivo or intracellular stability is desired or required. More specifically, D-peptides are resistant to endogenous peptidases and proteases, thereby providing better oral transepithelial and transdermal delivery of linked drugs and conjugates, improving the bioavailability of membrane-permanent complexes (see further discussion below), and extending the lifespan within the vascular and interstitial space (where such properties are desired). The use of D-isomer peptides can also enhance transdermal and oral transepithelial delivery of linked drugs and other cargo molecules. In addition, D-peptides cannot be efficiently processed for major histocompatibility complex class II restricted presentation to T helper cells and are therefore less likely to induce a humoral immune response in the whole organism. Therefore, peptide conjugates can be constructed using, for example, the D-isomer form of the cell penetrating peptide sequence, the L-isomer form of the cleavage site, and the D-isomer form of the therapeutic peptide.

[0107] 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 may also be considered 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 considered 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-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly); ornithine (Orn); citrulline (Cit); tert-butylalanine (t-BuA); tert-butylglycine (t-BuG); N-methylisoleucine (Melle); phenylglycine (Phg); norleucine (Nle); 4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F)) ; 3-fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); homoarginine (hArg); N-acetyllysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,4-diaminobutyric acid (Dab); p-aminophenylalanine (Phe(pNH2)); N-methylvaline (MeVal); homocysteine (hCys), homophenylalanine (hPhe) and homoserine (hSer); hydroxyproline (Hyp), homoproline (hPro), N-methylated amino acids and peptoids (N-substituted glycines).

[0108] Carboxyl terminal modifications include acylation with carboxylic acids: formic acid, acetic acid, propionic acid, fatty acids (myristic acid, palmitic acid, stearic acid), succinic acid, benzoic acid, benzyloxycarbonyl (Cbz); acetylation, and biotinylation. Amino-terminal modifications include: (i) acylation with carboxylic acids: formic acid, acetic acid, propionic acid, fatty acids (myristic acid, palmitic acid, stearic acid, etc.), succinic acid, benzoic acid, benzyloxycarbonyl (Cbz); (ii) biotinylation; (iii) amidation; (iv) attachment of dyes such as fluorescein (FITC, FAM, etc.), 7-hydroxy-4-methylcoumarin-3-acetic acid, 7-hydroxycoumarin-3-acetic acid, 7-methoxycoumarin-3-acetic acid, etc. coumarins; rhodamine (5-carboxyrhodamine 110 or 6G, 5(6)-TAMRA, ROX); dyes such as N-[4-(4-dimethylamino)phenylazo]benzoic acid (Dabcyl), 2,4-dinitrobenzene (Dnp), 5-dimethylaminonaphthalene-1-sulfonic acid (Dansyl); and (v) polyethylene glycol.

[0109] The polypeptides may be terminated at their N- and C-termini with an acyl group (abbreviated "Ac") and an amide group (abbreviated "Am"), respectively, such as an acetyl group (CH3CO-) at the N-terminus and an amide group (-NH2) at the C-terminus. A wide range of N-terminal capping functions are contemplated, preferably in conjunction with a terminal amino group, such as formyl;

[0110] Alkanoyl having 1 to 10 carbon atoms, such as acetyl, propionyl, butyryl;

[0111] an alkenoyl group having 1 to 10 carbon atoms, such as hex-3-enoyl;

[0112] an alkynoyl group having 1 to 10 carbon atoms, such as hex-5-ynoyl;

[0113] Aroyl, such as benzoyl or 1-naphthoyl;

[0114] heteroaroyl, such as 3-pyrrolyl or 4-quinolinyl;

[0115] Alkylsulfonyl, such as methylsulfonyl;

[0116] Arylsulfonyl, such as phenylsulfonyl or sulfonamide;

[0117] heteroarylsulfonyl, such as pyridine-4-sulfonyl;

[0118] Substituted alkanoyl having 1 to 10 carbon atoms, such as 4-aminobutyryl;

[0119] Substituted alkenoyl having 1 to 10 carbon atoms, such as 6-hydroxy-hex-3-enoyl;

[0120] Substituted alkynoyl having 1 to 10 carbon atoms, such as 3-hydroxy-hex-5-ynoyl;

[0121] substituted aroyl, such as 4-chlorobenzoyl or 8-hydroxy-naphthalen-2-yl;

[0122] Substituted heteroaroyl, such as 2,4-dioxo-1,2,3,4-tetrahydro-3-methyl-quinazolin-6-yl;

[0123] substituted alkylsulfonyl, such as 2-aminoethanesulfonyl;

[0124] Substituted arylsulfonyl, such as 5-dimethylamino-1-naphthalenesulfonyl;

[0125] substituted heteroarylsulfonyl, such as 1-methoxy-6-isoquinolinesulfonyl;

[0126] Carbamoyl or thiocarbamoyl;

[0127] 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;

[0128] Substituted carbamoyl (R'-NH-CO) and substituted thiocarbamoyl (R'-NH-CS), wherein R' is alkanoyl, alkenoyl, alkynyl, aroyl, heteroaroyl, substituted alkanoyl, substituted alkenoyl, substituted alkynoyl, substituted aroyl or substituted heteroaroyl, all as defined above.

[0129] The C-terminal capping function can be in an amide bond or an ester bond to the terminal carboxyl group. The capping function that provides the amide bond is called NR 1 R 2 , where R 1 and R 2 may be independently selected from the following groups: hydrogen;

[0130] Preferably, the alkyl group has 1 to 10 carbon atoms, such as methyl, ethyl, isopropyl;

[0131] Preferred are alkenyl groups having 1 to 10 carbon atoms, such as prop-2-enyl;

[0132] Preferred are alkynyl groups having 1 to 10 carbon atoms, such as prop-2-ynyl;

[0133] Substituted alkyl groups having 1 to 10 carbon atoms, such as hydroxyalkyl, alkoxyalkyl, mercaptoalkyl, alkylthioalkyl, haloalkyl, cyanoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkanoylalkyl, carboxyalkyl, carbamoylalkyl;

[0134] Substituted alkenyl groups having 1 to 10 carbon atoms, such as hydroxyalkenyl, alkoxyalkenyl, mercaptoalkenyl, alkylthioalkenyl, haloalkenyl, cyanoalkenyl, aminoalkenyl, alkylaminoalkenyl, dialkylaminoalkenyl, alkanoylalkenyl, carboxyalkenyl, carbamoylalkenyl;

[0135] Substituted alkynyl groups having 1 to 10 carbon atoms, such as hydroxyalkynyl, alkoxyalkynyl, mercaptoalkynyl, alkylthioalkynyl, haloalkynyl, cyanoalkynyl, aminoalkynyl, alkylaminoalkynyl, dialkylaminoalkynyl, alkanoylalkynyl, carboxyalkynyl, carbamoylalkynyl;

[0136] Aroylalkyl having up to 10 carbon atoms, such as benzoyl or 2-benzoylethyl;

[0137] aryl, such as phenyl or 1-naphthyl;

[0138] heteroaryl, such as 4-quinolinyl;

[0139] an alkanoyl group having 1 to 10 carbon atoms, such as an acetyl group or a butyryl group;

[0140] Aroyl groups, such as benzoyl;

[0141] heteroaroyl, such as 3-quinolinyl;

[0142] 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.

[0143] The capping functional group that provides the ester bond is designated OR, where R can be: alkoxy; aryloxy; heteroaryloxy; aralkyloxy; heteroaralkyloxy; substituted alkoxy; substituted aryloxy; substituted heteroaryloxy; substituted aralkyloxy; or substituted heteroaralkyloxy.

[0144] The capping functional group at the N-terminus or C-terminus, or both, can have a structure such that the capped molecule acts as a prodrug (a pharmacologically active derivative of a parent drug molecule), undergoing spontaneous or enzymatic conversion in vivo to release the active drug and having better delivery properties than the parent drug molecule (Bundgaard H, Ed: Design of Prodrugs, Elsevier, Amsterdam, 1985).

[0145] Judicious choice of capping groups allows for the addition of other activities to the peptide. For example, the presence of a sulfhydryl group attached to the N- or C-terminal cap will allow for the conjugation of the derivatized peptide to other molecules.

[0146] In another aspect, the peptide or its fragment or derivative can be a "retro-inverso peptide". A "retro-inverso peptide" refers to a peptide with a reversed peptide bond direction at at least one position, i.e., the amino and carboxyl termini are reversed relative to the side chains of the amino acids. Thus, a retro-inverso analog has reversed termini and peptide bonds in reversed directions while roughly maintaining the topology of the side chains in the native peptide sequence. Retro-inverso peptides can contain L-amino acids or D-amino acids, or a mixture of L-amino acids and D-amino acids, until all amino acids are D-isomers. Partial retro-inverso peptide analogs are polypeptides in which only part of the sequence is reversed and replaced by enantiomeric amino acid residues. Because the amino and carboxyl termini of the retro-inverso portion of such analogs have been reversed, the amino acid residues flanking the reversed portion are replaced by α-substituted gem-diaminomethane and malonate, respectively, with similar side chains. It has been found that the retro-inverso form of cell penetrating peptides is as effective as the native form in transmembrane transport. 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 some retro-inverse peptide analogs has been described (EP 97994-B), which is also incorporated herein by reference in its entirety.

[0147] "Sequence identity" or "homology" that a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (e.g., 80%, 85%, 90% or 95%) with another sequence refers to the percentage of bases (or amino acids) that are identical between the two sequences being compared when compared. Software programs known in the art can be used to determine the comparison and homology or sequence identity percentages, such as those described in Current Protocols In Molecular Biology (FM Ausubel et al., ed., 1987) Supplement 30, Section 7.7.18, Table 7.7.1. Preferably, the default parameters are used for the comparison. A preferred comparison program is BLAST using the default parameters. In particular, preferred programs are BLASTN and BLASTP using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort by = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR.

[0148] B. Polypeptides

[0149] Embodiments of the present disclosure also include longer polypeptides constructed from repeating units of the Cav-1 peptide. Polypeptide multimers may comprise different combinations of polypeptides. Such multimeric polypeptides can be prepared by chemical synthesis or by recombinant DNA techniques as discussed herein. When produced by chemical synthesis, the oligomers preferably have 2-5 repeats of the core polypeptide sequence, and the total number of amino acids in the multimer should not exceed about 160 residues, preferably not more than 100 residues (or their equivalent, when including linkers or spacers).

[0150] C. Peptoids

[0151] Cav-1 peptides can be peptidomimetic compounds that mimic the biological effects of natural Cav-1 polypeptides. Peptidomimetics can be non-natural peptides or non-peptide agents that reproduce the steric properties of the binding elements of natural Cav-1 polypeptides, thereby endowing them with the binding activity and biological activity of natural Cav-1 polypeptides. Similar to natural Cav-1 polypeptides or polypeptide multimers, peptidomimetics will have a binding surface (which interacts with any ligand bound by natural Cav-1) and a non-binding surface.

[0152] In some aspects, the present disclosure also includes compounds that retain some of the peptide characteristics. For example, any proteolytically unstable bond within the peptides of the present invention can be selectively replaced by a non-peptide element such as an isoelectric (N-methylated; D-amino acid) or a reduced peptide bond, while the remainder of the molecule retains its peptide nature.

[0153] Peptidomimetic compounds have been described for a variety of biologically active peptides / polypeptides (such as opioid peptides, VIP, thrombin, HIV protease, etc.), whether as 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). Certain mimetics that mimic secondary structures are described in Johnson et al., In: Biotechnology and Pharmacy, Pezzuto et al., Chapman and Hall (Eds.), NY, 1993. These methods are used to prepare peptidomimetics that have at least the binding capacity and specificity of native Cav-1 polypeptides and preferably also have biological activity. In view of the present disclosure, the knowledge of peptide chemistry and general organic chemistry available to those skilled in the art is sufficient for the design and synthesis of such compounds.

