Dry powder formulation of caveolin-1 peptide and method of use thereof

Abstract

The present invention provides a composition comprising a caveolin-1 (Cav-1) peptide. Also provided are methods of using the Cav-1 peptide to treat pulmonary infections or acute or chronic lung injury, particularly pulmonary fibrosis.

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 729,010, filed Sep. 10, 2018, which is hereby incorporated by reference in its entirety.

[0002] The present invention is the result of activities carried out within the scope of a joint research agreement that was in effect when the present invention was made. The parties to the joint research agreement are the University of Texas System and the Board of Regents of The University of Texas Health Science Center at Houston. FIELD OF THE INVENTION

[0003] The present invention generally relates 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 OF THE INVENTION

[0004] 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 tissue. For example, for treating inflammatory or fibrotic conditions, such as pulmonary fibrosis. Accordingly, 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

[0005] According to the present disclosure, there is provided a dry powder composition of a peptide comprising the amino acid sequence of SEQ ID NO:2.

[0006] In a first embodiment, a pharmaceutical composition is provided that comprises a dry powder of a peptide, the peptide comprising any one of the following sequences: SEQ ID NO: 2-20. In some aspects, the peptide is 7-20 amino acids in length. In a particular 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 C-terminus of the peptide of SEQ ID NO: 2. In certain aspects, the peptide may comprise L-amino acids or D-amino acids or both L-amino acids and D-amino acids. In other aspects, the peptide may comprise at least one non-standard amino acid. In several aspects, the peptide comprises 2 non-standard amino acids. In a particular aspect, the non-standard amino acid is ornithine.

[0007] In other aspects, the peptide may comprise an N-terminal modification or a C-terminal modification or both an N-terminal modification and a C-terminal modification. In a particular aspect, the N-terminal modification is acylation. In another aspect, the C-terminal modification is amidation.

[0008] 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 a particular aspect, 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), RQIKIWFONRRMKWKK (SEQ ID NO: 24) and GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 25).

[0009] 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 milling, ball milling or wet milling. In some aspects, the dry powder contains less than 10% (by weight) of water. In another aspect, the dry powder contains less than 1% (by weight) of water. In certain aspects, the pharmaceutical composition is substantially free of excipients. In a particular aspect, the pharmaceutical composition is free of excipients. In specific aspects, the pharmaceutical composition is formulated for pulmonary delivery. In another aspect, the pharmaceutical composition is formulated for dry powder inhalation. In other aspects, the pharmaceutical composition is formulated for inhalation by pressurized metered dose inhaler. In some aspects, the pharmaceutical composition is formulated for oral administration, topical administration or injection.

[0010] In certain aspects, the dry powder formulation of the examples contains a water content of less than about 10%, 9%, 8%, 7%, 6% or 5%. In other aspects, the composition contains a water content of from 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 examples has an average particle size of less than 10 μm. In certain aspects, the average particle size is from about 0.01 μm to about 10 μm; from about 0.1 μm to about 8 μm; from about 0.5 μm to about 7 μm or from 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 examples has a particle size of from about 1 μm to about 5 μm. In certain aspects, the dry powder formulation of the peptide of the examples (e.g., CSP7) consists of at least 70% (e.g., 70%-80%) of particles having a particle size of from about 1 μm to about 5 μm. In preferred aspects, at least about 70%, 75%, 80% or 85% (e.g., 75%-95%) of the particle size of the particles in the dry powder formulation is less than 5 μm.

[0011] Further embodiments of the invention provide a nebulizer device comprising the pharmaceutical composition of the above examples and aspects.

[0012] In yet another embodiment, a method of treating a subject is provided, the method comprising administering to the subject an effective amount of the pharmaceutical composition of the above embodiments and aspects. In certain aspects, the subject has an inflammatory disorder. In other aspects, the subject has a fibrotic condition. In several aspects, the subject has pulmonary inflammation, acute lung injury, pulmonary infection or the lungs. In another aspect, the subject has pulmonary inflammation. In a particular aspect, the subject has chronic obstructive pulmonary disease (COPD). In other aspects, the subject may have acute lung injury or infection, pulmonary infection, chemically induced lung injury, plastic bronchitis, asthma, acute respiratory distress syndrome (ARDS), inhalation smoke-induced acute lung injury (ISALI), bronchiolitis or bronchiolitis obliterans. In a particular aspect, the lung disease is a fibrotic condition of the lungs, interstitial lung disease or idiopathic pulmonary fibrosis (IPF) or lung scarring. In other aspects, the administration comprises dry powder inhalation. In other aspects, the administration comprises nebulizing a solution comprising the variant polypeptide.

[0013] 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 biologic response modifier or a bronchodilator. In several aspects, the subject is a human.

[0014] Yet another further embodiment of the invention provides a pharmaceutical composition comprising a peptide of SEQ ID No: 2-20, the peptide formulated as a milled dry powder having an inhalable particle size. For example, in certain aspects, the milled 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.

[0015] In yet another embodiment, a method of treating a subject is provided, the method comprising administering to the subject an effective amount of the composition of the embodiment by inhalation.

[0016] Other objects, features and advantages of the invention will become apparent from the following detailed description. However, it should be understood that while the preferred embodiments of the invention are indicated, the detailed description and specific examples are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The following figures form a part of this specification and are included to further illustrate certain aspects of the present invention. The present invention can be better understood by referring to one or more of these figures in combination with the detailed description of the specific embodiments presented herein.

[0018] Figure 1 : Scanning electron microscope image of CSP7 bulk powder. The powder sample was sputtered on a sample disk and dispersed by blowing in compressed nitrogen. 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.

[0019] Figure 2 : Optical microscope image of CSP7 bulk powder. The powder sample was sputtered on 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. The arrow points to the agglomerated particles of pure CSP7 powder.

[0020] Figure 3 : X-ray powder diffraction of CSP7 bulk powder particles. The CSP7 bulk powder particles were evaluated using X-ray powder diffraction to determine the crystallinity. The powder was measured between 2 and 40 2θ degrees with a step size of 0.025 2θ degrees and a rate of 2 degrees / min.

[0021] Figure 4 : Polarizing light microscope of CSP7 bulk powder. The crystallinity was evaluated by polarizing light microscope. The figure shows a representative image. The white arrow points to the crystalline region.

[0022] Figure 5 : Differential scanning calorimetry of CSP7 bulk powder. The bulk CSP7 powder was analyzed by differential scanning calorimetry using a TA Instruments Q20 differential scanning calorimeter. The figure shows the curve of modulated DSC with a frequency of 1 °C / 60 s and a rate of 2 °C / min, heating from 25 °C to 300 °C.

[0023] Figure 6 : Thermogravimetric analysis of CSP7 bulk powder. The thermogravimetric analysis was performed using a Mettler thermogravimetric analyzer model TGA / DSC. The figure shows the TGA curve. The heating rate was set at 10 °C / min, heating from 25 °C to 500 °C.

[0024] Figure 7 : Dynamic vapor sorption of CSP7 bulk powder. The bulk CSP7 powder was run on a surface measurement system DVS instrument for a complete adsorption / desorption cycle from 0% to 90% relative humidity in 10% steps at 25 °C. The water desorption at 0% humidity and the mass change at 90% humidity are shown.

[0025] Figure 8 : Particle size distribution of spray-dried CSP7. CSP7 was mixed with leucine, trehalose, sodium citrate, or leucine and trehalose and spray-dried. The particle size was evaluated by a Malvern Mastersizer 2000 (laser diffraction, Fraunhofer approximation; dispersion air pressure: 3.0 Bar). The curves of each spray-dried mixture are shown in the figure.

[0026] Figure 9 : Visual observation of homogeneous CSP7 suspension. A. Ethanol was treated with the maximum rotor-stator power for 1 minute; B. Untreated CSP7-ethanol suspension, with the red arrow indicating large particles / aggregates; C. The CSP7-ethanol suspension was treated with the maximum rotor-stator power for 1 minute, and the suspension turned dark gray; D. The CSP7-ethanol suspension was treated with the minimum rotor-stator power for 1 minute, and the suspension turned light gray.

[0027] Figure 10 : Optical microscope of jet-milled CSP7 powder. Powder samples collected from the designated position of the jet mill were imaged by an optical microscope. Representative images are shown in the figure.

[0028] Figure 11 : Scanning electron microscope of jet-milled CSP7 powder. Under the same conditions as the bulk CSP7 powder, the milled CSP7 powder (batch number 171027) was imaged by a scanning electron microscope (SEM). Representative SEM images of the milled CSP7 powder are shown in the figure.

[0029] Figure 12 : Optical microscope of CSP7 after thin film freezing. Powder samples were sputtered on glass slides and observed using a Leica optical microscope (Leica CTR6500). The scale bar is shown in the lower left corner of each image.

[0030] Figure 13 : Scanning electron microscope of spray-dried CSP7. The particle morphology of spray-dried CSP7 mixtures (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.

[0031] Figure 14 : X-ray powder diffraction of jet-milled CSP7 powder. X-ray powder diffraction patterns of the milled (batch number 171027) and untreated bulk CSP7 powder are shown. The diffraction curves indicate that the crystallinity of the milled CSP7 is reduced.

[0032] Figure 15: Physical state of spray-dried CSP7. The spray-dried CSP7 mixture was examined by X-ray diffraction. Curves indicating the crystallinity or lack of crystallinity of each spray-dried CSP7 mixture are shown.

[0033] Figure 16 : HPLC analysis of spray-dried CSP7. The purity of the spray-dried CSP7 mixture was examined by determining its chemical potency using HPLC.

[0034] Figure 17 : HPLC analysis of the stability of jet-milled CSP7. The stability of untreated bulk CSP7 powder and jet-milled CSP7 (batch no. 171027) was examined by determining its chemical potency using HPLC. Samples of each were stored under 3 different conditions of opening / closing the vial caps (4 °C, 25 °C / 60% RH, 40 °C / 75% RH), and then the chemical potency was determined after storage for 5, 15, and 32 days.

[0035] Figure 18 : Particle deposition of air-atomized 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). The powder deposited in the throat, pre-separator, and 1-MOC stage was extracted and measured separately. The percentage of untreated or jet-milled (collected from the collection vessel) (batch no. 171013) CSP7 powder deposited at specific locations is indicated.

[0036] Figure 19 : 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). The powder deposited in the capsule, device, adapter, throat, pre-separator, and 1-MOC stage was extracted and measured separately. The positions of untreated or jet-milled (batch no. 171027) CSP7 powder (collected from all fractions of the milled powder) in the mill were determined. The percentage of the milled powder present at each position is shown.

[0037] Figure 20 : Aerosol performance of 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). The powder deposited in the capsule, device, adapter, throat, pre-separator, and 1-MOC stage was extracted and measured separately. The percentage of powder deposited at specific locations is indicated.

[0038] Figure 21: Dynamic vapor sorption of jet-milled CSP7 powder. The milled CSP7 powder (batch number 171027) was run on a surface measurement system DVS instrument, and a complete adsorption / desorption cycle was carried out 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.

[0039] Figure 22 : Thermal analysis of jet-milled CSP7 powder. Differential scanning calorimetry (DSC) was performed on milled CSP7 (batch number 171027) using the calorimeter described above. Shown in the figure is the DSC curve with a frequency of 1 °C / 60 s and a rate of 2 °C / min, rising from 25 to 300 °C.

[0040] Figure 23 : Thermal properties of spray-dried 100% CSP7. Spray-dried CSP7 without excipients was analyzed by modulated differential scanning calorimetry. Shown in the figure is the mDSC curve.

[0041] 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. Shown in the figure is the mDSC curve.

[0042] 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. Shown in the figure is the mDSC curve.

[0043] 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. Shown in the figure is the mDSC curve.

[0044] 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. Shown in the figure is the mDSC curve.

[0045] Figure 28 : Thermal properties of all spray-dried CSP7 mixtures. Each manufactured spray-dried CSP7 mixture was analyzed by mDSC. Shown in the figure are all the mDSC curves of these powders.

[0046] Figure 29: Wet weight of mouse dissected lung tissue. Dissect euthanized mice, remove their lungs and weigh them. Mice were treated with saline, bleomycin to induce pulmonary fibrosis, or bleomycin and treated with CSP7 peptide for 12 minutes or 60 minutes.

[0047] Figure 30 : Collagen content of mouse lung tissue. Homogenize untreated, bleomycin-treated, or bleomycin- and CSP7-treated lung tissue and analyze the collagen content using the Quickzyme collagen assay. This figure depicts the total collagen content of the lung.

[0048] Figure 31 : Ashcroft score of mouse lung tissue. Homogenize untreated, bleomycin-treated, or bleomycin- and CSP7-treated lung tissue and analyze the collagen content using the Quickzyme collagen assay. The determination of the Ashcroft score is as described by Hubner et al. 2008, incorporated herein by reference. This figure depicts the total collagen content of the lung.

[0049] Figure 32 : Collagen content of mouse lung tissue. Homogenize untreated, bleomycin-treated, or bleomycin- and CSP7-treated lung tissue and analyze the collagen content using the Quickzyme collagen assay. This figure depicts the total collagen content of the lung.

[0050] Figure 33 : Stability of CSP7 (ammonium counterion) after up to 5 freeze-thaw cycles.

[0051] Figure 34 : Specific surface area of ground pure CSP7 (ammonium counterion) powder.

[0052] Figure 35 : Thermogravimetric analysis of ground pure CSP7 (ammonium counterion) powder.

[0053] Figure 36 : SEM image of ground pure CSP7 (ammonium counterion) powder.

[0054] Figure 37 : Appearance of ground CSP7 (ammonium counterion) powder in the stability study.

[0055] Figure 38 : Crystallinity of ground CSP7 (ammonium counterion) in the stability study. Detailed Description

[0056] Ⅰ. Definitions

[0057] As used herein, with respect to a particular component, "substantially free" is used herein to mean that the particular component is not deliberately formulated into the composition and / or 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 far less than 0.01%. Most preferably, a composition is used in which the amount of the particular component cannot be detected using standard analytical methods.

[0058] As used in this specification, "a" or "an" can mean one or more. As used in the claims herein, when used in conjunction with the word "comprising", the word "a" or "an" can mean one or more than one.

[0059] Unless expressly indicated to refer only to alternatives or the alternatives are mutually exclusive, the use of the term "or" in the claims refers to "and / or", although the present disclosure supports definitions that relate only to alternatives and "and / or". As used herein, "another" can mean at least a second or more.

[0060] Throughout this application, the term "about" is used to indicate that a value includes the inherent error variations of the equipment, method used to determine that value, or variations that exist among the subjects of study. Unless otherwise stated, "about" means + / - 10%.

[0061] As used herein, the term "peptide" generally refers to an amino acid sequence consisting of a single chain of amino acids linked by peptide bonds. Generally, unless otherwise defined, a peptide contains at least two amino acid residues and has a length of less than about 50 amino acids. In some aspects, counterions can be provided to the peptide. Also, in certain cases, a peptide can include N- and / or C-terminal modifications, such as blocking modifications that reduce degradation.

[0062] 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 following sequence: SEQ ID NO:1 (e.g., as measured by in vitro or in vivo assays). In some aspects, the biologically active peptide has the same or increased biological or biochemical activity compared to a native Cav-1 polypeptide.

[0063] The term "identity" or "homology" shall be construed to mean the percentage of amino acid residues in a candidate sequence that are identical to the residues of the corresponding sequence being compared, after aligning the sequences and introducing gaps (if necessary) to obtain the maximum percentage of identity over the entire length of the sequences, and without considering any conservative substitutions as part of sequence identity. Neither N- nor C-terminal extensions or insertions shall be construed to reduce identity or homology. Methods and computer programs for alignment are well known in the art. Sequence identity can be measured using sequence analysis software.

