Caveolin-1 peptide dried powder formulation and method of use

Abstract

To provide a dry powder formulation of caveolin-1 peptides and methods of use thereof.SOLUTION: Provided herein are compositions comprising caveolin-1 (Cav-1) peptides. Further provided are methods of using the Cav-1 peptides for the treatment of lung infections or acute or chronic lung injury, particularly lung fibrosis. Provided in a first embodiment is a pharmaceutical composition comprising a dry powder of a peptide, the peptide comprising a sequence of any one of SEQ ID NOs: 2-20. In some aspects, the peptide is 7-20 amino acids in length. In a specific aspect, the peptide comprises the amino acid sequence of SEQ ID NO: 2.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] This invention was made as a result of activities carried out within the scope of a collaborative research agreement that was in effect at the time the invention was made. The parties to the aforementioned collaborative research agreement were the Board of Regents of the University of Texas System and Lung Therapeutic.

[0003] This invention generally relates to the fields of molecular biology, pharmacology, and medicine. More specifically, this invention relates to compositions and methods for delivering dried powder therapeutic polypeptide compositions to a target, such as by delivery to the respiratory system. [Background technology]

[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 injury mechanism involves cell surface signaling interactions between uPA, uPAR, caveolin-1 ("Cav-1"), and β1-integrin (Shetty et al., 2005). Compositions modulating these interactions can be used in methods to inhibit apoptosis in injured, diseased, or damaged tissue, for example, to treat inflammation or fibrotic conditions such as pulmonary fibrosis. Therefore, there is a need for polypeptides that can be used to prevent or treat lung injury and disease, as well as, in particular, stable formulations and simple methods for the therapeutic delivery of such polypeptides. [Overview of the project] [Means for solving the problem]

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

[0006] In a first embodiment, a pharmaceutical composition is provided comprising a dry powder of a peptide, wherein the peptide comprises one of the sequences of SEQ ID NOs: 2 to 20. In some embodiments, the peptide has an amino acid length of 7 to 20. In a particular embodiment, the peptide comprises the amino acid sequence of SEQ ID NOs: 2. In a further embodiment, the peptide comprises at least one amino acid added to the N-terminus of the peptide of SEQ ID NOs: 2. In another embodiment, the peptide comprises at least one amino acid added to the C-terminus of the peptide of SEQ ID NOs: 2. In yet another embodiment, the peptide comprises at least one amino acid added to both the N-terminus and C-terminus of the peptide of SEQ ID NOs: 2. In a particular embodiment, the peptide may comprise an L-amino acid, a D-amino acid, or both L- and D-amino acids. In an additional embodiment, the peptide may comprise at least one non-standard amino acid. In some embodiments, the peptide comprises two non-standard amino acids. In a particular embodiment, the non-standard amino acid is ornithine.

[0007] In further embodiments, the peptide may include N-terminal modifications, C-terminal modifications, or both N-terminal and C-terminal modifications. In certain embodiments, the N-terminal modification is acylation. In other embodiments, the C-terminal modification is amidation.

[0008] In some embodiments, the peptide may include SEQ ID NOs: 3, 4, 6, 9, 5, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 10, and 20. In some embodiments, the peptide includes at least two repeats of any one sequence from SEQ ID NOs: 2 to 20. In certain embodiments, at least two repeats have the same amino acid sequence. In other embodiments, at least two repeats have different amino acid sequences. In yet another embodiment, the pharmaceutical composition further includes a cell-permeable peptide (CPP). In certain embodiments, the CPP includes an amino acid sequence selected from the group including GRKKRRQRRRPPQ (SEQ ID NO: 23), RQIKIWFQNRRMKWKK (SEQ ID NO: 24), and GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 25).

[0009] In additional embodiments, the dry powder is produced by a grinding process. In some embodiments, the dry powder is produced by a spray drying process. In other embodiments, the dry powder is produced by air jet grinding, ball grinding, or wet grinding. In some embodiments, the dry powder contains less than 10% (by weight) of water. In other embodiments, the dry powder contains less than 1% (by weight) of water. In certain embodiments, the pharmaceutical composition is essentially free of excipients. In certain embodiments, the pharmaceutical composition is free of excipients. In certain embodiments, the pharmaceutical composition is formulated for pulmonary delivery. In further embodiments, the pharmaceutical composition is formulated for dry powder inhalation. In other embodiments, the pharmaceutical composition is formulated for inhalation pressurized metered-dose inhalation. In some embodiments, the pharmaceutical composition is formulated for oral administration, topical administration, or injection.

[0010] In certain embodiments, the dry powder formulation of the embodiment contains a moisture content of about 10%, 9%, 8%, 7%, 6%, or less than 5%. In further embodiments, the composition contains a moisture content of about 0.01% to about 10%, 0.1% to about 10%, 1.0% to about 8%, or 1% to about 5%. In further embodiments, the dry powder formulation of the embodiment contains an average particle size of less than 10 μm. In certain embodiments, the average particle size is about 0.01 μm to about 10 μm, about 0.1 μm to about 8 μm, about 0.5 μm to about 7 μm, or about 1 μm to about 5 μm. In some embodiments, at least about 50%, 55%, 60%, 65%, or 70% of the dry powder composition of the embodiment contains a particle size of about 1 μm to about 5 μm. In certain embodiments, the dry powder formulation of the peptide of the embodiment (e.g., CSP7) consists of at least 70% (e.g., 70% to 80%) of particles having a size of about 1 μm to about 5 μm. In preferred embodiments, at least about 70%, 75%, 80%, or 85% (e.g., 75% to 95%) of the particles in the dry powder formulation are less than 5 μm in size.

[0011] Further embodiments of the present invention provide a nebulizer device comprising the embodiment and the pharmaceutical composition described above.

[0012] In yet another embodiment, a method is provided for treating a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of the embodiment and the embodiments described above. In certain embodiments, the subject has an inflammatory disorder. In other embodiments, the subject has a fibrotic condition. In some embodiments, the subject has pneumonia, acute lung injury, lung infection, or lung disease. In another embodiment, the subject has pneumonia. In certain embodiments, the subject has chronic obstructive pulmonary disease (COPD). In further embodiments, the subject may have acute lung injury or infection, lung infection, chemical-induced lung injury, cast bronchitis, asthma, acute respiratory distress syndrome (ARDS), inhalation smoke-induced acute lung injury (ISALI), bronchiolitis, or obstructive bronchiolitis. In certain embodiments, the lung disease is a pulmonary fibrotic condition, interstitial lung disease, or idiopathic pulmonary fibrosis (IPF) or pulmonary scarring. In additional embodiments, the administration comprises inhalation of a dry powder. In other embodiments, administration involves atomizing a solution containing the variant polypeptide.

[0013] In a further embodiment, the method further comprises administering at least one additional antifibrotic therapeutic agent. In some embodiments, the at least one additional antifibrotic agent is an NSAID, a steroid, a DMARD, an immunosuppressant, a bioresponse modulator, or a bronchodilator. In some embodiments, the subject is a human.

[0014] A further embodiment of the present invention provides a pharmaceutical composition comprising peptides of SEQ ID NOs: 2-20, formulated as a pulverized dry powder having a breathable particle size. For example, in certain embodiments, the pulverized dry powder has an aerodynamic median particle diameter (MMAD) of less than about 10 microns. A method for determining the MMAD is provided, for example, in Carvalho et al., 2011, which is incorporated herein by reference.

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

[0016] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, 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, it should be understood that the detailed description and specific examples are provided for illustrative purposes only, while illustrating preferred embodiments of the invention.

[0017] The following drawings form part of this specification and are included to further illustrate specific aspects of the invention. The invention will be better understood by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein. [Brief explanation of the drawing]

[0018] [Figure 1] Scanning electron microscope image of CSP7 bulk powder. The powder sample was sputtered onto a sample tray and spread out by blowing compressed nitrogen onto it. The sample was observed using a Hitachi S5500 SEM / STEM scanning electron microscope. The scale bar is drawn in the lower right of the image. [Figure 2] Optical microscope images of CSP7 bulk powder. Powder samples were sputtered onto glass slides and observed using a Leica optical microscope (Leica CTR6500). A scale bar is shown in the lower left corner of each image. Arrows point to aggregated particles of neat CSP7 powder. [Figure 3] X-ray powder diffraction of CSP7 bulk powder particles. CSP7 bulk powder particles were evaluated using X-ray powder diffraction to determine their crystallinity. The powder was measured from 2 to 40 2θ° using a step size of 0.025 2θ° and a rate of 2° / min. [Figure 4] Polarized light microscope image of CSP7 bulk powder. Crystallinity was evaluated using a polarized light microscope. The image is representative. White arrows indicate crystalline regions. [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 photograph shows a curve from modulated DSC that was ramped from 25 to 300 °C using a frequency of 1 °C / 60 seconds and a rate of 2 °C / minute. [Figure 6] Thermogravimetric Analysis of CSP7 Bulk Powder. Thermogravimetric analysis was performed using a Mettler model TGA / DSC thermogravimetric analyzer. The photograph shows the TGA curve. The heating rate was set at 10 °C / minute and was ramped from 25 to 500 °C. [Figure 7] Dynamic Vapor Sorption of CSP7 Bulk Powder. The bulk CSP7 powder was run on a Surface Measurement Systems DVS instrument at 25 °C with sufficient adsorption / desorption cycles at 10% increments from 0% to 90% relative humidity. Moisture desorption at 0% humidity and mass change at 90% humidity are shown. [Figure 8] Particle Size Distribution of Spray-Dried CSP7. CSP7 was mixed with either leucine, trehalose, sodium citrate, or leucine and trehalose and spray-dried. Particle size was evaluated by a Malvern Mastersizer 2000 (laser light diffraction, Fraunhofer approximation, dispersion air pressure: 3.0 Bar). The photograph shows the curve for each spray-dried mixture. [Figure 9] Visual Observation of Homogenized CSP7 Suspension. A. Ethanol treated for 1 minute with a rotor-stator at maximum output; B. Untreated CSP7-ethanol suspension, red arrow indicates large particles / aggregates; C. Ethanol suspension treated for 1 minute with a CSP7-rotor-stator at maximum output, the suspension turns dark gray; D. Ethanol suspension treated for 1 minute with a CSP7-rotor-stator at minimum output, the suspension turns light gray. [Figure 10] Optical Microscopy of Air-Jet Milled CSP7 Powder. A powder sample collected from the indicated position of an air-jet mill was imaged by optical microscopy. The photograph is a representative image. [Figure 11]Scanning electron microscope of air-jet milled CSP7 powder. The milled CSP7 powder (batch 171027) was imaged by a scanning electron microscope (SEM) under the same conditions as the bulk CSP7 powder. The photograph is a representative SEM image of the milled CSP7 powder. [Figure 12] Optical microscope of CSP7 after thin-film freezing. The powder sample was sputtered onto a slide glass and observed using a Leica optical microscope (Leica CTR6500). The scale bar is shown in the lower left corner of each image. [Figure 13] Scanning electron microscope of spray-dried CSP7. The particle morphology of the spray-dried CSP7 mixture (A = 100% LTI, B = leucine, C = trehalose, d = sodium citrate, e = leucine and trehalose) was examined using SEM. Representative images are depicted. [Figure 14] X-ray powder diffraction of air-jet milled CSP7 powder. The X-ray powder diffraction profiles of the milled (batch 171027) and untreated bulk CSP7 powders are shown. The diffraction curve indicates a decrease in the crystallinity of the milled CSP7. [Figure 15] Physical state of spray-dried CSP7. The spray-dried CSP7 mixture was examined by X-ray diffraction. Curves showing the crystallinity or lack thereof of each spray-dried mixture of CSP7 are shown. [Figure 16] HPLC analysis of spray-dried CSP7. The purity of the spray-dried CSP7 mixture was examined by assaying its chemical potency using HPLC. [Figure 17] HPLC analysis of the stability of air-jet milled CSP7. The stability of both the untreated bulk CSP7 powder and air-jet milled CSP7 (batch 171027) was examined by analyzing their chemical potency using HPLC. Each sample was stored under three different conditions (4 °C, 25 °C / 60% RH, 40 °C / 75% RH) using open / closed vials, and the chemical potency was analyzed after 5, 15, and 32 days of storage. [Figure 18]Deposition of aerosolized CSP7 particles. After aerosolization using a next-generation impactor (NGI), all collection surfaces were rinsed with a known amount of 20 mM Tris buffer (pH 10.3). The powder deposited in the throat, pre-separator, and stages 1-MOC was extracted and measured separately. The percentage of untreated or air-jet ground (batch 171013) CSP7 powder (collected from the collection container) deposited at specific locations is shown. [Figure 19] Aerodynamic particle size distribution of air-jet ground CSP7. After aerosolization using a next-generation impactor (NGI), all collection surfaces were rinsed with a known amount of 20 mM Tris buffer (pH 10.3). The capsules, apparatus, adapter, throat, pre-separator, and powder deposited in stages 1-MOC were extracted separately and measured. The positions in the grinder where either untreated or air-jet ground (batch 171027) CSP7 powder (collected from all fractions of ground powder) was measured. The percentage of ground powder present at each position is shown. [Figure 20] Aerosol performance of spray-dried CSP7 mixture. After aerosolization with NGI, all collection surfaces were rinsed with a known amount of 20 mM Tris buffer (pH 10.3). Powder deposited on the capsule, apparatus, adapter, throat, pre-separator, and stages 1-MOC was extracted and measured separately. The percentage of powder deposited at specific locations is shown. [Figure 21] Dynamic vapor adsorption of air-jet ground CSP7 powder. Ground CSP7 powder (batch 171027) was subjected to a complete adsorption / desorption cycle at 25°C in 10% increments from 0% to 90% relative humidity using a Surface Measurement Systems DVS instrument. Moisture desorption at 0% humidity and mass change at 90% humidity are shown. [Figure 22] Thermal analysis of air-jet pulverized CSP7 powder. Differential scanning calorimetry (DSC) was performed on CSP7 (batch 171027) pulverized using the calorimeter described above. DSC curves are plotted for temperatures ranging from 25 to 300°C, using a frequency of 1°C / 60 seconds and a speed of 2°C / min. [Figure 23] Thermal properties of spray-dried 100% CSP7. Spray-dried CSP7 without excipients was analyzed by modulated differential scanning calorimetry. The image shows the mDSC curve. [Figure 24] Thermal properties of spray-dried CSP7 containing leucine. A mixture of spray-dried CSP7 containing 25% leucine was analyzed by modulated differential scanning calorimetry. The image shows the mDSC curve. [Figure 25] Thermal properties of spray-dried CSP7 containing trehalose. A mixture of spray-dried CSP7 containing 25% trehalose was analyzed by modulated differential scanning calorimetry. The image shows the mDSC curve. [Figure 26] Thermal properties of spray-dried CSP7 using sodium citrate. A spray-dried CSP7 mixture containing 25% sodium citrate was analyzed by modulated differential scanning calorimetry. The image shows the mDSC curve. [Figure 27] Thermal properties of spray-dried CSP7 containing leucine and trehalose. A mixture of spray-dried CSP7 containing 15% leucine and 10% trehalose was analyzed by modulated differential scanning calorimetry. The image shows the mDSC curve. [Figure 28] Thermal properties of all spray-dried CSP7 mixtures. Each of the manufactured spray-dried CSP7 mixtures was analyzed by mDSC. All mDSC curves for these powders are plotted. [Figure 29] Wet weight of anatomical lung tissue from mice. Euthanized mice were dissected, their lungs removed, and their body weight measured. Mice were treated with saline or bleomycin to induce pulmonary fibrosis, or treated with bleomycin and then with CSP7 peptide for 12 or 60 minutes. [Figure 30] Collagen content of mouse lung tissue. Untreated, bleomycin-treated, or bleomycin and CSP7-treated lung tissue was homogenized, and collagen content was analyzed using the Quickzyme collagen assay. The graph shows the total collagen content of the lungs. [Figure 31]Ashcroft scores were obtained from mouse lung tissue. Untreated, bleomycin-treated, or bleomycin and CSP7-treated lung tissue was homogenized, and collagen content was analyzed using the Quickzyme collagen assay. Ashcroft scores were determined as described in Hubner et al. 2008, incorporated herein by reference. The graph shows the total collagen content of the lungs. [Figure 32] Collagen content of mouse lung tissue. Untreated, bleomycin-treated, or bleomycin and CSP7-treated lung tissue was homogenized, and collagen content was analyzed using the Quickzyme collagen assay. The graph shows the total collagen content of the lungs. [Figure 33] Stability of CSP7 (ammonium counterion) after up to 5 freeze-thaw cycles. [Figure 34] Specific surface area of ​​ground and neat CSP7 (ammonium counterion) powder. [Figure 35] Thermogravimetric analysis of ground and neat CSP7 (ammonium vs. ion) powder. [Figure 36] SEM images of ground and neat CSP7 (ammonium vs. ion) powder. [Figure 37] Appearance of pulverized CSP7 (ammonium counterion) powder in stability studies. [Figure 38] Crystallinity of pulverized CSP7 (ammonium counterions) in stability studies. [Modes for carrying out the invention]

[0019] I. Definition As used herein, “essentially not present” with respect to a particular component means that the particular component is not intentionally incorporated into the composition and / or is present only as a contaminant or in trace amounts. Therefore, the total amount of a particular component resulting from any unintentional contamination of the composition is far less than 0.01%. Most preferably, the amount of a specific component cannot be analyzed using standard analytical methods.