[0154] For example, this type of peptidomimetic can be identified by checking the three-dimensional structure of the polypeptide of the present invention that is free or compound-bound with a part (for example, soluble uPAR or its fragment). Alternatively, the structure of the polypeptide of the present invention that is bound to its part can be obtained by nuclear magnetic resonance spectroscopy. More knowledge of the interactive stereochemistry of this peptide and its part or receptor will allow the rational design of this peptidomimetic. In the absence of a part, the structure of the peptide of the present invention or polypeptide also can be provided for designing a support for simulation molecules.

[0155] D.PEGylation

[0156] Cav-1 peptides can be conjugated to heterologous polypeptide fragments or polymers, such as polyethylene glycol. Peptides can be linked to PEG to increase the hydrodynamic radius of the enzyme, thereby increasing serum persistence. Peptides can be conjugated to any targeting agent, such as a ligand that can specifically and stably bind to an external receptor (U.S. Patent Publication No. 2009 / 0304666).

[0157] In some aspects, the method and composition of the embodiment is relevant with the PEGylation of disclosed polypeptide.PEGylation is the process that poly (ethylene glycol) polymer chain is covalently linked to another molecule (normally medicine or therapeutic protein).PEGylation is normally achieved by hatching the reactive derivative of PEG with target macromolecule.The covalent attachment of PEG to medicine or therapeutic protein can " mask " this medicament to antagonize host immune system (immunogenicity and antigenicity that reduce), improve the hydrodynamic size (size in solution) of medicament, and this can prolong its circulation time by reducing kidney clearance.PEGylation can also provide water solubility to hydrophobic drugs and proteins.

[0158] The first step in PEGylation is the appropriate functionalization of the PEG polymer at one or both termini. PEGs that are activated at each terminus with the same reactive moiety 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.

[0159] The selection of suitable functional groups for PEG derivatives is based on the type of available reactive groups on the molecule to be coupled to the PEG. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. N-terminal amino groups and C-terminal carboxylic acids can also be used.

[0160] The techniques used to form first-generation PEG derivatives typically react PEG polymers with groups that can react with hydroxyl groups (usually anhydrides, acid chlorides, chloroformates, and carbonates). In second-generation PEGylation chemistry, more efficient functional groups (such as aldehydes, esters, amides, etc.) can be used for conjugation.

[0161] As the applications of PEGylation have become increasingly advanced and complex, the demand for heterobifunctional PEGs for conjugation has increased. These heterobifunctional PEGs are very useful for connecting two entities where a hydrophilic, flexible, and biocompatible spacer is required. Preferred end groups for heterobifunctional PEGs are maleimides, vinyl sulfones, disulfide pyridines, amines, carboxylic acids, and NHS esters.

[0162] The most common modifiers or linkers are based on methoxy PEG (mPEG) molecules. Their activity depends on the addition of protein-modifying groups to the alcohol termini. In some cases, polyethylene glycol (PEG diol) is used as a precursor molecule. The diol is then modified at both termini to produce heterodimeric or homodimeric PEG-linked molecules.

[0163] Proteins are typically PEGylated at nucleophilic sites, such as unprotonated thiols (cysteinyl residues) or amino groups. Examples of cysteinyl-specific modification reagents include PEG maleimide, PEG iodoacetate, PEG thiol, and PEG vinyl sulfone. All four have strong cysteinyl specificity under mild conditions and neutral to slightly alkaline pH, but each has some defects. The thioether formed by maleimide can be slightly unstable under alkaline conditions, so there may be some limitations in the formulation selection using this linker. The thiocarbamate bond formed by iodine PEG is more stable, but free iodine can modify tyrosine residues under some conditions. PEG thiol forms a disulfide bond with protein thiols, but this bond can also be unstable under alkaline conditions. The reactivity of PEG-vinyl sulfone is relatively slow compared to maleimide and iodine PEG; however, the thioether bond formed is extremely stable. Its slower reaction rate can also make the PEG-vinyl sulfone reaction easier to control.

[0164] Site-specific PEGylation at native cysteinyl residues is rarely performed because these residues are typically in the form of disulfide bonds or are required for biological activity. In another aspect, 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.

[0165] Amine-specific modifiers include PEG NHS esters, PEG triflates, PEG aldehydes, and PEG isothiocyanates. They all react under mild conditions and are highly specific for amino groups. PEG NHS esters may be a more reactive reagent; however, their high reactivity can make PEGylation reactions difficult to control on a large scale. PEG aldehydes react with amino groups to form imines, which are then reduced to secondary amines using sodium cyanoborohydride. Unlike sodium borohydride, sodium cyanoborohydride does not reduce disulfide bonds. However, this chemical is highly toxic and must be handled with caution, especially at low pH, where it is volatile.

[0166] Site-specific PEGylation is challenging due to the presence of multiple lysine residues on most proteins. Fortunately, because these reagents react with unprotonated amino groups, it is possible to direct PEGylation to amino groups with lower pK by performing the reaction at a lower pH. Generally speaking, the pK of the α-amino group is 1-2 pH units smaller than the pK of the ε-amino group of a lysine residue. By PEGylating the molecule at a pH of 7 or lower, high selectivity for the N-terminus can usually be achieved. However, this is only feasible if the N-terminal portion of the protein is not required for biological activity. In addition, the pharmacokinetic benefits of PEGylation are generally stronger than the significant loss of in vitro biological activity, resulting in products with greater in vivo biological activity, regardless of the PEGylation chemistry.

[0167] There are many parameters to consider when developing a PEGylation procedure. Fortunately, there are usually no more than four or five critical parameters. A "design of experiments" approach to optimizing PEGylation conditions is very useful. For thiol-specific PEGylation reactions, parameters to consider include: protein concentration, PEG to protein ratio (in moles), temperature, pH, reaction time, and in some cases, oxygen. (Oxygen can contribute to intermolecular disulfide formation by proteins, which will reduce the yield of the PEGylated product.) The same factors should be considered for amine-specific modifications (except oxygen), and pH may even be more critical, especially when targeting N-terminal amino groups.

[0168] For amine- and thiol-specific modifications, reaction conditions can affect protein stability. This can be limited by temperature, protein concentration, and pH. Additionally, the reactivity of the PEG linker should be known before initiating the PEGylation reaction. For example, if the PEGylation reagent is only 70% active, the amount of PEG used should ensure that only active PEG molecules are counted in the protein-PEG reaction stoichiometry.

[0169] E. Fusion protein

[0170] Certain embodiments of the present invention relate to fusion proteins of Cav-1 peptides. These molecules may have a polypeptide of the embodiment linked to a heterologous domain at the N- or C-terminus. For example, the fusion may also use a leader sequence from another species to allow recombinant expression of the protein in a heterologous host. The fusion protein may contain a half-life extender. Another useful fusion includes the addition of a protein affinity tag (such as a serum albumin affinity tag or six histidine residues) or an immunologically active domain (such as an antibody epitope, preferably 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).

[0171] In certain embodiments, the peptides of the embodiments may be linked to peptides that increase in vivo half-life, such as XTEN polypeptides (Schellenberger et al., 2009), IgG Fc domains, albumin, or albumin-binding peptides.

[0172] Methods for producing fusion proteins are well known to those skilled in the art. Such proteins can be produced by, for example, de novo synthesis of the complete fusion protein, or ligation of DNA sequences encoding heterologous domains and expression of the complete fusion protein.

[0173] The production of fusion proteins that restore the functional activity of the parent proteins can be facilitated by joining the genes with a bridging DNA fragment encoding a peptide linker that is spliced between the tandemly linked polypeptides. The linker should be of sufficient length to allow proper folding of the resulting fusion protein.

[0174] 2. Connector

[0175] In certain embodiments, the polypeptides of the embodiments can be chemically conjugated using bifunctional cross-linking reagents or fused to peptide linkers at the protein level.

[0176] Bifunctional cross-linking reagents have been widely used for various purposes, including the preparation of affinity matrices, modification and stabilization of various structures, identification of ligand and receptor binding sites, and structural studies. Suitable peptide linkers (such as Gly-Ser linkers) can also be used to link the polypeptides of the embodiments.

[0177] Homobifunctional reagents carrying two identical functional groups have been shown to be highly effective in cross-linking identical or dissimilar macromolecules or macromolecular subunits, and in attaching polypeptide ligands to their specific binding sites. Heterobifunctional reagents contain two different functional groups. By exploiting the differential reactivity of the two functional groups, cross-linking can be selectively and sequentially controlled. Based on the specificity of their functional groups, bifunctional cross-linking reagents can be categorized into groups with amino, sulfhydryl, guanidinyl, indolyl, and carboxyl specificity, for example. Among these, reagents targeting free amino groups are particularly popular due to their commercial availability, ease of synthesis, and applicable mild reaction conditions.

[0178] 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. Patent No. 5,889,155, more specifically, the entirety of which is incorporated herein by reference). The cross-linking reagent combines a nucleophilic hydrazide residue with an electrophilic maleimide residue, allowing, for example, the coupling of aldehydes to free thiols. The cross-linking reagent can be modified to cross-link a variety of functional groups.

[0179] In addition, any other linking / coupling agents and / or mechanisms known to those skilled in the art can be used to combine the polypeptides of the embodiments, such as, for example, antibody-antigen interactions, avidin-biotin bonds, amide bonds, ester bonds, thioester bonds, ether bonds, thioether bonds, phosphate bonds, phosphoramide bonds, anhydride bonds, disulfide bonds, ionic and hydrophobic interactions, bispecific antibodies and antibody fragments, or combinations thereof.

[0180] It is preferred to use a cross-linking agent that has reasonable blood stability. Many types of disulfide-containing linkers are known to be successfully used to conjugate targeting and therapeutic / prophylactic agents. Linkers containing sterically hindered disulfide bonds can be more stable in vivo. Therefore, these linkers form a group of linkers.

[0181] In addition to hindered cross-linkers, non-hindered linkers may also be used as described herein. Other useful cross-linkers (not believed to contain or generate protected disulfide bonds) include SATA, SPDP, and 2-imidothioether (Wawrzynczak and Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment relates to the use of flexible linkers.

[0182] Once chemically conjugated, the peptide is typically purified to separate the conjugate from non-conjugated agents and other contaminants. A wide variety of purification techniques are available to provide the conjugate of sufficient purity to be clinically useful.

[0183] Purification methods based on size separation (such as gel filtration, gel permeation or high performance liquid chromatography) are normally the most frequently used. Other chromatographic techniques (such as Blue-Sepharose separation) also are operable. Traditional methods for purifying fusion proteins from inclusion bodies can be useful, such as using weak detergents, such as sodium lauryl sarcosine (SLS).

[0184] 3. Cell-penetrating peptides and membrane-transporting peptides

[0185] In addition, in certain aspects, the Cav-1 peptide may further comprise a cell binding domain or a cell penetrating peptide (CPP). As used herein, the terms "cell penetrating peptide" and "membrane trafficking domain" are used interchangeably and refer to fragments of a polypeptide sequence that allow the polypeptide to cross a cell membrane (e.g., the plasma membrane in the case of eukaryotic cells). Examples of CPP fragments include, but are not limited to, fragments derived from HIV Tat (e.g., GRKKRRQRRRPPQ (SEQ ID NO: 23)), herpesvirus VP22, Drosophila antennal homeobox gene products, protegrin I, penetratin (RQIKIWFQNRRMKWKK (SEQ ID NO: 24)), or melittin (GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 25)). In certain aspects, the CPP comprises a T1 (TKIESLKEHG (SEQ ID NO:26)), T2 (TQIENLKEKG (SEQ ID NO:27)), 26 (AAEALAEAEALAEALEALAEAAAA (SEQ ID NO:28)), or INF7GLFEAIEGFIENGWEGMIEGWYGCG (SEQ ID NO:29)) CPP sequence.

