Compositions and methods for the treatment of acute lung injury

Abstract

To provide compositions and methods useful in the treatment of acute lung injury (ALI).SOLUTION: A composition comprises a polypeptide consisting of a sequence described below, wherein: X1 may be present or absent, and if present, is E; X2 may be present or absent, and if present, is L; X3 may be present or absent, and if present, is Q; X4 may be present or absent, and if present, is A or T; X5 may be present or absent, and if present, is T or E; X6 is H or Y; X7 is D or E; X8 is F or I; and X9 is R or K.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 719,217, filed August 17, 2018, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under 2R01 HL102016 awarded by the National Heart, Lung and Blood Institute. The government has certain rights in this invention. [Background technology]

[0003] Background of the Invention Pulmonary inflammation is a key component in the pathogenesis of acute lung injury (ALI) syndromes arising from multiple causes. Pulmonary inflammation, associated with the production of reactive oxygen species (ROS), is a key contributor to ALI syndrome. Activation of NADPH oxidase type 2 (NOX2), the main source of ROS in the lung, requires the phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6).

[0004] Current treatments for ALI are supportive, and there are currently no approved drugs specifically for its prevention or treatment. Thus, there is a need in the art for methods and compositions for preventing ALI. The present disclosure addresses this need. Summary of the Invention

[0005] In one aspect, A polypeptide consisting of TIFF0007822065000001.tif5128, X 1may or may not be present, and if present is E; X 2 is optionally present and, if present, is L; X 3 may or may not be present, and if present is Q; X 4 is optional and, if present, is A or T; X 5 is optional and, if present, is T or E; X 6 is H or Y; X 7 is D or E; X 8 is F or I; and X 9 is R or K, Polypeptides The present invention provides a composition comprising:

[0006] In various embodiments, the polypeptide is selected from the group consisting of: Selected from TIFF0007822065000002.tif12161.

[0007] In various embodiments, the polypeptide is selected from the group consisting of: Selected from TIFF0007822065000003.tif4164.

[0008] In various embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0009] In various embodiments, the polypeptide is encapsulated in one or more liposomes.

[0010] In various embodiments, the composition is formulated for aerosol inhalation or for intratracheal or intravenous infusion. In various embodiments, the pharmaceutical composition is administered to the subject by intravenous infusion.

[0011] In another aspect, A polypeptide consisting of TIFF0007822065000004.tif5128, X 1 may or may not be present, and if present is E; X 2 is optionally present and, if present, is L; X 3 may or may not be present, and if present is Q; X 4 is optional and, if present, is A or T; X 5 may be T or E; X 6 is H or Y; X 7 is D or E; X 8 is F or I; and X 9 is R or K, A polypeptide, a pharmaceutically acceptable carrier; The present invention provides a method of treating acute lung injury in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising:

[0012] In various embodiments, the polypeptide is selected from the group consisting of: Selected from TIFF0007822065000005.tif12161.

[0013] In various embodiments, the polypeptide is selected from the group consisting of: Selected from TIFF0007822065000006.tif4164.

[0014] In various embodiments, the polypeptide is encapsulated in one or more liposomes.

[0015] In various embodiments, the pharmaceutical composition is administered to the subject by aerosol inhalation or by intratracheal or intravenous infusion.

[0016] In another aspect, A polypeptide consisting of TIFF0007822065000007.tif5128, X 1 may or may not be present, and if present is E; X 2 is optionally present and, if present, is L; X 3 may or may not be present, and if present is Q; X 4 is optional and, if present, is A or T; X 5 may be T or E; X 6 is H or Y; X 7 is D or E; X 8 is F or I; and X 9 is R or K, A polypeptide, a pharmaceutically acceptable carrier; The present invention provides a method of treating sepsis in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising:

[0017] In various embodiments, the polypeptide is selected from the group consisting of: Selected from TIFF0007822065000008.tif12161.

[0018] In various embodiments, the polypeptide is selected from the group consisting of: Selected from TIFF0007822065000009.tif4164.

[0019] In various embodiments, the polypeptide is encapsulated in one or more liposomes.

[0020] In various embodiments, the pharmaceutical composition is administered to the subject by aerosol inhalation or by intratracheal or intravenous infusion. In various embodiments, the pharmaceutical composition is administered to the subject by intravenous infusion. [The present invention 1001] TIFF0007822065000010.tif5134 A polypeptide consisting of X 1 may or may not be present, and if present is E; X 2 is optionally present and, if present, is L; X 3 may or may not be present, and if present is Q; X 4 is optional and, if present, is A or T; X 5 is optional and, if present, is T or E; X 6 is H or Y; X 7 is D or E; X 8 is F or I; and X 9 is R or K, Polypeptides A composition comprising: [The present invention 1002] The polypeptide is TIFF0007822065000011.tif12133 1001. The composition of the present invention selected from the group consisting of: [The present invention 1003] The polypeptide is TIFF0007822065000012.tif12139 1001. The composition of the present invention selected from the group consisting of: [The present invention 1004] The composition of this invention 1001, further comprising a pharmaceutically acceptable carrier. [The present invention 1005] 1001. The composition of claim 1001, wherein the polypeptide is encapsulated in one or more liposomes. [The present invention 1006] A composition of the present invention formulated for aerosol inhalation or for intratracheal or intravenous infusion. [The present invention 1007] TIFF0007822065000013.tif5134 A polypeptide consisting of X 1 may or may not be present, and if present is E; X 2 is optionally present and, if present, is L; X 3 may or may not be present, and if present is Q; X 4 is optional and, if present, is A or T; X 5 may be T or E; X 6 is H or Y; X 7 is D or E; X 8 is F or I; and X 9 is R or K, A polypeptide, a pharmaceutically acceptable carrier; 10. A method of treating acute lung injury in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising: [The present invention 1008] The polypeptide is TIFF0007822065000014.tif12157 The method of the present invention 1007, selected from the group consisting of: [The present invention 1009] The polypeptide is TIFF0007822065000015.tif12140 The method of the present invention 1007, selected from the group consisting of: [The present invention 1010] 1007. The method of claim 10, wherein the polypeptide is encapsulated in one or more liposomes. [The present invention 1011] The method of claim 1007, wherein the pharmaceutical composition is administered to the subject by aerosol inhalation or by intratracheal or intravenous infusion. [The present invention 1012] TIFF0007822065000016.tif5134 A polypeptide consisting of X 1 may or may not be present, and if present is E; X 2 is optionally present and, if present, is L; X 3 may or may not be present, and if present is Q; X 4 is optional and, if present, is A or T; X 5 may be T or E; X 6 is H or Y; X 7 is D or E; X 8 is F or I; and X 9 is R or K, A polypeptide, a pharmaceutically acceptable carrier; 10. A method of treating sepsis in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising: [The present invention 1013] The polypeptide is TIFF0007822065000017.tif12134 The method of the present invention 1012, selected from the group consisting of: [The present invention 1014] The polypeptide is TIFF0007822065000018.tif12140 The method of the present invention 1012, selected from the group consisting of: [The present invention 1015] The method of claim 1012, wherein the polypeptide is encapsulated in one or more liposomes. [The present invention 1016] The method of claim 1012, wherein the pharmaceutical composition is administered to the subject by aerosol inhalation or by intratracheal or intravenous infusion. [Brief explanation of the drawings]