[0020] As used herein, “one (a)” or “one (an)” may mean one or more. As used in the claims, or in combination with the term “including,” terms such as “one (a)” or “one (an)” may mean one or more.

[0021] The use of the term “or” in the claims is used to mean “and / or,” although this disclosure supports the definitions of “alternatives only” and “and / or,” unless expressly indicated to mean that alternatives only or that the alternatives are mutually exclusive. As used herein, “another” may mean at least two or more.

[0022] Throughout this application, the term “approximately” is used to indicate that a value includes inherent variations in errors in the apparatus, methods, or other factors used to determine the value, or variations that exist between the test subjects. Unless otherwise noted, “approximately” means + / - 10%.

[0023] As used herein, the term “peptide” typically refers to a sequence of amino acids consisting of a single chain of amino acids linked by peptide bonds. Generally, a peptide contains at least two amino acid residues and, unless otherwise specified, is less than approximately 50 amino acids in length. In some embodiments, peptides may be provided with counterions. Similarly, in some cases, peptides may include N and / or C-terminal modifications, such as blocking modifications that reduce degradation.

[0024] A “biologically active” caveolin-1 (Cav-1) peptide refers to a peptide that increases p53 protein levels, decreases urokinase plasminogen activator (uPA) and uPA receptor (uPAR), and / or increases the expression of plasminogen activator inhibitor-1 (PAI-1) in cells such as fibrous lung fibroblasts. In some embodiments, a biologically active peptide has at least 20% of the biological or biochemical activity of the native Cav-1 polypeptide of SEQ ID NO: 1 (as measured, e.g., by in vitro or in vivo assay). In some embodiments, a biologically active peptide has the same or increased biological or biochemical activity compared to the native Cav-1 polypeptide.

[0025] The terms “identity” or “homology” should be interpreted as meaning 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, without considering conservative substitutions as part of sequence identity, and achieving the maximum percentage identity of the entire sequence as necessary. N-terminal or C-terminal extensions or insertions should not be interpreted as reducing identity or homology. Methods and computer programs for alignment are well known in the art. Sequence identity can be measured using sequence analysis software.

[0026] The terms “polypeptide” or “protein” are used in their broadest sense to refer to compounds of two or more subunit amino acids, amino acid analogs, or peptide mimics. Subunits may be linked by amide bonds. In other embodiments, subunits may be linked by other bonds, such as esters, ethers, etc. As used herein, the term “amino acid” refers to any natural and / or unnatural or synthetic amino acid, including glycine and both D or L optical isomers, as well as amino acid analogs and peptide mimics. The terms “peptidomimetic” or “peptide mimic” mean that the peptide according to the present invention is modified to include at least one non-peptide bond, such as a urea bond, carbamate bond, sulfonamide bond, hydrazine bond, or other covalent bond. When the peptide chain is short, peptides of three or more amino acids are generally referred to as oligopeptides. When the peptide chain is long (e.g., longer than 50 amino acids), the peptide is generally referred to as a polypeptide or protein.

[0027] The terms “subject,” “individual,” and “patient” are used interchangeably herein and refer to animals, e.g., humans or non-human animals (e.g., mammals), to which treatment including prophylactic treatment with the pharmaceutical compositions disclosed herein is provided. The term “subject” as used herein refers to humans and non-human animals. The term “non-human animals” includes all vertebrates, e.g., non-human primates (especially higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and non-mammals, e.g., chickens, amphibians, reptiles, etc. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal substitute as a disease model. Non-human mammals include mammals such as non-human primates (especially higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, and cattle. In some aspects, the non-human animal is a companion animal such as a dog or a cat.

[0028] "Treating" a disease or condition in a subject, or "treating" a patient with a disease or condition, means subjecting the individual to a drug treatment, such as the administration of a drug, resulting in the reduction or stabilization of at least one symptom of the disease or condition. Typically, when a peptide is administered therapeutically as a treatment, the peptide is administered to a subject exhibiting one or more symptoms of lung injury or pulmonary fibrosis.

[0029] "Isolated" means that the polypeptide has been separated from any natural environment, such as bodily fluids or blood, and separated from any naturally associated components of the polypeptide.

[0030] Isolated and “substantially pure” means a polypeptide that has been separated and purified, at least to some extent, from its naturally associated components. Typically, a polypeptide is substantially pure if it does not contain at least about 60% by weight, or at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, or even more than at least about 99% by weight, of the naturally associated proteins and naturally occurring organic molecules. 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 their proteins, or by chemical synthesis.

[0031] As used in this specification, the term “mutant” refers to a polypeptide that differs from the polypeptide by the deletion, addition, substitution, or side-chain modification of one or more amino acids, but still retains one or more specific functions or biological activities of a naturally occurring molecule. Amino acid substitutions include modifications in which an amino acid is replaced with a different naturally occurring or unconventional amino acid residue. Such substitutions may be classified as “conservative,” in which case an amino acid residue in the polypeptide is replaced with another naturally occurring amino acid having similar characteristics in terms of polarity, side-chain functional group, or size. Such conservative substitutions are well known in the art. Substitutions included in the present invention may also be “unconservative,” in which case an amino acid residue in the peptide is replaced with an amino acid having different properties, for example, a naturally occurring amino acid from a different group (e.g., replacing a charged or hydrophobic amino acid with alanine), or a naturally occurring amino acid is replaced with an unconventional amino acid. In some embodiments, amino acid substitutions are conservative. When used in reference to polynucleotides or polypeptides, the term "variant" may also encompass a polynucleotide or polypeptide whose primary, secondary, or tertiary structure may be altered compared to a reference polynucleotide or polypeptide (for example, compared to a wild-type polynucleotide or polypeptide).

[0032] The terms "insertion" or "deletion" typically refer to a range of approximately 1 to 5 amino acids. Acceptable mutations can be experimentally determined by synthetically producing peptides using recombinant DNA techniques, systematically inserting, deleting, or substituting nucleotides in the sequence.

[0033] In the context of peptides, the term "substitution" refers to a change in an amino acid, such as a different entity, or a change in an amino acid within an amino acid moiety. Substitutions can be conservative or non-conservative.

[0034] A “molecular analog” of a peptide or other molecule refers to a molecule that is functionally similar to the whole molecule or any fragment thereof. The term “analog” is also intended to include alleles and induced variants. Analogies typically differ from naturally occurring peptides at one or more positions, often by conserved substitutions. Analogies typically exhibit at least 80 or 90% sequence identity with the natural peptide. Some analogies also include modifications of unnatural amino acids or N- or C-terminal amino acids. Examples of unnatural amino acids include, but are not limited to, disubstituted amino acids, N-alkyl amino acids, lactate, 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, and σ-N-methylarginine. Fragments and analogs can be screened for prophylactic or therapeutic efficacy in transgenic animal models, as described below.

[0035] "Covalent bond" means being bonded directly or indirectly (for example, via a linker) by a covalent chemical bond. In some aspects of all embodiments of the present invention, the fusion peptides are covalently bonded.

[0036] 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 binding a nucleic acid sequence encoding one protein to a nucleic acid encoding another protein, so as to constitute a single open reading frame that can be translated in a cell into a single polypeptide containing all the proteins of interest. The order of protein arrangement can vary. A fusion protein may contain epitope tags or half-life extenders. Epitope tags include biotin, FLAG tags, c-myc, hemagglutinin, His6, digoxigenin, FITC, Cy3, Cy5, green fluorescent protein, V5 epitope tag, GST, β-galactosidase, AU1, AU5, and avidin. Half-life extenders include Fc domains and serum albumin.

[0037] The term “airway” as used herein refers to any part of the respiratory tract, including the upper airway, respiratory airway, and lungs. The upper airway includes the nose and nasal cavity, mouth, and throat. The respiratory airway includes the larynx, trachea, bronchi, and bronchioles. The lungs include the respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli.

[0038] The terms “Inhalation Smoke-Induced Acute Lung Injury” and “ISALI” are used interchangeably herein and refer to forms of acute lung injury (ALI) resulting from smoke inhalation. ALI is also referred to as “Mild Acute Respiratory Distress Syndrome; ARDS.” ARDS can be defined by finding one or more of the following conditions in a subject: 1) bilateral pulmonary infiltration on chest X-ray, 2) pulmonary capillary wedge pressure <18 mmHg (2.4 kPa) as measured by right heart catheterization as clinically indicated, and 3) PaO2 / FiO2 <300 mmHg (40 kPa). In some embodiments, treatment for ISALI includes treatment for one or more of the following conditions: reduced oxygen supply, airway obstruction (including severe airway obstruction), fibrinous airway cast or fragments, and alveolar fibrin deposition.

[0039] The term "air jet grinding" refers to a device or method for reducing particle size by using a jet of compressed gas to cause particles to collide with each other, thereby grinding the particles. Air jet grinding can be used to reduce the size of peptide particles. Other mechanical grinding devices that perform the same function can be used interchangeably with air jet grinders. Air jet grinding can be produced under a variety of environmental parameters such as temperature, pressure, relative / permissible humidity, and oxygen content.

[0040] The term "ball grinding" refers to a device or method for reducing particle size by adding the target particles and grinding medium inside a cylinder and rotating the cylinder. The target particles are broken down as the grinding medium moves up and down along the outside of the cylinder as it rotates. Ball grinding can be used to reduce the size of peptide particles. Other mechanical grinding devices that perform the same function can be used interchangeably with air jet grinding.

[0041] The terms "wet grinding" or "media grinding" refer to an apparatus or method for reducing particle size by adding desired particles to an apparatus equipped with a stirrer, which contains a medium including a liquid and a grinding medium. When the desired particles are added and the stirrer rotates, the energy dispersed by the stirrer causes the grinding medium and the desired particles to come into contact, and the desired particles are broken down. Other mechanical grinding apparatuses that perform the same function can be used interchangeably with air jet grinding.

[0042] The term "high-pressure homogenization" refers to a method of reducing particle size by adding target particles to a device that breaks down the target particles using a combination of pressure and mechanical force. Mechanical forces used in high-pressure homogenization may include, in particular, impact, shear, and cavitation. Other mechanical grinding devices that perform the same function can also be used interchangeably with air-jet grinding.

[0043] The term "cryogenic grinding" refers to an apparatus or method for reducing particle size by first cooling the target particles with dry ice, liquid nitrogen, or other cryogenic liquid, and then grinding the target particles to reduce their size. Other mechanical grinding apparatuses that perform the same function can be used interchangeably with air jet grinding.

[0044] The phrase “effective dose” or “therapeutically effective” means a dose of drug or agent sufficient to produce the desired therapeutic outcome. The desired therapeutic outcome may be subjective or objective improvement in the recipient of the administration, reduction of infection, reduction of inflammation, increased lung growth, increased lung repair, reduction of tissue edema, increased DNA repair, decreased apoptosis, reduction of tumor size, decreased rate of cancer cell proliferation, decreased metastasis, or any combination of the above.

[0045] As used herein, “excipient” refers to a pharmaceutically acceptable carrier, which is a relatively inert substance used to facilitate the administration or delivery of an active pharmaceutical ingredient (API) to a subject, or to facilitate the processing of an API into a drug formulation used pharmaceutically for delivery to the site of action of the subject. Excipients or pharmaceutically acceptable carriers include all inert components of the dosage form, excluding the active ingredient. Non-limiting examples of excipients include carriers, bulking agents, stabilizers, surfactants, surface modifiers, solubility enhancers, buffers, encapsulants, antioxidants, preservatives, nonionic wetting agents or clarifying agents, thickeners, and absorption enhancers. “Excipient-free” refers to the pharmaceutical composition of the target in a formulation that does not contain any excipients.

[0046] The phrase “pharmaceutically or pharmacologically acceptable” refers, where applicable, to molecular portions and compositions that, when administered to animals (e.g., humans), do not produce adverse reactions, allergic reactions, or other unfavorable reactions. The preparation of pharmaceutical compositions containing Cav-1 peptides such as CSP7 or additional active ingredients will be known to those skilled in the art in light of this disclosure. Furthermore, it is understood that, for administration to animals (e.g., humans), formulations must meet bioburden, sterility, pyrogenicity, general safety, and / or purity standards required by the FDA or other recognized regulatory authorities.

[0047] As used herein, “pharmaceutically acceptable carriers” include any and all aqueous solvents (e.g., water, alcohol / aqueous solutions, saline solutions, parenteral vehicles, e.g., sodium chloride, Ringer dextrose), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters, e.g., ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption retarders, salts, drugs, drug stabilizers, binders, disintegrants, lubricants, flavor modifiers (e.g., sweeteners, flavoring agents), such materials and combinations thereof, as known to those skilled in the art. The pH and actual concentrations of various components in the pharmaceutical composition are adjusted according to well-known parameters. In some embodiments, the carrier may encapsulate the therapeutic agent but is not consumed or administered to the subject itself (for example, a shell capsule enclosing a dry powder composition for use in a dry powder inhaler).

[0048] II. Caveolin-1 Peptide Embodiments of this disclosure provide a dry powder formulation of caveolin-1 (Cav-1) peptide. The caveolin-1 (Cav-1) scaffold domain or peptide interferes with Cav-1 interaction with Src kinase, mimicking the combined effect of uPA and anti-β1-integrin antibodies. Natural human Cav-1 has a length of 178 amino acids and a molecular weight of 22 kDa. The amino acid sequence of Cav-1 (SEQ ID NO: 1) is shown below: [ka]

[0049] In some embodiments, the peptide is a scaffold domain peptide containing an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 85%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, FTFTVT. The peptide may contain 1, 2, 3, 4 or more amino acid substitutions, deletions, or insertions compared to the sequence of SEQ ID NO: 1, for example, deriving a polypeptide of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 residues. In certain embodiments, the peptide is a cleaved form of the natural Cav-1 polypeptide, e.g., the exemplary peptides shown in Table 1. [Table 1]

[0050] The peptides provided in this disclosure are bioactive derivatives having the activity of the natural Cav-1 polypeptide in an in vitro or in vivo assay of binding or biological activity. In certain embodiments, the peptides inhibit or prevent bleomycin-induced apoptosis of lung epithelial cells (LECs) in vitro or in vivo with at least about 20% of the activity of the natural Cav-1 polypeptide, or at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, about 95%, 97%, 99%, and any range that can be derived therefrom, e.g., about 70% to about 80%, more preferably about 81% to about 90%, or even more preferably about 91% to about 99% of the activity of the natural Cav-1 polypeptide. The peptides may have more than 100% of the activity of the natural CAV-1 polypeptide. Assays for testing biological activity, such as anti-fibrotic activity, the ability to influence the expression of uPA, uPAR, and PAI-1 mRNA, or the ability to inhibit the proliferation of pulmonary fibroblasts, are well known in the art.

[0051] The peptides of this disclosure are natural Cav-1 polypeptides or modified versions thereof. The peptides may be synthetic, recombinant, or chemically modified peptides isolated or produced using methods well known in the art. Modifications may be made to the N-terminal, C-terminal, or internal amino acids. N-terminal modifications may include, but are not limited to, acylation, acetylation, or C-terminal amidation. Peptides may include conserved or non-conserved amino acid changes, as described below. Polynucleotide changes may result in amino acid substitutions, additions, deletions, fusions, and cleavages of the polypeptide encoded by the reference sequence. Peptides may also include amino acid insertions, deletions, or substitutions, including, for example, but not limited to, inserted amino acids, or non-standard amino acids not normally present in human proteins, such as ornithine, or amino acids (and other molecules) not normally present in the amino acid sequence underlying the peptide. The term "conservative substitution," when describing peptides, refers to a change in the amino acid composition of a peptide that does not substantially alter the peptide's activity. For example, a conserved substitution refers to the substitution of different amino acid residues with similar chemical properties. Conservative amino acid substitutions include the substitution of leucine with isoleucine or valine, the substitution of aspartic acid with glutamate, or the substitution of threonine with serine.

[0052] Conservative amino acid substitutions result from substituting one amino acid with another amino acid having similar structural and / or chemical properties, such as substituting leucine with isoleucine or valine, aspartic acid with glutamate, or threonine with serine. Therefore, a conservative substitution of a particular amino acid sequence refers to a substitution of an amino acid that is not critical to polypeptide activity, or an amino acid with another amino acid having similar properties (e.g., acidic, basic, positively or negatively charged, polar or nonpolar, etc.), so as not to reduce peptide activity even if the substitution of a critical amino acid does not. Tables of conservative substitutions providing functionally similar amino acids are well known in the art. For example, the following six groups contain amino acids that are conserved substitutions with each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W). (See also Creighton, Proteins, WH Freeman and Company (1984), which is incorporated in its entirety by reference.) In some embodiments, individual substitutions, deletions, or additions that change, add, or remove a single amino acid or a small proportion of amino acids may also be considered conserved substitutions if the change does not reduce the activity of the peptide. Insertions or deletions typically range from about 1 to 5 amino acids. The selection of conserved amino acids can be based on the position of the amino acid to be substituted within the peptide, for example, when the amino acid is outside the peptide and exposed to the solvent, or when it is inside the peptide and not exposed to the solvent.