[0186] III. Instructions for use

[0187] One aspect of the present invention relates to the use of the peptides described herein and their mutations, variants, analogs or derivatives. Specifically, these methods relate to administering any of the peptides described herein or pharmaceutically acceptable modifications thereof (such as dry powder) to a subject for treating or preventing a disease, injury or lung infection (e.g., a fibrotic condition of the lung), wherein the composition comprises a polypeptide of the embodiments in a pharmaceutically acceptable carrier.

[0188] A. Pharmaceutical Compositions

[0189] It is expected that the Cav-1 peptides provided herein can be administered systemically or topically to inhibit apoptosis and to treat and prevent damage to lung tissue. They can be administered intravenously, subcutaneously, intramuscularly, intrathecally and / or intraperitoneally. For example, dry powder formulations can be administered by instillation into a subject (e.g., subcutaneous instillation) or can be reconstituted in a liquid before injection. In particular aspects, the peptides are delivered locally to the airways, such as by administering a dry powder formulation using a dry powder inhaler. They can be administered alone or in combination with an anti-fibrotic compound.

[0190] The Cav-1 peptide dry powder can be combined with at least one additional therapeutic agent (e.g., a therapeutic agent for treating pulmonary fibrosis), administered simultaneously or sequentially. The other therapeutic agent can be an NSAID, a steroid, a DMARD, an immunosuppressant, a biological response modifier, a bronchodilator, or an antifibrotic agent such as pirfenidone (whose antifibrotic mechanism of action is not fully understood, but may involve blocking TGF-β), nintedanib (a broad tyrosine kinase blocker), or any other antifibrotic agent. Suitable NSAIDs are selected from the group consisting of non-selective COX-inhibitors: acetylsalicylic acid, mesalamine, ibuprofen, naproxen, flurbiprofen, fenoprofen, fenbufen, ketoprofen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, sirofenic acid, flurofenic acid ... Suitable steroids include fentiazac, cyclanac, etodolac, oxpinac, mefenamic acid, meclofenamic acid, flufenamic acid, niflumic acid, tolfenamic acid, diflunisal, flufenal, piroxicam, tenoxicam, lornoxicam and nimesulide and their pharmaceutically acceptable salts; selective COX 2-inhibitors: meloxicam, celecoxib and rofecoxib and their pharmaceutically acceptable salts. Suitable steroids include prednisone, prednisolone, methylprednisolone, dexamethasone, budesonide, fluocortolone and triamcinolone. Suitable DMARDs are sulfasalazine, olsalazine, chloroquine, gold derivatives (auranofin), D-penicillamine and cytostatics such as methotrexate and cyclophosphamide. Suitable immunosuppressants are cyclosporine A and its derivatives, mycophenolate mofetil, FK 506, OKT-3, ATG, 15-desoxyspergualin, mizoribine, misoprostol, rapamycin, reflunomide and azathioprine. Suitable biological response modifiers are interferon beta, anti-TNF-α (etanercept), IL-10, anti-CD3 or anti-CD25. Suitable bronchodilators are ipratropium bromide, bromotropium bromide, tiotropium bromide, epinephrine hydrochloride, salbutamol, terbutaline sulfate, fenoterol hydrobromide, salmeterol and formoterole.In such a combination, each active ingredient can be administered (e.g., orally or by inhalation) according to its usual dosage range or a dose below its usual dosage range. The dosage of the combined NSAID, steroid, DMARD, immunosuppressant and biological response modifier is suitably 1 / 50 of the usually recommended lowest dose to 1 / 1 of the usually recommended highest dose, preferably 1 / 20 to 1 / 2, and more preferably 1 / 10 to 1 / 5. For combination drugs, the usually recommended dosage should be understood as, for example, in Rote. 2002, EditioCantor Verlag Aulendorf, Germany or the dosages disclosed in the Physician's Desk Reference.

[0191] In the case of considering clinical application, it may be necessary to prepare a pharmaceutical composition comprising protein, antibody and drug in a form suitable for intended application. In general, pharmaceutical composition can include an effective amount of polypeptide or additional agent dissolved or dispersed in one or more embodiments in a pharmaceutical carrier. The term "pharmaceutical" refers to that molecular entities and compositions do not produce adverse, allergic or other adverse reactions when applied to animals (e.g., people) as appropriate. The preparation of a pharmaceutical composition containing the polypeptide of at least one embodiment separated by the method disclosed herein or another active ingredient will be known to those skilled in the art in view of the present disclosure, as illustrated by Remington's Pharmaceutical Sciences, 18th edition, 1990, which is incorporated herein by reference. In addition, for animal (e.g., people) use, it should be understood that the preparation should meet the bioburden, sterility, pyrogenicity, general safety and / or purity standards required by the FDA Office of Biological Standards or other appropriate regulatory agencies.

[0192] Certain embodiments of the present invention may include different types of carriers, depending on whether it is applied in solid, liquid or aerosol form, and whether sterility is required for routes of administration such as injection. The composition can be administered intravenously, intrathecally, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly, subcutaneously, mucosally, orally, topically, locally by inhalation (e.g., inhalation aerosol formulations), by injection, by infusion, by continuous infusion, by direct local perfusion of target cells, via catheters, via lavage, in lipid compositions (e.g., liposomes), or by other methods or any combination of the above methods known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, 1990, incorporated herein by reference). The choice of injection volume and needle size can be selected by those of ordinary skill in the art based on injection site, pushability, and injectability, including consideration of the viscosity of the solution or suspension to be injected and drug concentration, pH, and osmotic pressure. In some cases, the particle size of the active agent can be selected to provide a desired dissolution rate upon administration (eg, by subcutaneous injection).

[0193] The polypeptides provided herein can be formulated into compositions in the form of free alkali, neutral, zwitterionic or salt forms. Pharmaceutically acceptable salts include acid addition salts, such as those formed by the free amino groups of the protein composition, or those formed by inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid or mandelic acid). Salts formed with free carboxyl groups can also be derived from inorganic bases (such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide); or organic bases (such as isopropylamine, trimethylamine, histidine or procaine). After formulation, the solution is administered in a manner compatible with the dosage form and in a therapeutically effective amount. The preparation is easily administered in a variety of dosage forms, such as formulated for parenteral administration, such as injection solutions, or for delivery to the aerosol of the lung, or formulated for dietary administration, such as drug release capsules.

[0194] According to some aspects of the present invention further, the compositions that are suitable for use can be provided in a pharmaceutical carrier with or without an inert diluent. In some respects, carrier can include aerosol, gas, liquid, semisolid (that is, paste) or solid carrier. Unless any conventional media, reagent, diluent or carrier are harmful to the recipient or to the therapeutic effectiveness of the compositions contained therein, otherwise its purposes in the composition that can be used for practicing the method are suitable. The example of carrier or diluent includes fat, oil, water, saline solution, lipid, liposome, resin, adhesive, filler etc., or their combination. Composition also can include various antioxidants, to delay the oxidation of one or more components. In addition, the prevention of microbial action can be realized by preservatives, such as various antibacterials and antifungals, including but not limited to parahydroxybenzoate (for example, methyl parahydroxybenzoate, propyl parahydroxybenzoate), chlorobutanol, phenol, sorbic acid, thimerosal or their combination.

[0195] According to certain aspects of the present invention, the composition is combined with the carrier in any convenient and practical manner, ie, by solution, suspension, emulsification, admixture, encapsulation, absorption, etc. Such procedures are routine for those skilled in the art.

[0196] In a specific embodiment of the present invention, the composition is fully combined or mixed with a semisolid or solid carrier. Mixing can be carried out in any convenient way, such as grinding. Stabilizers can also be added during the mixing process to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers used in the composition include buffers, amino acids (such as glycine and lysine), carbohydrates, or lyoprotectants (such as glucose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.).

[0197] In some aspects, the pharmaceutical formulation comprises one or more surfactants. Surfactants used according to the disclosed methods include ionic and nonionic surfactants. Representative nonionic surfactants include polysorbates, such as and Surfactant (ICI Americas Inc. of Bridgewater, NJ); Poloxamer (e.g., Poloxamer 188); Surfactants (Sigma of St. Louis, Mo.); sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleoyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, panthenylpropyl-, or (e.g., lauramidopropyl); myristamidopropyl-, palmitoylpropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl- or disodium methyl oleoyl-taurate; MONAQUAT TM Surfactants (Mona Industries Inc. of Paterson, NJ); polyethylene glycol; polypropylene glycol; block copolymers of ethylene and propylene glycol, such as Surfactants (BASF of Mt. Olive, NJ); oligo(ethylene oxide) alkyl ethers; alkyl(thio)glucosides, alkyl maltosides; and phospholipids. For example, the surfactant can be present in the formulation in an amount of about 0.01% to about 5% (the weight of the surfactant relative to the total weight of the other solid components in the formulation; "w / w"), about 0.03% to about 0.5% (w / w), about 0.05% to about 0.5% (w / w), or about 0.1% to about 0.5% (w / w). However, in other aspects, the pharmaceutical formulations of the embodiments are substantially free of nonionic surfactants or substantially free of all surfactants.

[0198] Regarding the treatment methods of the present invention, it is not intended to administer one or more peptides disclosed herein or mutations, variants, analogs or derivatives thereof, and is not limited to a specific mode of administration, dosage or frequency of administration; the present invention encompasses all modes of administration, including intramuscular, intravenous, intraperitoneal, intracapsular, intraarticular, intralesional, subcutaneous or any other route sufficient to provide a dose sufficient to treat inflammation-related disorders. The therapeutic agent can be administered to the patient in a single dose or multiple doses. When multiple doses are administered, the doses can be separated from each other by, for example, one hour, three hours, six hours, eight hours, one day, two days, one week, two weeks or one month. For example, the therapeutic agent can be administered for, for example, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, 15 weeks, 20 weeks or more weeks. It should be understood that for any particular subject, a specific dosage regimen should be adjusted over time based on individual needs and the professional judgment of the person administering or supervising the administration of the composition. For example, if a lower dose does not provide sufficient therapeutic activity, the dose of the therapeutic agent can be increased.

[0199] While the attending physician will ultimately determine the appropriate amount and dosage regimen, a therapeutically effective amount of one or more polypeptides disclosed herein, or a mutation, variant, analog, or derivative thereof, can be provided at a dose of 0.0001, 0.01, 0.010.1, 1, 5, 10, 25, 50, 100, 500, or 1,000 mg / kg or g / kg. Effective doses can be extrapolated based on dose-response curves derived from in vitro or animal model test bioassays or systems.

[0200] Those of ordinary skill in the art can use conventional considerations (e.g., by means of appropriate conventional pharmacological protocols) to determine the dosage for a particular patient or subject. A doctor can, for example, first prescribe a relatively low dose and then increase the dose until an appropriate response is obtained. Depending on the application, the dosage administered to the patient is sufficient to affect the patient's beneficial therapeutic response over time, or, for example, to alleviate symptoms or other appropriate activity. The dosage is determined by the efficacy of the particular formulation and the activity, stability or serum half-life of one or more polypeptides disclosed herein, or their mutations, variants, analogs or derivatives, and the patient's condition and the body weight or surface area of the patient to be treated.