[0064] Broadly, the terms "polypeptide" or "protein" refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits can be joined by amide bonds. In another embodiment, the subunits can be joined by other bonds, such as by ester bonds, ether bonds. As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, which include both glycine and 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 called an oligopeptide. If the peptide chain is long (e.g., longer than 50 amino acids), the peptide is generally called a polypeptide or a protein.

[0065] The terms "subject", "individual", and "patient" are used interchangeably herein and refer to an animal, such as a human or non-human animal (e.g., a mammal), to which the pharmaceutical compositions disclosed herein are administered 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, e.g., mammals, such as non-human primates (especially higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, and non-mammals, such as chickens, amphibians, reptiles, etc. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or an animal substitute used as a disease model. Non-human mammals include mammals such as non-human primates (especially higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, and cows. In some aspects, the non-human animal is a companion animal, such as a dog or a cat.

[0066] "Treating" a subject's disease or disorder or "treating" a patient having a disease or disorder means causing an individual to receive a pharmaceutical treatment, such as administering a drug, such as to reduce or stabilize at least one symptom of the disease or disorder. Typically, when the peptide is therapeutically administered as a therapeutic agent, it is administered to a subject exhibiting one or more symptoms of lung injury or pulmonary fibrosis.

[0067] "Isolated" means that the polypeptide has been separated from any natural environment such as body fluids (e.g., blood) and separated from the components that naturally accompany the polypeptide.

[0068] Isolated and "substantially pure" means that the polypeptide has been separated and purified to at least some degree from the components that naturally accompany it. Typically, a polypeptide is substantially pure when it is at least about 60%, or at least about 70%, at least about 80%, at least about 90%, at least about 95%, or even at least about 99% by weight free of proteins and naturally occurring organic molecules that are naturally associated therewith. For example, substantially pure polypeptides can be obtained by extraction from natural sources, by expression of recombinant nucleic acids in cells that do not normally express the protein, or by chemical synthesis.

[0069] As used herein, the term "variant" refers to a polypeptide that is different from the polypeptide described, which differs by the deletion, addition, substitution, or modification of a side chain of one or more amino acids, 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 non-conventional 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 having 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 residue present in the peptide is replaced by an amino acid having different properties, such as a naturally occurring amino acid from a different group (e.g., replacing a charged or hydrophobic amino acid with alanine); alternatively, in which a naturally occurring amino acid is replaced by a non-conventional amino acid. In some embodiments, the amino acid substitutions are conservative. When used in reference to a polynucleotide or polypeptide, the term variant also encompasses a polynucleotide or polypeptide that can alter the primary, secondary, or tertiary structure as compared to a reference polynucleotide or polypeptide (e.g., as compared to a wild-type polynucleotide or polypeptide).

[0070] The terms "insertion" or "deletion" are generally in the range of about 1 to 5 amino acids. Permissible variants can be experimentally determined by synthesizing peptides and systematically making nucleotide insertions, deletions, or substitutions in the sequence using recombinant DNA techniques.

[0071] When referring to peptides, the term "substitution" means a change of an amino acid for a different entity (e.g., another amino acid or amino acid moiety). Substitutions can be conservative or non-conservative substitutions.

[0072] An "analogue" of a molecule such as a peptide is a molecule that has a function similar to the whole molecule or a fragment thereof. The term "analogue" is also intended to include allelic species and induced variants. Analogues typically differ from the naturally occurring peptide at one or several positions, which is usually due to conservative substitutions. Analogs typically exhibit at least 80% or 90% sequence identity with the native peptide. Some analogues also include non-natural amino acids or modifications of the N- or C-terminal amino acids. Examples of non-natural amino acids are, for example but not limited to: unsubstituted amino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine. The prophylactic or therapeutic efficacy of fragments and analogues can be screened in transgenic animal models as described below.

[0073] "Covalently bonded" means directly or indirectly (e.g., via a linker) connected by a covalent chemical bond. In some aspects of all embodiments of the present invention, the fusion peptides are covalently bonded.

[0074] As used herein, the term "fusion protein" refers to a recombinant protein of two or more proteins. A fusion protein can be produced, for example, by ligating a nucleic acid sequence encoding one protein to a nucleic acid encoding another protein such that they constitute a single open reading frame that can be translated in a cell into a single polypeptide with all the expected proteins. The order of arrangement of the proteins can vary. Fusion proteins can include epitope tags or half-life extenders. Epitope tags include biotin, FLAG tag, c-myc, hemagglutinin, His6, digoxin, FITC, Cy3, Cy5, green fluorescent protein, V5 epitope tag, GST, β-galactosidase, AU1, AU5, and avidin protein. Half-life extenders include the Fc domain and serum albumin.

[0075] The term "airway" as used herein refers to any part of the respiratory tract, including the upper respiratory tract, the respiratory airway, and the lungs. The upper respiratory tract includes the nose and nasal cavity, the mouth, and the throat. The respiratory airway includes the larynx, trachea, bronchi, and bronchioles. The lungs include respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli.

[0076] The terms "inhalation smoke-induced acute lung injury" and "ISALI" are used interchangeably herein and refer to a form of acute lung injury (ALI) caused by inhalation of smoke. ALI is also known as "mild acute respiratory distress syndrome; ARDS." ARDS can be defined by the presence of one or more of the following conditions in a subject: 1) chest X-ray showing bilateral pulmonary infiltrates; 2) pulmonary capillary wedge pressure < 18 mmHg (2.4 kPa) when measured by a right heart catheter as clinically indicated; and 3) PaO 2 / FiO 2 < 300 mmHg (40 kPa). In some embodiments, treatment of ISALI includes treatment of one or more of the following conditions: reduced oxygenation, airway obstruction (including severe airway obstruction), fibrinous airway casts or debris, and alveolar fibrin deposition.

[0077] The term "jet mill" refers to a device or method that reduces particle size by causing particles to collide with each other using a jet of compressed gas to break up the particles. Jet mills 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 jet mills. Jet milling can be carried out under various environmental parameters such as temperature, pressure, relative / absolute humidity, oxygen content, etc.

[0078] The term "ball mill" refers to a device or method that reduces particle size by adding the target particles and a grinding medium to the interior of a cylinder and rotating the cylinder. As the grinding medium rotates, the target particles break down as the grinding medium rises and falls along the outside of the cylinder. Ball mills 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 jet mills.

[0079] The term "wet mill" or "media mill" refers to a device or method that reduces particle size by adding the target particles to a device with a stirrer that contains a medium comprising a liquid and a grinding medium. As the target particles are added, as the stirrer rotates, the energy of its dispersion causes the grinding medium and the target particles to come into contact and break down the target particles. Other mechanical grinding devices that perform the same function can also be used interchangeably with jet mills.

[0080] The term "high-pressure homogenization" refers to a method of reducing particle size by adding the target particles to a device that combines pressure and mechanical forces to break down the target particles. The mechanical forces used in high-pressure homogenization may include impact, shear, and cavitation, etc. Other mechanical grinding devices that perform the same function can also be used interchangeably with jet mills.

[0081] The term "cryogenic grinder" 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 can also be used interchangeably with jet mills.

[0082] The phrase "effective amount" or "therapeutically effective" refers to the dose of a drug or pharmaceutical agent sufficient to produce the desired therapeutic outcome. The desired therapeutic outcome can be a subjective or objective improvement in the dose recipient, a reduction in infection, a reduction in inflammation, an increase in lung growth, an increase in lung repair, a reduction in tissue edema, an increase in DNA repair, a reduction in apoptosis, a reduction in tumor size, a reduction in the rate of cancer cell growth, a reduction in metastasis, or any combination of the above.

[0083] 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 the API into a pharmaceutically acceptable drug formulation for delivery to the site of action in the subject. Excipients or pharmaceutical carriers include all non-active components in a dosage form other than the active ingredient. Non-limiting examples of excipients include carrier agents, fillers, stabilizers, surfactants, surface modifiers, solubility enhancers, buffers, encapsulating agents, antioxidants, preservatives, non-ionic wetting or clarifying agents, thickeners, and absorption enhancers. "Excipient-free" refers to a form of the target drug composition that does not contain any excipients.

[0084] The term "pharmaceutical composition" or "medicinal composition" means that the molecular entity and composition, when appropriately administered to an animal such as a human, do not produce adverse, allergic, or other untoward reactions. According to the present disclosure, the preparation of a pharmaceutical composition 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 should be understood that the formulation should meet the bioburden, sterility, pyrogenicity, general safety, and / or purity standards required by the FDA or other certifying regulatory agencies.

[0085] As used herein, and as known to those of ordinary skill in the art, "pharmaceutical carrier" includes any and all excipients, processing aids, aqueous solvents (e.g., water, alcohol / aqueous solutions, saline solutions, parenteral carriers such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, disintegrants, lubricants, flavoring agents (e.g., sweeteners, flavorants), and the like, and combinations thereof. The pH value and exact concentration of each component in the pharmaceutical composition are adjusted according to known parameters. In some aspects, the carrier may encapsulate the therapeutic agent but is not itself consumed or administered to the subject (e.g., a shell capsule enclosing a dry powder composition, such as for use in a dry powder inhaler).

[0086] II. Caveolin-1 Peptide

[0087] Embodiments of the present disclosure provide dry powder formulations of caveolin-1 (Cav-1) peptides. The caveolin-1 (Cav-1) scaffolding domain or peptide interferes with the interaction between Cav-1 and Src kinase, mimicking the combined effect of uPA and anti-β1 integrin antibody. The length of native human Cav-1 is 178 amino acids and its molecular weight is 22 kDa. The amino acid sequence of Cav-1 is shown below (SEQ ID NO: 1).

[0088]

[0089] In some aspects, the peptide is a scaffolding domain peptide that comprises 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. Relative to the sequence of SEQ ID NO: 1, the peptide may comprise 1, 2, 3, 4, or more amino acid substitutions, deletions, or insertions, such as polypeptides having 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.

[0090] Table 1: Exemplary Cav-1 Peptides.

[0091]

[0092]

[0093] (a = D-alanine, O = ornithine)

[0094] The peptides provided in the present disclosure are biologically active derivatives that have the activity of the native Cav-1 polypeptide in in vitro or in vivo binding or biological activity assays. In particular aspects, the peptide inhibits or prevents apoptosis of bleomycin-induced lung epithelial cells (LECs) in vitro or in vivo, and its activity is at least about 20%, or at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, about 95%, 97%, 99% of the activity of the native Cav-1 polypeptide, and any range derivable therefrom, 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 peptide may have 100% of the activity of the native CAV-1 polypeptide or even higher activity. Assays for testing biological activities such as antifibrotic activity, the ability to affect the expression of uPA, uPAR, and PAI-1 mRNA, or the ability to inhibit lung fibroblast proliferation are well known in the art.

[0095] The peptides of the present disclosure are peptides of the native Cav-1 polypeptide or its modified forms. The peptides can be synthetic, recombinant, or chemically modified peptides isolated or produced using methods well known in the art. Modifications can be made to the N-terminus, C-terminus, or internal amino acids. N-terminal modifications can be, for example, but not limited to, acylation, acetylation, or C-terminal amidation. The peptides can include conservative or non-conservative amino acid changes, as described below. Changes in polynucleotides can result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptides encoded by the reference sequences. The peptides can 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, for example, but not limited to, the insertion of L-amino acids, or non-standard amino acids (such as ornithine), which do not normally occur in human proteins. When describing peptides, the term conservative substitution refers to an alteration in the amino acid composition of the peptide that substantially does not change the activity of the peptide. For example, a conservative substitution refers to replacing a different amino acid residue with an amino acid residue having similar chemical properties. Conservative amino acid substitutions include replacing leucine with isoleucine or valine, aspartic acid with glutamic acid, or threonine with serine.

[0096] Conservative amino acid substitutions are those that result from replacing one amino acid with another having a similar structure and / or chemical property, such as replacing leucine with isoleucine or valine, aspartic acid with glutamic acid, or threonine with serine. Thus, a conservative substitution of a particular amino acid sequence refers to the substitution of those amino acids that are not critical for polypeptide activity, or the substitution of an amino acid with another amino acid having similar properties (e.g., acidic, basic, positively or negatively charged, polar or nonpolar), such that even the substitution of a critical amino acid does not reduce the activity of the peptide. Tables of conservative substitutions providing functionally similar amino acids are well known in the art. For example, each of the following six groups contains 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, W.H. Freeman and Company (1984), which is incorporated herein by reference in its entirety.) In some embodiments, individual substitutions, deletions or additions of single amino acids or small percentages of amino acids can also be considered conservative substitutions if the change does not reduce the activity of the peptide. Insertions or deletions are typically in the range of about 1 to 5 amino acids. The choice of conservative amino acids can be based on the position of the amino acid being replaced in the peptide, e.g., if the amino acid is on the outside of the peptide and exposed to solvent, or on the inside and not exposed to solvent.

[0097] In alternative embodiments, the amino acid that will replace an existing amino acid can be selected based on the position of the existing amino acid, i.e., its exposure to the solvent (i.e., if the amino acid is exposed to the solvent or present on the outer surface of the peptide or polypeptide compared to an internal amino acid that is not exposed to the solvent). The selection of such conservative amino acid substitutions is well known in the art, for example, 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 suitable for the exterior of a protein or peptide (i.e., amino acids exposed to the solvent) can be selected, for example but not limited to, the following substitutions can be used: replace Y with F, replace T with S or K, replace P with A, replace E with D or Q, replace N with D or G, replace R with K, replace G with N or A, replace T with S or K, replace D with N or E, replace I with L or V, replace F with Y, replace S with T or A, replace R with K, replace G with N or A, replace K with R, replace A with S, K or P.

[0098] In alternative embodiments, conservative amino acid substitutions suitable for the interior of a protein or peptide can also be selected. For example, appropriate conservative substitutions can be used for amino acids inside the protein or peptide (i.e., amino acids not exposed to the solvent), for example but not limited to, the following conservative substitutions can be used: replace Y with F, replace T with A or S, replace I with L or V, replace W with Y, replace M with L, replace N with D, replace G with A, replace T with A or S, replace D with N, replace I with L or V, replace F with Y or L, replace S with A or T, replace A with S, G, T or V. In some embodiments, the term "variant" also encompasses non - conservative amino acid substitutions.

[0099] 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, such as but not limited to, by acetylation, ubiquitination, labeling, pegylation (derivatization with polyethylene glycol), lipidation, glycosylation, amidation, or addition of other molecules. A molecule is also a "derivative" of another molecule when it contains additional 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.

[0100] When used in conjunction with "derivative" or "variant", the term "functional" refers to a polypeptide of the present invention having a biological activity (functional or structural) that is substantially similar to the biological activity of that entity or molecule, which is a functional derivative or a functional variant thereof. The term functional derivative is intended to include fragments, analogs, or chemical derivatives of the molecule.

[0101] In some aspects, amino acid substitutions can be made at one or more positions of a polypeptide, where the substitution is for an amino acid with similar hydrophilicity. The importance of the hydrophilic amino acid index in conferring biological functions of protein interactions is well known in the art (Kyte and Doolittle, 1982). It is recognized that the relative hydrophilic properties of amino acids contribute to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules (such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Thus, such conservative substitutions can be made in the polypeptide and may have only a minor effect on its activity. As detailed in U.S. Patent 4,554,101, the following hydrophilic values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). These values can be used as a guide, and thus substitutions of amino acids with hydrophilic values within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. Thus, any polypeptide described herein can be modified by substituting an amino acid with a different but homologous amino acid having a similar hydrophilic value. Amino acids with hydrophilicity within + / -1.0 or + / -0.5 points are considered homologous.