[0021] The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments that are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown in the drawings. [Figure 1]Figures 1A and 1B: Eight- to ten-week-old wild-type C57Bl / 6 mice were infused with 2 μg / g body weight of PIP-2 either intratracheally (IT) or intravenously (IV). The infused peptide was dissolved in saline or incorporated into unilamellar liposomes. The liposomes consisted of dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (PC), phosphatidylglycerol (PG), and cholesterol (lipid molar ratio: 50:25:10:15). We determined that liposomes containing either 75% DPPC or 75% egg PC (with PG and cholesterol) were as effective as DPPC / PC / PG / cholesterol liposomes for intracellular delivery of PIP-2. Mice were sacrificed 5 min later, and the lungs were perfused until blood was removed. They then underwent recirculating perfusion in the presence of the fluorescent indicator Amplex Red and horseradish peroxidase (HRP) under temperature-controlled (37°C) conditions to monitor the oxidation of Amplex Red by HO. The fluorescence of perfusate aliquots was measured at the indicated times and expressed as arbitrary fluorescence units (AFU). Increased fluorescence over the time course of perfusion indicates the production of HO, which reflects the activation of cellular NADPH oxidase (NOX2). Administration of PIP-2 in saline had no effect on the rate of HO production, whereas PIP-2 in liposomes, injected either IT (Figure 1A) or IV (Figure 1B), significantly inhibited HO production. [Figure 2-1]Figures 2A–2F show the time course of lung injury after intratracheal instillation of lipopolysaccharide (LPS) by tracking various markers of tissue oxidation and lung inflammation. Bacterial (E. coli) lipopolysaccharide (LPS) was administered to wild-type C57Bl / 6 mice via intratracheal instillation at 5 μg / g body weight. Mice were sacrificed 12, 16, 24, or 48 h after LPS administration, as indicated. Lungs were excised and lavaged via the trachea with saline to obtain BALf; the lungs were then homogenized. Lung injury parameters included nucleated cells and protein in the BALf and the lung wet-to-dry weight ratio (W / D). Regarding W / D, the weight of the left upper lobe of the lung was measured before and after drying to constant weight in a desiccator. Indicators of tissue oxidative stress (bottom row) were thiobarbituric acid reactive substances (TBARS), 8-isoprostanes, and protein carbonyls measured in lung homogenates. Values ​​are means ± for n = 4. * P < 0.05 vs. all other values; § p < 0.05 vs. 12, 16, and 24 h. Figure 2A shows thiobarbituric acid reactive substances (TBARS). Figure 2B shows 8-isoprostanes. Figure 2C shows protein carbonyls in lung homogenates. [Figure 2-2] Figure 2D shows the number of cells in bronchoalveolar lavage fluid (BALF), Figure 2E shows the total protein in BALF, and Figure 2F shows the wet-to-dry lung weight ratio. [Figure 3-1]Figures 3A-3F. Acute lung injury (ALI) was induced by intratracheal LPS (5 mg / g body weight). PIP-2 in liposomes (2 μg / g body weight) was administered IT with LPS (0 h) or intravenously (IV) 12 or 16 h after LPS. PIP-2 was administered IV to avoid a second "attack" on the trachea. Mice were sacrificed at 24 h, and lungs were evaluated for lung injury and tissue oxidative stress. Results are means ± SE for n = 4. * P < 0.05 vs. all other groups. Figure 3A shows the number of cells in BALF. Figure 3B shows the total protein in BALF. Figure 3C shows the wet-to-dry lung weight ratio. [Figure 3-2] Figure 3D shows TBARS, Figure 3E shows 8-isoprostanes, and Figure 3F shows protein carbonyls in lung homogenates. [Figure 4A] Figures 4A and 4B: Prdx6 phospholipase A2 (aiPLA2) was measured by the release of palmitate from dipalmitoylphosphatidylcholine under acidic conditions (pH 4) in the absence of Ca2+. Figure 4A: Effect of increasing concentrations of PIP-2 on the aiPLA2 activity of recombinant human Prdx6. [Figure 4B] Figure 4B: Effect of treatment with LPS and PIP-2 on aiPLA2 activity in mouse lungs. Mice (n = 3 for each condition) were treated with intratracheal LPS (2 μg / g body weight) without or with PIP-2 in liposomes (2 μg / g body weight). Animals were sacrificed 6, 12, or 24 hours after LPS administration. Lungs were cleared of blood and homogenized. Control lungs were from mice that did not receive LPS. * P < 0.05 vs. corresponding control and vs. LPS + PIP-2; § P < 0.05 vs. 12 and 24 hours of LPS. [Figure 5A]Figures 5A and 5B: PIP-2 incorporated into liposomes was instilled intratracheally prior to lung isolation. Figure 5A: Isolated lungs were perfused in a recirculating system with artificial medium. NOX2 activity was stimulated by the addition of angiotensin II (Ang II). To detect ROS production, Amplex Red was added to the perfusate along with horseradish peroxidase. Aliquots of the perfusate were analyzed spectrophotometrically at intervals to determine Amplex Red oxidation, which indicates ROS production. Results are means ± SE for n = 3–4. [Figure 5B] Figure 5B: Mice were sacrificed 6, 12, or 24 hours after LPS administration (5 mg / g body weight), and the lungs were perfused in situ for 15 minutes with saline containing a fluorophore (difluorofluorescein diacetate, DFFDA). The lungs were then homogenized, and the fluorescence of the lung homogenate was determined as an index of ROS production. Results for Figures 5A and 5B are means ± SE for n = 4. * P < 0.05 vs. corresponding LPS and vs. LPS + PIP-2; § P < 0.05 vs. 12 and 24 hours of LPS; Δ P < 0.05 vs. corresponding LPS. [Figure 6] Figure 6: PIP-2 in liposomes was administered either intratracheally (IT) or intravenously (IV) to mice at time 0. Lungs were harvested at intervals between 4 and 72 hours after PIP-2 administration, homogenized, and analyzed for Prdx6-PLA2 activity. PIP-2 was effective by either the IT or IV route, with a calculated half-life of recovery of approximately 50 hours. Results are the mean + SE for n = 3-4. [Figure 7]Figures 7A and 7B: Kaplan-Meier plots of survival. LPS (15 mg / g body weight) was administered to all mice by either intratracheal (IT) infusion (Figure 7A) or intraperitoneal (IP) infusion (Figure 7B). As indicated by the arrows, PIP-2 in liposomes or placebo (liposomes alone) was administered intravenously (IV) 12 hours after LPS (this is the time zero treatment) and then at 12- or 24-hour intervals for a total of five doses. Figure 7A: 2 μg / g body weight PIP-2; N = 14 for each group. Figure 7B: 2 μg / g body weight or 20 μg / g body weight PIP-2; Placebo group, n = 8; 2 mg PIP-2 group, n = 7; 20 mg PIP-2 group, n = 10. [Figure 8] Figure 8: PLA2 inhibitory peptide (PIP-2) inhibits angiotensin II (Ang II)-stimulated ROS production in isolated perfused mouse lungs. PIP-2 (2 μg / g body weight) was administered intravenously to intact wild-type (WT) mice. WT basal, WT control, and NOX2 null mice received no peptide. Thirty minutes later, lungs were isolated from anesthetized mice and perfused in a recirculating system with Ang II (50 μM) as a Nox2 activator, as well as Amplex Red and horseradish peroxidase to detect ROS in the perfusate. WT basal lungs were not stimulated with Ang II. After a 15-minute equilibration period (referred to as time zero), aliquots were withdrawn at 15-minute intervals for fluorescence analysis. Each plotted point represents the mean ± SE for n = 3. Lines were plotted using the least mean squares method. The average rate of ROS reduction, calculated from the slope of each line, is shown in parentheses. *P < 0.05 vs. the other three slopes. [Figure 9]Figures 9A and 9B: PIP-2 inhibits increased pulmonary aiPLA2 activity and increased ROS production after LPS administration. LPS (5 μg / g body weight) was administered by intratracheal (IT) infusion with liposomes alone (denoted LPS) or with PIP-2 in liposomes (denoted +PIP-2). The control was liposomes alone without LPS (denoted Control). Mice were sacrificed 6, 12, or 24 hours after LPS, and lungs were perfused in situ for 15 minutes with saline containing the fluorophore difluorofluorescein diacetate (DFF-DA). Lungs were then homogenized and assayed for the following: Figure 9A: aiPLA2 activity; Figure 9B: fluorescence of lung homogenates as an indicator of ROS production. Results are shown as mean ± SE for N = 3 for A and mean ± SE for N = 4 for B. * P < 0.05 versus control and versus +PIP-2 at the same time points; P < 0.05 versus the corresponding value at Δ 6 h. [Figure 10] Figure 10: Effect of liposome composition on pulmonary delivery of PIP-2 after IV administration. Same protocol as in Figures 5A-5B for liposome-mediated PIP-2 delivery by intravenous infusion. *% of total lipid; all liposomes also contained 15% cholesterol. †Mean ± SE for n = 3 or range for n = 2. PC and DPPC in liposomes have similar efficacy for intracellular delivery of PIP-2. Omission of PG reduces efficacy by approximately 10%. [Figure 11] Figure 11: Percent "protection" from lung injury by PIP-2 assessed 24 hours after IT LPS. *Values ​​for the effect of PIP-2 administered 0, 12, or 16 hours after LPS. Percent protection from lung injury was calculated as [1 - (injury with PIP-2 - control) / (LPS only - control)]. PIP-2 "protection" is >75%. [Figure 12]Figure 12: Indices of lung injury in PIP-2-treated mice that survived high-dose LPS. Mice were instilled with LPS (15 μg / g body weight) either intratracheally (IT), row B; or intraperitoneally (IP), row C. PIP-2 in liposomes was instilled (IV) at 2 μg / g body weight or 20 μg / g body weight at the times indicated in Figure 7. Five surviving mice were sacrificed 108 hours after the start of treatment (120 hours after LPS administration). Results are compared with values ​​for historical control mice (no LPS) (row A). BALf, bronchoalveolar lavage fluid; TBARS, thiobarbituric-reactive substances. Values ​​are means ± SE for n = 4 for control and n = 5 for LPS + PIP-2. None of the means for LPS + PIP-2 were statistically different (p > 0.05) from the corresponding controls. PIP-2-treated mice that survived 5 days after LPS had normal lungs. [Figure 13] Figure 13: Effect of PIP-2 on ventilation-induced lung injury (VILI). Anesthetized mice were mechanically ventilated for 6 hours at a tidal volume of 12 ml / kg body weight, a respiratory rate of 120 breaths / min, and a positive end-expiratory pressure (PEEP) of 2 cm H2O. PIP-2 in liposomes (2 μg / g body weight) was administered by IT injection at the initiation of mechanical ventilation, and mice were sacrificed 6 hours later. Control values ​​represent values ​​for normal (non-ventilated) lungs. % protection was calculated as in Table 1. Results are means ± SE for n = 4. * P < 0.05 for VILI + PIP vs. VILI. PIP-2 protected against mechanical ventilation-associated lung injury. DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0023] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