[0053] In alternative embodiments, the amino acid to be substituted for 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., whether the amino acid is exposed to the solvent or located on the outer surface of the peptide or polypeptide, compared to an amino acid localized internally that is not exposed to the solvent). Such selection of conservative amino acid substitutions is disclosed, for example, in Dordo et al, J. Mol Biol, 1999, 217, 721-739 and Taylor et al, J. Theor. Biol. 119 (1986); 205-218, and S. French and B. Robson, J. Mol. Evol. 19 (1983) 171. Therefore, suitable conserved amino acid substitutions can be selected for the amino acids outside the protein or peptide (i.e., amino acids exposed to the solvent), and for example, the following substitutions can be used, but are not limited to: substitution of Y with F, substitution of T with S or K, substitution of P with A, substitution of E with D or Q, substitution of N with D or G, substitution of R with K, substitution of G with N or A, substitution of T with S or K, substitution of D with N or E, substitution of I with L or V, substitution of F with Y, substitution of S with T or A, substitution of R with K, substitution of G with N or A, substitution of K with R, substitution of A with S, substitution of K or P.

[0054] In alternative embodiments, conserved amino acid substitutions that are suitably contained within amino acids in a protein or peptide may be selected, for example, conserved substitutions suitable for amino acids within a protein or peptide (i.e., amino acids not exposed to a solvent) may be used, and for example, the following conserved substitutions may be used, but are not limited to these: Y is substituted with F, T is substituted with A or S, I is substituted with L or V, W is substituted with Y, M is substituted with L, N is substituted with D, G is substituted with A, T is substituted with A or S, D is substituted with N, I is substituted with L or V, F is substituted with Y or L, S is substituted with A or T, and A is substituted with S, G, T, or V. In some embodiments, non-conserved amino acid substitutions are also included within the terminology of variants.

[0055] In some embodiments, the polypeptide is a derivative of a natural Cav-1 polypeptide. As used herein, the term “derivative” refers to a peptide chemically modified by techniques such as, for example, acetylation, ubiquitination, labeling, pegylation (derivativeation with polyethylene glycol), lipidation, glycosylation, amidation, or addition of other molecules. A molecule is also a “derivative” of another molecule if it contains additional chemical parts that are not normally part of the molecule. Such parts can alter pH, or improve the stability, solubility, absorption, biological half-life, etc., of the molecule. Alternatively, such parts can reduce the toxicity of the molecule, eliminate or mitigate any undesirable side effects of the molecule. Parts capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences, 18th edition, ARGennaro, Ed., MackPubl., Easton, PA (1990), which is incorporated herein by reference in its entirety.

[0056] When used in combination with "derivative" or "mutant," the term "functional" refers to the polypeptide of the present invention having substantially similar biological activity (either functional or structural) to the biological activity of the entity or molecule that is a functional derivative or functional mutant. The term "functional derivative" is intended to include molecular fragments, analogs, or chemical derivatives.

[0057] In some embodiments, amino acid substitutions can be made in a polypeptide at one or more positions where the substitution is for amino acids with similar hydrophilicity. The importance of the hydropathic amino acid index in conferring interactive biological function to proteins is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydrophilic properties of amino acids contribute to the secondary structure of the resulting protein, which then governs the interaction between the protein and other molecules, such as enzymes, substrates, receptors, DNA, antibodies, and antigens. Therefore, such conservative substitutions can be made in polypeptides and are likely to have only a slight effect on their activity. As detailed in U.S. Patent No. 4,554,101, the following hydrophilic values ​​are 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), and tryptophan (-3.4). These values ​​can be used as guidelines, and therefore, substitutions of amino acids with a hydrophilicity value of ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more preferred. Thus, any polypeptide described herein can be modified by substituting amino acids with different but homologous amino acids having similar hydrophilicity values. Amino acids with hydrophilicity within ± / -1.0 or ± / -0.5 points are considered homologous.

[0058] The Cav-1 peptide may undergo co-translational and post-translational (C-terminal peptide cleavage) modifications, such as disulfide bond formation, glycosylation, acetylation, phosphorylation, and proteolytic cleavage (e.g., cleavage by furin or metalloproteinases), to the extent that such modifications do not affect the anti-inflammatory properties or ability to improve blood glucose control of the isolated peptide.

[0059] In some embodiments, Cav-1 peptides contain amino acids that do not exist naturally. The peptides may contain a combination of naturally occurring and non-naturally occurring amino acids, or may contain only non-naturally occurring amino acids. Non-naturally occurring amino acids may include synthetic non-natural amino acids, substituted amino acids, or one or more D-amino acids in the peptide (or other components of the composition, excluding the protease recognition sequence) may be desirable in certain situations. D-amino acid-containing peptides exhibit increased stability in vitro or in vivo compared to L-amino acid-containing forms. Therefore, the construction of peptides incorporating D-amino acids may be particularly useful when higher in vivo or intracellular stability is desired or required. More specifically, D-peptides are resistant to endogenous peptidases and proteases, thereby providing better oral transepithelial and transdermal delivery of conjugated drugs and conjugates, improved bioavailability of membrane-permanent complexes (see below for further details), and extended intravascular and interstitial lifespan where such properties are desirable. The use of D-isomerized peptides can also facilitate transdermal and oral transepithelial delivery of conjugated drugs and other cargo molecules. Furthermore, D-peptides are not efficiently able to process major histocompatibility complex class I restriction presentation to T helper cells, and therefore are less likely to induce a humoral immune response throughout the organism. Thus, peptide conjugates can be constructed, for example, using the D-isomerized form of a cell-permeable peptide sequence, the L-isomerized form of a cleavage site, and the D-isomerized form of a therapeutic peptide.

[0060] In addition to the 20 “standard” L-amino acids, D-amino acids or non-standard modified or abnormal amino acids as clearly defined in the art are also intended for use in this disclosure. Phosphorylated amino acids (Ser, Thr, Tyr), glycosylated amino acids (Ser, Thr, Asn), β-amino acids, GABA, and ω-amino acids are further intended for use in this disclosure. These include, for example, β-alanine (β-Ala) and other ω-amino acids, such as 3-aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid, etc.; α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly); ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (Mele); phenylglycine (Phg); norleucine (Nle); 4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine This includes fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); homoarginine (hArg); N-acetyllysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,4-diaminobutyric acid (Dab); p-aminophenylalanine (Phe(pNH2)); N-methylvaline (MeVal); homocysteine ​​(hCys), homophenylalanine (hPhe), and homoserine (hSer); hydroxyproline (Hyp), homoproline (hPro), N-methylated amino acids, and peptoids (N-substituted glycines).

[0061] Carboxylic acid terminal modifications include acylation, acetylation, and biotinylation using carboxylic acids, formic acid, acetic acid, propionic acid, fatty acids (myristic acid, palmitic acid, stearic acid), succinic acid, benzoic acid, and carbobenzoxy (Cbz). Amino-terminant modifications include (i) acylation with carboxylic acids, formic acid, acetic acid, propionic acid, fatty acids (such as myristic acid, palmitic acid, and stearic acid), succinic acid, benzoic acid, and carbobenzoxy (Cbz); (ii) biotinylation; (iii) amidation; (iv) addition of dyes such as fluorescein (FITC, FAM, etc.), 7-hydroxy-4-methylcoumarin-3-acetic acid, 7-hydroxycoumarin-3-acetic acid, 7-methoxycoumarin-3-acetic acid, and other coumarins; rhodamine (5-carboxyrhodamine 110 or 6G, 5(6)-TAMRA, ROX); N-[4-(4-dimethylamino)phenylazo]benzoic acid (Dabcyl), 2,4-dinitrobenzene (Dnp), 5-dimethylaminonaphthalene-1-sulfonic acid (Dansyl), and other dyes, as well as (v) polyethylene glycol.

[0062] Polypeptides can be capped at their N-terminus and C-terminus with acyl ("Ac") and amide ("Am") groups, respectively; for example, they can be capped with acetyl (CH3CO-) at the N-terminus and with amide (-NH2) at the C-terminus. A broad N-terminal capping function is desired, preferably through binding to terminal amino groups, for example, formyl; Alkanoyl compounds having 1 to 10 carbon atoms, such as acetyl, propionyl, and butyryl; Alkenoyls having 1 to 10 carbon atoms, for example, hexa-3-enoyl; Alkynoyls having 1 to 10 carbon atoms, for example, hexa-5-inoyl; Aroyl, for example, benzoyl or 1-naphthoyl; Heteroalloyl, e.g., 3-pyroyl or 4-quinoyl; Alkyl sulfonyl, for example, methanesulfonyl; Aryl sulfonyls, for example, benzenesulfonyl or sulfanillyl; Heteroarylsulfonyls, e.g., pyridine-4-sulfonyl; Substitutive alkanoyl compounds having 1 to 10 carbon atoms, e.g., 4-aminobutyryl; Substitutive alkenoyls having 1 to 10 carbon atoms, e.g., 6-hydroxy-hexa-3-enoyl; Substitutive alkinoyls having 1 to 10 carbon atoms, e.g., 3-hydroxy-hexa-5-inoyl; Substituted aroyls, e.g., 4-chlorobenzoyl or 8-hydroxy-naphtho-2-oil; Substituted heteroaloyl, e.g., 2,4-dioxo-1,2,3,4-tetrahydro-3-methylquinazoline-6-oil; Substitutive alkylsulfonyls, e.g., 2-aminoethanesulfonyl; Substitutive aryl sulfonyls, e.g., 5-dimethylamino-1-naphthalenesulfonyl; Substitutive heteroarylsulfonyls, e.g., 1-methoxy-6-isoquinoline sulfonyl; Carbamoyl or thiocarbamoyl; Substituted carbamoyl (R'-NH-CO) or substituted thiocarbamoyl (R'-NH-CS) (wherein R' is alkyl, alkenyl, alkynyl, aryl, heteroaryl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted aryl, or substituted heteroaryl); Substituted carbamoyl (R'-NH-CO) and substituted thiocarbamoyl (R'-NH-CS) (wherein R' is alkanoyl, alkenoyl, alkinoyl, aroyl, heteroaloyl, substituted alkanoyl, substituted alkenoyl, substituted alkinoyl, substituted aroyl, or substituted heteroaloyl), all as defined above.

[0063] The C-terminal capping function can be either an amide bond or an ester bond with the terminal carboxyl. The capping function that provides the amide bond is NR 1 R 2 It is defined as R1 and R 2 This can be cited independently from the following groups:

[0064] Alkyl atoms preferably having 1 to 10 carbon atoms, such as methyl, ethyl, and isopropyl; Alkenyls having 1 to 10 carbon atoms, for example, prop-2-enyl; Alkynnyls having 1 to 10 carbon atoms, for example, prop-2-inyl; Substitutive alkyl groups having 1 to 10 carbon atoms, for example, hydroxyalkyl, alkoxyalkyl, mercaptoalkyl, alkylthioalkyl, halogenoalkyl, cyanoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkanoylalkyl, carboxyalkyl, carbamoylalkyl; Substitutive alkenyls having 1 to 10 carbon atoms, for example, hydroxyalkenyls, alkoxyalkenyls, mercaptoalkenyls, alkylthioalkenyls, halogenoalkenyls, cyanoalkenyls, aminoalkenyls, alkylaminoalkenyls, dialkylaminoalkenyls, alkanoylalkenyls, carboxyalkenyls, carbamoylalkenyls; Substitutive alkynyls having 1 to 10 carbon atoms, for example, hydroxyalkynyls, alkoxyalkynyls, mercaptoalkynyls, alkylthioalkynyls, halogenoalkynyls, cyanoalkynyls, aminoalkynyls, alkylaminoalkynyls, dialkylaminoalkynyls, alkanoylalkynyls, carboxyalkynyls, carbamoylalkynyls; Aroylalkyl compounds having up to 10 carbon atoms, for example, phenacyl or 2-benzoylethyl; Aryl, for example, phenyl or 1-naphthyl; Heteroaryls, e.g., 4-quinolyl; Alkanoyl compounds having 1 to 10 carbon atoms, such as acetyl or butyryl; Aroyl, for example, benzoyl; Heteroalloyl, e.g., 3-quinoloyl; OR' or NR'R'', where R' and R'' are independently hydrogen, alkyl, aryl, heteroaryl, acyl, aroyl, sulfonyl, sulfumyl, or SO2-R'''' or SO-R'''', and R''' is a substituted or unsubstituted alkyl, aryl, heteroaryl, alkenyl, or alkynyl.

[0065] The capping function that provides the ester bond is indicated as OR, where R may be an alkoxy, aryloxy, heteroaryloxy, aralkyloxy, heteroaralkyloxy, substituted alkoxy, substituted aryloxy, substituted heteroaryloxy, substituted aralkyloxy, or substituted heteroaralkyloxy.

[0066] Either the N-terminal or C-terminal capping function, or both, may result in a structure in which the capped molecule spontaneously or enzymatically transforms in the body to release the active drug and functions as a prodrug (a pharmacologically inactive derivative of the parent drug molecule) with improved delivery properties compared to the parent drug molecule (Bundgaard H, Ed: Design of Prodrugs, Elsevier, Amsterdam, 1985).

[0067] A wise selection of capping groups allows for the addition of other activities to the peptide. For example, the presence of a sulfhydryl group attached to the N-terminal or C-terminal cap allows the derivatized peptide to be conjugated to other molecules.

[0068] In yet another embodiment, a peptide or a fragment or derivative thereof may be a “retroinversopeptide.” A “retroinversopeptide” refers to a peptide having a reversal of the direction of the peptide bond at at least one position, i.e., a reversal of the amino-terminus and carboxyl-terminus with respect to the amino acid side chain. Thus, retroinverso analogs have the ends reversed and the direction of the peptide bond reversed while largely maintaining the side chain topology as in the natural peptide sequence. Retroinversopeptides may contain L-amino acids, D-amino acids, or mixtures of L-amino acids and D-amino acids, where at most all amino acids are D-isomers. Partial retroinversopeptide analogs are polypeptides in which only a portion of the sequence is reversed and replaced with enantiomer amino acid residues. Because the retro-inverted portion of such analogs has the amino-terminus and carboxyl-terminus reversed, the amino acid residues adjacent to the retro-inverted portion are replaced with α-substituted geminal-diaminomethane and malonic acid, respectively, which have similar side chains. The retroinverso form of cell-permeable peptides has been found to function efficiently when moving across membranes, just as it does in its native form. The synthesis of retroinversopeptide analogs is described in Bonelli, F. et al., Int J Pept Protein Res. 24(6):553-6 (1984); Verdini, A and Viscomi, GC, J. Chem. Soc. Perkin Trans. 1:697-701 (1985), and U.S. Patent No. 6,261,569, which are incorporated herein by reference in their entirety. A process for the solid-phase synthesis of partial retroinversopeptide analogs is also described (EP97994-B), which is also incorporated herein by reference in its entirety.

[0069] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a specific percentage (e.g., 80%, 85%, 90%, or 95%) of “sequence identity” or “homology” to another sequence means, which means that when aligned, the proportion of bases (or amino acids) is the same in a comparison of the two sequences. This 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 (FMAusubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, which uses default parameters. Particularly preferred programs are BLASTN and BLASTP, which use the following default parameters. Genetic code=Standard;Filter=None;Stratum=Both;Cutoff=60;Expected=10;Matrix=BLOSUM62;Description=50 sequences;Screening=High score;Database=Non-redundant, GenBank+EMBL+DDBJ+PDB+GenBankCDS translations+SwissProtein+SPupdate+PIR.

[0070] B. Multimeric polypeptides Embodiments of this disclosure also include longer polypeptides constructed from repeating units of the Cav-1 peptide. Polypeptide polymers may comprise different combinations of polypeptides. Such polymer polypeptides can be produced by chemical synthesis or recombinant DNA techniques as discussed herein. When produced by chemical synthesis, the oligomers preferably have 2 to 5 repeats of the core polypeptide sequence, and the total number of amino acids in the polymer should not exceed about 160 residues, preferably 100 residues (or equivalent thereto, if linkers or spacers are included).

[0071] C. Peptide mimetic Cav-1 peptides can be peptide mimetic compounds that mimic the biological effects of the natural Cav-1 polypeptide. Peptide mimetic agents can be non-natural peptides or non-peptide agents that replicate the steric spatial properties of the binding elements of the natural Cav-1 polypeptide to have the binding activity and biological activity of the natural Cav-1 polypeptide. Like the natural Cav-1 polypeptide or polypeptide polymer, peptide mimetic agents have a binding surface (which interacts with any ligand to which natural Cav-1 binds) and a non-binding surface.

[0072] In some embodiments, the disclosure also includes compounds that retain partial peptide properties. For example, any protein-degradable bonds in the peptides of the present invention can be selectively replaced by non-peptide elements, such as equivalents (N-methylated; D-amino acids) or reduced peptide bonds, while the remainder of the molecule retains its peptide properties.

[0073] Peptide mimetic compounds that act as agonists, substrates, or inhibitors have been described for many bioactive peptides / polypeptides, including opioid peptides, VIP, thrombin, and HIV proteases. Methods for designing and preparing peptide mimetic 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). Specific mimics that mimic secondary structures are described in Johnson et al., In: Biotechnology and Pharmacy, Pezzuto et al., Chapman and These methods are described in Hall (Eds.), NY, 1993. These methods are used to produce peptide mimes that possess at least the binding ability and specificity of the natural Cav-1 polypeptide, and preferably also possess biological activity. Peptide chemistry and general organic chemistry knowledge available in the art is sufficient to design and synthesize such compounds, taking into account the present disclosure.