[0102] The Cav-1 peptide can contain co-translational and post-translational (C-terminal peptide cleavage) modifications, such as disulfide bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., cleavage 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 blood glucose control.

[0103] In some aspects, the Cav-1 peptides contain non-naturally occurring amino acids. The peptides can contain a combination of naturally occurring and non-naturally occurring amino acids, or can contain only non-naturally occurring amino acids. The non-naturally occurring amino acids in the peptides (or other components of the composition besides the protease recognition sequence) can include synthetic non-natural amino acids, substituted amino acids, or one or more D-amino acids, which are desired in certain cases. Peptides containing D-amino acids exhibit increased stability in vitro or in vivo compared to their L-amino acid-containing forms. Thus, the construction of peptides incorporating D-amino acids can be particularly useful when greater in vivo or intracellular stability is desired or required. More specifically, D-peptides are resistant to endogenous peptidases and proteases, thus providing better oral trans-epithelial and transdermal delivery of linked drugs and conjugates, improving the bioavailability of membrane-permanent complexes (see further discussion below), and extending the lifespan in the intravascular and interstitial spaces (when such properties are needed). The use of D-isomer peptides can also enhance the transdermal and oral trans-epithelial delivery of linked drugs and other cargo molecules. Additionally, D-peptides are not efficiently processed for major histocompatibility complex class II-restricted presentation to T helper cells and thus are less likely to induce a humoral immune response throughout the organism. Thus, for example, D-isomer forms of cell-penetrating peptide sequences, L-isomer forms of cleavage sites, and D-isomer forms of therapeutic peptides can be used to construct peptide conjugates.

[0104] In addition to the 20 “standard” L-amino acids, D-amino acids or non-standard, modified or unusual amino acids that are well-defined in the art may also be considered for 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 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-C1)); 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(pNH 2 )); N-methylvaline (MeVal); homocysteine (hCys), homophenylalanine (hPhe) and homoserine (hSer); hydroxyproline (Hyp), homoproline (hPro), N-methylated amino acids and peptoids (N-substituted glycines).

[0105] Carboxyl-terminal modifications include acylation with carboxylic acids: formic acid, acetic acid, propionic acid, fatty acids (myristic acid, palmitic acid, stearic acid), succinic acid, benzoic acid, benzyloxycarbonyl (Cbz); acetylation and biotinylation. Amino-terminal modifications include: (i) acylation with carboxylic acids: formic acid, acetic acid, propionic acid, fatty acids (myristic acid, palmitic acid, stearic acid, etc.), succinic acid, benzoic acid, benzyloxycarbonyl (Cbz); (ii) biotinylation; (iii) amidation; (iv) attachment of dyes such as fluorescein (FITC, FAM, etc.), 7-hydroxy-4-methylcoumarin-3-acetic acid, 7-hydroxycoumarin-3-acetic acid, 7-methoxycoumarin-3-acetic acid and other coumarins; rhodamine (5-carboxyrhodamine 110 or 6G, 5(6)-TAMRA, ROX); 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.

[0106] A polypeptide can be capped at its N- and C-termini with an acyl group (abbreviated as "Ac") and an amide group (abbreviated as "Am"), respectively, such as an acetyl group (CH 3 CO-) at the N-terminus and an amide group (-NH 2 ) at the C-terminus. A wide range of N-terminal capping functions are envisioned, preferably in connection with the terminal amino group, such as: formyl;

[0107] alkanoyl groups having 1 to 10 carbon atoms, such as acetyl, propionyl, butyryl;

[0108] enoyl groups having 1 to 10 carbon atoms, such as hex-3-enoyl;

[0109] alkynoyl groups having 1 to 10 carbon atoms, such as hex-5-ynoyl;

[0110] aroyl groups, such as benzoyl or 1-naphthoyl;

[0111] heteroaroyl groups, such as 3-pyrrolyl or 4-quinolyl;

[0112] alkylsulfonyl groups, such as methanesulfonyl;

[0113] arylsulfonyl groups, such as benzenesulfonyl or sulfamoyl;

[0114] heteroarylsulfonyl groups, such as pyridine-4-sulfonyl;

[0115] substituted alkanoyl groups having 1 to 10 carbon atoms, such as 4-aminobutyryl;

[0116] substituted enoyl groups having 1 to 10 carbon atoms, such as 6-hydroxy-hex-3-enoyl;

[0117] substituted alkynoyl groups having 1 to 10 carbon atoms, such as 3-hydroxy-hex-5-ynoyl;

[0118] substituted aroyl groups, such as 4-chlorobenzoyl or 8-hydroxy-naphthalene-2-carbonyl;

[0119] substituted heteroaroyl groups, such as 2,4-dioxo-1,2,3,4-tetrahydro-3-methyl-quinazoline-6-carbonyl;

[0120] substituted alkylsulfonyl groups, such as 2-aminoethanesulfonyl;

[0121] substituted arylsulfonyl groups, such as 5-dimethylamino-1-naphthalenesulfonyl;

[0122] substituted heteroarylsulfonyl groups, such as 1-methoxy-6-isoquinolinesulfonyl;

[0123] A carbamoyl or thiocarbamoyl group;

[0124] A substituted carbamoyl group (R'-NH-CO) or a substituted thiocarbamoyl group (R'-NH-CS), where R' is an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, a substituted alkyl group, a substituted alkenyl group, a substituted alkynyl group, a substituted aryl group or a substituted heteroaryl group;

[0125] A substituted carbamoyl group (R'-NH-CO) and a substituted thiocarbamoyl group (R'-NH-CS), where R' is an alkanoyl group, an alkenoyl group, an alkynoyl group, an aroyl group, a heteroaroyl group, a substituted alkanoyl group, a substituted alkenoyl group, a substituted alkynoyl group, a substituted aroyl group or a substituted heteroaroyl group, as defined above for all.

[0126] The C-terminal capping function can be in an amide bond or an ester bond linked to the terminal carboxyl group. The capping functional group providing the amide bond is called NR 1 R 2 where R 1 and R 2 can independently be selected from the following groups: hydrogen;

[0127] An alkyl group preferably having 1 to 10 carbon atoms, such as methyl, ethyl, isopropyl;

[0128] An alkenyl group preferably having 1 to 10 carbon atoms, such as prop-2-enyl;

[0129] An alkynyl group preferably having 1 to 10 carbon atoms, such as prop-2-ynyl;

[0130] A substituted alkyl group having 1 to 10 carbon atoms, such as a hydroxyalkyl group, an alkoxyalkyl group, a mercaptoalkyl group, an alkylthioalkyl group, a haloalkyl group, a cyanoalkyl group, an aminoalkyl group, an alkylaminoalkyl group, a dialkylaminoalkyl group, an alkanoylalkyl group, a carboxyalkyl group, a carbamoylalkyl group;

[0131] A substituted alkenyl group having 1 to 10 carbon atoms, such as a hydroxyalkenyl group, an alkoxyalkenyl group, a mercaptoalkenyl group, an alkylthioalkenyl group, a haloalkenyl group, a cyanoalkenyl group, an aminoalkenyl group, an alkylaminoalkenyl group, a dialkylaminoalkenyl group, an alkanoylalkenyl group, a carboxyalkenyl group, a carbamoylalkenyl group;

[0132] A substituted alkynyl group having 1 to 10 carbon atoms, such as a hydroxyalkynyl group, an alkoxyalkynyl group, a mercaptoalkynyl group, an alkylthioalkynyl group, a haloalkynyl group, a cyanoalkynyl group, an aminoalkynyl group, an alkylaminoalkynyl group, a dialkylaminoalkynyl group, an alkanoylalkynyl group, a carboxyalkynyl group, a carbamoylalkynyl group;

[0133] An aroylalkyl group having at most 10 carbon atoms, such as benzoyl or 2-benzoylethyl;

[0134] An aryl group, such as phenyl or 1-naphthyl;

[0135] A heteroaryl group, such as 4-quinolyl;

[0136] An alkanoyl group having 1 to 10 carbon atoms, such as acetyl or butyryl;

[0137] An aroyl group, such as benzoyl;

[0138] A heteroaroyl group, such as 3-quinolyl;

[0139] OR' or NR'R”, where R' and R” are independently hydrogen, alkyl, aryl, heteroaryl, acyl, aroyl, sulfonyl, sulfinyl or SO 2 -R”' or SO-R”', where R”' is a substituted or unsubstituted alkyl, aryl, heteroaryl, alkenyl or alkynyl.

[0140] A capping functional group providing an ester bond is designated as OR, where R can be: an alkoxy group; an aryloxy group; a heteroaryloxy group; an aralkyloxy group; a heteroaralkyloxy group; a substituted alkoxy group; a substituted aryloxy group; a substituted heteroaryloxy group; a substituted aralkyloxy group; or a substituted heteroaralkyloxy group.

[0141] The capping functional group at the N-terminus or C-terminus or both can have a structure such that the capped molecule serves as a prodrug (a pharmacologically active derivative of the parent drug molecule), which undergoes spontaneous or enzymatic conversion in vivo to release the active drug and has better delivery properties than the parent drug molecule (Bundgaard H, Ed: Design of Prodrugs, Elsevier, Amsterdam, 1985).

[0142] A judicious choice of the capping group allows the addition of other activities to the peptide. For example, the presence of a thiol group linked to the N- or C-terminal capping will allow the conjugation of the derivatized peptide with other molecules.

[0143] In another aspect, the peptide or fragment or derivative thereof can be a "retro-inverso peptide". A "retro-inverso peptide" refers to a peptide having 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 analogue has reversed termini and peptide bonds with reversed direction while generally maintaining the topology of the side chains in the native peptide sequence. A retro-inverso peptide can contain L-amino acids or D-amino acids, or a mixture of L-amino acids and D-amino acids, up to all amino acids being D-isomers. A partially retro-inverso peptide analogue is a polypeptide in which only a portion of the sequence is reversed and replaced by enantiomeric amino acid residues. Since the amino and carboxyl termini of the retro-inverso portion of such an analogue have been reversed, the amino acid residues flanking the reversed portion are replaced by a-substituted gem-diaminomethane and malonate esters with side chains similar to those of the amino acid residues, respectively. It has been found that cell-penetrating peptides in retro-inverso form are as effective as the native form in transmembrane transport. The synthesis of retro-inverso peptide analogues is described in Bonelli, F. et al., Int J Pept Protein Res. 24(6):553-6(1984); Verdini, A and Viscomi, G.C, J.Chem.Soc.Perkin Trans. 1:697-701(1985) and U.S. Patent No. 6,261,569, which are incorporated herein by reference in their entirety. A solid-phase synthesis method for partially retro-inverso peptide analogues (EP 97994-B) has also been described, which is also incorporated herein by reference in its entirety.

[0144] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (e.g., 80%, 85%, 90% or 95%) of "sequence identity" or "homology" with another sequence means the percentage of bases (or amino acids) that are the same in the two sequences being compared when aligned. Alignment and the percentage of homology or sequence identity can be determined using software programs known in the art, such as those described in Current Protocols In Molecular Biology (F.M. Ausubel et al., editors, 1987) Supplement 30, Section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort by = high score; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+SwissProtein+SPupdate+PIR.

[0145] B. Multimeric polypeptides

[0146] Embodiments of the present disclosure also include longer polypeptides constructed from repeating units of Cav-1 peptides. Polypeptide multimers can comprise different combinations of polypeptides. Such multimeric polypeptides can be prepared by chemical synthesis or by recombinant DNA techniques 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 exceed 100 residues (or its equivalent when linkers or spacers are included).

[0147] C. Peptidomimetics

[0148] The Cav-1 peptide can be a peptidomimetic compound that mimics the biological effects of the native Cav-1 polypeptide. Peptidomimetics can be non-natural peptides or non-peptide agents that can reproduce the steric characteristics of the binding elements of the native Cav-1 polypeptide, thereby conferring binding and biological activities of the native Cav-1 polypeptide. Similar to the native Cav-1 polypeptide or polypeptide multimer, the peptidomimetic will have a binding surface (which interacts with any ligand to which native Cav-1 binds) and a non-binding surface.

[0149] In some aspects, the present disclosure also includes compounds that retain some peptide characteristics. For example, any proteolytically labile bond within the peptides of the invention can be selectively replaced by non-peptide elements such as isosteres (N-methylation; D-amino acids) or reduced peptide bonds, while the remainder of the molecule retains its peptidic nature.

[0150] Peptidomimetic compounds have been described for a variety of bioactive peptides / polypeptides such as opioid peptides, VIP, thrombin, HIV protease, etc., whether agonists, substrates or inhibitors. Methods for designing and preparing peptidomimetic compounds are known in the art (Hruby, VJ, Biopolymers 33:1073-1082 (1993); Wiley, RA et al., Med. Res. Rev. 13:327-384 (1993); Moore et al., Adv. in Pharmacol 33:91-141 (1995); Giannis et al., Adv. in Drug Res. 29:1-78 (1997). Certain mimetics that mimic secondary structures are described in Johnson et al., In: Biotechnology and Pharmacy, Pezzuto et al., Chapman and Hall (Eds.), NY, 1993. These methods are used to prepare peptidomimetics that have at least the binding ability and specificity of the native Cav-1 polypeptide and preferably also have biological activity. Given 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.

[0151] For example, such peptidomimetics can be identified by examining the three-dimensional structure of the polypeptide of the invention that is bound either free or complexed to a ligand (e.g., soluble uPAR or a fragment thereof). Alternatively, the structure of the polypeptide of the invention bound to its ligand can be obtained by nuclear magnetic resonance spectroscopy techniques. More knowledge of the stereochemistry of the interaction of the peptide with its ligand or receptor will allow for the rational design of such peptidomimetic agents. In the absence of a ligand, the structure of the peptide or polypeptide of the invention can also provide a scaffold for the design of mimetic molecules.

[0152] D. PEGylation

[0153] The Cav-1 peptide can be conjugated to a heterologous polypeptide fragment or polymer such as polyethylene glycol. The peptide can be linked to PEG, increasing the hydrodynamic radius of the enzyme and thus increasing serum persistence. The polypeptide can be conjugated to any targeting agent such as a ligand that is capable of specifically and stably binding to an external receptor (U.S. Patent Publication 2009 / 0304666).

[0154] In some aspects, the methods and compositions of the embodiments relate to the pegylation of the disclosed polypeptides. Pegylation is the process of covalently attaching poly(ethylene glycol) polymer chains to another molecule, typically a drug or therapeutic protein. Pegylation is generally achieved by incubating a reactive derivative of PEG with the target macromolecule. Covalent attachment of PEG to the drug or therapeutic protein can "mask" the agent against the host immune system (reduced immunogenicity and antigenicity), increase the hydrodynamic size of the agent (size in solution), which can prolong its circulation time by reducing renal clearance. Pegylation can also confer water solubility to hydrophobic drugs and proteins.

[0155] The first step in pegylation is the appropriate functionalization of the PEG polymer at one or both ends. PEG with the same reactive moiety at each end is called "homo-bifunctional", while if the functional groups present are different, the PEG derivative is called "hetero-bifunctional" or "hetero-functional". Chemically active or activated derivatives of the PEG polymer are prepared to attach the PEG to the desired molecule.

[0156] The choice of suitable functional groups for the PEG derivative is based on the type of available reactive groups on the molecule to which the PEG will be conjugated. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. The N-terminal amino group and the C-terminal carboxylic acid can also be used.

[0157] Techniques for forming first-generation PEG derivatives generally involve reacting the PEG polymer with groups that can react with hydroxyl groups, typically acid anhydrides, acyl chlorides, chloroformates, and carbonates. In second-generation pegylation chemistry, more efficient functional groups, such as aldehydes, esters, amides, etc., can be used for conjugation.