[0024] As used herein, the articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0025] As used herein, "about," when referring to a measurable value such as an amount, duration, etc., is meant to encompass a ±20% or ±10% variation from the specified value, more preferably a ±5% variation, even more preferably a ±1% variation, and even more preferably a ±0.1% variation, such variation being appropriate for practicing the disclosed methods.

[0026] "Acute lung injury" or "ALI" as used herein refers to a syndrome characterized by the acute onset of bilateral pulmonary infiltrates accompanied by hypoxemia not associated with heart failure.

[0027] A disease or disorder is "alleviated" if the severity of a symptom of the disease or disorder, the frequency with which the patient experiences such symptoms, or both, is reduced.

[0028] As used herein, the term " composition " or " pharmaceutical composition " refers to the mixture of at least one compound useful in the present invention with a pharmaceutically acceptable carrier. Pharmaceutical compositions facilitate the administration of compounds to patients or subjects. In the art, there are multiple techniques for administering compounds, including but not limited to intravenous administration, oral administration, aerosol administration, parenteral administration, ocular administration, pulmonary administration and topical administration.

[0029] An "effective amount" or "therapeutically effective amount" of a compound is the amount of the compound sufficient to confer a beneficial effect on the subject to which it is administered. An "effective amount" of a delivery vehicle is an amount sufficient to effectively bind or deliver a compound.

[0030] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal, or cells thereof, suitable for the methods described herein, whether in vitro or in situ. In one non-limiting embodiment, the patient, subject, or individual is a human.

[0031] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not interfere with the biological activity or properties of the compound and that is relatively non-toxic, i.e., the material may be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0032] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, that is involved in carrying or transporting a compound useful within the present invention within or to a patient so that the compound can perform its intended function. Typically, such constructs are carried or transported from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the present invention, and not harmful to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; and other non-toxic and compatible substances used in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carriers" also includes any coatings, antibacterial and antifungal agents, absorption delaying agents, and the like, which are compatible with the activity of the compounds useful within the present invention and are physiologically acceptable to the patient. Supplementary active compounds can also be incorporated into the compositions. "Pharmaceutically acceptable carriers" can further include pharmaceutically acceptable salts of the compounds useful within the present invention.Other additional ingredients that may be included in pharmaceutical compositions used in practicing the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0033] As used herein, "PIP-2" refers to TIFF0007822065000019.tif4128 refers to the peptide with

[0034] As used herein, "PIP-4" refers to TIFF0007822065000020.tif4128 refers to the peptide with

[0035] As used herein, "PIP-5" refers to TIFF0007822065000021.tif4128 refers to the peptide with

[0036] As used herein, "treating a disease or disorder" means reducing the frequency with which a patient experiences symptoms of the disease or disorder. Disease and disorder are used interchangeably herein.

[0037] As used herein, "sepsis" is a potentially life-threatening condition caused by the body's response to infection, which can result in multiple organ failure.

[0038] As used herein, the term "treatment" or "treating" encompasses prevention and / or therapy. Thus, the compositions and methods of the present invention are not limited to therapeutic applications, but can also be used for prophylactic applications. Thus, "treating" a condition, disorder, or disorder, or "treatment" thereof, includes: (i) preventing or delaying the onset of clinical symptoms of an ongoing condition, disorder, or disorder in a subject who may have or be predisposed to the condition, disorder, or disorder, but who has not yet experienced or exhibited clinical or subclinical symptoms of the condition, disorder, or disorder; (ii) inhibiting a condition, disorder, or disorder, i.e., arresting or reducing the progression of the disease or at least one of its clinical or subclinical symptoms; or (iii) alleviating a disease, i.e., causing a reduction in the condition, disorder, or disorder, or at least one of its clinical or subclinical symptoms.

[0039] Ranges: Throughout this disclosure, various aspects of the invention may be expressed in range format. It should be understood that the description in range format is merely for convenience and brevity and is not to be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all individual numerical values ​​within that range, along with the possible subranges. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed each individual number within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6, along with subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. This applies regardless of the breadth of the range.

[0040] explanation composition The present invention is based in part on the generation of specific peptide inhibitors of aiPLA2 that can be used to treat ALI. The aiPLA2 inhibitory activity of several peptides of the invention is shown in Table 1 below.

[0041] Table 1. Effect of peptides on the aiPLA2 activity of recombinant hPrdx6. TIFF0007822065000022.tif179131

[0042] Table 2. Size optimization of inhibitory peptides with effect on aiPLA2 activity of human recombinant proteins. TIFF0007822065000023.tif148132

[0043] Table 3. Substitutions in PIP-2: Effect of human recombinant Prdx6 on inhibition of aiPLA2 activity TIFF0007822065000024.tif186154

[0044] Thus, in one respect, TIFF0007822065000025.tif5128, X 1 may or may not be present, and if present is E; X 2 is optionally present and, if present, is L; X 3 may or may not be present, and if present is Q; X 4 is optional and, if present, is A or T; X 5 is optional and, if present, is T or E; X 6 is H or Y; X 7 is D or E; X 8 is F or I; and X 9 is R or K, Polypeptides The present invention provides a composition comprising:

[0045] In various embodiments, the composition comprises: TIFF0007822065000026.tif12167. The compositions of the present invention can be provided to a subject as pharmaceutical compositions. Thus, in various embodiments, the compositions further comprise a pharmaceutically acceptable carrier. As shown in FIG. 1, the polypeptide can be effectively administered in liposomes. Thus, in various embodiments, the polypeptide is encapsulated in one or more liposomes. In various embodiments, the compositions are formulated for aerosol inhalation or for intratracheal or intravenous infusion. Suitable pharmaceutically acceptable carriers, along with inhalable or injectable formulations, are described elsewhere herein.