[0074] For example, such peptide mimetic compounds can be identified by examining the three-dimensional structure of the polypeptide of the present invention, either free or bound in complex with a ligand (e.g., a soluble uPAR or a fragment thereof). Alternatively, the structure of the polypeptide of the present invention bound to a ligand can be obtained by nuclear magnetic resonance spectroscopy. More knowledge about the stereochemistry of the interaction between the peptide and its ligand or receptor will enable the rational design of such peptide mimetic compounds. The structure of the peptide or polypeptide of the present invention in the absence of a ligand can also provide a scaffold for the design of mimetic molecules.

[0075] D.PEG conversion The Cav-1 peptide can be conjugated with heterogeneous polypeptide segments or polymers, such as polyethylene glycol. The polypeptide can be linked to PEG to increase the enzyme's hydrodynamic radius and thus increase serum persistence. The polypeptide can also be conjugated to any targeting agent, such as a ligand, that has the ability to specifically and stably bind to an external receptor (U.S. Patent Publication 20090304666).

[0076] In certain embodiments, the methods and compositions of the embodiments relate to the PEGylation of disclosed polypeptides. PEGylation is the process of covalently bonding a poly(ethylene glycol) polymer chain to another molecule (typically a drug or therapeutic protein). PEGylation is routinely achieved by incubation of reactive derivatives of PEG with a target macromolecule. By covalently bonding PEG to a drug or therapeutic protein, the active agent can be "masked" from the host immune system (reduced immunogenicity and antigenicity) or its hydrodynamic size (size in solution) can be increased, thereby extending circulation time by reducing renal clearance. PEGylation can also confer water solubility to hydrophobic drugs and proteins.

[0077] The first step in PEGylation is to suitably functionalize one or both ends of the PEG polymer. PEG in which each end is activated at the same reactive site is known as "homobifunctional," while PEG derivatives with different functional groups are referred to as "heterobifunctional" or "heterofunctional." Chemically active or activated derivatives of PEG polymers are prepared by adding desired molecules to PEG.

[0078] The selection of suitable functional groups in PEG derivatives is based on the types of reactive groups available in the molecule bound to PEG. Typical reactive amino acids for proteins include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. N-terminal amino groups and C-terminal carboxylic acids are also available.

[0079] The techniques used to form first-generation PEG derivatives typically involve reacting PEG polymers with hydroxyl groups, usually groups that react with anhydrides, acid chlorides, chloroformates, and carbonates. In second-generation PEGylation chemistry, more efficient functional groups, such as aldehydes, esters, and amides, are available for conjugation.

[0080] As the applications of PEGylation become more advanced and sophisticated, the need for heterobifunctional PEGs for conjugation is increasing. These heterobifunctional PEGs are highly useful for joining two components when a hydrophilic, flexible, and biocompatible spacer is required. Preferred terminal groups for heterobifunctional PEGs include maleimides, vinyl sulfones, pyridyl disulfides, amines, carboxylic acids, and NHS esters.

[0081] The most common modifiers, or linkers, are based on methoxy-PEG (mPEG) molecules. Their activity depends on the addition of protein-modifying groups to the alcohol terminus. In some cases, polyethylene glycol (PEG-diol) is used as a precursor molecule. The diol is then modified at both ends to create hetero- or homo-dimeric PEG-binding molecules.

[0082] Proteins are typically PEGylated at nucleophilic sites, such as aprotonated thiols (cysteinyl residues) or amino groups. Examples of cysteinyl-specific modifiers include PEG-maleimide, PEG-iodine acetate, PEG-thiol, and PEG-vinyl sulfone. All four are highly cysteinyl-specific under mild conditions and at neutral to slightly alkaline pH, but each has some drawbacks. The thioether formed with maleimide can be somewhat unstable under alkaline conditions, which may limit formulation options using this linker. Carbamothioate bonds formed with iodine-PEG are more stable, but free iodine can modify tyrosine residues under some conditions. PEG-thiol forms disulfide bonds with protein thiols, but these bonds can also be unstable under alkaline conditions. The PEG-vinyl sulfone reactivity is slower compared to maleimide and iodine-PEG, but the thioether bonds formed are extremely stable. This slower reaction rate also makes it easier to control the PEG-vinyl sulfone reaction.

[0083] Site-directed PEGylation of natural cysteinyl residues is rarely performed because these residues typically exist in the form of disulfide bonds or are required for biological activity. On the other hand, site-directed mutagenesis can be used to incorporate cysteinyl PEGylation sites for thiol-specific linkers. Cysteine ​​mutations must be designed so that the PEGylation reagent is reachable and remains biologically active after PEGylation.

[0084] Amine-specific modification reagents include PEG NHS esters, PEG torecylates, PEG aldehydes, PEG isothiocyanates, and several other reagents. All of these react under mild conditions and are highly specific to amino groups. PEG NHS esters are perhaps among the more reactive reagents, but their high reactivity can make controlling PEGylation reactions difficult on a large scale. PEG aldehydes form an imine with the amino group, which is then reduced to a secondary amine by sodium borohydride. Unlike sodium borohydride, sodium borohydride does not reduce disulfide bonds. However, this chemical is highly toxic and must be handled with care, especially at low pH levels where it becomes volatile.

[0085] Site-specific PEGylation can be difficult due to the presence of multiple lysine residues in most proteins. Fortunately, since these reagents react with unprotonated amino groups, PEGylation can be directed towards low-pK amino groups by carrying out the reaction at a low pH. Generally, the pK of α-amino groups is 1-2 pH units lower than that of ε-amino groups of lysine residues. High selectivity for the N-terminus can often be obtained by PEGylating molecules at pH 7 or below. However, this is only feasible if the N-terminal portion of the protein is not required for biological activity. Nevertheless, the pharmacokinetic benefits of PEGylation often outweigh the significant loss of in vitro biological activity, resulting in products with much greater in vivo biological activity regardless of the PEGylation chemical reaction.

[0086] When developing a PEGylation procedure, several parameters must be considered. Fortunately, there are usually four or five key parameters. A "design experiment" approach to optimizing PEGylation conditions can be very useful. For thiol-specific PEGylation reactions, parameters to consider include protein concentration, PEG-to-protein ratio (on a molar basis), temperature, pH, reaction time, and in some cases, oxygen exclusion. (Oxygen can contribute to intermolecular disulfide formation by the protein, which reduces the yield of the PEGylated product.) The same factors (excluding oxygen) must be considered for amine-specific modifications, except that pH can become even more important, especially when targeting the N-terminal amino group.

[0087] For both amine-specific and thiol-specific modifications, reaction conditions can affect protein stability. This can be limited by temperature, protein concentration, and pH. In addition, the reactivity of the PEG linker must be known before initiating the PEGylation reaction. For example, if the PEGylating agent is only 70% active, the amount of PEG used must be such that only active PEG molecules are reliably counted in the protein-PEG reaction stoichiometry.

[0088] E. Fusion protein Specific embodiments of the present invention relate to fused Cav-1 peptides. These molecules may have polypeptides of embodiments conjugated to heterologous domains at the N-terminus or C-terminus. For example, leader sequences from other species may also be used in the fusion to enable recombinant expression of the protein in a heterologous host. The fusion protein may contain a half-life extender. Other useful fusions include the addition of a protein affinity tag (e.g., a serum albumin affinity tag or six histidine residues), or, preferably, an immunoactive domain (e.g., an antibody epitope) that is cleavable and facilitates the purification of the fusion protein. Non-limiting affinity tags include polyhistidines, chitin-binding proteins (CBPs), maltose-binding proteins (MBPs), and glutathione-S-transferase (GST).

[0089] In certain embodiments, the peptide of the embodiment may be bound to peptides that increase the in vivo half-life, such as XTEN polypeptide (Schellenberger et al., 2009), IgG Fc domain, albumin, or albumin-binding peptides.

[0090] Methods for producing fusion proteins are well known to those skilled in the art. Such proteins can be produced, for example, by the novel synthesis of a complete fusion protein, or by the conjugation of DNA sequences encoding heterologous domains, followed by the expression of an intact fusion protein.

[0091] The creation of fusion proteins that restore the functional activity of the parent protein can be facilitated by linking a gene to a cross-linking DNA segment encoding a peptide linker spliced ​​between a series of linked polypeptides. The linker is long enough to allow for proper folding of the resulting fusion protein.

[0092] 2. Linker In certain embodiments, the polypeptide of the embodiment may be chemically conjugated using a bifunctional crosslinking reagent or fused with a peptide linker at the protein level.

[0093] Bifunctional crosslinking reagents are widely used for a variety of purposes, including affinity matrix preparation, modification and stabilization of diverse structures, identification of ligand and receptor binding sites, and structural studies. The polypeptides of the embodiments can be linked using suitable peptide linkers, such as Gly-Ser linkers.

[0094] Homodifunctional reagents, possessing two identical functional groups, have been found to be highly efficient in inducing crosslinking between identical and different polymers or polymer subunits, and in linking polypeptide ligands to their specific binding sites. Heterodifunctional reagents contain two different functional groups. By utilizing the different reactivity of the two different functional groups, crosslinking can be controlled both selectively and sequentially. Bifunctional crosslinking reagents can be classified according to the specificity of the functional group, e.g., amino, sulfhydryl, guanidine, indole, or carboxyl. Of these, reagents directed towards free amino acids have gained particular popularity due to their commercial availability, ease of synthesis, and the mild reaction conditions under which they can be applied.

[0095] Most heterobifunctional crosslinking reagents contain a primary amine-reactive group and a thiol-reactive group. Another example describes heterobifunctional crosslinking reagents and methods for using them (U.S. Patent No. 5,889,155, which is incorporated herein by reference in its entirety). The crosslinking reagents combine a nucleophilic hydrazide residue with an electrophilic maleimide residue to enable, in one example, the binding of an aldehyde to a free thiol. Crosslinking reagents can be modified to crosslink various functional groups.

[0096] Furthermore, any other linking / coupling agents and / or mechanisms known to those skilled in the art, such as antibody-antigen interactions, avidin-biotin linkages, amide links, ester links, thioester links, ether links, thioether links, phosphoester links, phosphoramide links, anhydrides, disulfide links, ionic interactions and hydrophobic interactions, bispecific antibodies and antibody fragments, or combinations thereof, may be used to combine the polypeptides of the embodiments.

[0097] It is preferable to employ crosslinking agents that have reasonable stability in the bloodstream. Numerous types of linkers containing disulfide bonds are known that can be successfully used to conjugate targeting agents and therapeutic / preventive agents. Linkers containing sterically obstructed disulfide bonds may be found to provide even greater stability in vivo. Therefore, these linkers constitute a group of linking agents.

[0098] In addition to interfering crosslinking agents, non-interfering crosslinking agents can also be employed according to this specification. Other useful crosslinking agents that are not considered to contain or produce protected disulfides include SATA, SPDP, and 2-iminothiolane (Wawrzynczak and Thorpe, 1987). The use of such crosslinking agents is well understood in the art. Another embodiment involves the use of flexible linkers.

[0099] Once chemically conjugated, the peptide is typically purified to separate the conjugate from the unconjugated agonist and other contaminants. Numerous purification methods are available to provide a conjugate of sufficient purity for clinical use.

[0100] For example, 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 methods, such as Blue-Sepharose separation, may also be used. Conventional methods for purifying fusion proteins from inclusion bodies using weak surfactants, such as sodium N-lauroyl sarcosinate (SLS), may also be useful.

[0101] 3. Cell permeability and membrane-transfer peptides Furthermore, in certain embodiments, the Cav-1 peptide may further comprise a cell-binding domain or a cell-permeable peptide (CPP). As used herein, the terms “cell-permeable peptide” and “membrane transition domain” are interchangeable and refer to a segment of the polypeptide sequence that enables the polypeptide to cross the cell membrane (e.g., the plasma membrane in eukaryotic cells). Examples of CPP segments include, but are not limited to, segments derived from HIV Tat (e.g., GRKKRRQRRRPPQ (SEQ ID NO: 23)), herpesvirus VP22, Drosophila Antennapedia homeobox gene products, protegrin I, penetratin (RQIKIWFQNRRMKWKK (SEQ ID NO: 24)), or melittin (GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 25)). In certain embodiments, CPPs include T1 (TKIESLKEHG (SEQ ID NO: 26)), T2 (TQIENLKEKG (SEQ ID NO: 27)), 26 (AALEALAEALEALAEALEALAEAAAA (SEQ ID NO: 28)), or INF7 (GLFEAIEGFIENGWEGMIEGWYGCG (SEQ ID NO: 29)) CPP sequences.

[0102] III.How to use One aspect of the present invention relates to the use of the peptides described herein, and their mutants, variants, analogs, or derivatives. Specifically, these methods relate to compositions for use in the treatment of a disease, injury, or infection of the lung (e.g., a fibrotic condition of the lung) by administering any one of the peptides described herein or a pharmaceutically acceptable modification thereof to a subject as a dry powder, the composition comprising the polypeptide of the embodiment in a pharmaceutically acceptable carrier.

[0103] A. Pharmaceutical Compositions The Cav-1 peptides provided herein can be administered systemically or topically to inhibit cell apoptosis and for the treatment and prevention of lung tissue damage. They can be administered intravenously, subcutaneously, intramuscularly, intrathecally, and / or intraperitoneally. For example, dry powder formulations can be administered to subjects by intravenous infusion (e.g., subcutaneous infusion) or can be reconstituted with liquid before injection. In certain embodiments, the peptides are delivered topically 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 anti-fibrous compounds.

[0104] Cav-1 peptide dry powder may be administered simultaneously or sequentially in combination with at least one additional therapeutic agent (e.g., a therapeutic agent for the treatment of pulmonary fibrosis). The additional therapeutic agent may be an NSAID, steroid, DMARD, immunosuppressant, bioresponse modulator, bronchodilator, or antifibrotic agent, such as pirfenedone, an agent whose antifibrotic mechanism is not fully understood but may involve TGF-beta blockade, nintedanib, a broad tyrosine kinase blocker, or any other antifibrotic agent. Suitable NSAIDs include non-selective COX inhibitors such as acetylsalicylic acid, mesalazine, ibuprofen, naproxen, fluviprofen, fenoprofen, fenbufen, ketoprofen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, thioxaprofen, suprofen, aluminoprofen, tiaprofenic acid, fluprofen, indomethacin, sulindac, tolmetine, zomepirac, nabumetone, diclofenac, fenclofenac, and alclofe Selected from Nac, bromfenac, ibufenac, aceclofenac, acemetacin, fentiazac, cridanac, etodolac, oxynac, mefenamic acid, meclofenamic acid, flufenamic acid, difluminic acid, tolfenamic acid, diflunisal, fluphenisal, piroxicam, tenoxicam, lornoxicam, and nimeslide, as well as their pharmaceutically acceptable salts, selective COX2 inhibitors meloxicam, celecoxib, and lofecoxib, as well as their pharmaceutically acceptable salts. Preferred steroids are prednisone, prednisolone, methylprednisolone, dexamethasone, budenoside, fluocortone, and triamcinolone. Suitable DMARDs include sulfasalazine, orsalazine, chloroquine, gold derivatives (Auranofin), D-penicillamine, and cell proliferation inhibitors such as methotrexate and cyclophosphamide. Suitable immunosuppressants include cyclosporine A and its derivatives, mycophenolate tomofetil, FK506, OKT-3, ATG, 15-desoxysperguarine, mizoribine, misoprostol, rapamycin, leflunomide, and azathioprine.Suitable bio-response modifiers are interferon-beta, anti-TNF-α (etanercept), IL-10, anti-CD3, or anti-CD25. Suitable bronchodilators are ipratropium bromide, oxytropium bromide, tiotropium bromide, epinephrine hydrochloride, salbutamol, terbutamol sulfate, fenoterol hydrobromide, salmeterol, and formoterol. In such combinations, each active ingredient can be administered (e.g., orally or by inhalation) according to either its usual dose range or a dose below the usual dose range. The combined dose of NSAIDs, steroids, DMARDs, immunosuppressants, and bio-response modifiers is from 1 / 50 of the minimum dose usually recommended to 1 / 1 of the usual dose, preferably 1 / 20 to 1 / 2, more preferably 1 / 10 to 1 / 5. The usual recommended doses of concomitant drugs are, for example, RoteListe® 2002, Edition. It should be understood that these are the dosages disclosed in Cantor Verlag Aulendorf, Germany, or in the Physician's Desk Reference.

[0105] When intended for clinical use, it may be necessary to prepare pharmaceutical compositions containing proteins, antibodies, and drugs in a form suitable for the intended use. Generally, pharmaceutical compositions may contain one or more polypeptides of effective embodiments, or additional drugs dissolved or dispersed in a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” means molecular elements and compositions that, when appropriately administered to animals, e.g., humans, do not produce adverse reactions, allergic reactions, or other side effects. The preparation of pharmaceutical compositions containing at least one polypeptide of an embodiment isolated by the methods disclosed herein, or additional active ingredients, is known to those skilled in the art in light of this disclosure, as exemplified in Remington's Pharmaceutical Sciences, 18th edition, 1990 (incorporated herein by reference). Furthermore, it is understood that, for animal (e.g., human) administration, formulations must meet bioburden, sterility, pyrogenicity, general safety, and / or purity standards required by the FDA Biological Standards Bureau or other appropriate regulatory authorities.