[0201] In some aspects, a single dose is administered to a subject, for example via instillation (by inhalation), once daily to treat a subject, preferably a mammal, more preferably a human suffering from or susceptible to pulmonary fibrosis, at a resulting dose of between about 0.2 mg / kg and about 250 mg / kg, such as between about 10 mg / kg and about 50 mg / kg. This dose may be administered daily for anywhere from about 3 days to one or more weeks.

[0202] Chronic administration is also possible, although the dosage may need to be adjusted downward as is well known in the art. However, the above ranges are suggestive, as the number of variables in an individual treatment regimen is large, and considerable deviation from these preferred values is to be expected.

[0203] For continuous administration, for example, by a pump system (such as the osmotic pump used in some of the experiments described below), the total dose over a period of about 1-2 weeks is preferably in the range of 1 mg / kg to 1 g / kg, preferably in the range of 20-300 mg / kg, more preferably in the range of 50-200 mg / kg. Following such a continuous dosage regimen, the total concentration of the active compound is preferably in the range of about 0.5 μM to about 50 μM, preferably in the range of about 1 μM to about 10 μM.

[0204] The effective concentration of active compounds for inhibiting or preventing apoptosis in vitro is in the range of about 0.5 nM to about 100 nM, more preferably in the range of about 2 nM to about 20 nM. The effective dose range and optimal dose range can be determined in vitro using the methods described herein.

[0205] B. Dry powder particle size reduction and dry powder inhalation devices.

[0206] The particle size of the preparation can be reduced by any suitable method, including but not limited to grinding, milling, thin film freezing, spray drying or crushing. Can be ground by any method known in the art, such as by jet mill, ball mill, wet grinding machine, media mill, high pressure homogenization or cryogenic grinding machine.

[0207] The stability of the peptide after particle size reduction can be assessed using techniques known in the art, including size exclusion chromatography; electrophoresis techniques; HPLC; mass spectrometry; spectroscopic techniques (such as UV spectroscopy and circular dichroism spectroscopy) and activity (measured in vitro or in vivo). For in vitro determination of protein stability, the aerosol composition can be collected and then distilled or absorbed onto a filter. For in vivo determination or administration of the composition to a subject through the lungs, the device for dry powder dispersion is suitable for inhalation by the subject. For example, protein stability can be assessed by determining the level of protein aggregation. Preferably, the dry powder composition of the present invention is substantially free of protein aggregates. The presence of soluble aggregates can be qualitatively determined using dynamic light scattering (DLS) (DynaPro-801TC, Protein Solutions Inc. of Charlottesville, Va.) and / or by ultraviolet spectrophotometry.

[0208] In certain embodiments, the CSP7 treatment patient of grinding can include the drug release of adjustment.In certain embodiments, the CS7 of grinding can be configured to slowly release or delayed release.In certain embodiments, the CSP7 of grinding can be configured to quickly release.In other embodiments, the CSP7 of grinding can be configured to slowly release and quickly release (that is, dual release curve).

[0209] In certain embodiments, the present disclosure provides a method for administering the inhalable CSP7 compositions provided herein. Administration can be, but is not limited to, administering the ground CSP7 by inhaling an inhaler. In certain embodiments, the inhaler is a passive dry powder inhaler (DPI), such as Plastiape RS01 single-dose DPI. In a dry powder inhaler, dry powder is stored in a container and delivered to the lungs by inhalation without the need for a propellant.

[0210] In some embodiments, the inhaler is a single-dose DPI, such as DoseOneTM 、Spinhaler、 Or Handihaler. In some embodiments, the inhaler is a multi-dose DPI, such as Plastiape RS02, Twisthaler TM 、 or Ellipta TM . In some embodiments, the inhaler is a multi-single-dose DPI (plurimonodose DPI) for the simultaneous delivery of multiple drugs in a single dose, such as Plastiape RS04 multi-single-dose DPI. Typically, a dry powder inhaler stores the drug in an internal reservoir and, with or without a propellant, delivers the drug by inhalation. Other types of dry powder inhalers store pre-distributed doses of drug in capsules (e.g., cellulose or gelatin matrices) or foil bags, which are pierced by a device to release the dose to the patient. A dry powder inhaler may require an inspiratory flow rate greater than 30 L / min to effectively deliver, such as between about 30-120 L / min. In some embodiments, the effective atomization of ground CSP7 is independent of the inspiratory force. In some embodiments, the flow resistance of the dry powder inhaler is 0.01 kPa. 0.5 min / L to 0.05kPa 0.5 min / L, such as 0.02kPa 0.5 min / L to 0.04kPa 0.5 min / L. Select a dry powder inhaler based on the patient population and their inspiratory capacity (e.g., high resistance, low resistance, passive, active).

[0211] In certain embodiments, inhaler can be a metered dose inhaler. The metered dose inhaler uses the short pulse form of the medicine of propellant auxiliary atomization to deliver a certain amount of medicine to the lungs. The metered dose inhaler comprises three main parts: tank, metering valve and actuator, and can utilize isolating device to make the particle of being emitted slow down and promote the patient to inhale atomized cloud. The pharmaceutical preparation comprising propellant and any required excipient is all stored in the tank. The metering valve allows the pharmaceutical preparation of dispensing limited amount. The actuator of metered dose inhaler or mouthpiece comprises the discharge nozzle of pairing, and usually comprises dust cover to prevent pollution. Use the required required inspiratory flow rate of metered dose inhaler to be less than 90L / min, such as between about 15-90L / min, preferably about 30L / min. In certain embodiments, the effective atomization of grinding CSP7 has nothing to do with inspiratory force.

[0212] In some embodiments, the inhaler is a nebulizer. The nebulizer is used to deliver medicine in the form of an atomized mist inhaled into the lungs. The drug preparation is atomized by compressed gas or ultrasonic waves. The jet nebulizer is connected to a compressor. The compressor discharges compressed gas at a high speed through the liquid drug preparation, thereby atomizing the drug preparation. The patient then inhales the atomized medicine. The ultrasonic nebulizer generates high-frequency ultrasonic waves, causing vibration of the internal components in contact with the liquid reservoir of the drug preparation, which causes the drug preparation to be atomized. The patient then inhales the atomized medicine. The nebulizer can utilize a flow rate between about 3-12L / min, such as about 6L / min. In some instances, the ground active substance (e.g., CSP7) can be suspended in a medicinal liquid carrier medium and applied by atomization (e.g., jet atomization). In other aspects, the composition of the embodiment can be applied by a vaporization method (e.g., rapid vaporization), such as by an electronic cigarette device.

[0213] In certain embodiments, compositions can be applied on a regular schedule. As used herein, a regular schedule refers to a predetermined specified time period. As long as the schedule is predetermined, a regular schedule can encompass time periods of identical or different lengths. For example, the application of a regular schedule may relate to twice a day, every day, every two days, every three days, every four days, every five days, every six days, once a week, once a month, or any set number of days or weeks therebetween. Alternatively, the application of a predetermined regular schedule may include twice a day in the first week, then every day in a few months. In certain embodiments, peptide (e.g., CSP7) is applied once a day. In a preferred embodiment, less than once a day, peptide is applied, such as every other day, every three days, or once a week. In certain embodiments, the peptide (e.g., CSP7) of a complete dose of an embodiment is between 1-100mg, such as 20-100mg, 50-100mg, 10-20mg, 20-40mg, 50-70mg, or 80-90mg.

[0214] In some embodiments, the peptide of the embodiment (e.g., CSP7) can be provided in unit dosage form (e.g., pre-divided dose), such as provided in the form of a capsule, blister or cartridge, wherein the unit dose comprises at least 1 mg of peptide, such as at least 5 mg, 10 mg, 15 mg or 20 mg of the peptide of the embodiment (e.g., CSP7) per dose. In some aspects, the unit dose is 1-10 mg (e.g., about 5 mg) of peptide. In specific aspects, the unit dosage form does not include the administration or addition of any excipient and is only used to hold a powder for inhalation (i.e., not administering a capsule, blister or cartridge). In some aspects, more than one unit dosage form is administered to the subject. For example, in the case of a dry powder inhaler, the peptide of the embodiment can be provided in a unit dose capsule, and more than one unit dose capsule (e.g., 3-4) can be administered to the subject by inhalation. In some embodiments, the peptide (e.g., CSP7) can be administered with a high emission dose, such as at least 10 mg, preferably at least 15 mg, even more preferably 20 mg. In some embodiments, administration of the milled peptide of the embodiments (e.g., CSP7) results in a high fine particle dose, such as greater than 5 mg, that enters the deep lung. Preferably, the fine particle dose that enters the deep lung is at least 10 mg, even more preferably at least 15 mg. In some aspects, the particle dose is produced by 1, 2, 3, 4, or 5 or more capsules containing a dose of the peptide of the embodiments (e.g., CSP7). In some aspects, the fine particle dose is at least 50%, such as at least 60%, 65%, 70%, 75%, or 80%, of the emitted dose.

[0215] In some embodiments, a change in the suction pressure drop results in a change in the emitted dose. In some embodiments, a change in the suction pressure of 3 kPa, such as from 4 kPa to 1 kPa, results in a reduction in the emitted dose of less than 25%, such as 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or less. In some embodiments, a change in the suction pressure results in a change in the fine particle dose. In some embodiments, a change in the suction pressure of 3 kPa, such as from 4 kPa to 1 kPa, results in a reduction in the fine particle dose of less than 15%, such as 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or less.

[0216] IV. Treatment of lung diseases

[0217] The peptides of the present invention can be used to treat a variety of lung conditions. The lung conditions treated may be acute or chronic. Acute lung conditions may be caused by acute lung injury, infection, or chemical induction. Chronic lung conditions may be the result of injury, infection, or disease.

[0218] A. Lung damage

[0219] In some respects, the subject suffers from acute lung injury (ALI) or infection or chemically induced lung injury.In particular aspects, the subject suffers from plastic bronchitis, asthma, chronic obstructive airway / lung (COPD), acute respiratory distress syndrome (ARDS), acute lung injury (ISALI) caused by smoke inhalation, bronchiectasis, the airway disease caused by inhalation toxins (for example, chlorine or other induced airway disease), exposure to mustard gas, exposure to particulate matter (for example, silica dust), bronchiolitis obliterans, bronchiolitis obliterans organizing pneumonia, collagen vascular lung disease (for example, from lupus, scleroderma or mixed connective tissue disease), interstitial lung disease (for example, idiopathic pulmonary fibrosis or sarcoidosis), drug-induced lung disease and accelerated pulmonary fibrosis (for example, occurring after the acute lung injury including ARDS). Lung diseases, including chronic obstructive pulmonary disease, asthma, infections, and acute and chronic lung injury leading to fibrosis, constitute the third leading cause of death worldwide (Murray et al., 1997; Rabe et al., 2007; Tsushima et al., 2009). Acute lung injury (ALI) is a serious medical problem among US military personnel. ALI in combat can be caused by a variety of etiologies.

[0220] Treatment of ALI caused by inhalation injury with inhaled anticoagulants, steroids, beta-agonists, high-frequency ventilation, and extracorporeal membrane oxygenation has variable and generally suboptimal results. There are no effective preventive measures other than obstruction with a respirator mask. Significant advances have been made in the management of ARDS, but watchful waiting for intrinsic healing mechanisms to take effect remains largely supported; in-hospital mortality remains above 40% (Matthay et al., 2012). Survivors of ALI often suffer from chronic respiratory impairment, accompanied by a decreased quality of life. Any modality that can accelerate recovery and / or prevent later complications, such as chronic respiratory insufficiency and pulmonary fibrosis, would be highly desirable. There is an urgent need to improve the early diagnosis of ALI and, more importantly, its prevention and treatment. The pathophysiology of ALI caused by direct inhalation lung injury or ARDS due to systemic disease is extremely complex and heterogeneous, involving systemic and local cardiopulmonary factors, such as increased membrane permeability, influx of inflammatory cytokines, oxidative cell damage, compartmental fluid flow, ion channel disturbances, and many other factors (Matthay et al., 2012). Clearly, new therapies are needed to treat and prevent lung diseases, such as ALI.