[0046] Methods for treating acute lung injury In another aspect, TIFF0007822065000027.tif5128, X 1 may or may not be present, and if present is E; X 2 is optionally present and, if present, is L; X 3 may or may not be present, and if present is Q; X 4 is optional and, if present, is A or T; X 5 may be T or E; X 6 is H or Y; X 7 is D or E; X 8 is F or I; and X9 is R or K, A polypeptide, a pharmaceutically acceptable carrier; The present invention provides methods of treating acute lung injury in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising: TIFF0007822065000028.tif4164. In various embodiments, the polypeptide administered to the subject is encapsulated in one or more liposomes. In various embodiments, the pharmaceutical composition is administered to the subject by aerosol inhalation or by intratracheal or intravenous injection.

[0047] Methods for Treating Sepsis In another aspect, TIFF0007822065000029.tif5128, X 1 may or may not be present, and if present is E; X 2 is optionally present and, if present, is L; X 3 may or may not be present, and if present is Q; X 4 is optional and, if present, is A or T; X 5 may be T or E; X 6 is H or Y; X 7 is D or E; X 8 is F or I; and X 9 is R or K, Polypeptides and a pharmaceutically acceptable carrier; The present invention provides methods of treating sepsis in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising: TIFF0007822065000030.tif4164. In various embodiments, the polypeptide administered to the subject is encapsulated in one or more liposomes. In various embodiments, the pharmaceutical composition is administered to the subject by aerosol inhalation or by intratracheal or intravenous infusion. In various embodiments, the pharmaceutical composition is administered to the subject by intravenous infusion.

[0048] Administration / Dosage / Formulation The dosage regimen can affect what constitutes an effective amount.The therapeutic preparation can be administered to the subject either before or after the occurrence of injury.In addition, the dosage can be divided into several portions, and the dosage can be administered daily or sequentially at different times, or the dosage can be continuously infused or bolus infused.In addition, the dosage of the therapeutic preparation can be proportionally increased or decreased when the urgent need of treatment or prevention situation is indicated.

[0049] The compositions of the present invention can be administered to patients, preferably mammals, more preferably humans, at dosages and for periods effective to treat lung injury in patients using known procedures. The effective amount of the therapeutic compound required to achieve a therapeutic effect can vary depending on factors such as the state of the disease or disorder in the patient, the patient's age, sex, and weight, and the ability of the therapeutic compound to treat or prevent acute lung injury in the patient. Dosage regimens can be adjusted to provide an optimal therapeutic response. For example, several divided doses can be administered daily, or the dose can be proportionally reduced as indicated by the urgent need of the therapeutic situation. A non-limiting example of an effective dose range for the therapeutic compounds of the present invention is approximately 1 to 5,000 mg / kg body weight / day. Those skilled in the art will be able to examine the relevant factors and make a determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0050] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to provide an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without causing toxicity to the patient.

[0051] In particular, the selected dosage level will vary depending on a variety of factors, including the activity of the particular compound used, the time of administration, the rate at which the compound is excreted or broken down, the duration of treatment, other drugs, compounds, or materials used in combination with the compound, the age, sex, weight, health, general fitness, and medical history of the patient being treated, and similar factors well known in the medical arts.

[0052] A medical doctor, e.g., a physician or veterinarian, having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can start doses of the compounds of the present invention used in the pharmaceutical composition at levels lower than those required to achieve the desired therapeutic effect, and can gradually increase the dosage until the desired effect is achieved.

[0053] In particular embodiments, it is particularly advantageous to formulate the compound into a unit dosage form for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to a physically discrete unit suitable as a unitary dosage for a patient to be treated; each unit contains a predetermined amount of a therapeutic compound calculated to produce a desired therapeutic effect in association with the required pharmaceutical vehicle. The unit dosage form of the present invention is defined by and directly varies according to (a) the characteristics specific to the therapeutic compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the art when compounding / formulating such a therapeutic compound for the treatment of lung injury in patients.

[0054] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.

[0055] In some embodiments, the compositions of the present invention are administered to patients at dosages ranging from 1 to 5 times per day or more. In other embodiments, the compositions of the present invention are administered to patients at dosage ranges including, but not limited to, once daily, once every two days, once every three days to once a week, and once every two weeks. It will be readily apparent to those skilled in the art that the frequency of administration of the various combination compositions of the present invention will vary from individual to individual depending on many factors, including, but not limited to, age, the disease or disorder being treated, gender, general health, and other factors. Therefore, the present invention should not be construed as limited to any particular dosing regimen, and the exact dosage and composition to be administered to any patient will be determined by the attending physician, taking into account all other factors relevant to the patient.

[0056] The compounds of the present invention for administration may range from about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about 40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg to about 7,500 mg, about 200 μg to about 7,000 mg, about 350 μg to about 6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 900 mg, about 75 mg to about 800 mg, about 80 mg to about 900 mg, about 90 mg to about 1000 mg, about 100 mg to about 1500 mg, about 15 ... The amount may be within the range of about 70 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.

[0057] In some embodiments, the dosage of the compound of the present invention is from about 1 mg to about 2,500 mg. In some embodiments, the dosage of the compound of the present invention used in the compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, the dose of a second compound described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0058] In certain embodiments, the present invention is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the present invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of acute lung injury in a patient.

[0059] The formulations may be used in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable administration mode known in the art. Pharmaceutical preparations may be sterilized and, if desired, mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic buffering, coloring substances, flavoring substances, and / or aromatic substances. They may also, if desired, be combined with other active agents, such as other analgesics.

[0060] The route of administration of any of the compositions of the present invention includes oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual, or topical. The compounds for use in the present invention can be formulated for administration by any suitable route, such as oral or parenteral administration, for example, transdermal, transmucosal (e.g., sublingual, lingual, buccal, urethral, ​​vaginal (e.g., vaginal and perivaginal), nasal and rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical administration.

[0061] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gelcaps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration, etc. It should be understood that the formulations and compositions that may be useful in the present invention are not limited to the particular formulations and compositions described herein.

[0062] Oral administration For oral application, tablets, sugar-coated tablets, liquids, drops, suppositories, or capsules, caplets, and gel caps are particularly suitable.The composition intended for oral use can be prepared according to any method known in the art, and such compositions can contain one or more substances selected from the group consisting of inert and non-toxic pharmaceutical excipients suitable for tablet manufacture.Such excipients include, for example, inert diluents such as lactose; granulating and disintegrating agents such as cornstarch; binders such as starch; and lubricants such as magnesium stearate.Tablets can be uncoated, or they can be coated by known techniques for aesthetic reasons or to delay the release of active ingredients.Preparations for oral use can also be presented as hard gelatin capsules, in which active ingredients are mixed with inert diluents.

[0063] The present invention also includes multi-layer tablets, which contain a layer that provides delayed release of one or more compounds of the present invention and an additional layer that provides immediate release of a drug for treating certain diseases or disorders. A wax / pH-sensitive polymer mixture can be used to obtain a stomach-insoluble composition in which the active ingredient is encapsulated, ensuring delayed release of the active ingredient.