[0106] Specific embodiments of the present invention may include different types of carriers depending on whether they are administered in solid, liquid, or aerosol form, and whether sterilization is required for the administration route, such as injection. The compositions may be administered intravenously, intrathecally, intradermally, percutaneously, intrathecally, intraarterially, intraperitoneally, intranasally, vaginally, intrarectally, intramuscularly, subcutaneously, mucous membranely, orally, topically, locally, by inhalation (e.g., inhalation of a spray formulation), by injection, infusion, continuous infusion, local perfusion directly immersing target cells, via catheter, by lavage, in a lipid composition (e.g., liposomes), or by other methods or any combination known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th edition, 1990, incorporated herein by reference). The selection of injection volume and needle size may be made by those skilled in the art based on the injection site, syringeability, and injectability, and includes considering the viscosity of the solution or suspension to be injected, as well as the drug concentration, pH, and osmotic pressure. In some cases, the particle size of the activator can be selected to provide a desired dissolution rate at the time of administration (e.g., by subcutaneous injection).

[0107] The peptides presented herein can be formulated in compositions in the form of free bases, neutrals, zwitterions, or salts. Pharmaceutically acceptable salts include acid addition salts, such as those formed with free amino groups of protein-like compositions, or those formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids (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, potassium, ammonium, calcium, or ferric hydroxide, or organic bases such as isopropylamine, trimethylamine, histidine, or procaine. During formulation, the solution is administered in a therapeutically effective dose in a manner compatible with the dosage formulation. The formulations are readily administered in various dosage forms, for example, for parenteral administration, such as injectable solutions or aerosols for pulmonary delivery, or for gastrointestinal administration, such as drug-release capsules.

[0108] Further according to a particular aspect of the present invention, a composition suitable for administration may be provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier may, in some aspects, include an aerosol, gas, liquid, semi-solid, i.e., paste, or solid carrier. It is appropriate to use an administerable composition for use in the practice of this method unless any conventional culture medium, active substance, diluent, or carrier is detrimental to the therapeutic effect of the recipient or the composition contained therein. Examples of carriers or diluents include fats, oils, water, saline, lipids, liposomes, resins, binders, fillers, or combinations thereof. The composition may also include various antioxidants to delay the oxidation of one or more components. Furthermore, preservatives such as various antimicrobial and antifungal agents, including but not limited to parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof, can provide prevention of microbial action.

[0109] According to certain aspects of the present invention, the composition is combined with the carrier in any convenient and practical manner, namely by solution, suspension, emulsification, mixing, encapsulation, and adsorption. Such procedures are commonplace for those skilled in the art.

[0110] In certain embodiments of the present invention, the composition is completely combined or mixed with a semi-solid or solid carrier. Mixing can be carried out by any conventional method, for example, by grinding. Stabilizers may also be added in 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 composition include buffers, amino acids, e.g., glycine and lysine, carbohydrates or freeze-drying agents, e.g., dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, and the like.

[0111] In some embodiments, the pharmaceutical formulation comprises one or more surfactants. Surfactants used according to the disclosed method include ionic and nonionic surfactants. Typical nonionic surfactants include polysorbates, e.g., TWEEN®-20 and TWEEN®-80 surfactants (ICI Americas, Bridgewater, New Jersey). Inc.); Poloxamer (e.g., Poloxamer 188); TRITON® surfactant (Sigma, St. Louis, Missouri); Sodium dodecyl sulfate (SDS); Sodium octyl glycoside; Lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; Lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; Linoleyl-, myristyl-, or cetyl-betaine; Lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or (e.g., lauroamidopropyl); Myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; Sodium methyl cocoyl-, or disodium methyl oleyl-taurate; MONAQUAT® surfactant (Mona Industries, Patterson, New Jersey) This includes polyethyl glycol; polypropylene glycol; block copolymers of ethylene and propylene glycol, such as PLURONIC® surfactant (BASF, Mount Olive, New Jersey); oligo(ethylene oxide) alkyl ethers; alkyl(thio) glucosides, alkyl maltosides; and phospholipids. For example, surfactants may be present in the formulation in amounts of about 0.01% to about 5% (weight of surfactant relative to the total weight of other solid components of the formulation; "w / w"), about 0.03% to about 5%, 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 further embodiments, the pharmaceutical formulation of the embodiment is essentially free of nonionic surfactants or essentially free of all surfactants.

[0112] With respect to the therapeutic methods of the present invention, the administration of one or more peptides or their mutants, variants, analogs, or derivatives disclosed herein is not intended to be limited to any particular mode of administration, dosage, or frequency of administration. The present invention envisions all modes of administration, including intramuscular, intravenous, intraperitoneal, intravesical, intra-articular, intralesional, subcutaneous, or any other route sufficient to provide a dose sufficient to treat inflammation-related disorders. The therapeutic agent may be administered to a patient in a single dose or in multiple doses. When multiple doses are administered, the doses may be separated from each other, for example, by 1 hour, 3 hours, 6 hours, 8 hours, 1 day, 2 days, 1 week, 2 weeks, or 1 month. For example, the therapeutic agent may be administered for, for example, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20 weeks or more. It should be understood that for a particular subject, a particular administration regimen may need to be adjusted over time according to the 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 dosage of the therapeutic agent can be increased.

[0113] While the attending physician ultimately determines the appropriate dose and administration regimen, the therapeutically effective dose of one or more polypeptides or their mutants, variants, analogs, or derivatives disclosed herein may be provided in doses of 0.0001, 0.01, 0.01, 0.1, 1, 5, 10, 25, 50, 100, 500, or 1,000 mg / kg or g / kg. The effective dose may be extrapolated from dose-response curves obtained from bioassays or systems in in vitro or animal model studies.

[0114] The dosage for a particular patient or subject can be determined by those skilled in the art using conventional considerations (e.g., by appropriate conventional pharmacological protocols). A physician may, for example, prescribe a relatively low dose initially and then increase the dose until an appropriate response is obtained. The dose administered to a patient is sufficient, depending on the application, to influence the patient's beneficial therapeutic response over time, or, for example, depending on the application, to reduce symptoms or other appropriate activity. The dose is determined by the potency of the particular formulation, as well as the activity, stability, or serum half-life of one or more polypeptides or their mutants, variants, analogs, or derivatives disclosed herein, the patient's condition, and the patient's body weight or surface area being treated.

[0115] In some embodiments, the subject is given a single dose once daily to treat a subject, preferably a mammal, more preferably a human suffering from or susceptible to pulmonary fibrosis resulting therefrom, for example via intravenous infusion (by inhalation), in a dose of about 0.2 mg / kg to about 250 mg / kg, for example, about 10 mg / kg to about 50 mg / kg. Such a dose may be administered daily for any period of about 3 days to 1 week or more.

[0116] As is well understood in this field, dose adjustments may be necessary, but long-term administration is possible. However, the aforementioned range is suggestive, given the large number of variables in individual treatment regimes and the expectation of significant deviations from these preferred values.

[0117] In the case of continuous administration, for example, using a pump system such as an osmotic pump used in some of the experiments described below, the total dose over a period of about 1 to 2 weeks is preferably in the range of 1 mg / kg to 1 g / kg, preferably 20 to 300 mg / kg, and more preferably 50 to 200 mg / kg. After such a continuous administration regimen, the total concentration of the active compound is preferably in the range of about 0.5 to about 50 μM, preferably about 1 to about 10 μM.

[0118] The effective concentration of the active compound for inhibiting or preventing apoptosis in vitro is in the range of about 0.5 nM to about 100 nM, more preferably in the range of about 2 nM to about 20 nM. The effective dose and optimal dose range can be determined in vitro using the method described herein.

[0119] B. Reduced particle size of dry powder and dry powder suction device. The particle size of the formulation can be reduced by any suitable method, including but not limited to milling, grinding, thin-film freezing, spray drying, or crushing. Grinding can be carried out by any method known in the art, such as air jet grinding, ball grinding, wet grinding, media grinding, high-pressure homogenization, or cryogenic grinding.

[0120] Peptide stability after particle size reduction can be evaluated using techniques known in the art, including spectroscopic techniques such as size exclusion chromatography, electrophoresis, HPLC, mass spectrometry, UV spectroscopy, and circular dichroism spectroscopy, and activity (measured in vitro or in vivo). To perform an in vitro assay of protein stability, the aerosol composition can be collected and then distilled or adsorbed onto a filter. For in vivo assays or for pulmonary administration of the composition to a subject, the apparatus for dry powder dispersion can be adapted 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 qualitatively determined using dynamic light scattering (DLS) (DynaPro-80lTC, Protein Solutions Inc., Charlottesville, Virginia) and / or ultraviolet spectrophotometry.

[0121] In some embodiments, patient treatment with crushed CSP7 may involve regulated drug release. In some embodiments, crushed CSP7 may be formulated for sustained or delayed release. In some embodiments, crushed CSP7 may be formulated for immediate release. In further embodiments, crushed CSP7 may be formulated for both sustained and immediate release (i.e., a dual-release profile).

[0122] In some embodiments, the Disclosure provides a method for administering an inhalable CSP7 composition provided herein. Administration may, but is not limited to, inhalation of pulverized CSP7 using an inhaler. In some embodiments, the inhaler is a passive dry powder inhaler (DPI), such as a Plastiape RSOl single-dose DPI. In a dry powder inhaler, the dry powder is stored in a reservoir and delivered to the lungs by inhalation without the use of a propellant.

[0123] In some embodiments, the inhaler is a single-dose DPI such as DoseOne®, Spinhaler, Rotohaler®, Aerolizer®, or Handihaler. In some embodiments, the inhaler is a multi-dose DPI such as Plastiape RS02, Turbuhaler®, Twisthaler®, Diskhaler®, Diskus®, or Ellipta®. In some embodiments, the inhaler is a multi-dose DPI for the simultaneous delivery of single doses of multiple drugs, such as Plastiape RS04 multi-dose (plurimonodose) DPI. Typically, a dry powder inhaler has the drug stored in an internal reservoir, and the drug is delivered by inhalation with or without a propellant. Other types of dry powder inhalers have pre-divided doses of the drug stored in capsules (e.g., cellulose or gelatin-based) or foil pouches, each of which is punctured by the device to release the dose to the patient. Dry powder inhalers may require an inspiratory flow rate of more than 30 L / min, for example, about 30–120 L / min, for effective delivery. In some embodiments, the efficient aerosolization of the pulverized CSP7 is independent of the inspiratory force. In some embodiments, the dry powder inhaler is 0.01 kPa 0.5 min / L~0.05kPa 0.5 minutes / L, for example, 0.02 kPa 0.5 min / L~0.04kPa 0.5 It has a flow resistance of min / L. Dry powder inhalers (e.g., high resistance, low resistance, passive, active) are selected based on the patient population and their inspiratory capacity.

[0124] In some embodiments, the inhaler may be a metered-dose inhaler. A metered-dose inhaler delivers a defined amount of drug to the lungs in a short burst of aerosolized drug aided by the use of a propellant. A metered-dose inhaler comprises three main parts: a canister, a metering valve, and an actuator, and may utilize a spacer device to slow down the released particles and facilitate the inhalation of the aerosolized cloud by the patient. The pharmaceutical formulation, including the propellant and any necessary excipients, is stored in the canister. The metering valve allows for the dispensing of a specified amount of formulation. The actuator or mouthpiece of the metered-dose inhaler includes a fitting dispensing nozzle and typically includes a dust cap to prevent contamination. The required inspiratory flow rate for using a metered-dose inhaler may be less than 90 L / min, e.g., about 15–90 L / min, preferably about 30 L / min. In some embodiments, the efficient aerosolization of the pulverized CSP7 is independent of the inspiratory force.

[0125] In some embodiments, the inhaler is a nebulizer. Nebulizers are used to deliver drugs in the form of aerosolized mist that is inhaled into the lungs. The formulation is aerosolized by compressed gas or ultrasound. Jet nebulizers are connected to a compressor. The compressor releases compressed gas at high speed through the liquid pharmaceutical formulation, aerosolizing it. The aerosolized drug is then inhaled by the patient. Ultrasonic nebulizers generate high-frequency ultrasound, causing vibrations in internal elements that come into contact with the liquid reservoir of the pharmaceutical formulation, thereby aerosolizing the pharmaceutical formulation. The aerosolized drug is then inhaled by the patient. Nebulizers can utilize flow rates of about 3 to 12 L / min, for example, about 6 L / min. In some examples, a pulverized active substance (e.g., CSP7) can be suspended in a pharmaceutically acceptable liquid carrier vehicle and administered by atomization (e.g., air jet atomization). In further embodiments, the compositions of the embodiments can be administered by vaporization methods (e.g., rapid vaporization) such as those using an e-cigarette device.

[0126] In some embodiments, the composition may be administered on a regular schedule. As used herein, a regular schedule refers to a predetermined specified period. A regular schedule may encompass periods of the same or different lengths, as long as the schedule is predetermined. For example, a regular schedule may include administration twice daily, daily, every two days, every three days, every four days, every five days, every six days, weekly, monthly, or any set number of days or weeks in between. Alternatively, a predetermined regular schedule may include administration twice daily for the first week, followed by daily administration for several months. In some embodiments, the peptide (e.g., CSP7) is administered once daily. In preferred embodiments, the peptide is administered less than once daily, for example, every other day, every three days, or once a week. In some embodiments, the total dose of the peptide (e.g., CSP7) of the embodiment is 1 to 100 mg, for example, 20 to 100, 50 to 100, 10 to 20, 20 to 40, 50 to 70, or 80 to 90 mg.

[0127] In some embodiments, the peptide of the embodiment (e.g., CSP7) may be provided in unit dosage forms (e.g., pre-divided doses) such as capsules, blisters, or cartridges, where the unit dose contains at least 1 mg of the peptide per dose, e.g., at least 5 mg, 10 mg, 15 mg, or 20 mg of the peptide of the embodiment (e.g., CSP7). In some embodiments, the unit dose is 1 to 10 mg (e.g., about 5 mg) of the peptide. In certain embodiments, the unit dosage form does not involve the administration or addition of any excipients and is simply used to hold powder for inhalation (i.e., capsules, blisters, or cartridges are not administered). In some embodiments, two or more unit dosage forms are administered to the subject. For example, in the case of a dry powder inhaler, the peptide of the embodiment may be provided in unit dose capsules, and two or more unit dose capsules (e.g., 3-4) may be administered to the subject by inhalation. In some embodiments, the peptide, such as CSP7, may be administered in high-release doses such as at least 10 mg, preferably at least 15 mg, and even more preferably 20 mg. In some embodiments, administration of the pulverized peptide of the embodiment (e.g., CSP7) results in a particulate dose to the deep lungs, such as more than 5 mg. Preferably, the particulate dose to the deep lungs is at least 10 mg, and more preferably at least 15 mg. In some embodiments, the particulate dose is prepared from one, two, three, four, or five or more capsules containing the dose of the peptide of the embodiment (e.g., CSP7). In some embodiments, the particulate dose is at least 50% of the released dose, for example, at least 60, 65, 70, 75, or 80%.

[0128] In some embodiments, a change in inhalation pressure results in a change in the amount released. In some embodiments, a 3 kPa change in inhalation pressure, for example, from 4 kPa to 1 kPa, results in a reduction in the amount released of less than 25%, for example, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or less.

[0129] In some embodiments, a change in inhalation pressure results in a change in particulate matter dose. In some embodiments, a change in inhalation pressure of 3 kPa, for example, from 4 kPa to 1 kPa, results in a decrease in particulate matter dose of less than 15%, for example, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or less.

[0130] IV. Lung condition for treatment The peptides of the present invention can be used to treat a variety of lung conditions. The lung conditions for treatment may be acute or chronic. Acute lung conditions may be acute lung injury, infection, or chemical-induced. Chronic lung diseases may be the result of injury, infection, or disease.

[0131] A. Lung injury In some embodiments, subjects have acute lung injury (ALI) or infection, or chemical-induced lung injury. In certain embodiments, subjects have cast bronchitis, asthma, chronic obstructive airway / lung disease (COPD), acute respiratory distress syndrome (ARDS), inhalation smoke-induced acute lung injury (ISALI), bronchiectasis, inhalation toxin-induced airway disease (e.g., chlorine or other induced airway disease), exposure to mustard gas, exposure to particulate matter (e.g., silica dust), obstructive bronchiolitis, obstructive bronchiolitis with organizing pneumonia, collagen vascular lung disease (e.g., from lupus, scleroderma, or mixed connective tissue disease), interstitial lung disease (e.g., idiopathic pulmonary fibrosis or sarcoidosis), drug-induced lung disease, and accelerated pulmonary fibrosis (e.g., occurring after acute lung injury, including ARDS). Lung diseases, including chronic obstructive pulmonary disease, asthma, infections, and acute and chronic lung injury leading to fibrosis, constitute the third leading cause of death worldwide (Murray et al., 1997; Rabe et al., 2007; Tsushima et al., 2009). Acute lung injury (ALI) is a serious medical problem among U.S. military personnel. Combat-related ALI can result from a very wide range of etiologies.