[0221] In some embodiments, a method of treating or preventing acute lung injury, lung infection, or lung disease in a subject is provided, comprising administering to the subject an effective amount of a peptide comprising the amino acid sequence of FTTFTVT (SEQ ID NO: 2) or a variant thereof, wherein the peptide maintains the biological activity of caveolin-1 (Cav-1). In some aspects, the method of administering the pharmaceutical formulation of the peptide comprises dry powder inhalation. In certain aspects, the subject is a human.

[0222] B. Lung disease

[0223] Lung diseases include pulmonary fibrosis, lung inflammation, idiopathic pulmonary fibrosis, cystic fibrosis, chronic obstructive pulmonary disease (COPD), bronchitis, bronchiolitis, bronchiolitis obliterans, asthma and lung infections, as well as acute and chronic lung injury leading to fibrosis (Murray et al., 1997; Rabe et al., 2007; Tsushima et al., 2009). These diseases constitute the third leading cause of death worldwide.

[0224] Cystic fibrosis is a hereditary disease of the exocrine glands and eccrine sweat glands that primarily affects the digestive and respiratory systems. This disease is typically characterized by chronic respiratory infections, pancreatic insufficiency, abnormal mucus secretion, and premature death. Cystic fibrosis (CF) is characterized by progressive airflow obstruction. Individual subpopulations of individuals with CF also develop airway hyperresponsiveness to inhaled cholinergic agonists (Weinberger, 2002 and Mitchell et al., 1978), and reversibility of airflow limitation to responses to bronchodilators (van Haren et al., 1991 and van Haren et al., 1992). The presence of bronchial hyperresponsiveness and airway obstruction suggests that there may be a common cause of disease between CF and other airway narrowing diseases, such as asthma or chronic obstructive pulmonary disease (COPD), in which airway smooth muscle dysfunction is considered to cause the disease process.

[0225] Lung infections may be bacterial. The infectious bacteria may be Pseudomonas aeruginosa, Bacillus anthracis, Listeria monocytogenes, Staphylococcus aureus, Salmonella, Yersinia pestis, Mycobacterium leprae, Mycobacterium africanum, M. asiaticum, M. aviuin-intracellulaire, M. chelonei abscessus, M. fallax, Pseudomonas fallaxii, Mycobacterium fortuitum, Mycobacterium kansasii, Mycobacterium leprae, M. malmoense, M. shimoidei, M. simiae, M. szulgai, M. xenopi, Mycobacterium tuberculosis, Brucella melitensis, Brucella suis, Brucella canis, Legionella pneumophila, Francisella tularensis, Pneumocystis carinii, Mycoplasma, or Burkholderia. Bacterial infections may cause pneumonia.

[0226] Chronic obstructive pulmonary disease (COPD) is a term used to classify two major airflow obstruction disorders: chronic bronchitis and emphysema. Approximately 16 million Americans have COPD, 80-90% of whom are lifetime smokers. COPD is the leading cause of death in the United States, causing 122,283 deaths in 2003. Direct healthcare costs for COPD in the United States were approximately $20.9 billion in 2003. Chronic bronchitis is an inflammation of the bronchial airways, which connect the trachea to the lungs. When inflamed, the bronchi secrete mucus, causing a chronic cough.

[0227] In emphysema, the alveolar sacs overinflate because the lung's elastin skeleton is damaged. Inflammatory cells in the emphysematous lung release elastase, an enzyme that degrades or destroys elastin fibers within the lung matrix. Emphysema has various causes, including smoking, exposure to environmental pollutants, alpha-antitrypsin deficiency, and aging.

[0228] Bronchiolitis is most commonly caused by a viral lower respiratory tract infection and is characterized by acute inflammation, edema, necrosis of small airway epithelial cells, 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, accessory muscle use, and / or nasal dilation.

[0229] Bronchiolitis obliterans is a progressive decrease in airflow due to abnormal remodeling of the small airways in the lungs (Meyer et al., 2014). Bronchiolitis obliterans syndrome is a major complication of lung transplantation and is often used to describe delayed allograft dysfunction resulting in a persistent decrease in forced expiratory volume and force that is not due to other known causes (Meyer et al., 2014).

[0230] The term "asthma" may refer to acute asthma, chronic asthma, intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma, chronic persistent asthma, mild to moderate asthma, mild to moderate persistent asthma, mild to moderate chronic persistent asthma, allergic (extrinsic) asthma, non-allergic (intrinsic) asthma, nocturnal asthma, bronchial asthma, exercise-induced asthma, occupational asthma, seasonal asthma, asymptomatic asthma, gastroesophageal asthma, idiopathic asthma, and cough variant asthma. During asthma, the airways are constantly inflamed and may occasionally experience spasms.

[0231] In some embodiments, a method for treating or preventing a lung infection or lung disease in a subject is provided, comprising administering to the subject an effective amount of a dry powder peptide comprising the amino acid sequence of FTTFTVT (SEQ ID NO: 2; herein referred to as CSP7), wherein the dry powder peptide maintains the biological activity of caveolin-1 (Cav-1). In some aspects, the method of administering the pharmaceutical formulation of the embodiments comprises dry powder inhalation of the peptide. In certain aspects, the subject is a human.

[0232] V. Examples

[0233] The following examples are included to illustrate preferred embodiments of the present invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the present invention and, therefore, can be considered to constitute preferred modes for its practice. However, in light of the present disclosure, it should be understood by those skilled in the art that many changes may be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention.

[0234] Example 1 - Methods and Materials

[0235] Preparation of dry powder peptide: CSP7 peptide (SEQ ID NO: 2), batch #: AHF66 / / 470103 was synthesized by Polypeptide Laboratories (San Diego, USA).

[0236] Preparation and spray drying of CSP7 mixtures. CSP7 formulations containing CSP7 alone (CSP7), or a 75% / 25% mixture of CSP7 / leucine, CSP7 / trehalose, or CSP7 / sodium citrate, or a 75% / 15% / 10% mixture of CSP7 / leucine / trehalose, were prepared in water at pH 10 (adjusted with NH4OH) and spray dried using a BLD-35 with a 2-inch cyclone.

[0237] The particle size of dry powder CSP7 was reduced by thin-film freezing (TFF). 0.3 mg / ml CSP7 bulk powder and 0.9 mg / ml mannitol (mass ratio of 1:3) were dissolved in 10 mM Tris buffer and the pH was adjusted to 8.05. The solution was then filtered through a 0.45 μm membrane and dripped into a rolling chamber filled with liquid nitrogen. The metered freezing temperature was between -55°C and -65°C. The frozen flakes were then lyophilized in a VirTis Advantage freeze dryer (VirTis Company Inc., NY, US). Lyophilization conditions were as follows: equilibration: -55°C, 100 mTorr for 30 minutes; primary drying: temperature increased to -30°C, 100 mTorr over 250 minutes; maintained at -30°C, 100 mTorr for 660 minutes; secondary drying: temperature increased to 30°C, 100 mTorr over 720 minutes; maintained at 30°C, 100 mTorr for 240 minutes. The TFF-treated sample is designated as Lot 171014.

[0238] The particle size of dry powder CSP7 was reduced by cryogenic grinding. One gram of CSP7 bulk powder was added to a small cryogenic grinding tube and then loaded into a 6870 Freeze / Grinding Machine (SPEX Certiprep TM , NJ, USA). Milling was performed in five cycles with a 10-minute precooling period, each cycle running at 10 CPS for 5 minutes followed by a 2-minute cooldown. The milled sample was retrieved and weighed, and the yield was calculated to be 73.5% based on the ratio of the retrieved weight to the loaded weight.

[0239] The particle size of CSP7 powder was reduced by ball milling (BM). Bulk CSP7 powder was suspended in its antisolvent, ethanol (anhydrous), to a concentration of 1 mg / ml. Approximately half the solvent volume of zirconium balls (2 mm) was added to the suspension. The suspension was then ground in an 8000M mixer / grinder (SPEX SamplePrep, NJ, USA). Samples were collected from the grinding process and tested at 5, 10, and 30 minutes.

[0240] Use a rotor-stator to reduce the particle size of CSP7 powder. Disperse CSP7 bulk powder in ethanol to a concentration of 1 mg / ml. Immerse the tip of the rotor-stator (5 mm x 75 mm flat bottom) in the suspension and homogenize to reduce the particle size.

[0241] HPLC analysis. The samples were dissolved in 20 mM Tris buffer (pH 10.3) and analyzed by HPLC. The chromatographic column was run on a C18(2) liquid chromatography column with a particle size of 5 μm and a pore size of Phenomenex Security Guard cartridge kit was used as a guard column. Mobile phase A was H 2 The HPLC-MS / MS was performed using a flow rate of 1 mL / min. The column was maintained at 25°C for 25 minutes and the sample was detected at a wavelength of 220 nm. The buffer gradient was set to the conditions specified in Table 2.

[0242]

[0243]

[0244] Determine the aerodynamic particle size distribution of the ground CSP7 bulk powder. Approximately 3.5 mg of ground CSP7 powder was manually filled into size 3 HPMC capsules (Capsugel, Peapack, NJ). The CSP7 capsules were then atomized using an RS01 single-dose dry powder inhaler (high resistance) and the aerodynamic particle size distribution was measured by a next-generation impactor (NGI, MSP Corp., Shoreview, MN). The inhaler generated an aerosol within 4 seconds at an air flow rate of 60 L / min to achieve an inhaled volume of 4 L and a 4 kPa pressure drop on the device. Before each run, the NGI collection surface was coated with a 5% (v / v) methanol solution of polysorbate 20. Each run ejected one capsule, and each sample was repeated three times (three capsules). After atomization, all collection surfaces were rinsed with a specific volume of 20 mM Tris buffer (pH 10.3) to collect the drug. The powder deposited in the capsule, device, adapter, throat, pre-separator, and 1-MOC stage was extracted separately.

[0245] For each test, the delivered dose was defined as the mass of CSP7 entering the NGI. Based on the dose deposited on the 1-MOC level of the NGI, Copley Inhaler Test Data Analysis Software (CITDAS, Copley Scientific, Nottingham UK) was used to calculate and analyze the geometric standard deviation (GSD), mass median aerodynamic diameter (MMAD), and fine particle fraction % (FPF%). FPF is defined as the mass fraction of particles less than 5.0 μm of the delivered dose.