[0064] Parenteral administration For parenteral administration, the compounds of the invention may be formulated for injection or infusion, for example, intravenous, intramuscular, or subcutaneous injection or infusion, or may be formulated for administration as a bolus dose and / or for continuous infusion. Suspensions, solutions, or emulsions in oily or aqueous vehicles may be used, optionally containing other formulatory agents such as suspending, stabilizing, and / or dispersing agents.

[0065] Additional Dosage Forms Additional dosage forms of the present invention include those described in the following U.S. Patents: 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of the present invention also include those described in the following U.S. Patent Applications: 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of the present invention also include those described in the following PCT application numbers: WO 03 / 35041; WO 03 / 35040; WO 03 / 35029; WO 03 / 35177; WO 03 / 35039; WO 02 / 96404; WO 02 / 32416; WO 01 / 97783; WO 01 / 56544; WO 01 / 32217; WO 98 / 55107; WO 98 / 11879; WO 97 / 47285; WO 93 / 18755; and WO 90 / 11757.

[0066] Controlled-Release Formulations and Drug Delivery Systems In certain embodiments, the formulations of the present invention may be, but are not limited to, short-acting, fast-releasing, or controlled release formulations, including, for example, sustained release, delayed release, and pulsed release formulations.

[0067] The term sustained release is used in its ordinary sense to refer to a drug formulation that provides for the gradual release of drug over an extended period of time and that can, but does not necessarily, provide substantially constant blood levels of drug over an extended period of time, which may be a month or longer and should be a longer release than the same amount of agent administered as a bolus.

[0068] For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material that provides sustained release properties to the compound. Thus, compounds for use in the methods of the invention may be administered in the form of microparticles, e.g., by injection, or in the form of wafers or discs, by implantation.

[0069] In one embodiment of the invention, the compounds of the invention are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

[0070] The term delayed release is used herein in its ordinary sense to refer to a drug formulation that provides an initial release of drug after some delay following administration of the drug, and which may, but does not necessarily, include a delay of from about 10 minutes to about 12 hours.

[0071] The term pulsatile release is used herein in its ordinary sense to refer to a drug formulation that provides for release of the drug in such a manner as to produce a pulsatile plasma profile of the drug following administration of the drug.

[0072] The term immediate release is used in its ordinary sense to refer to a drug formulation that provides for release of the drug immediately after administration of the drug.

[0073] As used herein, short-term refers to any time period up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes after drug administration, as well as any and all whole or partial increments thereof.

[0074] As used herein, rapid elimination refers to any time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes after drug administration, and any and all whole or partial increments thereof.

[0075] dosage The therapeutically effective amount or dose of the compounds of the present invention will vary depending on the age, sex, and weight of the patient, the patient's current health condition, and the progression of acute lung injury in the patient being treated. Those skilled in the art will be able to determine the appropriate dosage depending on these and other factors.

[0076] Suitable doses of the compounds of the present invention can range from about 0.01 mg to about 5,000 mg per day, such as about 0.1 mg to about 1,000 mg per day, for example, about 1 mg to about 500 mg per day, or about 5 mg to about 250 mg per day. Doses can be administered as a single dose per day or multiple doses per day, for example, 1 to 4 times per day or more. When multiple doses are used, each dose can be the same or different. For example, a 1 mg dose per day can be administered as two 0.5 mg doses, with an interval of about 12 hours between doses.

[0077] It is understood that the amount of compound divided into daily doses can be administered, for example, every day, every other day, every two days, every three days, every four days, or every five days. For example, if every other day administration is used, a 5 mg dose per day can be started on Monday, the first subsequent 5 mg dose per day can be administered on Wednesday, the next subsequent 5 mg dose per day can be administered on Friday, and so on.

[0078] If the patient's condition does improve, at the physician's discretion, the inhibitors of the present invention are optionally administered continuously; alternatively, the administered drug may be temporarily reduced in dosage or temporarily discontinued for a period of time (i.e., a "drug holiday"). The length of the drug holiday optionally varies from 2 days to 1 year, and includes, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. Dose reduction amounts during drug holidays include 10% to 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0079] Once the patient's health condition improves, maintenance doses are administered if necessary.Subsequently, the dosage or frequency, or both, of the administered dose are reduced according to the viral load until the improved disease is maintained.In some embodiments, the patient requires long-term intermittent treatment due to any recurrence of symptoms and / or infection.

[0080] The compounds for use in the methods of the present invention can be formulated as unit dosage forms. The term "unit dosage form" refers to a physically discrete unit suitable as a unitary dosage for a patient to be treated, each unit containing a predetermined amount of active material calculated to produce a desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form can be for a single daily dose, or can be one of multiple daily doses (e.g., about 1 to 4 times per day or more). When multiple daily doses are used, the unit dosage form can be the same for each dose, or different for each dose.

[0081] The toxicity and therapeutic efficacy of such treatment regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, LD 50 (the dose that is lethal to 50% of the population), and ED 50 This involves determining the LD (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which is defined as the LD 50 and ED 50 The data obtained from cell culture assays and animal studies are optionally used to derive a range of dosage for use in humans. The dosage of such compounds is preferably determined to be at an ED that results in minimal toxicity. 50 The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.

[0082] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of the present invention and are encompassed by the claims appended hereto. For example, it should be understood that modifications of reaction conditions, using art-recognized substitutes and no more than routine experimentation, including but not limited to reaction time, reaction size / volume, and experimental reagents such as solvents, catalysts, atmospheric conditions such as pressure and nitrogen atmosphere, and reducing / oxidizing agents, are within the scope of this application.

[0083] Whenever values ​​and ranges are provided herein, it should be understood that all values ​​and ranges encompassed by those values ​​and ranges are intended to be encompassed within the scope of the present invention. Moreover, all values ​​that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the application. [Example]

[0084] Experimental example The present invention is described in further detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and, unless otherwise stated, are not intended to be limiting. Therefore, the present invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations that become evident as a result of the teachings provided herein.

[0085] Without further description, one of ordinary skill in the art will be able, using the foregoing description and the following illustrative examples, to make and utilize the compounds of the present invention, and to practice the claimed methods. The following examples, therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0086] Example 1: Activity of PIP peptides in vivo Mice were injected with PIP-2 (2 μg / g body weight) in liposomes (see Figure 1) either IT (Figure 1A) or IV (Figure 1B); these liposomes contained a tracer [ 3 The lungs were excised from the mice and tested in isolation. The slope of the line indicates the production of the oxidant (H2O2).

[0087] PIP-2 in liposomes, administered IV or IT, inhibited Prdx6 activity in lung homogenates. Maximum inhibition was observed within 4 hours after PIP-2 administration. Recovery from inhibition began approximately 36 hours and was complete by 48 hours. Results were similar for PIP-1, PIP-2, and PIP-4 (PIP-3 and PIP-5 were not tested). Based on mouse results, PIP-2 or PIP-4 could be administered once every 24–36 hours to maintain maximal inhibition of Prdx6 activity and NOX2 activation. Efficacy requires liposomes for peptide delivery. Inhibition after IT and IV administration was similar. Data are presented in Tables 4–6 below.

[0088] Table 4. aiPLA2 activity in mouse lungs 24 hours after IV injection of PIP-4 with and without liposomes for delivery. TIFF0007822065000031.tif89128PIP-4 = 2μg / g mouse body weight. Mean + / - SE; n = 3.

[0089] Table 5. aiPLA2 activity in mouse lung homogenates over time following IT or IV infusion of PIP-2: persistence in vivo TIFF0007822065000032.tif106155

[0090] Table 6. Prdx6-PLA2 activity in mouse lung homogenates over time following IT or IV PIP infusion. TIFF0007822065000033.tif148162* To avoid possible influence of non-internalized SP-A peptide, lungs were lavaged prior to the assay.