[0132] ALI from inhalation injury is treated with inhaled anticoagulants, steroids, beta-agonists, high-frequency ventilation, and extracorporeal membrane oxygenation, but outcomes are variable and generally suboptimal. There are no effective preventive measures other than the barrier of a respiratory mask. Although management of ARDS has advanced significantly, it remains largely symptomatic, and patients must carefully wait for endogenous healing mechanisms to take effect. In-hospital mortality remains above 40% (Matthay et al., 2012). ALI survivors often suffer from chronic respiratory diseases that reduce their quality of life. Any modality that can accelerate recovery and / or prevent late complications such as chronic respiratory failure and pulmonary fibrosis is highly desirable. Early diagnosis of ALI, and more importantly, improved prevention and treatment, are urgently needed. The pathophysiology of ALI from direct inhalation lung injury or ARDS resulting from systemic disease is highly complex and heterogeneous, encompassing systemic and local cardiopulmonary factors such as increased membrane permeability, influx of inflammatory cytokines, oxidative cell damage, fluid movement into compartments, ion channel dysfunction, and many others (Matthay et al., 2012). Clearly, new treatment approaches are needed to manage and prevent lung injuries such as ALI.

[0133] In some embodiments, a method is provided for treating or preventing acute lung injury, lung infection, or lung disease in a subject, comprising administering an effective amount of a peptide containing the amino acid sequence of FTFTVT (SEQ ID NO: 2) or a variant thereof to the subject, wherein the peptide maintains the biological activity of caveolin-1 (Cav-1). In some embodiments, the method of administering the pharmaceutical formulation of the peptide includes inhalation of a dry powder. In certain embodiments, the subject is human.

[0134] B. Lung diseases Lung diseases include pulmonary fibrosis, pneumonia, idiopathic pulmonary fibrosis, cystic fibrosis, chronic obstructive pulmonary disease (COPD), bronchitis, bronchiolitis, bronchiolitis obstructive, asthma, and lung infections, as well as acute and chronic lung injury leading to fibrosis (Murray et al., 1997; Rabe et al., 2007; Tsushima et al., 2009). These diseases are the third leading cause of death worldwide.

[0135] Cystic fibrosis is a genetic disorder of the exocrine and exocrine sweat glands that primarily affects the digestive and respiratory systems. The disease is typically characterized by chronic respiratory infections, pancreatic insufficiency, abnormally mucinous mucus discharge, and premature death. Cystic fibrosis (CF) is characterized by progressive airflow obstruction. A subset of patients with CF also develop airway hyperresponsiveness to inhaled cholinergics (Weinberger, 2002 and Mitchell et al., 1978) and reversibility of airflow limitation in response to bronchodilators (van Haren et al., 1991 and van Haren et al., 1992). The presence of airway hyperresponsiveness and airway obstruction suggests a possible common etiology between CF and other airway narrowing diseases such as asthma or chronic obstructive pulmonary disease (COPD), in which airway smooth muscle dysfunction is thought to contribute to the disease process.

[0136] Lung infections can be caused by bacterial infections. Infectious bacteria include Pseudomonas aeruginosa, Bacillus anthracis, Listeria monocytogenes, Staphylococcus aureus, Salmenellosis, Yersina pestis, Mycobacterium leprae, M.africanum, M.asiaticum, M.aviuin-intracellulaire, M.chelonei abscessus, M.fallax, M.fortuitum, M.kansasii, M.leprae, M.malmoense, M.shimoidei, M.simiae, M.szulgai, M.xenopi, M.tuberculosis, Brucella melitensis, Brucella suis, Brucella abortus, Brucella canis, Legionella pneumonophilia, Francisella tularensis, Pneumocystis carinii, mycoplasma, or Burkholderia cepacia. Bacterial infections can lead to pneumonia.

[0137] Chronic obstructive pulmonary disease (COPD) is a term used to classify two major airflow obstruction disorders: chronic bronchitis and emphysema. Approximately 16 million Americans have COPD, and 80-90% of them were smokers for much of their lives. COPD is the leading cause of death in the United States, with 122,283 deaths in 2003. The cost of COPD in the United States was approximately $20.9 billion in direct medical costs in 2003. Chronic bronchitis is inflammation of the bronchial airways. The bronchial airways connect the trachea to the lungs. When inflamed, the bronchi secrete mucus, causing a chronic cough.

[0138] In emphysema, the alveolar sacs become excessively swollen as a result of damage to the elastin skeleton of the lungs. Inflammatory cells in emphysematous lungs release elastase enzymes, which break down or damage elastin fibers in the lung matrix. There are many causes of emphysema, including smoking, exposure to environmental pollutants, alpha-1 antitrypsin deficiency, and aging.

[0139] Bronchiolitis is most commonly caused by viral lower respiratory tract infections and is primarily characterized by acute inflammation, edema, necrosis of epithelial cells forming the inner walls of the small airways, and increased mucus production (Ralston et al., 2014). Signs and symptoms typically begin with rhinitis and cough and may progress to tachypnea, wheezing, rales, use of accessory muscles, and / or nasal flaring.

[0140] Obstructive bronchiolitis is a progressive decrease in airflow resulting from abnormal remodeling of the small airways in the lungs (Meyer et al., 2014). Obstructive bronchiolitis syndrome is a major complication of lung transplantation and is often used to explain delayed allograft failure, resulting in a persistent decline in forced expiratory vital capacity and vital energy not caused by other known causes (Meyer et al., 2014).

[0141] The term "asthma" can refer to acute asthma, chronic asthma, intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent asthma, chronic persistent asthma, mild to moderate asthma, mild to moderate persistent asthma, mild to moderate chronic persistent asthma, allergic (exogenous) asthma, non-allergic (endogenous) asthma, nocturnal asthma, bronchial asthma, exercise-induced asthma, occupational asthma, seasonal asthma, asymptomatic asthma, gastroesophageal asthma, idiopathic asthma, and cough variant asthma. During asthma, the airways are persistently inflamed and may occasionally experience spasms.

[0142] In some embodiments, a method is provided for treating or preventing a lung infection or lung disease in a subject, comprising administering to the subject an effective amount of a dry powder peptide containing the amino acid sequence of FTFTVT (amino acid sequence 2, referred to herein as CSP7), wherein the dry powder peptide maintains the biological activity of caveolin-1 (Cav-1). In some embodiments, the method of administering the pharmaceutical formulation of the embodiment involves inhalation of the dry powder peptide. In certain embodiments, the subject is human. [Examples]

[0143] V. Examples The following embodiments are included to illustrate preferred embodiments of the present invention. Those skilled in the art should understand that the techniques disclosed in subsequent embodiments demonstrate that the techniques discovered by the inventors are fully functional in carrying out the present invention and therefore constitute a preferred mode of implementation. However, those skilled in the art should understand that, in terms of this disclosure, many modifications can be made in the specific embodiments disclosed, without departing from the spirit and scope of the present invention, while still yielding the same or similar results.

[0144] Example 1 - Method and Materials Preparation of dried powder peptide. CSP7 peptide (SEQ ID NO: 2), lot number: AHF66 / / 470103 was synthesized by Polypeptide Laboratories (San Diego, USA).

[0145] Preparation and spray drying of CSP7 mixtures. CSP7 formulations containing either CSP7 alone (CSP7), or a 75% / 25% mixture of CSP7 / leucine, CSP7 / trehalose, or CSP7 / sodium citrate, or a 75% / 15% / 10% mixture of CSP7 / leucine / trehalose, were prepared in pH 10 water (adjusted with NH4OH) and spray-dried using BLD-35 equipped with a 2'' cyclone.

[0146] Decreased particle size of dried CSP7 powder 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 -55 to -65°C. The frozen thin sections were then processed using VirTis The samples were freeze-dried using an Advantage Freeze Dryer (VirTis Company Inc., New York, USA). The freeze-drying conditions were as follows: Equilibrium: Hold at -55°C for 30 minutes at 100 mTorr; Primary drying: Increase temperature to -30°C for 250 minutes at 100 mTorr; Hold at -30°C for 660 minutes at 100 mTorr; Secondary drying: Increase temperature to 30°C for 720 minutes at 100 mTorr; and hold at 30°C for 240 minutes at 100 mTorr. The samples treated with TFF are referred to as batch 171014.

[0147] Particle size reduction of dry powder CSP7 by cryogenic grinding. 1 gram of CSP7 bulk powder was added to a small cryomill tube and filled into a 6870Freeze / Mill (SPEX Certiprep®, New Jersey, USA). Grinding was performed in 5 cycles with 10 minutes of pre-cooling, each cycle having a 5-minute run time at 10 CPS followed by 2 minutes of cooling. The ground sample was collected and weighed, and the yield was calculated to be 73.5% based on the recovered weight relative to the filled weight.

[0148] Particle size reduction of CSP7 powder by ball grinding (BM). CSP7 bulk powder was suspended in its poor solvent, anhydrous ethanol, to a concentration of 1 mg / ml. Approximately half the amount of zirconium balls (2 mm) were added to the suspension. The suspension was then filled into an 8000 M Mixer / Mill (SPEX SamplePrep, New Jersey, USA) and ground. Samples were collected from the grinding process and tested at 5 minutes, 10 minutes, and 30 minutes, respectively.

[0149] Reduction in particle size of CSP7 powder using a rotor-stator. The 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.

[0150] High-performance liquid chromatography analysis samples were dissolved in 20 mM Tris buffer (pH 10.3) and analyzed using a Phenomenex Luna® C18(2) liquid chromatography column with a particle size of 5 μm and a pore size of 100 Å. A Phenomenex Security Guard Guard cartridge kit was used as the guard column. Mobile phase A was H 2 O + 0.1% (v / v) trifluoroacetic acid (TFA), and mobile phase B was 80% acetonitrile + 20% H2O + 0.09% (v / v) TFA. Samples were injected in an amount of 20 μL. Samples were each run for 25 minutes, and the column was held at 25 °C at a flow rate of 1 ml / min. Samples were detected at a wavelength of 220 nm. The buffer gradient was set to the conditions specified in Table 2.

Table 2

[0151] Determination of the aerodynamic particle size distribution of pulverized CSP7 bulk powder. Approximately 3.5 mg of pulverized CSP7 powder was manually filled into size 3 HPMC capsules (Capsugel, Peapack, NJ). The CSP7 capsules were then aerosolized 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). Aerosols were generated for the inhaler at an airflow rate of 60 L / min for 4 seconds, achieving an inhalation volume of 4 L and a pressure drop of 4 kPa across the entire instrument. Before each run, the NGI collection surface was coated with 5% (v / v) polysorbate 20 in methanol. One capsule was photographed per run, and each sample was repeated three times (3 capsules). After aerosolization, all collection surfaces were rinsed with a specific amount of 20 mM Tris buffer (pH 10.3) and the drug was collected. The capsules, apparatus, adapter, throat, pre-separator, and powder deposited in stages 1-MOC were extracted separately.

[0152] For each test, the delivered dose is defined as the mass of CSP7 that entered the NGI. Geometric standard deviation (GSD), aerodynamic median particle size (MMAD), and particulate matter percentage (FPF%) were calculated using Copley Inhaler Testing Data Analysis Software (CITDAS, Copley Scientific, Nottingham, UK) based on the dose accumulated in stages 1 to MOC of the NGI. FPF is defined as the mass fraction of particles smaller than 5.0 μm, using the delivered dose.

[0153] Preparation and lysis of lung tissue. Female mice aged 6-8 weeks were ordered from Jackson Laboratories, storage: 000664 C57BL / 6J, cared for and housed according to IACUC guidelines. The following week, the mice were weighed, anesthetized with IP injections of 80 mg / kg ketamine and 6 mg / kg xylazine (approximately 115 ul / mouse), and given a single intratracheal infusion of bleomycin. Briefly, a 26G plastic catheter was inserted into the trachea, and the mice received 2 x 20 ul infusions of 0.8 U / kg bleomycin (Biotang, catalog no. RB003) via pipette (at 30-second intervals to allow for removal from the airway). The subjects received only the same volume of saline. Weight was tracked (approximately 10% weight loss occurred in injured animals), and the animals were given a dry powder inhalation protocol (CH technologies) daily for one week. The dry powder administration was based on the minimum effective dose of a previous atomized formulation, estimated to be 0.7 mcg / animal lung delivery dose (Tepper et al., 2016, which is incorporated herein by reference). In summary, an exposure time of 12 minutes / day corresponded to the "1X" dose, while the "5X" dose specified a 60-minute / day treatment equivalent to 3.5 mcg / animal. Animals were exposed for 7 consecutive days (days 14–20, during the bleomycin injury fibrosis period) and sacrificially killed 24 hours after the final lethal dose of heparinized ketamine / xyaridin cocktail (25% heparin). A subset of the whole lung was taken for histology. Briefly, blood was removed from the lung by transcardiac perfusion with 10 ml of saline. The lung was then inflated with saline for 1 minute, followed by 4% PFA for 1 minute, 20 cm above the dissection stage. The trachea was tied off, the lung was resected, fixed, embedded, and sectioned into 4-micron sections to visualize the maximum surface area, and stained with hematoxylin and eosin. Images were captured using an AperioAT2 high-capacity digital full-slide scanner, and the lung was scored for fibrous injury according to a modified Ashcroft scoring protocol (Huebner et al., 2008, incorporated herein by reference).For molecular analysis, the entire lung was homogenized in RIPA buffer and a protease inhibitor (Santa Cruz), as well as 1% DTT (to inhibit RNAses), and homogenized in parallel at 4°C (Precellys Evolution, Bertin Instruments) for downstream assays. The collagen content of the lung homogenate was assayed using the Total Collagen Assay (Quickzyme) with collagen standards provided by the manufacturer, according to the manufacturer's instructions. Colorimetric analysis was read using a microplate reader (FilterMax F5, Molecular Devices, 580 nm). Furthermore, RNA was extracted from the homogenate (Zymogen Research) and reverse transcribed into cDNA (Qiagen, QuantiNova reverse transcription 205413). The results of these studies are shown in Figures 29–32.

[0154] Homogenization buffer for 28 samples was prepared by adding 224 μL of cocktail inhibitor, 224 μL of NaOV4, 224 μL of PMSF, and 0.22 g of DTT to 22.4 mL of RIPA buffer. 800 μL of homogenization buffer was added per sample. Samples were homogenized using a Precellys Evolution equipped with CK28 beads. The homogenization protocol used was for hard tissues and was performed twice at 4°C for each sample. The homogenized samples were then equalized, with 400 μL set aside for BCA concentration determination and protein assays, 200 μL set aside for RNA isolation, and 200 μL set aside for collagen assays.

[0155] Collagen assay. Collagen standards were prepared for use by adding 125 μL of Quickzyme collagen standard to 125 μL of 12M HCl and 200 μL of each sample to 200 μL of 12M HCl. Samples and standard solutions were incubated at 95°C for 20 hours and vortexed briefly after 20 minutes. After incubation, samples were centrifuged at 13,000 xg for 10 minutes. Standards were prepared according to the manufacturer's instructions (Quickzyme). Next, 100 μL of each sample was diluted in 50 μL of water. Next, 10 μL of each diluted sample was further diluted in 100 μL of 4M HCl. Replications of the standards and each sample were pipetted into plates. 75 μL of assay buffer was added to each well, the plate was covered, and shaken for 20 minutes. 75 μL of detection reagent mixture was added per well, the plate was mixed, and incubated at 60°C for 1 hour. Next, the plate was read as described above.

[0156] RNA isolation. RNA isolation was performed using the QiagenRNeasy kit, following the manufacturer's instructions. Briefly, 25 μL of RLT buffer and 75 μL of 70% ethanol were added to 50 μL of sample in RIP A buffer to obtain a total of 150 μL of starting material. Next, 50 μL of the starting material for each sample was added to 50 μL of RNase-free water. Then, 350 μL of RLT buffer was added and the samples were thoroughly mixed. Next, 250 μL of 95-100% ethanol was added to each and the samples were mixed again. Then, 700 μL of each sample was added to the respective spine column and centrifuged at 8000 x g, discarding the pass-through fraction. 500 μL of RPE was added to each column and the columns were centrifuged again. Another 500 μL of RPE was added, and this time the samples were centrifuged at 8000 x g for 2 minutes. The sample was transferred to a new microcentrifuge tube and centrifuged at 8000xg for 1 minute, after which RNA was eluted with 40 μL of RNase-free water. The sample was quantified using nanodrop and analyzed as described above.

[0157] Example 2 - Characterization of CSP7 bulk powder Scanning electron microscopy. A bulk powder sample of CSP7 was sputtered onto a sample tray and spread by blowing compressed nitrogen onto it. The sample was imaged using scanning electron microscopy (Figure 1). The SEM shows the presence of large particles (>5 μm). Furthermore, most of the particles appeared to be large (>5 μm) and therefore outside the breathable range.