[0158] Since the applications of pegylation have become increasingly advanced and complex, the demand for hetero-bifunctional PEGs for conjugation has increased. These hetero-bifunctional PEGs are well-suited for linking two entities where a hydrophilic, flexible, and biocompatible spacer is required. Preferred end groups for hetero-bifunctional PEGs are maleimide, vinyl sulfone, pyridyl disulfide, amine, carboxylic acid, and NHS ester.

[0159] The most common modifiers or linkers are based on methoxy PEG (mPEG) molecules. Their activity depends on adding a protein-modifying group to the alcohol end. In some cases, polyethylene glycol (PEG diol) is used as a precursor molecule. The diol is then modified at both ends to produce hetero-dimeric or homo-dimeric PEG linker molecules.

[0160] Proteins are generally PEGylated at nucleophilic sites such as unprotonated thiols (cysteine residues) or amino groups. Examples of cysteine - specific modifying reagents include PEG maleimide, PEG iodoacetate, PEG thiol, and PEG vinyl sulfone. All four are highly cysteine - specific under mild conditions and neutral to slightly alkaline pH, but each has some drawbacks. The thioether formed by maleimide can be slightly unstable under basic conditions, so there may be some limitations in formulation choices using this linker. The thiocarbamate bond formed by iodo - PEG is more stable, but the 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 basic conditions. The reactivity of PEG - vinyl sulfone is relatively slow compared to maleimide and iodo - PEG; however, the thioether bond formed is extremely stable. Its slower reaction rate can also make the PEG - vinyl sulfone reaction easier to control.

[0161] Site - specific PEGylation at native cysteine residues is rarely performed because these residues are usually in the form of disulfide bonds or are required for biological activity. On the other hand, site - directed mutagenesis can be used to incorporate cysteine PEGylation sites for thiol - specific linkers. Cysteine mutations must be designed so that they are accessible to the PEGylation reagent and still have biological activity after PEGylation.

[0162] Amine - specific modifiers include PEG NHS ester, PEG triflate, PEG aldehyde, PEG isothiocyanate, etc. They all react under mild conditions and are highly specific for amino groups. PEG NHS ester may be a more reactive reagent; however, its high reactivity can make the PEGylation reaction difficult to control on a large scale. PEG aldehyde forms an imine with the amino group, which is then reduced to a secondary amine with sodium cyanoborohydride. Unlike sodium borohydride, sodium cyanoborohydride will not reduce disulfide bonds. However, this chemical is highly toxic and must be disposed of carefully, especially at a pH where its volatility is low.

[0163] Site-specific PEGylation is challenging due to the presence of multiple lysine residues on most proteins. Fortunately, since these reagents react with unprotonated amino groups, it is possible to direct PEGylation towards the lower pK amino groups by performing the reaction at a lower pH. Generally, the pK of the α-amino group is 1-2 pH units lower than that of the ε-amino group of lysine residues. High selectivity for the N-terminus can usually be achieved by PEGylating the molecule at pH 7 or lower. However, this is only feasible when the N-terminal portion of the protein is not required for biological activity. Additionally, the pharmacokinetic beneficial effects of PEGylation are usually stronger than the significant loss of in vitro biological activity, resulting in a product with greater in vivo biological activity, regardless of the PEGylation chemistry.

[0164] Multiple parameters need to be considered when developing a PEGylation procedure. Fortunately, the key parameters usually do not exceed four or five. The "design of experiments" approach for optimizing PEGylation conditions is very useful. For thiol-specific PEGylation reactions, the parameters to consider include: protein concentration, the ratio of PEG to protein (in moles), temperature, pH, reaction time, and in some cases, excluding oxygen. (Oxygen can contribute to the formation of intermolecular disulfides through the protein, which will reduce the yield of the PEGylated product.) The same factors (excluding oxygen) should be considered for amine-specific modifications, and pH may be even more critical, especially when targeting the N-terminal amino group.

[0165] For amine- and thiol-specific modifications, the reaction conditions can affect the stability of the protein. This can limit the temperature, protein concentration, and pH. Additionally, the reactivity of the PEG linker should be known before starting the PEGylation reaction. For example, if the PEGylation reagent is only 70% active, then the amount of PEG used should ensure that only the active PEG molecules are counted in the protein-PEG reaction stoichiometry.

[0166] E. Fusion Proteins

[0167] Certain embodiments of the present invention relate to fusion proteins of Cav-1 peptides. These molecules can have polypeptides of the embodiments linked to heterologous domains at the N- or C-terminus. For example, a leader sequence from another species can also be used for fusion to allow recombinant expression of the protein in a heterologous host. The fusion protein can include a half-life extender. Another useful fusion includes the addition of a protein affinity tag (such as a serum albumin affinity tag or six histidine residues) or an immunoreactive domain (such as an antibody epitope, preferably cleavable) to facilitate the purification of the fusion protein. Non-limiting affinity tags include polyhistidine, chitin-binding protein (CBP), maltose-binding protein (MBP), and glutathione-S-transferase (GST).

[0168] 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.

[0169] Methods for generating fusion proteins are well known to those skilled in the art. Such proteins can be generated, for example, by de novo synthesis of a complete fusion protein, or by ligation of DNA sequences encoding heterologous domains and expression of the complete fusion protein.

[0170] By linking a gene to a bridging DNA fragment encoding a peptide linker that splices between tandemly linked polypeptides, the generation of fusion proteins that restore the functional activity of the parental protein can be facilitated. The linker should have sufficient length to allow proper folding of the resulting fusion protein.

[0171] 2. Linker

[0172] In certain embodiments, the polypeptides of the embodiments can be chemically conjugated with bifunctional crosslinking reagents or fused with peptide linkers at the protein level.

[0173] Bifunctional crosslinking 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.

[0174] Homobifunctional reagents carrying two identical functional groups have been shown to efficiently cause crosslinking between the same or different macromolecules or macromolecular subunits and to link polypeptide ligands to their specific binding sites. Heterobifunctional reagents contain two different functional groups. By taking advantage of the different reactivity of the two different functional groups, crosslinking can be selectively and sequentially controlled. According to the specificity of their functional groups, bifunctional crosslinking reagents can be classified, for example, as amino-, thiol-, guanidyl-, indolyl-, carboxyl-specific groups. Among them, reagents directed at free amino groups are particularly popular because of their commercial availability, ease of synthesis, and mild reaction conditions applicable.

[0175] Most heterobifunctional crosslinking reagents contain a primary amine-reactive group and a thiol-reactive group. In another example, a heterobifunctional crosslinking reagent and a method of using the crosslinking reagent are described (U.S. Patent No. 5,889,155, which is hereby incorporated by reference in its entirety). The crosslinking reagent combines a nucleophilic hydrazide residue with an electrophilic maleimide residue, allowing, for example, the coupling of aldehydes with free thiols. The crosslinking reagent can be modified to crosslink various functional groups.

[0176] In addition, any other linker / conjugating agent and / or mechanism 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 ester bonds, phosphoramide bonds, anhydride bonds, disulfide bonds, ionic and hydrophobic interactions, bispecific antibodies and antibody fragments, or combinations thereof.

[0177] Cross-linking agents with reasonable blood stability are preferably used. Many types of linkers containing disulfide bonds are known to be successfully used for conjugating targeting and therapeutic / prophylactic agents. Linkers containing sterically hindered disulfide bonds can be more stable in vivo. Thus, these linkers are a group of conjugating agents.

[0178] In addition to the hindered cross-linking agents, unhindered linkers can also be used as provided herein. Other useful cross-linking agents (not considered to contain or generate protected disulfide bonds) include SATA, SPDP, and 2-iminothiolane (Wawrzynczak and Thorpe, 1987). The use of such cross-linking agents is well understood in the art. Another embodiment relates to the use of flexible linkers.

[0179] Once chemically conjugated, the peptides are typically purified to separate the conjugate from the unconjugated agent and other contaminants. A number of purification techniques are available to provide a conjugate of sufficient purity for clinical use.

[0180] Purification methods based on size separation (such as gel filtration, gel permeation, or high performance liquid chromatography) are generally the most commonly used. Other chromatographic techniques (such as Blue-Sepharose separation) can also be used. Traditional methods for purifying fusion proteins from inclusion bodies can be useful, such as using a mild detergent, such as sodium lauroyl sarcosinate (SLS).

[0181] 3. Cell-Penetrating Peptides and Membrane-Translocating Peptides

[0182] 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 translocation domain" are used interchangeably and refer to a fragment of a polypeptide sequence that allows the polypeptide to cross the cell membrane (e.g., the plasma membrane in the case of eukaryotic cells). Examples of CPP fragments include, but are not limited to, fragments derived from: HIV Tat (e.g., GRKKRRQRRRPPQ (SEQ ID NO:23)), herpes simplex virus VP22, Drosophila antennapedia gene product, protegrin I, penetratin (RQIKIWFQNRRMKWKK (SEQ ID NO:24)), or melittin (GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO:25)). In certain aspects, the CPP comprises T1 (TKIESLKEHG (SEQ ID NO:26)), T2 (TQIENLKEKG (SEQ ID NO:27)), 26 (AALEALAEALEALAEALEALAEAAAA (SEQ ID NO:28)), or INF7 (GLFEAIEGFINGWEGMIEGWYGCG (SEQ ID NO:29)) CPP sequences.

[0183] III. Methods of Use

[0184] One aspect of the invention relates to the use of the peptides described herein and their mutants, variants, analogs, or derivatives. Specifically, these methods involve administering to a subject any one of the peptides described herein or a pharmaceutical modification thereof (such as a dry powder) for the treatment or prevention of a disease, injury, or pulmonary infection (e.g., fibrotic conditions of the lung) in a composition comprising the polypeptide of an embodiment in a pharmaceutical carrier.

[0185] A. Pharmaceutical Compositions

[0186] The Cav-1 peptides provided herein are expected to be administered systemically or locally to inhibit apoptosis and for the treatment and prevention of damage to lung tissue. They can be administered intravenously, subcutaneously, intramuscularly, intrathecally, and / or intraperitoneally. For example, the dry powder formulation can be administered by infusion into the subject (e.g., subcutaneous infusion), or it can be reconstituted in a liquid prior to injection. In certain aspects, the peptide is delivered locally to the airways, such as by administering the dry powder formulation using a dry powder inhaler. They can be administered alone or in combination with an anti-fibrotic compound.

[0187] The Cav-1 peptide dry powder can be administered in combination with, simultaneously or sequentially with at least one additional therapeutic agent (e.g., a therapeutic agent for treating pulmonary fibrosis). The other therapeutic agents can be NSAIDs, steroids, DMARDs, immunosuppressants, biologic response modifiers, bronchodilators or anti-fibrotic agents such as pirfenidone (the mechanism of its anti-fibrotic action is not fully understood but may involve the blockade of TGF-β), nintedanib (a broad tyrosine kinase blocker) or any other anti-fibrotic agent. Suitable NSAIDs are selected from non-selective COX-inhibitors: acetylsalicylic acid, mesalazine, ibuprofen, naproxen, flurbiprofen, fenoprofen, fenbufen, ketoprofen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, celecoxib acid, fluprofen, indomethacin, sulindac, tolmetin, zomepirac, nabumetone, diclofenac, fenclofenac, alclofenac, bromfenac, ibuprofen acid, aceclofenac, acemetacin, fentiazac, clidanac, etodolac, oxpinac, mefenamic acid, meclofenamic acid, flufenamic acid, niflumic acid, tolfenamic acid, diflunisal, flufenisal, piroxicam, tenoxicam, lornoxicam and nimesulide and their pharmaceutically acceptable salts; selective COX 2-inhibitors: meloxicam, celecoxib and rofecoxib and their pharmaceutically acceptable salts. Suitable steroids are prednisone, prednisolone, methylprednisolone, dexamethasone, budenoside, flucortolone and triamcinolone. Suitable DMARDs are sulfasalazine, olsalazine, chloroquine, gold derivatives (auranofin), D-penicillamine and cytostatic agents such as methotrexate and cyclophosphamide. Suitable immunosuppressants are cyclosporin A and its derivatives, mycophenolate mofetil, FK 506, OKT-3, ATG, 15-desoxyspergualin, mizoribine, misoprostol, rapamycin, reflunomide and azathioprine. Suitable biologic response modifiers are interferon β, anti-TNF-α (etanercept), IL-10, anti-CD3 or anti-CD25. Suitable bronchodilators are ipratropium bromide, bromioxifium bromide, tiotropium bromide, adrenaline hydrochloride, salbutamol, terbutaline sulfate, fenoterol hydrobromide, salmeterol and formoterole.In such combinations, each active ingredient can be administered (e.g., orally or by inhalation) at its usual dosage range or at a dosage below its usual dosage range. The dosages of the combination of NSAIDs, steroids, DMARDs, immunosuppressants, and biologic response modifiers are suitably from 1 / 50 of the lowest usually recommended dosage to 1 / 1 of the highest usually recommended dosage, preferably from 1 / 20 to 1 / 2, and more preferably from 1 / 10 to 1 / 5. For combination drugs, the usually recommended dosages should be understood as, for example, the dosages disclosed in Rote. 2002, Editio Cantor Verlag Aulendorf, Germany or the Physician's Desk Reference.

[0188] In view of clinical applications, it may be necessary to prepare pharmaceutical compositions containing proteins, antibodies, and drugs in a form suitable for the intended application. Generally, the pharmaceutical compositions may contain an effective amount of one or more embodiments of polypeptides or additional agents dissolved or dispersed in a pharmaceutical carrier. The term "pharmaceutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when administered, as appropriate, to an animal (e.g., a human). The preparation of pharmaceutical compositions containing at least one embodiment of a polypeptide isolated by the methods disclosed herein, or additional active ingredients, will be known to those of skill in the art in view of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Edition, 1990, which is incorporated herein by reference. In addition, for administration to an animal (e.g., a human), it should be understood that the formulation should meet the bioburden, sterility, pyrogenicity, general safety, and / or purity standards required by the FDA Office of Biologics Standards or other appropriate regulatory agencies.

[0189] Certain embodiments of the present invention may include different types of carriers, depending on whether it is administered in solid, liquid or aerosol form and whether sterility is required for administration routes such as injection. The compositions can be administered intravenously, intrathecally, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, rectally, intramuscularly, subcutaneously, mucosally, orally, topically, locally by inhalation (e.g., inhaled aerosolized formulations), by injection, by infusion, by continuous infusion, by direct local perfusion bathing the target cells, via a catheter, via lavage, in lipid components (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 the injection site, pushability and injectability, which includes considering the viscosity of the solution or suspension to be injected as well as the drug concentration, pH and osmolarity. In some cases, the particle size of the active agent can be selected to provide the desired dissolution rate upon administration (e.g., by subcutaneous injection).

[0190] The polypeptides provided herein can be formulated into compositions in free base, neutral, zwitterionic or salt form. 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 formulations are readily administered in a variety of dosage forms, such as formulated for parenteral administration, such as injection solutions, or for delivery to the lungs as an aerosol, or formulated for dietary administration, such as drug release capsules, etc.

[0191] In accordance with certain aspects of the present invention, the compositions suitable for administration can be provided in a pharmaceutical carrier with or without an inert diluent. In some aspects, the carrier can include an aerosol, gas, liquid, semi-solid (i.e., paste) or solid carrier. The use of any conventional medium, reagent, diluent or carrier in the administrable compositions for practicing the methods is appropriate unless it is detrimental to the recipient or to the therapeutic effectiveness of the composition contained therein. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers, etc., or combinations thereof. The compositions can also contain various antioxidants to retard the oxidation of one or more components. Additionally, prevention of microbial action can be achieved by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0192] In accordance with certain aspects of the present invention, the composition and the carrier are combined in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption, etc. Such procedures are conventional to those skilled in the art.