[0091] Example 2: Time course of injury after IT LPS Bacterial (Escherichia coli) lipopolysaccharide (LPS) was administered to wild-type C57Bl / 6 mice via intratracheal (IT) instillation at 5 μg / g body weight. Mice were sacrificed 12, 16, 24, or 48 hours after LPS administration, as indicated. Lungs were excised and lavaged via the trachea with saline to obtain BALF; the lungs were then homogenized. Measured parameters included nucleated cells and protein in BALF, thiobarbituric acid reactive substances (TBARS), 8-isoprostane, and protein carbonyl in lung homogenates, and lung wet-dry weight ratio (W / D). Values ​​are means ± SD for n = 4.

[0092] As shown in Figure 2, increased cells in BALF reflect inflammation, protein in BALF and increased wet / dry weight ratio reflect altered alveolar permeability, TBARS and 8-isoprostanes reflect peroxidation of cell membrane lipids, and protein carbonyls reflect oxidation of tissue proteins. All of these effects are characteristic of the ALI syndrome. Following a single dose of LPS, significant lung injury is present, which remains essentially unchanged between 12 and 24 hours post-LPS. Partial recovery is observed at 48 hours. LPS-induced lung injury remains relatively stable between 12 and 24 hours; this likely reflects an equilibrium between ongoing lung injury and the recovery process. The data are presented in Table 7 below.

[0093] Table 7. Time course of injury after IT LPS TIFF0007822065000034.tif78166LPS 5μg / g

[0094] Example 3: PIP-2 protects against lung injury An IT model of acute lung injury (ALI), shown in Table 9, was used to test the effects of PIP-2. PIP-2 in liposomes (2 μg / g body weight) was administered IT with LPS (0 h) or intravenously (IV) 12 or 16 h after LPS. IV administration of PIP-2 was used to prevent a second "attack" on the trachea. Mice were sacrificed at 24 h, and lungs were evaluated for injury as described in Table 9. % protection from lung injury was calculated as [1 - (injury with PIP-2 - control) / (LPS only - control)]. Results are mean ± SE for n = 4. * P < 0.01 vs. control. † P < 0.01 vs. no PIP, 24 hours.

[0095] As shown in Figure 3, administration of PIP-2 simultaneously with LPS completely prevented lung injury when assessed 24 hours after LPS administration. PIP-2 administered 12 or 16 hours after LPS provided approximately 85-95% protection from lung injury when assessed 24 hours after LPS administration. The effect of PIP-2 is quite dramatic. Both PIP-2 and PIP-4 prevented lung injury when administered at time 0 and further injury when administered at 12-16 hours, allowing the damaged lungs to heal during the 12-16 hour period between LPS administration and sacrifice 24 hours later. Thus, PIP-2 and PIP-4 can both prevent and treat lung injury. Data for various markers of lung injury are presented in Tables 8-11.

[0096] Table 8. Effect of PIP-2 on inflammation and edema after LPS TIFF0007822065000035.tif107147n = 4, PIP-2 concentration (2 μg / g mouse body weight), LPS 5 μg / g

[0097] Table 9. Effect of PIP-2 on lung tissue oxidation after LPS TIFF0007822065000036.tif120159n = 4, PIP concentration (2 μg / g mouse body weight), LPS 5 μg / g

[0098] Table 10. Effect of PIP-4 on inflammation and edema after LPS TIFF0007822065000037.tif107147n = 4; PIP concentration 2 μg / g mouse body weight, LPS 5 μg / g

[0099] Table 11. Effect of PIP-4 on lung tissue oxidation after LPS TIFF0007822065000038.tif135164n = 4, PIP concentration (2 μg / g mouse body weight), LPS 5 μg / g

[0100] Example 4: PIP-2 is stable as a dry powder. aiPLA2 activity was measured at intervals to determine how long the peptide maintained its potency as an inhibitor of aiPLA2 activity. The peptide was stable over a 4-month observation period.

[0101] (Table 12) PIP-2 activity during 4 months of storage at room temperature as a dry powder demonstrates stability. TIFF0007822065000039.tif129128

[0102] Example 5: The materials and methods used in the following examples are described herein.

[0103] animal C57Bl / 6J mice and NADPH oxidase (Nox2) null mice were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained in the University of Pennsylvania Laboratory Animal Resources (ULAR) facility under HEPA-filtered air and a 12-h light / dark cycle.

[0104] reagent The estimated purity of the peptides, assessed by mass spectrometry, was >89%. Lipopolysaccharide (LPS) extracted from Escherichia coli 0111:B4 cell membranes and purified by gel filtration chromatography was obtained from Sigma-Aldrich (St. Louis, MO, USA, catalog number L3012). Amplex Red / horseradish peroxidase (HRP) assay kit (catalog number A22188) and reduced difluorofluorescein diacetate carboxy adduct (DFF-DA, catalog number 13293) were purchased from Life Technologies, Grand Island, NY, USA (through Thermo-Fisher Scientific). Angiotensin II (Ang II) was obtained from Bachem, Torrance, CA, USA (catalog number 4095850.0005). Raw lipids were purchased from Sigma-Aldrich, St. Louis, MO, USA, and liposomes were prepared by evaporation to dryness followed by reconstitution in saline, reflecting the composition of pulmonary surfactant as previously described; the liposome composition was 0.5: dipalmitoylphosphatidylcholine (DPPC), 0.25: egg phosphatidylcholine (PC), 0.10: phosphatidylglycerol (PG), and 0.15: cholesterol. The PIP-2 loading was 0.15 μg PIP-2 / μg lipid.

[0105] Administration of LPS and PIP-2 Anesthetized mice received LPS (either 5 μg / g body weight or 15 μg / g body weight) in 20 μl saline, which was instilled into the lungs through an intratracheal catheter placed at the level of the tracheal carina. We previously demonstrated that PIP-2 is ineffective when injected alone, but when encapsulated in liposomes, it inhibits aiPLA2 activity with a half-life of approximately 50 hours. PIP-2 in liposomes was suspended in 20 μl saline for IV or IT infusion. For studies evaluating the effects of PIP-2 at time zero, LPS administration was followed by liposomes ± PIP-2, also administered IT. For studies evaluating the effects of PIP-2 administered later after LPS, liposomes ± PIP-2 were administered by injection into the retinal artery. This change in administration route was used to minimize damage to the mouse trachea, which can occur with repeated tracheotomy and can lead to adverse effects on the lungs. The dose of PIP-2 used for treatment after intratracheal LPS was 2 μg / g mouse body weight; in control mice, this dose of PIP-2 has been shown to maximally inhibit pulmonary aiPLA2 activity for at least 24 hours. To ensure maximal protection, we administered a second dose of PIP-2 at 12 hours and continued PIP-2 administration every 24 hours thereafter. For the sepsis model, LPS (15 μg / g body weight) in 20 μl saline was injected intraperitoneally, and mice were treated with IV PIP-2 at either 2 μg / g body weight or 20 μg / g body weight; note that for this sepsis model, the first dose of PIP-2 was administered IV, not IT. We used the same number of PIP-2 doses in the sepsis model as in the IT LPS model. After recovery from anesthesia, all mice were maintained in a vivarium with free access to food and water.

[0106] Assessment of lung injury At the end of each experiment using IT LPS (either 24 or 120 hours), surviving mice were sacrificed by exsanguination under anesthesia. The lungs were perfused in situ via the pulmonary artery to remove blood and then lavaged via the trachea with saline. The lungs were then excised from the thoracic cavity for histological analysis. We assessed the effect of LPS on lung injury by measuring the number and protein content of nucleated cells in the bronchoalveolar lavage fluid (BALf), the wet-to-dry lung weight ratio in the left upper lobe, and thiobarbituric acid reactive substances (TBARS), 8-isoprostanes, and protein carbonyls in lung homogenates to determine the oxidation of lipid and protein components in lung tissue. For mouse mortality studies, survival plots were constructed using the Kaplan-Meier estimator.