[0158] CSP7 particle size evaluation. Particle size was investigated using Spraytec laser diffraction and the Sympatec HELOS-R laser diffraction system equipped with solid or wet dispersion attachments to determine whether the bulk powder was within the breathable range. Using the dry dispersion method, the size of CSP7 bulk powder particles was determined to be larger than the breathable particle size. Table 3 shows the particle sizes of particles evaluated as Dv10, Dv50 (median), and Dv90 within the distribution. As shown in Table 3, more than 50% of all analyzed CSP7 particles had a particle size of 5.3 μm or larger, which is larger than the breathable range. [Table 3]

[0159] Next, particle size was determined by Sympatec laser diffraction using the wet dispersion method. CSP7 was dissolved in ethanol + 0.05% Tween® 80 as the dispersion medium and sonicated for 10 minutes. The size measurement results of CSP7 particles using wet dispersion are shown in Table 4. As shown in Table 4, the average particle size (Dv50) of the wet-dispersed particles was 29.0 ± 0.8, which was well above the breathable range. [Table 4]

[0160] The average particle size was further evaluated using a dry dispersion method and a Spraytec laser diffractometer. The CSP7 bulk powder was dispersed at 40 PSI, and the average particle size (8.6 ± 1.5 μm) was above the breathable range (Table 5). [Table 5]

[0161] The proportion of dry powder particles found to be smaller than 5 μm was found to be only 34.5 ± 4.1%. Considering that each laser diffraction method revealed that the majority of the bulk powder has particle sizes outside the breathable range, the dry powder used in treatment needs to be treated in some way to reduce its particle size.

[0162] Morphology of bulk CSP7 powder. A bulk CSP7 powder sample was sputtered onto a glass slide and observed with an optical microscope (Figure 2). SEM was confirmed by the optical microscope, indicating the presence of large particles (>5 μm). Optical microscopy also shows the presence of particle aggregates, indicated by the arrows in Figure 2.

[0163] Determination of the crystallinity of CSP7 bulk powder particles. CSP7 bulk powder particles were evaluated using X-ray powder diffraction (Figure 3). Neat CSP7 was found to exhibit some crystallinity by X-ray powder diffraction (Figure 3). To confirm the results of X-ray diffraction, the crystallinity was evaluated by polarized light microscopy (Figure 4). As seen in Figure 4, crystalline CSP7 is present in the bulk CSP7 powder, and specific typical crystalline morphologies are indicated by the white arrows in the image.

[0164] Thermal analysis of CSP7 bulk powder. The melting point of neat CSP7 was determined using differential scanning calorimetry (DSC) (Figure 5). The melting point of CSP7 was determined to be 211.03°C, as determined by DSC (Figure 5). Analysis was continued using thermogravimetric analysis (TGA) (Figure 6). TGA shows that the weight of neat CSP7 begins to decrease dramatically above 216°C.

[0165] Moisture content of CSP7 bulk powder. Moisture content of bulk powder CSP7 measured by Mettler The water content was evaluated using the Karl Fischer-Volumetric (KF-V) titration with the Toledo Karl Fischer Titrator, and the test was performed in a triad. Table 6 shows the water content of each test and the average water content of the three tests. [Table 6]

[0166] Next, the moisture adsorption of the CSP7 bulk powder was analyzed using dynamic vapor adsorption (DVS) (Figure 7). The CSP7 sample was subjected to a sufficient adsorption / desorption cycle, and it was found that the sample had 6.32% moisture desorption at zero relative humidity. A mass change of 10.54% was found at 90% relative humidity (Figure 7).

[0167] Example 3 - Characterization of CSP7 powder after particle size reduction Particle size reduction of CSP7 powder. For the powder to be effectively inhaled and deposited in the lungs, the particle size generally needs to have an aerodynamic median particle diameter of less than approximately 5 μm. Various techniques were employed to reduce the particle size of the neat material, including air jet pulverization (AJM), ball pulverization (BM), cryogenic pulverization (CM), thin film freezing (TFF), and spray drying. First, AJM was performed to reduce the particle size of the CSP7 bulk powder, and the pulverized CSP7 was collected from several positions within the pulverizer. Tables 5 and 6 show the yield and particle size distribution of the first batch (batch 171013) of the pulverized powder collected from the indicated positions. The particle size distribution was determined as described above by Sympatec laser diffraction dry dispersion (Table 8) or Sympatec laser diffraction wet dispersion (Table 9). [Table 8] [Table 9-1]

[0168] Using the same conditions as above, the second batch of CSP7 (batch 171027) was ground from 10 grams of neat bulk powder. Again, the powder size distribution and yield were evaluated from the same location and are listed in Table 10. [Table 10]

[0169] A third batch of CSP7 dried powder was subjected to thin-film freezing (TFF) (batch 171014) and analyzed as described above using both Sympatec laser diffraction dry dispersion (Table 11) and wet dispersion (Table 12). [Table 11-1] [Table 12]

[0170] Another batch of CSP7 dry powder was subjected to cryogenic grinding (CM) to reduce particle size. The particle size of CM CSP7 was evaluated as described above by laser diffraction using dry dispersion (Table 13) and wet dispersion (Table 14). [Table 13] [Table 14]

[0171] Another batch of CSP7 bulk powder was subjected to ball grinding (BM) to reduce particle size. Table 15 shows the particle size distribution of the BMCSP7 powder obtained at several points during the grinding process. [Table 15]

[0172] Further batches were prepared using CSP7 (also referred to as CSP7, based on the designated development acronym) and leucine, trehalose, sodium citrate, or a mixture of leucine and trehalose, and subjected to spray drying to reduce particle size. The size of the spray-dried particles was examined by dry dispersion laser diffraction (Figure 8, Table 16). Again, compared to bulk CSP7, spray drying significantly reduced the size of the CSP7 particles. [Table 16]

[0173] CSP7 particle size, when measured by solid dispersion and wet dispersion, decreased dramatically after each method for reducing particle size (air jet grinding, thin film freezing, cryogenic grinding, ball grinding, and spray drying) (compared to Tables 1 and 2, 5, 6, 10, 13, 14, 16, 17, 18, and 19). After air jet grinding, the majority of CSP7 particles remained within the breathable range, but thin film freezing, cryogenic grinding, and ball grinding were less effective, and the proportion of ground powder within the breathable range was lower when measured by laser diffraction using solid or liquid dispersion.

[0174] Particle size was also reduced using a rotor-stator handheld homogenizer (Figure 9). CSP7 changed the color of the ethanol to either light gray or dark gray after homogenization, depending on the duration and force of homogenization (Figure 9). Although this method was not pursued further due to the observed color change, it is clear that homogenization reduced particle size.

[0175] Morphology of the pulverized CSP7 powder. The morphology of each pulverized sample was examined using optical microscopy or scanning electron microscopy. Powder samples from air jet pulverization were examined as described above, and optical microscopy showed that the particle size had decreased to 1 μm > 5 μm and was homogeneous (Figure 10). Furthermore, the pulverized CSP7 particles did not contain aggregates (Figure 10). SEM showed homogenization of the air jet pulverized particles and a reduced particle size between 1 and 5 μm (Figure 11). Powder samples obtained after TFF were also examined, and although the particle size was larger, the samples were found to be free of aggregates (Figure 12). Further analysis included evaluation of the particle morphology of the spray-dried formulation by scanning electron microscopy. Representative SEM images of the formulation are shown in Figure 13.

[0176] Crystallinity of AJMCSP7. The crystallinity of pulverized CSP7 powder (batch 171027) was evaluated by X-ray diffraction, and the diffraction pattern is shown in Figure 14. The crystallinity of spray-dried CSP7 was also examined by X-ray diffraction, and the curve is plotted in Figure 15. Formulations of CSP7 alone or CSP7 conjugated with trehalose or sodium citrate are amorphous, but CSP7 conjugated with leucine or leucine and trehalose appears to contain crystalline leucine properties, as indicated by the sharp peaks in Figure 15.

[0177] HPLC evaluation of pulverized or spray-dried CSP7 powder. To determine whether pulverization of CSP7 bulk powder affects its chemical potency, samples of pulverized CSP7 powder were collected from various parts of the pulverizer and evaluated using HPLC performed under the conditions listed in Table 2. Potency was evaluated using the following formula:

number

[0178] As is evident from Table 26, grinding did not adversely affect the potency of the collected samples. Similarly, when batch 171027 was tested, its chemical potency was determined to be 100.14%. [Table 26-1]

[0179] To determine its purity, the spray-dried CSP7 mixture was examined by RP-HPLC (Table 17, Figure 16). Similar to air-jet ground CSP7 powder, the spray-dried CSP7 retained approximately 100% purity. [Table 17]

[0180] Stability of CSP7. The stability of both untreated bulk CSP7 powder and air-jet ground CSP7 (batch 171027) was investigated by analyzing their chemical potency using HPLC. Each sample was stored under three different conditions, and its chemical potency was assayed at 5, 15, and 32 days of storage (Figure 17). The 24-hour stability of spray-dried CSP7 was also investigated by HPLC (as described in the specifications) 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). [Table 31-1]

[0181] Aerodynamic particle size distribution of pulverized CSP7. To determine the aerodynamic particle size distribution of pulverized CSP7, the amount of powder deposited at various locations in the NGI collector was extracted separately. The delivered dose was measured as the mass of CSP7 that entered the NGI collector after aerosolization, and the amount of CSP7 deposited on individual surfaces was extracted and measured separately. The amount of either untreated or air-jet pulverized (batch 171013) CSP7 remaining in the capsule or deposited in the device, adapter, throat, pre-separator, and stage 1-MOC is shown in Figure 18 as a percentage of the total amount of CSP7 delivered. The percentage of fine particles (FPF%), mass-mean aerodynamic diameter (MMAD), and geometric standard deviation (GSD) of both pulverized and untreated (e.g., unpulverized or untreated) CSP7 is shown in Table 9. [Table 9-2]

[0182] The aerodynamic particle size distribution of the second pulverized CSP7 batch (171027) was determined as described above, except that approximately 4.25 mg of powder was used per HPMC capsule of size 3. The GSD, FPF%, and MMAD are shown in Table 11, and the percentage of CSP7 deposited at each location, again as the percentage of the total amount of CSP7 delivered, is shown in Figure 19. [Table 11-2]

[0183] The aerosolization of the spray-dried formulations was also investigated in a similar manner (Figure 20). Each formulation showed a fine powder fraction of over 60%, and each formulation had MMAD of 2.5 μm to 3 μm (Table 25). A summary of the analytical results of the spray-dried formulations, including moisture content, is shown in Table 25. [Table 25-1]

[0184] Determination of moisture content of pulverized CSP7. Air-jet pulverized CSP7 powder (batch 171027) was analyzed using dynamic vapor adsorption under the same conditions used to analyze bulk CSP7 (Figure 21). Similar to neat bulk powder, the pulverized CSP7 had 4.61% moisture desorption at 0% relative humidity (Figure 21). KF-V analysis revealed a moisture content of 4.9% (Table 7), but at 90% relative humidity, the mass change increased to 13.59% (Figure 21). [Table 7]

[0185] Thermogravimetric analysis of CSP7 powder with reduced particle size. Thermogravimetric analysis was performed on pulverized CSP7 (batch 171027) using the same method as for bulk CSP7, and it was found that the pulverized CSP7 had properties very similar to untreated CSP7 (Figure 22). The thermal properties of the spray-dried formulation were also evaluated and are shown in Table 24 and Figures 23-27 (summarized in Figure 28). In particular, the midpoint Tg of the mixed formulation was significantly lower than that of spray-dried CSP7 alone (comparison of 001C-F and 001B in Table 24). [Table 24]

[0186] The results presented herein demonstrate that various methods are effective in reducing the particle size of CSP7 powder, and that the resulting powders exhibit very similar properties.

[0187] Example 4 - Treatment of bleomycin-induced pulmonary fibrosis by inhalation of CSP7 dry powder Induction of fibrosis and treatment with CSP7 in mice. Pulmonary fibrosis was induced in mice by treatment with bleomycin. Mice were intranasally administered 0.8 U / kg of bleomycin and allowed to wait 14 days to induce disease before treatment. The mice were then left untreated, treated with 12 minutes of inhalation of dry CSP7 powder, or treated with 60 minutes of inhalation of dry CSP7 powder. On the final day of treatment, the mice were euthanized, their lungs were removed, rapidly frozen, and stored at -80°C. The weight of the flash-frozen lungs was measured before further analysis (Figure 29).

[0188] Lung tissue was homogenized and collagen content was analyzed using the Quickzyme collagen assay (Figure 30). Bleomycin-induced fibrosis resulted in a significant increase in collagen in the lungs compared to saline treatment (P=0.0062) (Figure 30). The Ashcroft score, a measure of pulmonary fibrosis in mice, was lower in mice treated with CSP7 after bleomycin-induced pulmonary fibrosis (Figure 31).

[0189] RNA was also prepared from homogenized lung tissue and used as described above.

[0190] Example 5 - Suspension of formulation for intramuscular / subcutaneous injection [Table 25-2]

[0191] Regarding preparation: 1.1. Prepare a 20 mM Tris buffer solution (the pH should be approximately 10.3). 2.2.1. Dissolve 5% (w / w) CMC in 20 mM Tris buffer and add 0.2% (w / w) poloxamer 188. Stir overnight at approximately 600 rpm. 3.3.0.7% (w / w) NaCl is added to the CMC solution. 4.4. Adjust the pH of the solution to 7 by adding approximately 28.5 μL / ml of 1N HCl. 5.5. Weigh and add a specified amount of air-jet ground CSP7 powder (collected from a collection bag to obtain a fraction of small particle size) into a clean vial. 6.6. Using a clean rod, first grind the powder and ensure that there are no visible aggregated particles. 7.7. Gradually add the prepared solution to the vial by milling / grinding using a rod. 8.8. If the powder is completely moistened and visible aggregated particles are found, add an appropriate amount to reach the target volume. result [Table 26-2]

[0192] Example 6 - Formulation solution for intramuscular / subcutaneous injection Regarding preparation: 1.1. Prepare a 20 mM Tris buffer solution (the pH should be approximately 10.3). 2.2.1. Dissolve 5% (w / w) CMC in 20 mM Tris buffer and add 0.2% (w / w) poloxamer 188. Stir overnight at approximately 600 rpm. 3.3.0.7% (w / w) NaCl is added to the CMC solution. 4.4.1.2~1.4 mg / ml CSP7 (untreated powder) is added to the solution and dissolved by vortexing. The pH should be approximately 9, then -50 μL 1N HCl is added to adjust the pH to 8.2~8.5. [Table 27] result [Table 28]

[0193] Example 7 - Pre-formulation study of polypeptide variants The solubility study of the variant was conducted by adding the variant powder to the solvent at a concentration of 5 mg / ml and then vortexing for 3 minutes. Observe the appearance of the solution at 5 minutes. Further add the powder (about 5 mg / ml each time), repeat vortexing, and observe until precipitation or gelation occurs. Results:

Table 29

Table 30

Table 31-2

Table 32

Table 33

[0194] Pre-formulation study of Example 8 - CSP7 (ammonium counter ion) form The dissolution of the neat (i.e., non-milled) CSP7 ammonium counter ion form (Table 35) was carried out by adding an excess amount of peptide powder to 3 mL of different pH buffers (Table 36) and mixing on an orbital shaker at 100 rpm for 24 minutes.

[0195] For the freeze-thaw stability study (for Figure 33), 0.1 mg / mL CSP7 (ammonium counter ion) peptide in phosphate buffer system (PBS, pH 7.4) was aliquoted into 15 mL / vial for rapid and slow freezing, respectively. For rapid 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 completely freeze the aliquots in the vials, and then thawed at room temperature. Each sample was subjected to 5 freeze-thaw cycles. The recovery rate % represents the ratio of each sample concentration compared to the original (untreated) concentration.

[0196] After filtering the solubility and freeze-thaw samples through a 0.45 μm membrane, high-performance liquid chromatography (HPLC, Thermo Fisher Scientific, Fair) was performed. The assay was performed by Lawn, NJ). Briefly, samples were analyzed using a Dionex 3000 HPLC system equipped with a Waters® reversed-phase C18 column, 2.5 μm, 150 mm x 4.60 mm. The HPLC column was heated to 60°C for testing, and peptides were detected at a wavelength of 215 nm and a flow rate of 1 mL / min. Two mobile phases were used: A (0.1% acetic acid in water) and B (0.1% acetic acid in acetonitrile). The injection volume was 20 μL, and standard curves were plotted from 0.01 to 1 mg / ml. [Table 34] [Table 35] [Table 36]

[0197] Example 9: Characterization and stability study of pulverized CSP7 (ammonium counterion) powder as lot number UTA181028 CSP7 peptides were ground using a Model 00 Jet-O-Mizer (trademark) (also known as Aljet mill, Fluid Energy, Telford, Pennsylvania). The feed rate, extrusion pressure, and grinding 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 various sections of the jet grinding process, including the tube after the grinding chamber (bfC), cyclone (C), collection container adapter (D), collection bag adapter (E), collection bag (H), and collection container (G). A Turbula mixer (Glen Mills) was also used. The collected powder was mixed for 10 minutes using Clifton Inc. (New Jersey, USA). [Table 37]

[0198] Specific surface area of ​​ground and CSP7 ammonium ionized neat powder. The specific surface area of ​​ground and untreated CSP7 powder is analyzed using a Monosorb rapid surface area analyzer Model MS-21 (Quantachrome Instruments, Boynton Beach, Florida) and the single-point BET method (Figure 34). Samples are gas-released for 20-24 hours using nitrogen gas at 20 psi at 25°C to remove water and other impurity molecules from the surface. A nitrogen / helium mixture (50:50 v / v) is used as the adsorbent, and the equipment is calibrated with nitrogen before testing.