[0193] In a specific embodiment of the present invention, the composition is thoroughly combined or mixed with a semi-solid or solid carrier. The mixing can be carried out in any convenient manner, 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 for use in the compositions include buffering agents, amino acids (such as glycine and lysine), carbohydrates or lyoprotectants (such as glucose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.).

[0194] In some aspects, the pharmaceutical formulation contains one or more surfactants. The surfactants used in accordance with the methods of the present disclosure include ionic and non-ionic surfactants. Representative non-ionic surfactants include polysorbates, such as and Surfactant (ICI Americas Inc. of Bridgewater, N.J.); poloxamers (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-, palmitamidopropyl- or (e.g., lauramidopropyl); myristoyl aminopropyl-, palmitoylpropyl- or isostearoyl aminopropyl-dimethylamine; sodium methyl cocoyl- or sodium methyl oleoyl-taurine; MONAQUAT TM Surfactants (Mona Industries Inc. of Paterson, N.J.); polyethylene glycol; polypropylene glycol; block copolymers of ethylene and propylene glycol, such as Surfactants (BASF of Mt. Olive, N.J.); oligomeric (ethylene oxide) alkyl ethers; alkyl (thio) glucosides, alkyl maltosides; and phospholipids. For example, the amount of surfactant present in the formulation can be from about 0.01% to about 5% (weight of surfactant relative to the total weight of other solid components in the formulation; "w / w"), from about 0.03% to about 0.5% (w / w), about 0.05% to about 0.5% (w / w), or about 0.1% to about 0.5% (w / w). However, in other respects, the pharmaceutical formulations of the examples are substantially free of nonionic surfactants or substantially free of all surfactants.

[0195] Regarding the treatment methods of the present invention, it is not intended to administer one or more of the peptides or their mutants, variants, analogs or derivatives disclosed herein, and is not limited to a specific mode of administration, dose or dosing frequency; the present invention encompasses all modes of administration, including intramuscular, intravenous, intraperitoneal, intracapsular, intra-articular, 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, for example, by one hour, three hours, six hours, eight hours, one day, two days, one week, two weeks or one month. For example, the therapeutic agent can be administered for, for example, 2 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, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition. For example, if a lower dose does not provide sufficient therapeutic activity, the dose of the therapeutic agent can be increased.

[0196] Although the attending physician will ultimately determine the appropriate amount and dosing regimen, a therapeutically effective amount of one or more of the polypeptides or their mutants, variants, analogs, or derivatives disclosed herein 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. The effective dose can be extrapolated from a dose-response curve derived from in vitro or animal model test bioassays or systems.

[0197] One of ordinary skill in the art can determine the dose for a particular patient or subject using routine considerations (e.g., by means of appropriate conventional pharmacological regimens). The physician can, for example, initially prescribe a relatively low dose and then increase the dose until an appropriate response is obtained. Depending on the application, the dose administered to the patient is sufficient to affect a beneficial therapeutic response in the patient over time, or to alleviate symptoms or other appropriate activities, for example. The dose is determined by the efficacy of the particular formulation and the activity, stability, or serum half-life of one or more of the polypeptides or their mutants, variants, analogs, or derivatives disclosed herein, as well as the condition of the patient and the weight or surface area of the patient to be treated.

[0198] In some aspects, a single dose is administered to the subject, once daily, for example via infusion (by inhalation), to treat the subject, which is preferably a mammal, more preferably a human being suffering from or susceptible to pulmonary fibrosis, and the resulting dose is 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 can be administered at any location daily for about 3 days to one or more weeks.

[0199] Chronic administration is also possible, although as is well known in the art, the dose may need to be downregulated. However, the above ranges are suggestive because the number of variables in an individual treatment regimen is large, and significant deviations from these preferred values are expected.

[0200] For continuous administration, for example, via a pump system (such as the osmotic pump used in some of the experiments described below), the total dose within a time range 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. After such a continuous dose 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.

[0201] The effective concentration of the active compound that inhibits or prevents 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 the optimal dose range can be determined in vitro using the methods described herein.

[0202] B. Dry powder particle size reduction and dry powder inhaler devices.

[0203] The particle size of the formulation can be reduced by any suitable method, including but not limited to grinding, milling, thin film freezing, spray drying, or crushing. Grinding can be carried out by any method known in the art, such as by jet mills, ball mills, wet mills, media mills, high pressure homogenization, or cryogenic mills.

[0204] The stability of the peptide after particle size reduction can be evaluated using techniques known in the art, including size exclusion chromatography; electrophoretic 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 for administering the composition to a subject via the lungs, a device for dry powder dispersion is suitable for inhalation by the subject. For example, protein stability can be evaluated 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 determined qualitatively using dynamic light scattering (DLS) (DynaPro-801TC, Protein Solutions Inc. of Charlottesville, Va.) and / or by ultraviolet spectrophotometry.

[0205] In some embodiments, treating a patient with milled CSP7 can include controlled drug release. In some embodiments, milled CS7 can be formulated for slow release or delayed release. In some embodiments, milled CSP7 can be formulated for rapid release. In other embodiments, milled CSP7 can be formulated for both slow release and rapid release (i.e., dual release profile).

[0206] In some embodiments, the present disclosure provides methods for administering the inhalable CSP7 compositions provided herein. Administration can be, but is not limited to, inhalation of milled CSP7 using an inhaler. In some embodiments, the inhaler is a passive dry powder inhaler (DPI), such as the Plastiape RSOl single-dose DPI. In a dry powder inhaler, the dry powder is stored in a container and delivered to the lungs by inhalation without the use of a propellant.

[0207] In some embodiments, the inhaler is a single-dose DPI, such as the 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 plurimonodose DPI for simultaneous delivery of multiple drugs in single doses, such as the Plastiape RS04 plurimonodose DPI. Generally, a dry powder inhaler stores the drug in an internal reservoir and delivers the drug by inhalation, with or without a propellant. Other types of dry powder inhalers store pre-dosed drugs in capsules (e.g., cellulose or gelatin matrix) or foil pouches and pierce them individually through the device to release the dose to the patient. A dry powder inhaler may require an inspiratory flow rate greater than 30 L / min for effective delivery, such as between approximately 30 - 120 L / min. In some embodiments, the effective atomization of milled CSP7 is independent of the inspiratory force. In some embodiments, the flow resistance of the dry powder inhaler is between 0.01 kPa 0.5 min / L and 0.05 kPa 0.5 min / L, such as between 0.02 kPa 0.5 min / L and 0.04 kPa 0.5 min / L. The dry powder inhaler is selected according to the patient population and their inspiratory ability (e.g., high resistance, low resistance, passive, active).

[0208] In some embodiments, the inhaler can be a metered-dose inhaler. A metered-dose inhaler uses a short pulse form of a propellant-assisted aerosolized drug to deliver a defined amount of drug to the lungs. A metered-dose inhaler includes three main parts: a canister, a metering valve, and an actuator, and may utilize a spacer device to slow down the emitted particles and facilitate the patient's inhalation of the aerosol cloud. The drug formulation, including the propellant and any required excipients, is stored in the canister. The metering valve allows the dispensing of a limited amount of the drug formulation. The actuator of the metered-dose inhaler or mouthpiece contains paired discharge nozzles and typically includes a dust cap to prevent contamination. The required inspiratory flow rate for using a metered-dose inhaler can be less than 90 L / min, such as between about 15 - 90 L / min, preferably about 30 L / min. In some embodiments, the effective atomization of milled CSP7 is independent of the inspiratory force.

[0209] In some embodiments, the inhaler is a nebulizer. The nebulizer is used to deliver a drug in the form of an aerosolized mist inhaled into the lungs. The pharmaceutical formulation is atomized by a compressed gas or ultrasonically. A jet nebulizer is connected to a compressor. The compressor discharges compressed gas at a high speed through the liquid pharmaceutical formulation, thereby atomizing the pharmaceutical formulation. The patient then inhales the atomized drug. An ultrasonic nebulizer generates high-frequency ultrasonic waves, causing vibration of an internal element in contact with the reservoir of the pharmaceutical formulation, which results in atomization of the pharmaceutical formulation. The patient then inhales the atomized drug. The nebulizer can utilize a flow rate between about 3 - 12 L / min, such as about 6 L / min. In some instances, a ground active substance (e.g., CSP7) can be suspended in a pharmaceutical liquid carrier vehicle and administered by atomization (e.g., jet atomization). In other aspects, the compositions of the embodiments can be administered by a vaporization method (e.g., rapid vaporization), such as through an e-cigarette device.

[0210] In some embodiments, the composition can be administered 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 the same or different lengths. For example, administration on a regular schedule may involve twice a day, daily, 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 in between. Alternatively, administration on a predetermined regular schedule can include twice a day in the first week and then daily for several months, etc. In some embodiments, the peptide (e.g., CSP7) is administered once a day. In a preferred embodiment, the peptide is administered less than once a day, such as once every other day, once every three days, or once a week. In some embodiments, the full dose of the peptide of the embodiments (e.g., CSP7) is between 1 - 100 mg, such as 20 - 100 mg, 50 - 100 mg, 10 - 20 mg, 20 - 40 mg, 50 - 70 mg, or 80 - 90 mg.

[0211] In some embodiments, the peptides of the embodiments (e.g., CSP7) can be provided in unit dosage forms (e.g., pre-divided doses), such as in the form of capsules, blisters or cartridges, wherein the unit dose contains at least 1 mg of the peptide, such as at least 5 mg, 10 mg, 15 mg or 20 mg of the peptide of the embodiments (e.g., CSP7) per dose. In some aspects, the unit dose is 1 - 10 mg (e.g., about 5 mg) of the peptide. In certain aspects, the unit dosage form does not include the administration or addition of any excipients and is only used to contain the powder for inhalation (i.e., the capsule, blister or cartridge is not administered). 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 peptides of the embodiments can be provided in unit dose capsules, and more than one unit dose capsule (e.g., 3 - 4) can be administered to the subject by inhalation. In some embodiments, the peptide (such as, CSP7) can be administered at a high emission dose, such as at least 10 mg, preferably at least 15 mg, even more preferably 20 mg. In some embodiments, administering the milled peptide of the embodiments (e.g., CSP7) results in a high fine particle dose entering the deep lung, such as greater than 5 mg. Preferably, the fine particle dose entering 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 the dose of the peptide of the embodiments (e.g., CSP7). In some aspects, the fine particle dose is at least 50% of the emission dose, such as at least 60%, 65%, 70%, 75% or 80%.

[0212] In some embodiments, a change in the inhalation pressure drop results in a change in the emission dose. In some embodiments, a 3 kPa change in the inhalation pressure, such as from 4 kPa to 1 kPa, results in a reduction in the emission 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 inhalation pressure results in a change in the fine particle dose. In some embodiments, a 3 kPa change in the inhalation pressure, 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.

[0213] Ⅳ. Treatment of Pulmonary Disorders

[0214] The peptides of the present invention can be used to treat a variety of pulmonary disorders. The pulmonary disorders to be treated may be acute or chronic. Acute pulmonary disorders may be acute lung injury, infection or chemically induced. Chronic pulmonary disorders may be the result of injury, infection or disease.

[0215] A. Lung Injury

[0216] In some aspects, the subject has acute lung injury (ALI) or infection or chemically-induced lung injury. In certain aspects, the subject has plastic bronchitis, asthma, chronic obstructive airway / lung (COPD), acute respiratory distress syndrome (ARDS), inhalation smoke-induced acute lung injury (ISALI), bronchiectasis, inhalation toxin-induced airway diseases (e.g., chlorine gas or other airway disease-inducing agents), exposure to mustard gas, exposure to particulate matter (e.g., silica dust), bronchiolitis obliterans, bronchiolitis obliterans organizing pneumonia, collagen vascular lung diseases (e.g., from lupus, scleroderma or mixed connective tissue disease), interstitial lung diseases (e.g., idiopathic pulmonary fibrosis or sarcoidosis), drug-induced lung diseases and accelerated pulmonary fibrosis (e.g., occurring after acute lung injury including ARDS). Lung diseases including chronic obstructive pulmonary disease, asthma, infection and acute and chronic lung injuries leading to fibrosis constitute the third leading cause of death globally (Murray et al., 1997; Rabe et al., 2007; Tsushima et al., 2009). In United States military personnel, acute lung injury (ALI) is a serious medical problem. ALI in combat may be caused by multiple etiologies.

[0217] Treatment of ALI caused by inhalation injury with inhaled anticoagulants, steroids, beta agonists, high frequency ventilation and extracorporeal membrane oxygenation has variables and generally unsatisfactory results. Except for obstacles with respiratory masks, there are no effective preventive measures. Considerable progress has been made in the management of ARDS, but it still largely supports observational waiting for the endogenous healing mechanism to take effect; the in-hospital mortality rate remains higher than 40% (Matthay et al., 2012). Survivors of ALI often have chronic respiratory disorders with a decreased quality of life. Any means that can accelerate recovery and / or prevent subsequent complications such as chronic respiratory insufficiency and pulmonary fibrosis would be highly desirable. There is an urgent need to improve the early diagnosis of ALI and, more importantly, to prevent and treat ALI. The pathophysiology of direct inhalation lung injury caused by systemic diseases or ALI caused by ARDS is extremely complex and heterogeneous, including systemic and local cardiopulmonary factors such as increased membrane permeability, influx of inflammatory cytokines, oxidative cell damage, compartmental fluid flow, ion channel disarray and many other factors (Matthay et al., 2012). Clearly, new therapies are needed to treat and prevent pulmonary disorders such as ALI.

[0218] In some embodiments, a method of treating or preventing acute lung injury, pulmonary infection, or pulmonary disease in a subject is provided, which comprises 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.

[0219] B. Pulmonary Diseases

[0220] Pulmonary diseases include pulmonary fibrosis, pulmonary inflammation, idiopathic pulmonary fibrosis, cystic fibrosis, chronic obstructive pulmonary disease (COPD), bronchitis, bronchiolitis, bronchiolitis obliterans, asthma, and pulmonary infections, as well as acute and chronic lung injuries that lead to fibrosis (Murray et al., 1997; Rabe et al., 2007; Tsushima et al., 2009). These diseases constitute the third leading cause of death worldwide.

[0221] Cystic fibrosis is a hereditary disease of the exocrine glands and exocrine 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. A subset of individuals with CF also exhibit airway hyperresponsiveness to inhaled cholinergic agonists (Weinberger, 2002 and Mitchell et al., 1978), and a reversibility of airflow limitation in response to bronchodilators (van Haren et al., 1991 and van Haren et al., 1992). The presence of bronchial hyperresponsiveness and airway obstruction suggests a possible common disease etiology between CF and other airway narrowing diseases such as asthma or chronic obstructive pulmonary disease (COPD), where airway smooth muscle dysfunction is thought to contribute to the disease process.

[0222] Lung infections can be bacterial infections. Infectious bacteria can be Pseudomonas aeruginosa, Bacillus anthracis, Listeria monocytogenes, Staphylococcus aureus, Salmonella, Yersinia pestis, Mycobacterium leprae, Mycobacterium africanum, M. asiaticum, M. avium-intracellulaire, M. chelonei abscessus, M. fallax, Pseudomonas faecalis, 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 can lead to pneumonia.

[0223] Chronic obstructive pulmonary disease (COPD) is a term used to classify two major obstructive airway disorders: chronic bronchitis and emphysema. Approximately 16 million Americans have COPD, and 80 - 90% of them were smokers at some point in their lives. COPD is a leading cause of death in the United States, causing 122,283 deaths in 2003. In 2003, the direct healthcare costs for COPD in the United States were approximately $20.9 billion. Chronic bronchitis is an inflammation of the bronchial airways. The bronchial airways connect the trachea to the lungs. When inflamed, the bronchi secrete mucus, causing a chronic cough.