[0107] Measurement of pulmonary ROS production and aiPLA2 activity The effect of PIP-2 on ROS production in control (untreated) lungs was determined in vitro using isolated perfused lungs. PIP-2 in liposomes was administered intravenously at 2 μg / g mouse body weight. After 30 min, mice were anesthetized, and the lungs were isolated, purged, and perfused in a recirculating system with a perfusion solution containing Ang II (50 μM) as a Nox2 activator, as well as Amplex Red and horseradish peroxidase to detect ROS. Lungs from wild-type mice and NOX2-null mice not treated with PIP-2 served as controls. The basal rate of ROS production was assessed using WT lungs perfused in the absence of Ang II. The perfusion protocol included a 15-min equilibration period followed by a 60-min experimental period. Aliquots of perfusate were withdrawn at 15-minute intervals and analyzed for resorufin (λ), a product of the oxidation of Amplex Red. 励起 568 nm, λ 蛍光The perfusion of the perfusate was analyzed by fluorescence at 581 nm. The rate of Amplex Red oxidation was calculated and expressed as arbitrary fluorescence units (AFU) normalized to mouse body weight. In the absence of HRP in the perfusate, there was a slow rate of Amplex Red oxidation (approximately 7% of Ang II-stimulated fluorescence), indicating non-ROS-mediated oxidation of the fluorophore; this value was subtracted to obtain the values ​​shown.

[0108] To determine lung ROS production after LPS treatment, intact mice were treated with LPS (5 μg / g) ± PIP-2 (2 μg / g). Mice were anesthetized 6, 12, or 24 hours after LPS treatment, and the lungs were in situ evacuated and then perfused for 10 minutes with saline containing the fluorophore DFF-DA, which is intracellularly hydrolyzed to DFF. Lungs were then homogenized, and the fluorescence of the homogenate was measured at Ex 495 nm and Em 525 nm. Lung fluorescence was expressed as AFU per minute of perfusion, normalized to mouse body weight.

[0109] statistical analysis Data were expressed as mean ± standard error (SE). The slope of the linear plots was calculated by the least mean squares method. SigmaStat software (Jandel Scientific, San Jose, CA) was used to assess statistical significance. Mean differences between groups were assessed by one-way ANOVA followed by Bonferroni post-hoc test. For comparisons of two groups, means were compared by Student's t-test. Differences between means were considered statistically significant when P < 0.05.

[0110] result: Inhibition of pulmonary ROS production by PIP. To confirm the inhibitory effect of PIP compounds on NOX2 activation, we examined ROS production by isolated perfused lungs in the presence of Ang II, a known activator of NOX2. Amplex Red oxidation was used as an indicator of ROS production. Under control conditions, i.e., without the addition of stimulators of NOX2 activity, perfused lungs exhibited a very low baseline rate of ROS production (Figure 8, WT basal). ROS production significantly increased when Ang II was added to the perfusate to activate NOX2 (Figure 8, WT control). ROS production was reduced by 76% in NOX2-null lungs compared with WT lungs, indicating that NOX2 is the primary source of ROS generated in the perfusate after Ang II stimulation. As previously shown, addition of PIP-2 (in liposomes) to WT lungs inhibited ROS production (approximately 75%), similar to NOX2-null lungs. Thus, PIP-2 resulted in essentially complete inhibition of NOX2-mediated ROS production.

[0111] We next determined the effects of PIP-2 (in liposomes) on aiPLA2 activity and ROS production after LPS in the lung. These parameters were determined 6, 12, and 24 h after administration of intravenous LPS. aiPLA2 activity in lung homogenates increased by approximately 50% compared to controls at 6 h after treatment with LPS, and by a further 50% at 12 and 24 h (Figure 9A). To determine pulmonary ROS production, we used an intracellular fluorophore (DFF-DA). ROS-induced fluorescence was very low in the lungs of LPS-untreated controls, whereas it increased approximately 10-fold at 6 h and approximately 20-fold at both 12 and 24 h in the lungs of LPS-treated mice (Figure 9B). This increase in pulmonary DFF fluorescence after LPS may have been slightly underestimated due to signal dilution by the presence of edema in these lungs (see below). Pretreatment of mice with PIP-2 before LPS administration resulted in dramatic reductions in aiPLA2 activity and ROS-generated fluorescence at all three time points, to values ​​similar to those of untreated controls. These results indicate that intratracheal administration of LPS leads to increased ROS production in the lungs, which is maintained for at least 24 hours and can be almost completely inhibited by pretreatment of the lungs with PIP-2.

[0112] Time course of LPS-induced lung injury Susceptibility to LPS-induced lung injury varies significantly among mouse strains. For this study, we determined the course of lung injury in C57Bl / 6J mice administered IT LPS at 5 μg / g body weight (Figures 2A–2F). When assessed 12 h after LPS, the lungs showed significant damage, as indicated by increased nucleated cells in the BALf, increased BALf protein, and increased lung wet-dry weight ratio (p < 0.05). These results are consistent with pulmonary inflammation (cells in the BALf), altered alveolar-capillary barrier permeability (BALf protein), and lung fluid accumulation (lung wet / dry weight). Increases in TBARS, 8-isoprostanes, and protein carbonyls in lung tissue indicate oxidative stress due to oxidation of lipid and protein components in lung tissue. These indices of lung injury showed similar values ​​at 12, 16, and 24 h after LPS (Figures 2A-2F), indicating that the extent of lung injury was essentially stable between 12 and 24 h after this nonlethal dose of LPS. A partial recovery (approximately 50%, p < 0.05) in the indices of lung injury was observed at 48 h, but they were still elevated compared to controls (p < 0.05).

[0113] Effect of PIP-2 on LPS-induced lung injury To test the effect of PIP-2 administration on lung injury, mice were treated with IT-administered LPS (5 μg / g body weight). The LPS dose was selected based on our previous study using the same batch of LPS, which showed relatively low levels of lung injury at 1 μg / g body weight and more severe damage without significant mortality when used at 5 μg LPS / g body weight. PIP-2 (2 μg / g body weight, in liposomes) was administered at 0, 12, or 16 hours after LPS administration. We have previously shown that this dose of PIP-2 can inhibit pulmonary aiPLA2 activity by approximately 90% for at least 24 hours. To avoid excessive damage to the trachea, PIP-2 was administered IT at time 0 and IV at 12 or 16 hours. Animals were sacrificed, and lungs were examined 24 hours after LPS administration. All indicators of lung injury, reflecting pulmonary inflammation, alveolar-capillary barrier dysfunction, lung fluid accumulation, and tissue oxidative stress, were elevated in LPS-treated mice compared with controls (p < 0.05). PIP-2 administered at time 0 completely prevented lung injury when assessed 24 h after LPS (Figures 3A–3F). Indicators of tissue injury in the lungs of mice treated with PIP-2 at 12 and 16 h were also significantly reduced compared with LPS alone, and values ​​were not significantly different from control values ​​(Figures 3A–3F). Because lung injury was present in the lungs at 12 and 16 h after LPS, the normal values ​​at 24 h in the lungs of LPS-treated mice administered PIP-2 at 12 or 16 h can only mean that the lungs were able to fully recover from that injury during the 8–12 h period between PIP-2 administration and lung examination.

[0114] PIP-2 treatment prevents death in mice due to high doses of LPS. Mice treated with a low dose of LPS (5 μg / g body weight) suffered significant lung injury, but it was transient, and essentially all mice recovered from the injury (not shown). To test the effect of PIP-2 treatment using a more severe injury model, mice were administered a higher dose of LPS (15 μg / g body weight). Survival data are plotted with the initial PIP-2 treatment as time 0; LPS was administered 12 hours before PIP-2 (-12 hours, which is outside the graphs in Figures 7A and 7B). At this higher dose of LPS, mice treated with placebo (liposomes alone) showed a 73% mortality rate within 24 hours after LPS and a 100% mortality rate by 48 hours. For the treatment groups, PIP-2 was administered to mice 12, 24, 48, 72, and 96 hours after LPS, and the mice were sacrificed at 120 hours; PIP-2-treated mice showed only a 17% mortality rate (83% survival) 36 hours after the start of PIP-2 treatment and no further mortality occurred during the observation period. In addition to the effect on mortality, a significant difference was observed in the behavior of mice administered PIP-2 after LPS, in that most mice returned to normal physical activity by 12 hours after PIP-2 administration. Indicators of lung injury in treated mice sacrificed 120 hours after LPS showed no abnormalities (Table 13).