[0199] Thermogravimetric analysis of pulverized and CSP7 ammonium vs. ionized neat powder. The method described in paragraph

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

[0200] Scanning electron microscope (SEM) images of ground and neat CSP7 (ammonium counterion) powder. The morphology of CSP7 (Figure 36) is analyzed using a Zeiss Supra 40VP SEM (Carl Zeiss Microscopy GmbH, Jena, Germany). Samples are mounted on an aluminum SEM stub by carbon conductive tape and coated with 12 nm platinum / palladium (Pt / Pd) using a Cressington sputter coater 208HR (Cressington Scientific Instruments Ltd., Watford, UK). Images are taken for neat (i.e., untreated) CSP7 samples and ground CSP7 samples.

[0201] Stability study of pulverized CSP7 (ammonium counterion) from lot number UTA181028. The stability of pulverized CSP7 powder was investigated under various storage conditions for up to 6 months. Pulverized CSP7 peptide is packaged in two forms: bulk pulverized powder and encapsulated pulverized powder. When stored as bulk pulverized powder, 0.21–0.24 g of peptide was filled into 20 mL scintillation vials (Kimble®, DWK Life Sciences, Millville, New Jersey, USA) and stored in heat-sealed foil pouches (Impak Corp, Los Angeles, California, USA) containing two bags of 1 g silica gel desiccant (Tyvek® and Sorbco Packaging LLC, Belen, New Jersey, USA) in each pouch. The pulverized peptide powder was then encapsulated in size 3 HPMC capsules (Capsugel, Morristown, New Jersey, USA) at a weight of approximately 11 ± 5% mg. 22–26 capsules were then placed in HDPE bottles (Drug Plastic, Boyertown, Pennsylvania, USA), and the HDPE bottles were sealed in foil pouches (without desiccant). The packages were kept in a stable chamber under the following storage conditions: -20°C, 25°C / 60%RH, and 40°C / 75%RH. Samples were taken out for testing at 1, 3, and 6 months (Table 38). For tests other than aerodynamic particle size distribution, the encapsulated powder was removed from the capsules and mixed in glass vials by rotating the vials. [Table 38]

[0202] Appearance of the crushed powder. The appearance of the crushed powder was recorded by taking a photograph with a standard camera (Figure 30).

[0203] Chemical stability of pulverized peptides. The powder was assayed using the HPLC method described in Example 8. The results are shown in Table 39 below. Percentages represent assay volume compared to mass balance. The assay was adjusted for moisture content.

Table 39

[0204] The moisture content of the CSP powder. The moisture content in the peptide powder is measured using a Coulometric Karl Fischer (Mettler Toledo C20 Leicester, Ohio, USA) (Table 40). The reliability of the instrument is tested with a Karl Fischer moisture content standard (Hydranal™ water standard, Honeywell, Charlotte, North Carolina, USA). A known amount of powder is suspended in anhydrous methanol (Sigma, St. Louis, Missouri), and the suspension is injected into the anolyte (Hydranal™-Coulomat AG, Honeywell, Charlotte, North Carolina, USA) to cause a titration to occur in the presence of the catholyte (Hydranal™-Coulomat CG, Honeywell, Charlotte, North Carolina, USA). The results are recorded as the difference in the moisture content in the sample minus the blank anhydrous methanol solution.

Table 40

[0205] Geometric particle size distribution. The GPSD of the CSP7 powder before and after milling is analyzed using a Sympatec HELOS laser diffraction instrument with RODOS dispersion (Sympatec GmbH, Germany). The measurements are taken every 10 milliseconds after powder dispersion at 3 bar. To determine the particle size distribution, the measured values of optical density from 5 to 25% are averaged. The particle size by volume is reported as the 10th, 50th, and 90th percentiles (e.g., Dv10, Dv50, and Dv90), respectively, and the percentage of particles falling within the size range of 1 - 5 μm. The results are shown in Table 41.

Table 41

[0206] Aerodynamic particle size distribution analysis was performed by conducting the NGI described in paragraph

[0215] , with the powder weight in the tested capsule being 11 ± 5 mg, except that the pre-separator was removed during the NGI assembly in the stability study. The results are shown in Table 42. [Table 42]

[0207] 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.

[0208] All methods disclosed and claimed herein may be made and performed without excessive experimentation in the view of this disclosure. While the compositions and methods of the present invention have been described in the view of preferred embodiments, it will be apparent to those skilled in the art that variations may be made to the methods, steps, or sequence of steps described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant active substances may be substituted with the active substances described herein while achieving the same or similar results. All such similar substitutions and modifications, apparent to those skilled in the art, are considered to be within the spirit, scope, and concept of the invention as defined in the appended claims. References

[0209] The following references are incorporated herein by reference to the extent that they provide supplementary exemplary procedures or other details to those presented herein. Carvalho et al., “Influence of particle size on regional lung deposition - What evidence is there?”Int.J.Pharma.406:1-10,2011. Huebner, R.-H.; Gitter, W.; El Mokhtari, NE; Mathiak, M.; Both, M.; Bolte, EL; Freitag-Wolf, S.; Bewig, B. Standardized quantification of pulmonary fibrosis in histological samples.Biotechniques, 44, 507-11, 514-7, 2008. Surasarang et al., “Optimization of Formulation for a Novel Inhaled Candidate Therapeutic for Idiopathic Fibrosis,” Drug Development and Industrial Pharmacy, 44(2):184-198, 2017. Tepper,JS;Kuehl,PJ;Cracknell,S.;Nikula,KJ;Pei,L.;Blanchard,JDSymposium Summary:“breathe In,Breathe Out,Its Easy:What You Need to Know about Developing Inhaled Drugs.”Int.J.Toxicol.35,376-392,2016. The present invention provides, for example, the following items. (Item 1) A pharmaceutical composition comprising a dried peptide powder, wherein the peptide comprises one of the sequences from SEQ ID NOs: 2 to 20. (Item 2) The pharmaceutical composition according to item 1, wherein the peptide has an amino acid length of 7 to 20. (Item 3) The pharmaceutical composition according to item 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 2. (Item 4) The pharmaceutical composition according to item 3, wherein the peptide comprises at least one amino acid added to the N-terminus of the peptide of SEQ ID NO: 2. (Item 5) The pharmaceutical composition according to item 3, wherein the peptide comprises at least one amino acid added to the C-terminus of the peptide of Sequence ID No. 2. (Item 6) The pharmaceutical composition according to item 3, wherein the peptide comprises at least one amino acid added to the N-terminus and C-terminus of the peptide of SEQ ID NO: 2. (Item 7) The pharmaceutical composition according to item 1, wherein the peptide comprises an L-amino acid. (Item 8) The pharmaceutical composition according to item 1, wherein the peptide comprises a D-amino acid. (Item 9) The pharmaceutical composition according to item 1, wherein the peptide comprises both L-amino acids and D-amino acids. (Item 10) The pharmaceutical composition according to item 1, wherein the peptide comprises at least one deuterated residue. (Item 11) The pharmaceutical composition according to item 1, wherein the peptide comprises at least one non-standard amino acid. (Item 12) The pharmaceutical composition according to item 11, wherein the peptide comprises two non-standard amino acids. (Item 13) The pharmaceutical composition according to item 11, wherein the non-standard amino acid is ornithine. (Item 14) The pharmaceutical composition according to item 1, wherein the peptide includes an N-terminal modification. (Item 15) The pharmaceutical composition according to item 1, wherein the peptide includes a C-terminal modification. (Item 16) The pharmaceutical composition according to item 1, wherein the peptide comprises an N-terminal modification and a C-terminal modification. (Item 17) The pharmaceutical composition according to item 14, wherein the N-terminal modification is acylation. (Item 18) The pharmaceutical composition according to item 15, wherein the C-terminal modification is amidation. (Item 19) The pharmaceutical composition according to item 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 3. (Item 20) The pharmaceutical composition according to item 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 4. (Item 21) The pharmaceutical composition according to item 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 6. (Item 22) The pharmaceutical composition according to item 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 9. (Item 23) The pharmaceutical composition according to item 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 5. (Item 24) The pharmaceutical composition according to item 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 7. (Item 25) The pharmaceutical composition according to item 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 8. (Item 26) The pharmaceutical composition according to item 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 11. (Item 27) The pharmaceutical composition according to item 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 12. (Item 28) The pharmaceutical composition according to item 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 13. (Item 29) The pharmaceutical composition according to item 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 14. (Item 30) The pharmaceutical composition according to item 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 15. (Item 31) The pharmaceutical composition according to item 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 16. (Item 32) The pharmaceutical composition according to item 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 17. (Item 33) The pharmaceutical composition according to item 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 18. (Item 34) The pharmaceutical composition according to item 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 19. (Item 35) The pharmaceutical composition according to item 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 10. (Item 36) The pharmaceutical composition according to item 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 20. (Item 37) The pharmaceutical composition described in item 1, further comprising cell-permeable peptides (CPPs). (Item 38) The pharmaceutical composition according to item 37, wherein the CPP comprises an amino acid sequence selected from the group including GRKKRRQRRRPPQ (SEQ ID NO: 23), RQIKIWFQNRRMKWKK (SEQ ID NO: 24), and GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 25). (Item 39) The pharmaceutical composition according to item 1, wherein the peptide comprises at least two repeats of any one sequence from sequence numbers 2 to 20. (Item 40) The pharmaceutical composition according to item 39, wherein at least two of the repeats have the same amino acid sequence. (Item 41) The pharmaceutical composition according to item 39, wherein at least two of the repeats have different amino acid sequences. (Item 42) The pharmaceutical composition according to item 1, wherein the dried powder is produced by a grinding process. (Item 43) The pharmaceutical composition according to item 1, wherein the dried powder is produced by a spray-drying process. (Item 44) The pharmaceutical composition according to item 1, wherein the dried powder is produced by air jet pulverization. (Item 45) The pharmaceutical composition according to item 1, wherein the dried powder is produced by ball grinding. (Item 46) The pharmaceutical composition according to item 1, wherein the dried powder is produced by wet grinding. (Item 47) The pharmaceutical composition according to item 1, wherein the dried powder contains less than 10% (by weight) of water. (Item 48) The pharmaceutical composition according to item 1, wherein the dried powder contains less than 1% (by weight) of water. (Item 49) The pharmaceutical composition according to item 1, wherein the pharmaceutical composition is essentially free of excipients. (Item 50) The pharmaceutical composition according to item 49, wherein the pharmaceutical composition does not contain an excipient. (Item 51) The pharmaceutical composition according to item 1, wherein the pharmaceutical composition is formulated for pulmonary delivery. (Item 52) The pharmaceutical composition according to item 51, wherein the pharmaceutical composition is formulated for inhalation as a dry powder. (Item 53) The pharmaceutical composition according to item 51, wherein the pharmaceutical composition is formulated for inhalation pressurized metered-dose inhalation. (Item 54) The pharmaceutical composition according to item 1, wherein the pharmaceutical composition is formulated for oral administration, topical administration, or injection. (Item 55) A nebulizer device containing the pharmaceutical compositions described in items 1 to 51. (Item 56) A method for treating a subject, comprising administering to the subject an effective amount of a pharmaceutical composition described in items 1 to 51. (Item 57) The method described in item 56, wherein the subject has an inflammatory disorder. (Item 58) The method according to item 56, wherein the subject has a fibrous state. (Item 59) The method according to item 56, wherein the subject has pneumonia, acute lung injury, lung infection, or lungs. (Item 60) The method described in item 59, wherein the subject has pneumonia. (Item 61) The method described in item 56, wherein the subject has chronic obstructive pulmonary disease (COPD). (Item 62) The method described in item 56, wherein the subject has acute lung injury or infection. (Item 63) The method described in item 56, wherein the subject has a pulmonary infection. (Item 64) The method described in item 56, wherein the subject has chemical-induced lung injury. (Item 65) The method according to item 56, wherein the subject has cast bronchitis. (Item 66) The method described in item 56, wherein the subject has asthma. (Item 67) The method described in item 56, wherein the subject has acute respiratory distress syndrome (ARDS). (Item 68) The method according to item 56, wherein the subject has inhalation smoke-induced acute lung injury (ISALI). (Item 69) The method according to item 56, wherein the subject has bronchiolitis. (Item 70) The method according to item 56, wherein the subject has obstructive bronchiolitis. (Item 71) The method according to item 56, wherein the lung disease is a fibrotic state of the lung. (Item 72) The method according to item 56, wherein the lung disease is an interstitial lung disease. (Item 73) The method according to item 56, wherein the lung disease is idiopathic pulmonary fibrosis (IPF) or pulmonary scarring. (Item 74) The method according to item 56, wherein the administration includes inhalation of a dry powder. (Item 75) The method according to item 56, wherein the administration comprises atomizing a solution containing a mutant polypeptide. (Item 76) The method according to item 56, further comprising administering at least one additional antifibrotic agent. (Item 77) The method according to item 76, wherein the at least one additional antifibrotic agent is an NSAID, steroid, DMARD, immunosuppressant, biological response modulator, or bronchodilator. (Item 78) The method described in item 56, wherein the subject is a human. (Item 79) A pharmaceutical composition comprising peptides of sequence numbers 2-20, formulated as pulverized dry powders having a breathable particle size. (Item 80) A method for treating a subject, comprising administering an effective amount of the composition described in item 79 to the subject by inhalation.

Claims

1. A dry powder inhaler comprising a pharmaceutical composition containing a peptide or a pharmaceutically acceptable salt thereof, wherein the peptide consists of the amino acid sequence FTTFTVT (SEQ ID NO: 2).

2. The dry powder inhaler according to claim 1, wherein the pharmaceutically acceptable salt is an ammonium salt or an acetate salt.

3. The dry powder inhaler according to claim 1 or claim 2, wherein the pharmaceutical composition comprises leucine, trehalose, sodium citrate, or a combination thereof.

4. The dry powder inhaler according to claim 3, wherein the pharmaceutical composition contains leucine.

5. The dry powder inhaler according to any one of claims 1 to 4, wherein the pharmaceutical composition comprises a lubricant.

6. The dry powder inhaler according to any one of claims 1 to 5, wherein the pharmaceutical composition is enclosed in a capsule.

7. The dry powder inhaler according to any one of claims 1 to 6, wherein the pharmaceutical composition is enclosed in a shell capsule.

8. The dry powder inhaler according to any one of claims 1 to 7, wherein the pharmaceutical composition contains particles having a particle size of less than 5 μm.

9. The dry powder inhaler according to any one of claims 1 to 8, wherein the pharmaceutical composition contains particles having a particle size between about 1 μm and about 5 μm.

10. The dry powder inhaler according to any one of claims 1 to 8, wherein the pharmaceutical composition contains particles having a particle size of about 2 μm, about 2.5 μm, or about 3 μm.

11. The dry powder inhaler according to any one of claims 1 to 8, wherein the pharmaceutical composition comprises particles, and at least 70% of the particles have a particle size of about 1 μm to about 5 μm.

12. The dry powder inhaler according to any one of claims 1 to 8, wherein the pharmaceutical composition contains particles, and 75% to 95% of the particles have a particle size of less than 5 μm.

13. The dry powder inhaler according to any one of claims 8 to 12, wherein the particle size of the aforementioned particles is the aerodynamic median particle diameter (MMAD).

14. The dry powder inhaler according to any one of claims 1 to 13, wherein the pharmaceutical composition is stable for at least one month, at least three months, or at least six months.

15. The dry powder inhaler according to any one of claims 1 to 14, wherein the pharmaceutical composition is stable for at least six months.

16. The dry powder inhaler according to any one of claims 1 to 15, wherein the pharmaceutical composition contains less than 10% (by weight) of water.

17. The dry powder inhaler according to any one of claims 1 to 15, wherein the pharmaceutical composition contains less than 1% (by weight) of water.

18. A dry powder inhaler according to any one of claims 1 and 2, or any one of claims 3 to 5, where the pharmaceutical composition is essentially free of excipients or free of excipients.

19. A dry powder inhaler according to any one of claims 1 to 18 for treating a subject in need thereof.

20. The dry powder inhaler according to claim 19, wherein the subject has an inflammatory disorder.

21. The dry powder inhaler according to claim 19, wherein the subject has a fibrous state.

22. The dry powder inhaler according to claim 19, wherein the subject has pneumonia, chronic obstructive pulmonary disease (COPD), acute lung injury, lung infection, chemical-induced lung injury, cast bronchitis, asthma, acute respiratory distress syndrome (ARDS), inhaled smoke-induced acute lung injury (ISALI), bronchiolitis, obstructive bronchiolitis, fibrotic pulmonary condition, interstitial lung disease, idiopathic pulmonary fibrosis (IPF), or pulmonary scarring.

23. The dry powder inhaler according to claim 19, wherein the subject has interstitial lung disease.

24. The dry powder inhaler according to claim 19, wherein the subject has idiopathic pulmonary fibrosis.

25. The dry powder inhaler according to any one of claims 19 to 24, wherein the pharmaceutical composition is administered to the subject by inhalation of the dry powder.

26. The dry powder inhaler according to any one of claims 19 to 25, wherein the pharmaceutical composition is administered to the subject together with at least one additional therapeutic agent.

27. The dry powder inhaler according to claim 26, wherein the at least one additional therapeutic agent is a nonsteroidal anti-inflammatory drug (NSAID), a steroid, a disease-modifying antirheumatic drug (DMARD), an immunosuppressant, a biological response modulator, or a bronchodilator.

28. The dry powder inhaler according to any one of claims 19 to 27, wherein the subject is a human.

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