[0224] In emphysema, the alveolar sacs become over-inflated due to damage to the elastic protein framework of the lungs. Inflammatory cells in the emphysematous lungs release elastase, an enzyme that degrades or destroys the elastic protein fibers within the lung matrix. Emphysema has multiple causes, including smoking, exposure to environmental pollutants, alpha-1 antitrypsin deficiency, and aging.

[0225] Bronchiolitis is most commonly caused by a viral lower respiratory tract infection and is characterized by acute inflammation, edema, necrosis of the 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, use of accessory muscles, and / or nasal flaring.

[0226] Bronchiolitis obliterans results in a progressive reduction 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 commonly used to describe late allograft dysfunction, resulting in a persistent decline in forced expiratory volume and force that is not caused by other known reasons (Meyer et al., 2014).

[0227] The term "asthma" can refer to acute asthma, chronic asthma, intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma, chronic persistent asthma, mild-to-moderate asthma, mild-to-moderate persistent asthma, mild-to-moderate chronic persistent asthma, allergic (extrinsic) asthma, non-allergic (intrinsic) asthma, nocturnal asthma, bronchial asthma, exercise-induced asthma, occupational asthma, seasonal asthma, asymptomatic asthma, gastroesophageal asthma, idiopathic asthma, and cough-variant asthma. During asthma, the airways are continuously inflamed and may occasionally spasm.

[0228] In some embodiments, a method of treating or preventing a pulmonary infection or a pulmonary disease in a subject is provided, which comprises 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 embodiment comprises dry powder inhalation of the peptide. In certain aspects, the subject is a human.

[0229] Ⅴ. Examples

[0230] The following examples are included to illustrate the preferred embodiments of the present invention. Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques that the inventors have found to function well in the practice of the present invention and can thus be considered to constitute a preferred mode of its practice. However, based on the present disclosure, those skilled in the art should understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the present invention and still obtain the same or similar results.

[0231] Example 1 - Methods and Materials

[0232] Preparation of dry powder peptide. The CSP7 peptide (SEQ ID NO:2), lot number #: AHF66 / / 470103 was synthesized by Polypeptide Laboratories (San Diego, USA).

[0233] Manufacture of CSP7 mixtures and spray drying. CSP7 formulations containing either individual CSP7 (CSP7), or 75% / 25% mixtures of CSP7 / leucine, CSP7 / trehalose, or CSP7 / sodium citrate, or 75% / 15% / 10% mixtures of CSP7 / leucine / trehalose were prepared in water at pH 10 (adjusted with NH 4 OH) and spray dried using a BLD-35 with a 2-inch cyclone separator.

[0234] Reducing the particle size of dry powder CSP7 by thin film freezing (TFF). 0.3 mg / ml of CSP7 bulk powder and 0.9 mg / ml of mannitol (mass ratio 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 dropped into a rolling chamber filled with liquid nitrogen. The measured 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). The lyophilization conditions were as follows: Equilibration: held at -55 °C, 100 mTorr for 30 minutes; Primary drying: temperature raised to -30 °C, 100 mTorr in 250 minutes; held at -30 °C, 100 mTorr for 660 minutes; Secondary drying: temperature raised to 30 °C, 100 mTorr in 720 minutes; and held at 30 °C, 100 mTorr for 240 minutes. The sample treated by TFF was designated as batch number 171014.

[0235] Reducing the particle size of dry powder CSP7 by cryogenic milling. One gram of CSP7 bulk powder was added to a small cryogenic grinding tube and then loaded into a 6870 freezer / mill (SPEX Certiprep TM , NJ, USA). Milling was carried out in 5 cycles, pre-cooled for 10 minutes, and each cycle was run at 10 CPS for 5 minutes and then cooled for 2 minutes. The milled sample was retrieved and weighed, and the yield calculated based on the ratio of the retrieved weight to the loaded weight was 73.5%.

[0236] Reducing the particle size of CSP7 powder by ball mill (BM). CSP7 bulk powder was suspended in its anti-solvent ethanol (anhydrous) to a concentration of 1 mg / ml. Approximately half of the zirconia ball solvent volume (2 mm) was added to the suspension. The suspension was then loaded into an 8000M mixer / mill (SPEX SamplePrep, NJ, USA) for milling. Samples were collected from the milling process and tested at 5 minutes, 10 minutes, and 30 minutes respectively.

[0237] Reducing the particle size of CSP7 powder using a rotor-stator. CSP7 bulk powder was dispersed in ethanol to a concentration of 1 mg / ml. The tip of the rotor-stator (5 mm * 75 mm flat bottom) was immersed in the suspension and homogenized to reduce the particle size.

[0238] High performance liquid chromatography analysis. Samples were dissolved in 20 mM Tris buffer (pH 10.3) and run on a Phenomenex C18(2) liquid chromatography column with a particle size of 5 μm and a pore size of The Phenomenex Security Guard guard column kit was used as a guard column. Mobile phase A was H 2 O + 0.1% (v / v) trifluoroacetic acid (TFA), and mobile phase B was 80% acetonitrile + 20% H 2 O + 0.09% (v / v) TFA. The injection volume of the sample was 20 μL. Each sample was run for 25 minutes, and the column was maintained at 25 °C with a flow rate of 1 ml / min. The sample was detected at a wavelength of 220 nm. The buffer gradient was set to the specific conditions in Table 2.

[0239]

[0240]

[0241] Determine the aerodynamic particle size distribution of the milled CSP7 bulk powder. Approximately 3.5 mg of the milled CSP7 powder was manually filled into size 3 HPMC capsules (Capsugel, Peapack, NJ). The CSP7 capsules were then nebulized using an RS01 single-dose dry powder inhaler (high resistance), and the aerodynamic particle size distribution was measured using a Next Generation Impactor (NGI, MSP Corp., Shoreview, MN). The inhaler generated an aerosol at an air flow rate of 60 L / min in 4 s to achieve an inhalation volume of 4 L and a pressure drop of 4 kPa across the device. Before each run, the NGI collection surface was coated with a methanol solution of 5% (v / v) polysorbate 20. One capsule was ejected per run, and each sample was run three times (three capsules) in duplicate. After nebulization, 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.

[0242] For each test, the delivered dose was defined as the mass of CSP7 entering the NGI. Based on the dose deposited in the 1-MOC stage 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 was defined as the mass fraction of particles smaller than 5.0 μm of the delivered dose.

[0243] Prepare and lyse lung tissue. Female mice, 6 - 8 weeks old, were ordered from Jackson Laboratories, stock: 000664 C57BL / 6J, and were cared for and housed according to IACUC guidelines. The following week, the mice were weighed, anesthetized by intraperitoneal (IP) injection with 80 mg / kg ketamine and 6 mg / kg xylazine (approx. 115 μl / mouse), and instilled with bleomycin intratracheally. Briefly, a 26G plastic catheter was inserted into the trachea, and the mice received 2 x 20 μl instillations of 0.8 U / kg bleomycin (Biotang, Cat#RB003) via pipette (30 s apart to clear from the airway). Controls received only the same volume of saline. Body weight was tracked (approx. 10% weight loss in injured animals), and the animals were subjected to a dry powder inhalation protocol (CH technology) daily for one week. The dry powder dose was based on the minimum effective dose of a previous nebulized formulation, which was estimated to be 0.7 mcg / dose of lung delivery per animal (Tepper et al., 2016, incorporated herein by reference). In summary, an exposure time of 12 minutes / day corresponded to a '1X' dose, while a '5X' dose corresponded to a 60 - minute / day treatment, equivalent to 3.5 mcg / animal. The animals were exposed continuously for seven days (days 14 - 20; during the fibrotic phase of bleomycin injury), and were sacrificed 24 hours after the last dose with a lethal dose of heparinized ketamine / xylazine mixture (25% heparin). A portion of the whole lung was collected for histological examination. Briefly, 10 ml of saline was perfused through the heart to clear the blood from the lungs. Then, the lungs were inflated with saline for 1 minute at 20 cm above the dissection area, and then inflated with 4% PFA for 1 minute. The trachea was ligated, and the lungs were excised, fixed, embedded, and sectioned to 4 μm for visualization of the maximum surface area, and stained with hematoxylin and eosin. Images were captured with an Aperio AT2 high - volume digital whole - slide scanner, and the lungs were scored for fibrotic injury according to a modified Ashcroft scoring scheme (Hübner et al., 2008, incorporated herein by reference). For molecular analysis, the whole lungs were homogenized in RIPA buffer and protease inhibitor (Santa Cruz) and 1% DTT (to inhibit RNase), and were homogenized in parallel at 4°C (Precellys Evolution, Bertin Instruments) and subjected to downstream assays. The collagen content of the lung homogenates was determined according to a total collagen assay (Quickzyme), using a collagen standard provided by the manufacturer, and according to the manufacturer's instructions. The colorimetric assay was 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. Figures 29 - 32 .

[0244] Homogenization buffer for 28 samples was prepared by adding 224 μL of a mixture 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 the homogenization buffer was added to each sample. The samples were homogenized using a Precellys Evolution with CK28 beads. The homogenization protocol was for hard tissues and was performed twice for each sample at 4 °C. And then the homogenized samples were aliquoted, with 400 μL stored for BCA concentration determination and protein assay, 200 μL for RNA isolation, and 200 μL for collagen determination.

[0245] Collagen determination. The collagen standards used were prepared by adding 125 μL of the Quickzyme collagen standard to 125 μL of 12 M HCl and adding 200 μL of each sample to 200 μL of 12 M 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 13,000 x g for 10 minutes. The standard was prepared according to the manufacturer's instructions (Quickzyme). Then 100 μL of each sample was diluted to 50 μL of water. Then 10 μL of each diluted sample was further diluted to 100 μL of 4 M HCl. The standards and replicates of each sample were pipetted into the plate. 75 μL of the assay buffer was added to each well and the plate was covered before shaking for 20 minutes. 75 μL of the detection reagent mixture was added to each well and the plate was mixed before incubating at 60 °C for one hour. Then the plate was read as shown above.

[0246] 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 the sample in RIP A buffer, resulting in a total of 150 μL of starting material. Then, 50 μL of the starting material of each sample was added to 50 μL of RNase-free water. Then, 350 μL of buffer RLT was added, and the samples were mixed well. Then, 250 μL of 95 - 100% ethanol was added to each of them, and the samples were mixed again. Then, 700 μL of each sample was added to its respective nucleic acid purification column and centrifuged at 8000 X g, and the flow-through was discarded. 500 μL of RPE was added to each column, and the column was centrifuged again. 500 μL of RPE was added again, and this time the sample was centrifuged at 8000x g for 2 minutes. The sample was transferred to a new microcentrifuge tube, and the RNA was eluted with 40 μL of RNase-free water by centrifugation at 8000x g for 1 minute. The samples were quantified by nanodrop and analyzed as described above.

[0247] Example 2 - Characterization of CSP7 Bulk Powder

[0248] Scanning Electron Microscopy. Samples of the bulk powder of CSP7 were sputtered on a sample disk and dispersed by blowing in 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 were thus outside the inhalable range.

[0249] CSP7 Particle Size Assessment. The particle size was examined using a Spraytec laser diffractometer equipped with a solid-state or wet dispersion attachment and a Sympatec laser diffractometer HELOS-R system to determine whether the bulk powder was within the inhalable range. Using the dry dispersion method, it was determined that the CSP7 bulk powder particle size was larger than the inhalable particle size. Table 3 shows the particle sizes of the particles evaluated as Dv 10, Dv 50 (median), and Dv 90 within the distribution. As shown in Table 3, for all CSP7 particles analyzed, more than 50% had a particle size of 5.3 μm or larger, which is greater than the inhalable range.

[0250]

[0251] Next, the particle size was determined by the wet dispersion method using a Sympatec laser diffractometer. CSP7 was dissolved in ethanol + 0.05% Tween 80 as the dispersion medium and sonicated for 10 minutes. The results of determining the CSP7 particle size using the wet dispersion method 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, far outside the inhalable range.

[0252]

[0253] The Spraytec laser diffraction instrument was used again, with the dry dispersion method, to further evaluate the average particle size. The CSP7 bulk powder was dispersed at 40 PSI, and the average particle size (8.6 ± 1.5 μm) was again higher than the respirable range (Table 5).

[0254]

[0255] The percentage of dry powder particles smaller than 5 μm was found to be only 34.5 ± 4.1%. Considering that the laser diffraction method found that the particle size of most of the bulk powder exceeded the respirable range, any dry powder used for treatment needs to be processed in some way to reduce the particle size.

[0256] The morphology of the bulk CSP7 powder. The bulk powder CSP7 sample was 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 aggregates, visible in Figure 2 and indicated by the arrows.

[0257] Determine the crystallinity of the CSP7 bulk powder particles. The CSP7 bulk powder particles were evaluated using X-ray powder diffraction ( Figure 3 ). Using X-ray powder diffraction, pure CSP7 was found to exhibit a certain degree of crystallinity ( Figure 3 ). To confirm the X-ray diffraction results, the crystallinity was evaluated by polarized light microscopy ( Figure 4 ). As shown in Figure 4 , crystalline CSP7 was present in the bulk CSP7 powder, and the white arrows in the image indicate some typical crystalline forms.

[0258] Thermal analysis of the 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 ). The analysis was continued using thermogravimetric analysis (TGA) ( Figure 6 ). TGA showed that the weight of pure CSP7 began to decrease significantly above 216 °C ( Figure 6 ).

[0259] Moisture content of CSP7 bulk powder. The moisture content of CSP7 bulk powder was evaluated by Karl Fischer - volumetric (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 these 3 tests.

[0260]

[0261] Next, the water 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 it was found that the sample had a water desorption of 6.32% at zero relative humidity. When the relative humidity was 90%, a mass change of 10.54% was found ( Figure 7 ).

[0262] Example 3 - Characterization of CSP7 powder with reduced particle size

[0263] Reduction of the particle size of CSP7 powder. To effectively inhale the powder and deposit it into the lungs, the particle size should generally have a mass median aerodynamic diameter of less than about 5 μm. A variety of techniques were performed to reduce the particle size of the pure material, including air jet milling (AJM), ball milling (BM), cryogenic milling (CM), thin film freezing (TFF), and spray drying. First, AJM was performed to reduce the particle size of CSP7 bulk powder and the milled CSP7 was collected from multiple positions inside the mill. Tables 5 and 6 show the yield and particle size distribution of the first batch of milled powder (batch number 171013) collected from the indicated positions. As described above, the particle size distribution was determined by the dry dispersion method (Table 8) or the wet dispersion method (Table 9) of the Sympatec laser diffractometer.

[0264]

[0265]

[0266] Using the same conditions as above, a second batch of CSP7 (batch number 171027) was milled from 10 g of pure bulk powder. Again, the particle size distribution and yield of the powder were evaluated from the same positions and are listed in Table 10.

[0267]

[0268] The third batch of CSP7 dry powder was subjected to thin film freezing (TFF) (batch number 171014) and analyzed as above using the dry dispersion method (Table 11) and the wet dispersion method (Table 12) of the Sympatec laser diffractometer.

[0269]

[0270]

[0271] Another batch of CSP7 dry powder was cryomilled (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.

[0272]

[0273]

[0274] Another batch of CSP7 bulk powder was ball milled (BM) to reduce the particle size. Table 15 shows the particle size distribution of BM CSP7 powder taken at several time points during the milling process.