[0246] Preparation and lysis of lung tissue. Female mice aged 6-8 weeks were ordered from Jackson Laboratories, stock: 000664C57BL / 6J and were cared for and housed according to IACUC guidelines. Over the next week, mice were weighed, anesthetized with 80mg / kg of ketamine and 6mg / kg of xylazine (approximately 115ul / mouse) by intraperitoneal (IP) injection, and bleomycin was instilled into the trachea. Briefly, a 26G plastic catheter was inserted into the trachea, and mice received 2x 20ul of 0.8U / kg bleomycin (Biotang, Cat#RB003) instillations (30s apart to clear from the airway) via a pipette. Controls received only the same volume of saline. Body weight was tracked (weight loss was approximately 10% in injured animals), and animals were subjected to a dry powder inhalation regimen (CH technology) for one week every day. The dry powder dose was based on the minimum effective dose of a previous nebulized formulation, which was estimated to be a pulmonary delivery dose of 0.7 mcg / animal (Tepper et al., 2016, incorporated herein by reference). Overall, an exposure time of 12 minutes / day corresponds to a '1X' dose, while a '5X' dose corresponds to a 60-minute / day treatment, equivalent to 3.5 mcg / animal. Animals were exposed for seven consecutive days (days 14-20; during the fibrotic phase of bleomycin injury) and sacrificed 24 hours after the final lethal dose of a heparinized ketamine / xylazine mixture (25% heparin). A portion of the whole lung was collected for histological examination. Briefly, 10 ml of saline was transcardially perfused to clear the lungs of blood. Then, 20 cm above the dissection area, the lungs were inflated with saline for 1 minute, followed by 4% PFA for 1 minute. The trachea was ligated, and the lungs were excised, fixed, embedded, and sectioned to 4 microns to maximize surface area visualization and stained with hematoxylin and eosin. Images were captured using an Aperio AT2 high-capacity digital whole-slice scanner, and lung fibrosis injury scores were performed according to a modified Ashcroft scoring scheme (Hübner et al., 2008, incorporated herein by reference). For molecular analysis, whole lungs were homogenized in RIPA buffer and protease inhibitors (SantaCruz) and 1% DTT (to inhibit RNAse), and homogenized in parallel at 4C (Precellys Evolution, Bertin Instruments) and downstream assayed. The collagen content of lung homogenates was determined according to the total collagen assay (Quickzyme) using collagen standards provided by the manufacturer and according to the manufacturer's instructions. Colorimetric assays were read on a microplate reader (FilterMax F5, Molecular Devices, 580 nm).In addition, RNA was extracted from the homogenate (Zymogen Research) and then reverse transcribed into cDNA (Qiagen, QuantiNova Reverse Transcription 205413). The results of these studies are shown in. Figure 29-32 .

[0247] Homogenization buffer was prepared for 28 samples by adding 224 μL of cocktail inhibitor, 224 μL of NaOV4, 224 μL of PMSF, and 0.22 g of DTT to 22.4 mL of RIPA buffer. 800 μL of homogenization buffer was added to each sample. The samples were homogenized using a Precellys Evolution with CK28 beads. The homogenization protocol was used for hard tissues, and each sample was performed twice at 4°C. The homogenized samples were then aliquoted, with 400 μL stored for BCA concentration determination and protein assay, 200 μL for RNA isolation, and 200 μL for collagen assay.

[0248] Collagen assay. The collagen standard used was prepared by adding 125 μL of Quickzyme collagen standard to 125 μL of 12M HCl and adding 200 μL of each sample to 200 μL of 12M HCl. The samples and standards were incubated at 95°C for 20 hours and briefly vortexed after 20 minutes. After incubation, the samples were centrifuged at 13,000xg for 10 minutes. The standard was prepared according to the manufacturer's instructions (Quickzyme). 100 μL of each sample was then diluted into 50 μL of water. 10 μL of each diluted sample was then further diluted into 100 μL of 4M HCl. Repeats of the standard and each sample were pipetted into the plate. 75 μL of assay buffer was added to each well and the plate was covered before shaking for 20 minutes. 75 μL of detection reagent mixture was added to each well and the plate was mixed before incubating at 60°C for one hour. The plate was then read as shown above.

[0249] RNA isolation. RNA isolation was performed using the Qiagen RNeasy kit according to the manufacturer's instructions. Briefly, 25 μL of RLT buffer and 75 μL of 70% ethanol were added to 50 μL of sample in RIP A buffer, yielding a total of 150 μL of starting material. 50 μL of starting material for each sample was then added to 50 μL of RNase-free water. Then, 350 μL of Buffer RLT was added, and the samples were mixed thoroughly. 250 μL of 95-100% ethanol was then added to each column, and the samples were mixed again. 700 μL of each sample was then added to its respective nucleic acid purification column and centrifuged at 8000 × g, with the flow-through discarded. 500 μL of RPE was added to each column, and the columns were centrifuged again. 500 μL of RPE was added again, and this time the samples were centrifuged at 8000 × g for 2 minutes. The samples were transferred to new microcentrifuge tubes, and the RNA was eluted with 40 μL of RNase-free water by centrifugation at 8000 × g for 1 minute. Samples were quantified by nanodrop and analyzed as described above.

[0250] Example 2 - Characterization of CSP7 Bulk Powder

[0251] Scanning electron microscopy. Bulk powder samples of CSP7 were sputtered onto a sample tray and spread by blowing compressed nitrogen. The samples were imaged by scanning electron microscopy ( Figure 1 ). SEM showed the presence of large particles (>5 μm). In addition, most of the particles appeared to be large (>5 μm) and therefore not in the respirable range.

[0252] CSP7 Particle Size Evaluation. Particle size was checked using a Spraytec laser diffractometer and a Sympatec laser diffractometer HELOS-R system equipped with either a solid or wet dispersion accessory to determine if the bulk powder was within the respirable range. The CSP7 bulk powder particle size was determined to be larger than the respirable size using a dry dispersion method. Table 3 shows the particle sizes of the particles within the distribution assessed as Dv 10, Dv 50 (median), and Dv 90. As shown in Table 3, of all CSP7 particles analyzed, more than 50% had a particle size of 5.3 μm or larger, which is larger than the respirable range.

[0253]

[0254] Next, particle size was determined using a Sympatec laser diffractometer using a wet dispersion method. CSP7 was dissolved in ethanol + 0.05% Tween 80 as the dispersion medium and sonicated for 10 minutes. The results of the wet dispersion method for determining CSP7 particle size are shown in Table 4. As shown in Table 4, the average particle size (Dv 50) of the wet-dispersed particles was 29.0 ± 0.8, well outside the respirable range.

[0255]

[0256] The average particle size was further evaluated using a dry dispersion method, again using a Spraytec laser diffractometer. The CSP7 bulk powder was dispersed at 40 PSI and the average particle size (8.6 ± 1.5 μm) was again above the respirable range (Table 5).

[0257]

[0258] The percentage of dry powder particles smaller than 5 μm was found to be only 34.5 ± 4.1%.Given that the laser diffraction methods each found that the majority of the bulk powder had a particle size outside the respirable range, any dry powder used for processing would need to be processed in some way to reduce the particle size.

[0259] The morphology of bulk CSP7 powder. Bulk powder CSP7 samples were sputtered onto a glass slide and observed by optical microscopy ( Figure 2 ). Optical microscopy confirmed the SEM, indicating the presence of large particles (>5 μm). Optical microscopy also showed the presence of particle agglomerates, Figure 2 As can be seen in the figure, it is indicated by the arrow.

[0260] Determine the crystallinity of CSP7 bulk powder particles. X-ray powder diffraction was used to evaluate the crystallinity of CSP7 bulk powder particles ( Figure 3 Using X-ray powder diffraction, it was found that pure CSP7 showed a certain degree of crystallinity ( Figure 3 To confirm the X-ray diffraction results, the crystallinity was evaluated by polarizing microscopy ( Figure 4 ).like Figure 4 As shown in FIG, crystalline CSP7 is present in bulk CSP7 powder, and the white arrows in the image indicate some typical crystalline forms.

[0261] Thermal analysis of CSP7 bulk powder. Differential scanning calorimetry was used to determine the melting point of pure CSP7 ( Figure 5 As determined by DSC, the melting point of CSP7 was determined to be 211.03°C ( Figure 5 ). Thermogravimetric analysis (TGA) was used to analyze ( Figure 6 ). TGA shows that the weight of pure CSP7 begins to decrease significantly above 216℃ ( Figure 6 ).

[0262] Moisture content of CSP7 bulk powder. The moisture content of bulk powder CSP7 was assessed by Karl Fischer-Volume (KF-V) titration using a Mettler Toledo Karl Fischer titrator and repeated three times. Table 6 shows the moisture content of each test and the average moisture content of the three tests.

[0263]

[0264] Next, the moisture uptake of CSP7 bulk powder was analyzed using dynamic vapor sorption (DVS). Figure 7 The CSP7 sample was run through a complete adsorption / desorption cycle and was found to have a water desorption of 6.32% at zero relative humidity. When the relative humidity was 90%, a mass change of 10.54% ( Figure 7 ).

[0265] Example 3 - Characterization of CSP7 Powder After Particle Size Reduction

[0266] The particle size of CSP7 powder is reduced. In order to effectively inhale the powder and deposit it in the lungs, the particle size should generally have a mass median aerodynamic diameter less than about 5 μm. A variety of techniques have been performed to reduce the particle size of pure material, including air jet milling (AJM), ball milling (BM), cryogenic milling (CM), thin film freezing (TFF) and spray drying. First, AJM is performed to reduce the particle size of CSP7 bulk powder, and the ground CSP7 is collected from multiple positions in the grinder. Tables 5 and 6 show the yield and particle size distribution of the first batch of powder (batch number 171013) collected from the positions shown. As mentioned above, particle size distribution is determined by Sympatec laser diffractometer dry dispersion method (Table 8) or Sympatec laser diffractometer wet dispersion method (Table 9).

[0267]

[0268]

[0269] A second batch of CSP7 (batch number 171027) was milled from 10 grams of pure bulk powder using the same conditions as above. Similarly, the particle size distribution and yield of the powder were evaluated from the same location and are listed in Table 10.

[0270]

[0271] The third batch of CSP7 dry powder (Batch No. 171014) was thin film frozen (TFF) using the Sympatec laser diffractometer dry dispersion method (Table 11) and wet dispersion method (Table 12) and analyzed as described above.

[0272]

[0273] Another batch of CSP7 dry powder was cryogenically ground (CM) to reduce the particle size. The particle size of CM CSP7 was evaluated by laser diffraction using the dry dispersion method (Table 13) and the wet dispersion method (Table 14) as described above.

[0274]

[0275] Another batch of CSP7 bulk powder was ball milled (BM) to reduce the particle size. Table 15 shows the particle size distribution of the BM CSP7 powder taken at several time points during the milling process.

[0276]

[0277] Further batches were produced with CSP7 (also referred to by the designated development abbreviation, CSP7) and leucine, trehalose, sodium citrate, or a mixture of leucine and trehalose and spray dried to reduce particle size. The spray dried particle size was examined by dry dispersion laser diffraction ( Figure 8 , Table 16). Again, spray drying significantly reduced the particle size of CSP7 relative to bulk CSP7.

[0278]

[0279]

[0280] The CSP7 particle size was significantly reduced with each of the particle size reduction methods (jet milling, thin film freezing, cryogenic grinding, ball milling, and spray drying) when measured by both solid dispersion and wet dispersion methods (compare Tables 1 and 2 with Tables 5, 6, 10, 13, 14, 16, 17, 18, and 19). The majority of the CSP7 particles fell within the respirable range after jet milling, as measured by either solid dispersion or liquid dispersion methods by laser diffraction, but thin film freezing, cryogenic grinding, and ball milling were less effective, with a smaller percentage of the milled powder falling within the respirable range.

[0281] Particle size can also be reduced using a rotor-stator handheld homogenizer ( Figure 9 After homogenization, the color of CSP7 in ethanol will change to light gray or dark gray, depending on the duration and efficacy of homogenization. Figure 9 , and it is apparent that homogenization reduced the particle size, although the process was not pursued further due to the observed color change.

[0282] Morphology of the ground CSP7 powder. The morphology of each of the ground samples was examined using optical microscopy or scanning electron microscopy. Powder samples from the jet mill were examined as above, and optical microscopy showed that the particle size was reduced to a size of 1 μm > particle size > 5 μm and was homogeneous ( Figure 10 In addition, the ground CSP7 particles do not contain agglomerates ( Figure 10 SEM showed that the homogenization and reduction of the jet milled particles were between 1 μm and 5 μm ( Figure 11 The powder samples obtained after TFF were also examined, and although the particle size was larger, no agglomerates were found in the samples ( Figure 12 ). Further analysis included evaluation of the particle morphology of the spray-dried formulations by scanning electron microscopy. Representative SEM images of the formulations are shown in Figure 13 .