[0275]

[0276] Further batches were produced with CSP7 (also referred to as the designated development abbreviation, CSP7) and leucine, trehalose, sodium citrate, or a mixture of leucine and trehalose, and spray dried to reduce the particle size. The particle size of the spray dried product was examined by laser diffraction using the dry dispersion method ( Figure 8 , Table 16). Again, spray drying significantly reduced the particle size of CSP7 relative to the bulk CSP7.

[0277]

[0278]

[0279] When measured by the solid dispersion method and the wet dispersion method, for each method of reducing the particle size (jet milling, thin film freezing, cryomilling, ball milling, and spray drying), the particle size of CSP7 was significantly reduced (compare Tables 1 and 2 with Tables 5, 6, 10, 13, 14, 16, 17, 18, and 19). When measured by laser diffraction using the solid dispersion method or the liquid dispersion method, after jet milling, most CSP7 particles fell within the respirable range, but for thin film freezing, cryomilling, and ball milling, the results were less satisfactory, with a smaller percentage of the milled powder reduced to the respirable range.

[0280] The particle size can also be reduced using a rotor-stator hand-held homogenizer ( Figure 9 ). After homogenization, depending on the duration and efficacy of the homogenization, the color of CSP7 in ethanol changes to light grey or dark grey, Figure 9 , and it was evident that although this method was not pursued further due to the observed color change, the homogenization reduced the particle size.

[0281] Morphology of milled CSP7 powder. The morphology of each milled sample was examined using an optical microscope or a scanning electron microscope. The powder samples from the jet mill were examined as above, and optical microscopy indicated that the particle size was reduced to a size where 1 μm > particle size > 5 μm and was homogeneous ( Figure 10 ). In addition, the milled CSP7 particles were free of agglomerates ( Figure 10 ). SEM indicated homogenization and reduced particle size of the jet-milled particles 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 assessment of the particle morphology of the spray-dried formulation by scanning electron microscopy. Representative SEM images of the formulation are shown in Figure 13 .

[0282] Crystallinity of AJM CSP7. The milled CSP7 powder (batch no. 171027) was evaluated by X-ray diffraction and showed crystallinity in the diffractogram ( Figure 14 ). The crystallinity of spray-dried CSP7 was also examined by X-ray diffraction, and the curves are shown in Figure 15 . Formulations of CSP7 alone or conjugated with trehalose or sodium citrate were amorphous, while CSP7 conjugated with leucine or leucine and trehalose appeared to contain crystalline leucine features, as shown by the spikes in Figure 15 .

[0283] HPLC evaluation of milled or spray-dried CSP7 powder. To determine whether the milled CSP7 bulk powder had an effect on chemical potency, the milled CSP7 powder samples collected from various parts of the mill were evaluated by HPLC under the conditions listed in Table 2. Potency was evaluated using the following formula:

[0284]

[0285] As is clearly visible from Table 26, milling had no detrimental effect on the potency of any of the samples collected. Similarly, when batch no. 171027 was tested, the chemical potency was determined to be 100.14%

[0286]

[0287] The spray-dried CSP7 mixture was examined by RP-HPLC to check purity (Table 17, Figure 16 ). Similar to the jet-milled CSP7 powder, the spray-dried CSP7 retained approximately 100% purity.

[0288]

[0289]

[0290] CSP7 Stability. The stability of the untreated bulk CSP7 powder and jet-milled CSP7 (Lot No. 171027) was examined by determining its chemical potency using HPLC. Each sample was stored under 3 different conditions, and then the chemical potency was determined at storage times of 5 days, 15 days, and 32 days ( Figure 17 ). The stability of the spray-dried CSP7 within 24 hours was also examined by HPLC (the method described earlier in the specification) to understand its short-term stability. Each formulation was found to be stable with no increase in impurities after 2 hours or 24 hours (Table 31).

[0291]

[0292] Aerodynamic Particle Size Distribution of the Milled CSP7. To determine the aerodynamic particle size distribution of the milled CSP7, the amounts of powder deposited at various positions of the NGI collector were separately extracted. The delivered dose was measured as the mass of CSP7 entering the NGI collector after atomization, and the amounts of CSP7 deposited on individual surfaces were separately extracted and measured. The amounts of untreated or jet-milled (Lot No. 171013) CSP7 remaining in the capsule or deposited within the device, adapter, throat, pre-separator, and 1-MOC stage are shown in Figure 18 , as a percentage of the total amount of CSP7 delivered. The percentage of fine particle fraction (FPF%), mass median aerodynamic diameter (MMAD), and geometric standard deviation (GSD) of the milled and untreated (e.g., un-milled or untreated) CSP7 are listed in Table 9.

[0293]

[0294] The aerodynamic particle size distribution of the second batch of milled CSP7 Lot No. (171027) was determined as above, except that approximately 4.25 mg of powder was used for each size 3 HPMC capsule. The GSD, FPF%, and MMAD are shown in Table 11, and the percentage of CSP7 deposited at each position is shown in Figure 19 , again as a percentage of the total amount of CSP7 delivered.

[0295]

[0296] The atomization of the spray-dried formulations was also examined ( Figure 20 ). Each formulation showed a fine powder fraction greater than 60%, and the MMAD of each formulation was between 2.5 μm and 3 μm (Table 25). Table 25 shows a summary of the analytical results of the spray-dried formulations, which includes the water content.

[0297]

[0298]

[0299] Determine the moisture content of milled CSP7. Under the same conditions used for the analysis of bulk CSP7, the jet-milled CSP7 powder (lot number 171027) was analyzed using dynamic vapor sorption ( Figure 21 ). Similar to the bulk pure powder, the milled CSP7 had 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 ).

[0300]

[0301] Thermogravimetric analysis of the reduced particle size CSP7 powder. The milled CSP7 (lot number 171027) was subjected to thermogravimetric analysis in the same manner as bulk CSP7, and the milled CSP7 was found to have very similar properties to the untreated CSP7 ( Figure 22 ). The thermal properties of the spray-dried formulations were also evaluated and are shown in Table 24 and Figures 23 - 27 (summarized in Figure 28 ). Notably, the midpoint Tg of the mixed formulation was significantly lower than that of the individual spray-dried CSP7 (compare 001C-F with 001B in Table 24).

[0302]

[0303]

[0304] The results presented herein demonstrate that multiple methods for reducing the particle size of CSP7 powder are effective and that the resulting powders exhibit very similar characteristics.

[0305] Example 4 - Treatment of Bleomycin-Induced Pulmonary Fibrosis by Inhalation of Dry Powder of CSP7

[0306] Induce and treat fibrosis in mice with CSP7. Mice were treated with bleomycin to induce pulmonary fibrosis. Mice were administered 0.8 U / kg bleomycin intranasally and waited for 14 days before treatment to develop the disease. Then the mice were left untreated, or treated by inhalation of dry powder of CSP7 for 12 minutes, or treated by inhalation of dry powder of CSP7 for 60 minutes. The mice were euthanized on the last day of treatment, and the lungs were removed, snap-frozen, and stored at -80 °C. The snap-frozen lungs were weighed before further analysis ( Figure 29 ).

[0307] Homogenize the lung tissue and analyze the 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 pulmonary fibrosis, the Ashcroft score (a measure of pulmonary fibrosis in mice) was lower in mice treated with CSP7( Figure 31 ).

[0308] RNA was also prepared from the homogenized lung tissue and used as described above.

[0309] Example 5 - Formulation Suspension for Intramuscular / Subcutaneous Injection

[0310]

[0311] *: Jet-milled CSP7 showed a geometric particle size of Dv(10) = 0.75 μm; Dv(50) = 1.93 μm; Dv(90) = 4.29 μm, measured by laser diffraction.

[0312] For preparation:

[0313] 1. Prepare 20 mM Tris buffer (should be pH ~ 10.3)

[0314] 2. Dissolve 1.5% (w / w) CMC in 20 mM Tris buffer and add 0.2% (w / w) poloxamer 188. Stir overnight at ~600 rpm

[0315] 3. Add 0.7% (w / w) NaCl to the CMC solution

[0316] 4. Adjust the pH of the solution to 7 by adding ~28.5 μl / ml of 1N HCl

[0317] 5. Weigh and add a certain amount of jet-milled (collected from the collection bag to obtain a fraction with a smaller particle size) CSP7 powder in a clean vial

[0318] 6. First, mill the powder with a clean rod to ensure there are no obvious agglomerated particles

[0319] 7. Gradually add the prepared solution to the vial and grind / mill with a rod

[0320] 8. When the powder is completely wet and no visible agglomerated particles are found up to the target volume

[0321] Results:

[0322]

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

[0324] For preparation:

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

[0326] 2. Dissolve 1.5% (w / w) CMC in 20 mM Tris buffer, and add 0.2% (w / w) poloxamer 188. Stir overnight at ~ 600 rpm

[0327] 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 value should be about 9, and then adjust the pH value to 8.2 - 8.5 by adding ~ 50 μl 1N HCl

[0329]

[0330] Results:

[0331]

[0332] Example 7 - Pre - formulation Study of Polypeptide Variants

[0333] Conduct solubility studies of the variants by adding the variant powder to the solvent at a concentration of 5 mg / ml, and then vortex for 3 minutes. Observe the appearance of the solution within 5 minutes. Add more powder (each time ~ 5 mg / ml) and repeat vortexing and observation until precipitation or gelation occurs.

[0334] Results:

[0335]

[0336]

[0337] a Precipitation: Centrifuge the solution, then aliquot the supernatant and dilute it with buffer.

[0338] b High - viscosity solution: Take an aliquot of the solution and dilute it with buffer.

[0339]

[0340]

[0341]

[0342]

[0343]

[0344] Pre - formulation studies of Example 8 - CSP7 (ammonium counterion) form

[0345] The solubility of the pure (i.e., un - milled) CSP7 ammonium counterion form (Table 35) was performed by adding an excess of peptide powder to 3 mL of different pH buffers (Table 36) and mixing at 100 rpm on an orbital shaker for 24 hours at room temperature.

[0346] For the freeze - thaw stability study ( Figure 33 ), 0.1 mg / mL CSP7 (ammonium counterion) peptide in phosphate buffer system (PBS, pH 7.4) was aliquoted into 15 mL / vials and subjected to rapid freezing and slow freezing, respectively. For rapid freezing and slow freezing, the samples were immersed in liquid nitrogen for at least 5 minutes or placed in a - 20 °C freezer for at least 1.5 hours to ensure complete freezing of the aliquots in the vials, and then thawed to room temperature. Each sample underwent 5 freeze - thaw cycles. The recovery % represents the percentage of each sample concentration relative to the original (untreated) concentration.

[0347] Before performing the determination by high - performance liquid chromatography (HPLC, Thermo Fisher Scientific, Fair Lawn, NJ), the solubility samples and freeze - thaw samples were filtered through a 0.45 μm membrane. Briefly, the samples were analyzed using a Dionex 3000 HPLC system equipped with a reversed - phase C18 column 2.5 μm, 150 mm x 4.60 mm. The HPLC column was heated to 60 °C for testing, and the peptide was detected at a wavelength of 215 nm and a flow rate of 1 mL / min. The two mobile phases were A (aqueous solution of 0.1% acetic acid) and B (acetonitrile solution of 0.1% acetic acid). The injection volume was 20 μL, and a standard curve was plotted from 0.01 - 1 mg / ml.

[0348] Table 34. Details of the HPLC method.

[0349]

[0350] Table 35: pH solubility curve of CSP7 (ammonium counterion).

[0351] 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

[0352] Table 36. Buffer systems and the amount of CSP7 peptide added to 3 mL of buffer at each pH value

[0353]

[0354] Example 9. Characterization and Stability Study of Ground CSP7 (Ammonium Counterion) Powder Lot UTA181028

[0355] The CSP7 peptide was ground using a Model 00 Jet - O - Mizer TM (also known as Aljet mill, Fluid Energy, Telford, PA). The feed rate, propulsion pressure, and milling pressure were 1 g / min, 60 psi, and 70 psi, respectively (Table 37). The batch size was 20 g, and the ground powder was collected from different parts of the jet mill, which included: the tube after the milling chamber (bfC), cyclone separator (C), collection container adapter (D), collection bag adapter (E), collection bag (H), and collection container (G). The collected powder was mixed for 10 minutes using a Turbula mixer (Glen Mills Inc., Clifton, NJ, USA).

[0356] Table 37. Parameters for Grinding CSP7 (Ammonium Counterion) Powder

[0357] Parameter Value Feed rate 1 g / min Pushing pressure 60 psi Grinding pressure 70 psi

[0358] Specific surface area of the ground CSP7 ammonium counterion pure powder. The specific surface areas of the ground and untreated CSP7 powders were analyzed by the single - point BET method using a Monosorb MS - 21 rapid surface area analyzer (Quantachrome Instruments, Boynton Beach, FL) Figure 34 ). The samples were degassed with nitrogen at 20 psi for 20 - 24 hours at 25 °C to remove water and other impurity molecules on the surface. A mixture of nitrogen / helium (50:50 v / v) was used as the adsorbate, and the apparatus was calibrated with nitrogen before testing.

[0359] Thermogravimetric analysis of the ground CSP7 ammonium counterion pure powder. 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 .

[0360] Scanning electron microscope (SEM) images of the ground and pure CSP7 (ammonium counterion) powder. The morphology of CSP7 Figure 36) Analyzed using a Zeiss Supra 40VP SEM (Carl Zeiss Microscopy GmbH, Jena, Germany). The samples were fixed on aluminum SEM specimen stubs with 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 pure (i.e., untreated) and milled CSP7 samples.

[0361] Stability study of milled CSP7 (ammonium counterion) from lot UTA181028. The stability of milled CSP7 powder was studied over a period of up to 6 months under different storage conditions. The milled CSP7 peptide was packaged in two forms: bulk milled powder and encapsulated milled powder. For storage in the form of bulk milled powder, 0.21 - 0.24 g of the peptide was loaded into 20 mL scintillation vials ( DWK Life Sciences, Millville, NJ, US), and stored in heat-sealed aluminum foil pouches (Impak Corp, Los Angeles, CA, US), with two bags of 1 g silica gel desiccant in each pouch ( Sorbco Packaging LLC, Belen, NM, US). The milled peptide powder was also encapsulated in size 3 HPMC capsules (Capsugel, Morristown, NJ, US), weighing approximately 11 ± 5% mg, and then 22 - 26 capsules were packaged in an HDPE bottle (DrugPlastic, Boyertown, PA, US), and the HDPE bottle was then sealed in an aluminum foil pouch (without desiccant). The packages were stored in a stability chamber under the following storage conditions: -20 °C, 25 °C / 60% RH, and 40 °C / 75% RH. Samples were taken 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 a glass vial by rotating the vial.

[0362] Table 38. Test schedule for the stability study.

[0363]

[0364] * Abbreviation: HPLC: High Performance Liquid Chromatography

[0365] 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.

[0366] Appearance of the milled powder. The appearance of the milled powder was recorded by taking a photograph using a conventional camera ( Figure 30 ).

[0367] Chemical stability of the milled peptide. The powder was assayed using the HPLC method described in Example 8. The results are shown in Table 39 below. The percentages represent the measured amounts compared to the mass balance. The assays were adjusted for moisture content.

[0368] Table 39. Stability study of the milled CSP7 ammonium counterion by HPLC assay.

[0369]

[0370] Moisture content of the CSP powder. The moisture content in the peptide powder was determined using the Karl Fischer coulometric method (Mettler Toledo C20, Leicester, OH, US) (Table 40). The reliability of the instrument was tested using a Karl Fischer water content standard (Hydranal™ water standard, Honeywell, Charlotte, NC, US). A known amount of the powder was suspended in anhydrous methanol (Sigma, St. Louis, MO), and the suspension was injected into the anolyte (Hydranal TM -Coulomat AG, Honeywell, Charlotte, NC, US) and titration was initiated in the presence of the catholyte (Hydranal TM -Coulomat CG, Honeywell, Charlotte, NC, US). The results were recorded as the difference between the water content in the sample minus the blank anhydrous methanol solution.