[0283] Crystallinity of AJM CSP7. Milled CSP7 powder (batch 171027) was evaluated by X-ray diffraction and showed crystallinity ( Figure 14 The crystallinity of spray-dried CSP7 was also examined by X-ray diffraction, and the curve is shown in Figure 15 Preparations of CSP7 alone or coupled to trehalose or sodium citrate were amorphous, whereas CSP7 coupled to leucine or leucine and trehalose appeared to contain crystalline leucine features, as shown in Figure 2. Figure 15 As shown by the spike in .

[0284] HPLC Evaluation of Milled or Spray-Dried CSP7 Powders. To determine whether milling CSP7 bulk powder had an effect on chemical potency, milled CSP7 powder samples collected from various locations on the mill were evaluated using HPLC under the conditions listed in Table 2. Potency was estimated using the following formula:

[0285]

[0286] It is clear from Table 26 that grinding had no detrimental effect on the potency of any of the samples collected. Likewise, when batch number 171027 was tested, the chemical potency was determined to be 100.14%.

[0287]

[0288] The spray-dried CSP7 mixture was checked by RP-HPLC to check the purity (Table 17, Figure 16 ). Similar to the jet-milled CSP7 powder, the spray-dried CSP7 retained approximately 100% purity.

[0289]

[0290]

[0291] CSP7 stability. The stability of untreated bulk CSP7 powder and jet-milled CSP7 (lot 171027) was examined by measuring their chemical potency using HPLC. Each sample was stored under three different conditions and then the chemical potency ( Figure 17 The stability of the spray-dried CSP7 was also examined by HPLC (method described earlier in the specification) over a 24-hour period to understand its short-term stability. Each formulation was found to be stable, with no increase in impurities after 2 or 24 hours (Table 31).

[0292]

[0293] Aerodynamic particle size distribution of ground CSP7. To determine the aerodynamic particle size distribution of ground CSP7, the amount of powder deposited at various locations in the NGI collector was extracted separately. The delivered dose was measured as the mass of CSP7 entering the NGI collector after atomization, and the amount of CSP7 deposited on individual surfaces was extracted and measured separately. The amount of untreated or jet-milled (batch 171013) CSP7 remaining in the capsule or deposited in the device, adapter, throat, pre-separator, and 1-MOC stage is shown in Figure 18 , as a percentage of the total amount of CSP7 delivered. Table 9 lists the fine particle fraction percentage (FPF%), mass mean aerodynamic diameter (MMAD), and geometric standard deviation (GSD) of ground and untreated (eg, unground or untreated) CSP7.

[0294]

[0295] The aerodynamic particle size distribution of the second milled batch of CSP7 lot number (171027) was determined as above, except that approximately 4.25 mg of powder was used per size 3 HPMC capsule. The GSD, FPF%, and MMAD are shown in Table 11, and the percentage of CSP7 deposited in each location is shown in Table 12. Figure 19 , again as a percentage of the total amount of CSP7 delivered.

[0296]

[0297] The atomization of the spray-dried formulation was also examined ( Figure 20 Each formulation showed a fine powder fraction greater than 60%, with the MMAD for each formulation ranging from 2.5 μm to 3 μm (Table 25). Table 25 shows a summary of the analytical results for the spray-dried formulations, including water content.

[0298]

[0299]

[0300] Determination of moisture content of ground CSP7. Air jet milled CSP7 powder (Batch 171027) was analyzed using dynamic vapor sorption under the same conditions used for analysis of bulk CSP7 ( Figure 21 ). Similar to the bulk pure powder, the ground CSP7 has a water desorption rate of 4.61% at 0% relative humidity ( Figure 21 ). KF-V analysis found the moisture content to be 4.9% (Table 7), although the mass change increased to 13.59% when the relative humidity was 90% ( Figure 21 ).

[0301]

[0302] Thermogravimetric analysis of CSP7 powder with reduced particle size. Thermogravimetric analysis of ground CSP7 (batch 171027) was performed in the same manner as bulk CSP7 and found that the ground CSP7 had very similar properties to the untreated CSP7 ( Figure 22 The thermal properties of the spray-dried formulations were also evaluated and are shown in Tables 24 and Figure 23-27 (Summary in Figure 28 ). It is noteworthy that the midpoint Tg of the mixed formulation is significantly lower than that of spray-dried CSP7 alone (compare 001C-F to 001B in Table 24).

[0303]

[0304]

[0305] The results presented here demonstrate that various methods for reducing the particle size of CSP7 powders are effective and that the resulting powders exhibit very similar properties.

[0306] Example 4 - Treatment of bleomycin-induced pulmonary fibrosis by inhalation of CSP7 dry powder

[0307] Induction and CSP7 treatment of mice with fibrosis. Mice were treated with bleomycin to induce pulmonary fibrosis. Mice were given 0.8 U / kg bleomycin intranasally and allowed to develop disease for 14 days before treatment. Mice were then left untreated, treated with inhalation of CSP7 dry powder for 12 minutes, or treated with inhalation of CSP7 dry powder for 60 minutes. On the last day of treatment, mice were euthanized and their lungs removed, snap-frozen, and stored at -80°C. The snap-frozen lungs were weighed ( Figure 29 ).

[0308] Lung tissue was homogenized and analyzed for collagen content using the Quickzyme collagen assay ( Figure 30 Bleomycin-induced fibrosis resulted in a significant increase in collagen in the lung compared to saline treatment (P=0.0062) ( Figure 30 After bleomycin-induced lung fibrosis, mice treated with CSP7 had lower Ashcroft scores (a measure of lung fibrosis in mice) ( Figure 31 ).

[0309] RNA was also prepared from homogenized lung tissue and used as described above.

[0310] Example 5 - Formulation Suspension for Intramuscular / Subcutaneous Injection

[0311]

[0312] *: Jet-milled CSP7 showed geometric particle sizes of Dv(10) = 0.75 μm; Dv(50) = 1.93 μm; Dv(90) = 4.29 μm, measured by laser diffraction.

[0313] For preparation:

[0314] 1. Prepare 20mM Tris buffer (should be pH ~10.3)

[0315] 2. Dissolve 1.5% (w / w) CMC in 20mM Tris buffer and add 0.2%

[0316] (w / w) Poloxamer 188. Stir overnight at 600 rpm. 3. Add 0.7% (w / w) NaCl to the CMC solution.

[0317] 4. Adjust the pH of the solution to 7 by adding ~28.5 μl / ml of 1N HCl

[0318] 5. Weigh and add a certain amount of jet-milled CSP7 powder (collected from the collection bag to obtain a smaller particle size fraction) into a clean vial.

[0319] 6. First use a clean rod to grind the powder to ensure there are no obvious agglomerated particles

[0320] 7. Gradually add the prepared solution to the vial and grind / mill with a rod

[0321] 8. When the powder is fully wetted, no visible agglomerated particles reaching the target volume are observed.

[0322]

[0323] Example 6 - Formulation Solution for Intramuscular / Subcutaneous Injection

[0324] For preparation:

[0325] 1. Prepare 20mM Tris buffer (should be pH ~10.3)

[0326] 2. Dissolve 1.5% (w / w) CMC in 20mM Tris buffer and add 0.2%

[0327] (w / w) Poloxamer 188. Stir overnight at ~600 rpm 3. Add 0.7% (w / w) NaCl to the CMC solution.

[0328] 4. Add 1.2-1.4 mg / ml CSP7 (untreated powder) to the solution and vortex to dissolve. The pH should be approximately 9, then adjust the pH to 8.2-8.5 by adding ~50 μl 1N HCl.

[0329]

[0330] result:

[0331]

[0332] Example 7 - Pre-formulation studies of polypeptide variants

[0333] Solubility studies of the variants were performed by adding the variant powder to the solvent at a concentration of 5 mg / ml and then vortexing for 3 minutes. The appearance of the solution was observed within 5 minutes. More powder was added (~5 mg / ml each time) and the vortexing and observation were repeated until precipitation or gelation occurred.

[0334] result:

[0335]

[0336]

[0337] a Precipitation: Centrifuge the solution, then aliquot the supernatant and dilute with buffer. b High viscosity solutions: Take an aliquot and dilute with buffer.

[0338]

[0339]

[0340] Example 8 - Pre-formulation studies of CSP7 (ammonium counterion) forms

[0341] Solubility of the pure (i.e., unground) CSP7 ammonium counterion form (Table 35) was performed by adding excess peptide powder to 3 mL of different pH buffers (Table 36) and mixing on an orbital shaker at 100 rpm at room temperature for 24 hours.

[0342] For freeze-thaw stability studies ( Figure 33 ), 0.1 mg / mL CSP7 (ammonium counterion) peptide in a phosphate buffer system (PBS, pH 7.4) was aliquoted into 15 mL / vial and subjected to quick freezing and slow freezing, respectively. For quick freezing and slow freezing, the sample was immersed in liquid nitrogen for at least 5 minutes, or placed in a -20 ° C freezer for at least 1.5 hours to ensure that the aliquots in the vial were completely frozen and then thawed to room temperature. Each sample underwent 5 freeze-thaw cycles. The recovery rate (%) represents the percentage of each sample concentration to the original (untreated) concentration.

[0343] Solubility samples and freeze-thaw samples were filtered through a 0.45 μm membrane before being assayed by high performance liquid chromatography (HPLC, Thermo Fisher Scientific, Fair Lawn, NJ). Samples were analyzed on a Dionex 3000 HPLC system equipped with a 2.5 μm reverse-phase C18 column, 150 mm x 4.60 mm. The HPLC column was heated to 60°C for testing, and peptides were detected at a wavelength of 215 nm and a flow rate of 1 mL / min. The two mobile phases were A (0.1% acetic acid in water) and B (0.1% acetic acid in acetonitrile). The injection volume was 20 μL, and a standard curve was generated from 0.01 to 1 mg / mL.

[0344] Table 34. HPLC method details.

[0345]

[0346] Table 35: pH solubility curve of CSP7 (ammonium counterion).

[0347] pH Concentration (mg / mL) 3 0.17 4 0.07 5 0.05 6 0.07 7 0.10 8 0.38 9 2.30 10 7.87 11 14.43

[0348] Table 36. Buffer systems and the amount of CSP7 peptide added to 3 mL of buffer at each pH value

[0349]

[0350] Example 9. Characterization and stability study of milled CSP7 (ammonium counterion) powder batch number UTA181028

[0351] Use Model 00Jet-O-Mizer TM The CSP7 peptide was ground using a jet mill (also known as Aljet mill, Fluid Energy, Telford, PA). Feed rate, driving pressure, and milling pressure were respectively 1 g / min, 60 psi, and 70 psi (Table 37). 20 g was used in batches, and the powder ground was collected from the different parts of the jet mill, which included a tube (bfC) after the milling chamber, a cyclone separator (C), a collecting container adapter (D), a collecting bag adapter (E), a collecting bag (H), and a collecting container (G). The collected powder was mixed for 10 minutes using a Turbula mixer (Glen Mills Inc., Clifton, NJ, USA).

[0352] Table 37. Parameters used for milling CSP7 (ammonium counterion) powder.

[0353] parameter value Feed speed 1g / min Push pressure 60psi Grinding pressure 70psi

[0354] The specific surface area of the ground and untreated CSP7 powders was analyzed by the single-point BET method using a Monosorb MS-21 rapid surface area analyzer (Quantachrome Instruments, Boynton Beach, FL). Figure 34 The sample was degassed with 20 psi nitrogen at 25°C for 20-24 hours to remove water and other impurity molecules on the surface. A nitrogen / helium mixture (50:50 v / v) was used as the adsorbent, and the apparatus was calibrated with nitrogen before testing.