[0371] Table 40. Moisture content of the powder samples in the stability study.

[0372]

[0373]

[0374] Geometric particle size distribution. The GPSD of CSP7 powder was analyzed before and after milling using a Sympatec HELOS laser diffraction instrument (Sympatec GmbH, Germany) equipped with RODOS dispersion. After the powder was dispersed at 3 bar, measurements were taken every 10 ms. Measurements between 5% and 25% light density were averaged to determine the particle size distribution. The particle sizes reported by volume are represented by the 10th, 50th, and 90th percentiles (e.g., Dv 10, Dv 50, and Dv 90), respectively, and the percentage of particles falling within the 1 - 5 μm particle size range. The results are shown in Table 41.

[0375] Table 41. Geometric particle size distribution of milled CSP7 (ammonium counterion) in the stability study

[0376]

[0377] Aerodynamic particle size distribution: The aerodynamic particle size distribution was evaluated in the stability study by performing 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.

[0378] Table 42. Aerodynamic particle size distribution of milled CSP7 (ammonium counterion) in the stability study.

[0379]

[0380]

[0381] Note: IP: Inlet port; FPD: Fine particle dose (particles < 5 μm); FPF(%) : Fine particle dose in the delivered dose

[0382] 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 .

[0383] ***

[0384] In view of the present disclosure, all of the methods disclosed and claimed herein can be made and executed without undue experimentation. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods as well as to the steps of the methods or the sequence of said steps without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications which are obvious to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0385] References

[0386] The following references, to the extent that they provide exemplary procedures or other details supplementary to those described herein, are expressly incorporated herein by reference.

[0387] Carvalho et al., “Influence of particle size on regional lung deposition - What evidence is there?” Int. J. Pharma. 406:1 - 10, 2011.

[0388] Hübner, R.-H.; Gitter, W.; El Mokhtari, N.E.; Mathiak, M.; Both, M.; Bolte, E.L.; Freitag-Wolf, S.; Bewig, B. Standardized quantification of pulmonary fibrosis in histological samples. Biotechniques, 44, 507 - 11, 514 - 7, 2008.

[0389] 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.

[0390] Tepper, J.S.; Kuehl, P.J.; Cracknell, S.; Nikula, K.J.; Pei, L.; Blanchard, J.D. Symposium Summary: “Breathe In, Breathe Out, It's Easy: What You Need to Know about Developing Inhaled Drugs.” Int. J. Toxicol. 35, 376 - 392, 2016。 Sequence Listing <110> The University of Texas System Board of Regents Lung Therapeutics, Inc. Williams, Robert O. III Watts, Alan B. Zhang, Yajie Sahakijpijarn, Sawittree Koleng, John J. Christensen, Dale <120> Dry Powder Formulation of Caveolin - 1 Peptide and Method of Use Thereof <130> UTFB.P1188WO <150> US 62 / 729,010 <151> 2018 - 09 - 10 <160> 29 <170> PatentIn version 3.5 <210> 1 <211> 178 <212> PRT <213> homo sapiens <400> 1 Met Ser Gly Gly Lys Tyr Val Asp Ser Glu Gly His Leu Tyr Thr Val 1 5 10 15 Pro Ile Arg Glu Gln Gly Asn Ile Tyr Lys Pro Asn Asn Lys Ala Met 20 25 30 Ala Asp Glu Leu Ser Glu Lys Gln Val Tyr Asp Ala His Thr Lys Glu 35 40 45 Ile Asp Leu Val Asn Arg Asp Pro Lys His Leu Asn Asp Asp Val Val 50 55 60 Lys Ile Asp Phe Glu Asp Val Ile Ala Glu Pro Glu Gly Thr His Ser 65 70 75 80 Phe Asp Gly Ile Trp Lys Ala Ser Phe Thr Thr Phe Thr Val Thr Lys 85 90 95 Tyr Trp Phe Tyr Arg Leu Leu Ser Ala Leu Phe Gly Ile Pro Met Ala 100 105 110 Leu Ile Trp Gly Ile Tyr Phe Ala Ile Leu Ser Phe Leu His Ile Trp 115 120 125 Ala Val Val Pro Cys Ile Lys Ser Phe Leu Ile Glu Ile Gln Cys Ile 130 135 140 Ser Arg Val Tyr Ser Ile Tyr Val His Thr Val Cys Asp Pro Leu Phe 145 150 155 160 Glu Ala Val Gly Lys Ile Phe Ser Asn Val Arg Ile Asn Leu Gln Lys 165 170 175 Glu Ile <210> 2 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 2 Phe Thr Thr Phe Thr Val Thr 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 3 Ala Ser Phe Thr Thr Phe Thr Val Thr 1 5 <210> 4 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 4 Lys Ala Ser Phe Thr Thr Phe Thr Val Thr Lys Gly Ser 1 5 10 <210> 5 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 5 Lys Ala Ser Phe Thr Thr Phe Thr Val Thr Lys Gly Ser 1 5 10 <210> 6 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <220> <221> MISC_FEATURE <222> (1)..(2) <223> X = D-alanine <220> <221> MISC_FEATURE <222> (18)..(19) <223> X = D-alanine <400> 6 Xaa Xaa Glu Gly Lys Ala Ser Phe Thr Thr Phe Thr Val Thr Lys Gly 1 5 10 15 Ser Xaa Xaa <210> 7 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <220> <221> MISC_FEATURE <222> (1)..(2) <223> X = D-alanine <220> <221> MISC_FEATURE <222> (18)..(19) <223> X = D-alanine <220> <221> MISC_FEATURE <222> (19)..(19) <223> NH2 functional group present <400> 7 Xaa Xaa Glu Gly Lys Ala Ser Phe Thr Thr Phe Thr Val Thr Lys Gly 1 5 10 15 Ser Xaa Xaa <210> 8 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <220> <221> MISC_FEATURE <222> (1)..(1) <223> Acetyl group present <220> <221> MISC_FEATURE <222> (1)..(2) <223> X = D-alanine <220> <221> MISC_FEATURE <222> (18)..(19) <223> X = D-alanine <220> <221> MISC_FEATURE <222> (19)..(19) <223> NH2 functional group present <400> 8 Xaa Xaa Glu Gly Lys Ala Ser Phe Thr Thr Phe Thr Val Thr Lys Gly 1 5 10 15 Ser Xaa Xaa <210> 9 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <220> <221> MISC_FEATURE <222> (1)..(1) <223> X = ornithine <220> <221> MISC_FEATURE <222> (11)..(11) <223> X = ornithine <400> 9 Xaa Ala Ser Phe Thr Thr Phe Thr Val Thr Xaa Ser 1 5 10 <210> 10 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <220> <221> MISC_FEATURE <222> (1)..(1) <223> X = ornithine <220> <221> MISC_FEATURE <222> (11)..(11) <223> X = ornithine <220> <221> MISC_FEATURE <222> (12)..(12) <223> NH2 functional group present <400> 10 Xaa Ala Ser Phe Thr Thr Phe Thr Val Thr Xaa Ser 1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <220> <221> MISC_FEATURE <222> (7)..(7) <223> NH2 functional group present <400> 11 Phe Thr Thr Phe Thr Val Thr 1 5 <210> 12 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <220> <221> MISC_FEATURE <222> (8)..(8) <223> Presence of NH2 functional group <400> 12 Phe Thr Thr Phe Thr Val Thr Lys 1 5 <210> 13 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <220> <221> MISC_FEATURE <222> (11)..(11) <223> Presence of NH2 functional group <400> 13 Lys Ala Ser Phe Thr Thr Phe Thr Val Thr Lys 1 5 10 <210> 14 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <220> <221> MISC_FEATURE <222> (1)..(1) <223> Presence of acetyl group <220> <221> MISC_FEATURE <222> (11)..(11) <223> Presence of NH2 functional group <400> 14 Lys Ala Ser Phe Thr Thr Phe Thr Val Thr Lys 1 5 10 <210> 15 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Amino Acid <220> <221> MISC_FEATURE <222> (1)..(1) <223> X = Ornithine <220> <221> MISC_FEATURE <222> (11)..(11) <223> NH2 Functional Group Present <400> 15 Xaa Ala Ser Phe Thr Thr Phe Thr Val Thr Lys 1 5 10 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Amino Acid <220> <221> MISC_FEATURE <222> (1)..(1) <223> Acetyl Group Present <220> <221> MISC_FEATURE <222> (1)..(1) <223> X = Ornithine <220> <221> MISC_FEATURE <222> (11)..(11) <223> NH2 Functional Group Present <400> 16 Xaa Ala Ser Phe Thr Thr Phe Thr Val Thr Lys 1 5 10 <210> 17 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <220> <221> MISC_FEATURE <222> (1)..(1) <223> Acetyl group present <220> <221> MISC_FEATURE <222> (13)..(13) <223> NH2 functional group present <400> 17 Lys Ala Ser Phe Thr Thr Phe Thr Val Thr Lys Gly Ser 1 5 10 <210> 18 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <220> <221> MISC_FEATURE <222> (14)..(14) <223> NH2 functional group present <400> 18 Asp Ser Gly Lys Ala Ser Phe Thr Thr Phe Thr Val Thr Lys 1 5 10 <210> 19 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <220> <221> MISC_FEATURE <222> (1)..(1) <223> Presence of acetyl group <220> <221> MISC_FEATURE <222> (14)..(14) <223> Presence of NH2 functional group <400> 19 Asp Ser Gly Lys Ala Ser Phe Thr Thr Phe Thr Val Thr Lys 1 5 10 <210> 20 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <220> <221> MISC_FEATURE <222> (1)..(1) <223> Presence of acetyl group <220> <221> MISC_FEATURE <222> (1)..(1) <223> X = Ornithine <220> <221> MISC_FEATURE <222> (11)..(11) <223> X = Ornithine <220> <221> MISC_FEATURE <222> (12)..(12) <223> Presence of NH2 functional group <400> 20 Xaa Ala Ser Phe Thr Thr Phe Thr Val Thr Xaa Ser 1 5 10 <210> 21 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <220> <221> MISC_FEATURE <222> (9)..(9) <223> Presence of NH2 functional group <400> 21 His Asp Gly Ile Trp Lys Ala Ser Phe 1 5 <210> 22 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <220> <221> MISC_FEATURE <222> (9)..(9) <223> Presence of NH2 functional group <400> 22 His Val Thr Lys Tyr Trp Phe Tyr Arg 1 5 <210> 23 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 23 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln 1 5 10 <210> 24 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 24 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <210> 25 <211> 26 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 25 Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu 1 5 10 15 Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln 20 25 <210> 26 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 26 Thr Lys Ile Glu Ser Leu Lys Glu His Gly 1 5 10 <210> 27 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 27 Thr Gln Ile Glu Asn Leu Lys Glu Lys Gly 1 5 10 <210> 28 <211> 26 <212> PRT <213> Artificial sequence <220> <223> Synthetic amino acid <400> 28 Ala Ala Leu Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Glu Ala 1 5 10 15 Leu Glu Ala Leu Ala Glu Ala Ala Ala Ala 20 25 <210> 29 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Amino Acid <400> 29 Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Glu Gly Trp Tyr Gly Cys Gly 20 25

Claims

1. A dry powder pharmaceutical composition comprising a peptide, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the peptide is FTTFTVT (SEQ ID NO: 2), wherein the composition is produced by jet milling, wherein at least 70% of the dry powder particles in the composition have a particle size of 1 µm to 5 µm, and wherein the pharmaceutical composition is formulated for use in a dry powder inhaler.

2. The dry powder pharmaceutical composition according to claim 1, wherein the composition comprises a lubricant.

3. The dry powder pharmaceutical composition according to claim 1, wherein the composition is encapsulated in a capsule.

4. The dry powder pharmaceutical composition according to claim 1, wherein the composition is enclosed in a hard capsule.

5. The dry powder pharmaceutical composition according to claim 1, wherein the peptide is at least 95% pure by weight.

6. The dry powder pharmaceutical composition according to claim 1, wherein the peptide is at least 99% pure by weight.

7. The dry powder pharmaceutical composition according to claim 1, wherein the composition comprises less than 10% water by weight.

8. The dry powder pharmaceutical composition according to claim 1, wherein the composition comprises less than 1% water by weight.

9. The dry powder pharmaceutical composition according to claim 1, wherein the pharmaceutical composition consists of the peptide.

10. The dry powder pharmaceutical composition according to any one of claims 1-9, characterized in that, it is for treating a subject in need thereof, the treatment comprising administering to the subject an effective amount of the pharmaceutical composition.

11. The dry powder pharmaceutical composition according to claim 10, wherein the subject has an inflammatory disorder.

12. The dry powder pharmaceutical composition according to claim 10, wherein the subject has a fibrotic condition.

13. The dry powder pharmaceutical composition according to claim 10, wherein the subject has a lung inflammation.

14. The dry powder pharmaceutical composition according to claim 10, wherein the subject has chronic obstructive pulmonary disease (COPD).

15. The dry powder pharmaceutical composition according to claim 10, wherein the subject has acute lung injury.

16. The dry powder pharmaceutical composition according to claim 10, wherein the subject has a lung infection.

17. The dry powder pharmaceutical composition according to claim 10, wherein the subject has chemically induced lung injury.

18. The dry powder pharmaceutical composition according to claim 10, wherein the subject has plastic bronchitis.

19. The dry powder pharmaceutical composition according to claim 10, wherein the subject has asthma.

20. The dry powder pharmaceutical composition according to claim 10, wherein the subject has acute respiratory distress syndrome (ARDS).

21. The dry powder pharmaceutical composition according to claim 10, wherein the subject has inhalation smoke-induced acute lung injury (ISALI).

22. The dry powder pharmaceutical composition according to claim 10, wherein the subject has bronchiolitis.

23. The dry powder pharmaceutical composition according to claim 10, wherein the subject has bronchiolitis obliterans.

24. The dry powder pharmaceutical composition according to claim 12, wherein the subject has a fibrotic disorder of the lungs.

25. The dry powder pharmaceutical composition according to claim 10, wherein the subject has an interstitial lung disease.

26. The dry powder pharmaceutical composition according to claim 25, wherein the interstitial lung disease is idiopathic pulmonary fibrosis (IPF).

27. The dry powder pharmaceutical composition according to claim 10, which further comprises the administration of at least one additional anti-fibrotic therapeutic agent.

28. The dry powder pharmaceutical composition according to claim 27, wherein the at least one additional anti-fibrotic therapeutic agent is a non-steroidal anti-inflammatory drug (NSAID).

29. The dry powder pharmaceutical composition according to claim 27, wherein the at least one additional anti-fibrotic therapeutic agent is a steroid.

30. The dry powder pharmaceutical composition according to claim 27, wherein the at least one additional anti-fibrotic therapeutic agent is a disease-modifying anti-rheumatic drug (DMARD).

31. The dry powder pharmaceutical composition according to claim 27, wherein the at least one additional anti-fibrotic therapeutic agent is an immunosuppressant.

32. The dry powder pharmaceutical composition according to claim 27, wherein the at least one additional anti-fibrotic therapeutic agent is a biologic response modifier.

33. The dry powder pharmaceutical composition according to claim 27, wherein the at least one additional anti-fibrotic therapeutic agent is a bronchodilator.

34. The dry powder pharmaceutical composition according to claim 10, wherein the subject is a human.

35. The dry powder pharmaceutical composition according to claim 1, wherein the composition does not contain excipients.

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