[0355] Thermogravimetric analysis of ground pure powder of CSP7 ammonium counterion was performed using the method described in paragraph

[0021] except that the starting temperature was 35°C instead of 25°C. The results are shown in Figure 35 .

[0356] Scanning electron microscope (SEM) image of ground and pure CSP7 (ammonium counterion) powder. The morphology of CSP7 ( Figure 36) were analyzed using a Zeiss Supra 40VP SEM (Carl Zeiss Microscopy GmbH, Jena, Germany). The samples were mounted on aluminum SEM sample holders using carbon conductive tape and coated with 12 nm of platinum / palladium (Pt / Pd) using a Cressington sputter coater 208HR (Cressington Scientific Instruments Ltd., Watford, UK). Images were taken of both pure (i.e., untreated) and milled CSP7 samples.

[0357] Stability study of ground CSP7 (ammonium counterion) from lot number UTA181028. The stability of ground CSP7 powder was studied under different storage conditions for up to 6 months. Ground CSP7 peptide was packaged in two forms: bulk ground powder and packaged ground powder. For storage in bulk ground powder form, 0.21-0.24 g of peptide was packed into 20 mL scintillation vials ( DWK Life Sciences, Millville, NJ, USA) and stored in heat-sealed aluminum foil pouches (Impak Corp, Los Angeles, CA, USA), each containing two 1-gram bags of silica gel desiccant ( Sorbco Packaging LLC, Belen, NM, US). The ground peptide powder was also encapsulated in size 3 HPMC capsules (Capsugel, Morristown, NJ, US) weighing approximately 11 ± 5% mg, and 22-26 capsules were then packaged in HDPE bottles (Drug Plastic, Boyertown, PA, US), which were then sealed in aluminum foil pouches (without desiccant). The packages were stored in a stability chamber at the following storage conditions: -20°C, 25°C / 60% RH, and 40°C / 75% RH. Samples were removed at 1 month, 3 months, and 6 months for testing (Table 38). For tests other than aerodynamic particle size distribution, the encapsulated powder was removed from the capsules and mixed into glass vials by rotating the vials.

[0358] Table 38. Testing schedule for stability studies.

[0359]

[0360] *Abbreviations: HPLC: High Performance Liquid Chromatography; KF-C: Karl Fischer Coulometric; GPSD: Geometric Particle Size Distribution; LD: Laser Diffraction; APSD: Aerodynamic Particle Size Distribution; NGI: Next Generation Impactor; SEM: Scanning Electron Microscope; XRPD: X-ray Powder Diffraction.

[0361] Appearance of the ground powder. The appearance of the ground powder was recorded by taking pictures using a conventional camera ( Figure 30 ).

[0362] Chemical stability of the ground peptide. The powder was assayed using the HPLC method described in Example 8. The results are shown in Table 39 below. Percentages represent the assayed amount compared to the mass balance. The assay was adjusted for moisture content.

[0363] Table 39. HPLC analysis of the stability of the ammonium counterion of milled CSP7.

[0364]

[0365] Moisture content of CSP powder. The moisture content of the peptide powder was determined using the Karl Fischer coulometric method (Mettler Toledo C20 Leicester, OH, US) (Table 40). The reliability of the device was tested using a Karl Fischer water content standard (Hydranal™ water standard, Honeywell, Charlotte, NC, US). A known amount of powder was suspended in anhydrous methanol (Sigma, St. Louis, MO) and the suspension was injected into the anolyte (Hydranal™ water standard). TM -Coulomat AG, HHoneywell, Charlotte, NC, US) in the catholyte (Hydranal TMThe titration was initiated in the presence of 1% Coulomat CG, H Honeywell, Charlotte, NC, US). The results were recorded as the difference between the water content in the sample and the blank anhydrous methanol solution.

[0366] Table 40. Moisture content of powder samples from stability studies.

[0367]

[0368]

[0369] Geometric Particle Size Distribution. The GPSD of CSP7 powder was analyzed before and after grinding using a Sympatec HELOS laser diffractometer (Sympatec GmbH, Germany) equipped with a RODOS disperser. After the powder was dispersed at 3 bar, measurements were taken every 10 ms. The measured values between 5% and 25% optical density were averaged to determine the particle size distribution. The reported particle size by volume is expressed as the 10th, 50th, and 90th percentiles (e.g., Dv 10, Dv 50, and Dv 90), as well as the percentage of particles falling within the 1-5 μm size range. The results are shown in Table 41.

[0370] Table 41. Geometric Particle Size Distribution of Milled CSP7 (Ammonium Counterion) in Stability Studies

[0371]

[0372] Aerodynamic Particle Size Distribution: Aerodynamic particle size distribution was evaluated in the stability study by performing the NGI as described in paragraph

[00215] , except that the powder weight in the capsules tested in the stability study was 11 ± 5% mg and the pre-separator was removed from the NGI assembly. The results are shown in Table 42.

[0373] Table 42. Aerodynamic particle size distribution of CSP7 (ammonium counterion) milled in stability studies.

[0374]

[0375]

[0376] Note: IP: air inlet; FPD: fine particle dose (particles < 5 μm); FPF (%): fine particle dose in delivered dose

[0377] Crystallinity of CSP7 powder. The crystallinity of the powder was evaluated by the method described in paragraph

[0018] and the results are shown in Figure 38 .

[0378] ***

[0379] In view of the present disclosure, all methods disclosed and claimed herein can be performed and executed without undue experimentation. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be made to the methods described herein and the steps of the methods or the order of the steps without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related can replace the agents described herein while obtaining the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0380] References

[0381] To the extent that the following references provide exemplary procedures or other details supplementary to those described herein, they are expressly incorporated herein by reference.

[0382] Carvalho et al., "Influence of particle size on regional lung deposition—Whatevidence is there?" Int. J. Pharma. 406:1-10, 2011.

[0383] Hübner, R.-H.; Gitter, W.; El Mokhtari, NE; Mathiak, M.; Both, M.; Bolte, EL; Freitag-Wolf, S.; Bewig, B. Standardized quantification of pulmonary fibrosis inhistological samples. Biotechniques, 44, 507-11, 514-7, 2008. Surasarang et al., “Optimization of Formulation for a Novel Inhaled Candidate Therapeutic for Idiopathic Fibrosis,” Drug Development and Industrial Pharmacy, 44(2):184-198, 2017.

[0384] Tepper,J.S.;Kuehl,P.J.;Cracknell,S.;Nikula,K.J.;Pei,L.;Blanchard,J.D.

[0385] Symposium Summary:“breathe In,Breathe Out,Its Easy:What You NeedtoKnow about Developing Inhaled Drugs.”Int.J.Toxicol.35,376-392,2016。

Claims

1. A dry powder composition comprising particles, wherein the particle comprises a peptide comprising the amino acid sequence FTTFTVT (SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof, and The dry powder composition is formulated for use in a dry powder inhaler.

2. The dry powder composition according to claim 1, wherein the peptide has the amino acid sequence of FTTFTVT (SEQ ID NO: 2) and 1 to 5 additional amino acids at the N-terminus and / or C-terminus of FTTFTVT (SEQ ID NO: 2).

3. The dry powder composition of claim 1, wherein the peptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 3-20. The dry powder composition according to claim 1 , wherein the peptide consists of the amino acid sequence of FTTFTVT (SEQ ID NO: 2).

5. The dry powder composition of any one of claims 1 to 3, wherein the peptide comprises a cell penetrating peptide (CPP), optionally wherein the CPP comprises an amino acid sequence selected from the group consisting of GRKKRRQRRRPPQ (SEQ ID NO: 23), RQIKIWFQNRRMKWKK (SEQ ID NO: 24), and GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 25).

6. The dry powder composition according to any one of claims 1 to 5, wherein the dry powder composition is encapsulated.

7. The dry powder composition according to any one of claims 1 to 6, wherein the pharmaceutically acceptable salt is an ammonium salt or an acetate salt.

8. The dry powder composition of any one of claims 1-7, wherein the particles comprise leucine, trehalose, sodium citrate, or a combination thereof.

9. The dry powder composition according to any one of claims 1 to 8, wherein the dry powder composition comprises a lubricant.

10. The dry powder composition of any one of claims 1-7, wherein the dry powder composition does not comprise a pharmaceutically acceptable carrier or excipient.

11. The dry powder composition of any one of claims 1-10, wherein the dry powder composition is at least about 95% (w / v) pure.

12. The dry powder composition according to any one of claims 1 to 11, wherein the dry powder composition comprises less than 10% (by weight) water.

13. The dry powder composition according to any one of claims 1 to 12, wherein the particles have a particle size of less than 5 μm.

14. The dry powder composition of any one of claims 1-13, wherein the particles have a particle size of about 1 μm to about 5 μm.

15. The dry powder composition of any one of claims 1-14, wherein the dry powder composition is stable for at least 1 month, at least 3 months, or at least 6 months.

16. The dry powder composition of any one of claims 1-15, wherein the dry powder composition is stable for at least 6 months.

17. The dry powder composition according to any one of claims 1 to 16, wherein the dry powder composition is produced by a grinding process or a spray drying process.

18. The dry powder composition of claim 17, wherein the dry powder is produced by jet milling, ball milling, wet milling or cryogenic milling.

19. A pharmaceutical composition comprising the dry powder composition according to any one of claims 1 to 18.

20. Use of the dry powder composition according to any one of claims 1 to 18 or the pharmaceutical composition according to claim 19 in the preparation of a medicament for treating a subject in need thereof, wherein the composition is administered to the subject using a dry powder inhaler.

21. The use of claim 20, wherein the dry powder inhaler provides an aerosol comprising a fine particle fraction (FPF) of at least 50%, at least 60%, at least 70% or at least 80% of the emitted dose.

22. The use according to claim 20 or 21, wherein the dry powder inhaler provides an aerosol comprising an emission fraction of at least 80% or at least 90%.

23. The use according to any one of claims 20 to 22, wherein the dry powder inhaler provides an aerosol comprising a mass median aerodynamic diameter of less than 5 μm.

24. The use of any one of claims 20-23, wherein the emitted dose to the subject is at least 10 mg, at least 15 mg, or at least 20 mg.

25. The use according to any one of claims 1 to 24, wherein the dose of fine particles administered to the subject is greater than 5 mg.

26. The use according to any one of claims 20-25, wherein the subject suffers from an inflammatory disorder.

27. The use according to any one of claims 20-25, wherein the subject has a fibrotic disorder.

28. The method of any one of claims 20 to 25, wherein the subject has lung inflammation, acute lung injury, lung infection, chronic obstructive pulmonary disease (COPD), chemically induced lung injury, plastic bronchitis, asthma, acute respiratory distress syndrome (ARDS), smoke inhalation induced acute lung injury (ISALI), bronchiolitis, bronchiolitis obliterans, fibrotic conditions of the lung, interstitial lung disease, idiopathic pulmonary fibrosis, or lung scarring.

29. The use according to claim 28, wherein the interstitial lung disease is idiopathic pulmonary fibrosis.

30. The use according to any one of claims 20 to 29, wherein the medicament further comprises at least one additional therapeutic agent, optionally wherein the at least one additional therapeutic agent is a nonsteroidal anti-inflammatory drug (NSAID), a steroid, a disease-modifying antirheumatic drug (DMARD), an immunosuppressant or a bronchodilator.

31. A dry powder inhaler comprising the dry powder composition according to any one of claims 1 to 18 or the pharmaceutical composition according to claim 19.

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