[0115] (Table 13) Lung injury is repaired in mice that survive high dose LPS. TIFF0007822065000040.tif49131

[0116] Mice were injected IT with LPS (15 μg / g body weight); PIP-2 in liposomes (2 μg / g body weight) was injected IV at the times indicated in Figure 7A. Five surviving mice were sacrificed 120 h after LPS; control mice received liposomes but not LPS. BALf, bronchoalveolar lavage fluid; TBARS, thiobarbituric reactive substances. Values ​​are means ± SE for N = 4 for control and N = 5 for LPS + PIP-2. None of the means for LPS + PIP-2 were statistically different (p > 0.05) from the corresponding controls.

[0117] We next evaluated the effects of PIP-2 in mice administered LPS (15 μg LPS / g body weight) by the intraperitoneal route as a model of ALI associated with systemic sepsis. Based on our previous studies, we selected a dose of LPS that resulted in 60% mortality at 10 μg LPS / g body weight; our goal was to produce 100% mortality in placebo-treated mice, similar to that observed in the high-dose IT LPS study. The survival rate of placebo-treated mice (liposomes only) was less than 40% at 24 hours post-LPS, and 100% of the mice had died by 48 hours (Figure 7B). In contrast, treatment of mice with PIP-2 (2 μg / g body weight) increased survival to 86% at 36 hours post-LPS, with 43% of the mice fully recovering. With a higher dose of PIP-2 (20 μg / g body weight), long-term survival was significantly higher at 70%. Thus, PIP-2 significantly increased mouse survival in this model of systemic sepsis-associated ALI.

[0118] ALI is a severe disease syndrome with an approximately 40% mortality rate. Inflammation is a key factor that can amplify lung damage associated with the initial insult. To date, there are no approved pharmacological treatments for the inflammatory component of the syndrome. The mechanism of lung injury during pulmonary inflammation is complex, but excessive ROS production appears to play a major role. We previously demonstrated that the aiPLA2 activity of Prdx6 is required for the activation of ROS production by NOX2 and described several nonapeptides derived from the pulmonary surfactant protein A (SP-A) sequence that inhibit aiPLA2 activity and thereby inhibit NOX2 activation in lung cells. This study confirms that these peptides, designated PLA2 inhibitory peptides (PIP-2, PIP-4, and PIP-5), inhibit ROS production by Ang II-activated NOX2 in isolated mouse lungs. Although PIP-2 appeared to be slightly more active than the other two, all three PIP compounds were effective inhibitors, likely reflecting in part the high conservation of the Prdx6 amino acid sequence across species. We demonstrate that the binding site of the 16-amino acid precursor of PIP is to the amino acid sequence encompassing amino acids 195-204 of Prdx6. The sequence of this segment of human Prdx6 is: TIFF0007822065000041.tif4128; the corresponding mouse sequence is identical at 8 of 10 amino acids, has a Q instead of a K at position 200, and a C instead of an L at position 201. For further studies, we chose PIP-2, a PIP derived from the corresponding sequence in human SP-A. The amino acid sequence of PIP-2 is: The file is TIFF0007822065000042.tif4128.

[0119] The primary goal of this study was to evaluate the effect of PIP-2 on lung injury associated with intratracheal administration of LPS. We first demonstrated that PIP-2 significantly inhibited Ang II-mediated ROS production; Ang II is a known activator of NOX2, and as we previously showed, activation requires aiPLA2 activity. We then showed that treatment with LPS resulted in both a significant increase in pulmonary aiPLA2 activity and, through NOX2 activation, a significant increase in ROS production; the LPS-induced increases in both aiPLA2 activity and ROS production were also inhibited by PIP-2.

[0120] The first test of PIP-2 efficacy in a lung injury model was coadministration of PIP-2 with LPS, which significantly protected against subsequent lung injury. Measurements to assess acute lung injury after LPS included: a) nucleated cells in BALf (inflammation); b) protein in BALf (alveolar-capillary permeability); c) lung wet-dry weight ratio (pulmonary edema); and d) pulmonary TBARS, 8-isoprostanes, and protein carbonyls (tissue lipid and protein oxidation). All of these indicators of injury were significantly elevated in the lungs assessed 12–24 h after LPS administration. However, none of these indicators of tissue injury were altered in the lungs when PIP-2 was coadministered with LPS. Thus, PIP-2 can prevent ALI associated with LPS administration in mice.

[0121] The next study investigated the effect of PIP-2 administered 12 or 16 hours after LPS administration as a treatment (not preventative) modality. As shown in Figures 3A-3F, tissue damage associated with nonlethal LPS is maximal at this time point. Administration of PIP-2 at either 12 or 16 hours after LPS essentially restored lung injury parameters to normal when examined 24 hours after LPS. Our conclusion from this study is that PIP-2 prevented ongoing LPS-associated lung injury and allowed the lungs to repair themselves during the 8- to 12-hour period between PIP-2 administration and animal sacrifice.

[0122] Our final study evaluated the effects of PIP-2 on lung function and survival in mice after a lethal dose of LPS. Administration of PIP-2 every 12 to 24 hours after LPS administration dramatically improved mouse behavior, significantly reduced mouse mortality, and restored indicators of lung injury to normal levels. Thus, a nonapeptide inhibitor of Prdx6's PLA2 activity prevented ROS production following NOX2 activation and prevented the death associated with a lethal dose of LPS. These results demonstrate that PIP-2 can both prevent and treat LPS-induced ALI in a mouse model.

[0123] Our results with PIP-2 lead to similar conclusions as our previous studies, which demonstrated protection from LPS-induced ALI using several different means to inhibit aiPLA2 activity and subsequent NOX2 activation. These included: a) administration of MJ33, a lipid inhibitor of aiPLA2 activity; b) use of Prdx6-null mice (not a perfect model because the peroxidase activity of Prdx6 is also lost); and c) mice with a mutation in amino acid D140 in Prdx6, which is an essential component of the aiPLA2 active site. MJ33-inhibited mice, D140A mutant mice, and PIP-2-treated mice all retained the peroxidase activity of Prdx6, whereas this activity was abolished in Prdx6-null mice. In these previous studies, LPS was administered intravenously in a) and b) as a model of direct lung injury, and intraperitoneally in c) as a model of non-infectious sepsis. We propose that the mechanism of protection provided by PIP-2 is its inhibition of the aiPLA2 activity of Prdx6 through an allosteric effect resulting from the peptide's binding to Prdx6. As demonstrated experimentally and as expected based on the dissimilarity of its potential binding site to other proteins, the PIP peptide does not inhibit other pulmonary PLA2 enzymes. Inhibition of aiPLA2 activity prevents the production of lyso-PC and its downstream products, thereby preventing the activation of Rac, an essential cofactor for Nox2 activation. Interestingly, the cholesterol-lowering drug simvastatin also inhibits Rac activation, and simvastatin has been shown to inhibit ROS production by endothelial cells and to be protective in a mouse model of LPS-induced ALI. Although the evidence is currently inconclusive, inhibition of Rac activation may have beneficial effects on non-ROS-mediated ALI symptoms in addition to its effect on NOX2 activation.

[0124] This and previous studies demonstrate that NOX2 is the primary source of ROS in the lung and that the enzyme is activated in the presence of LPS. In addition to the LPS model, ROS production by NOX2 has been shown to play a central role in several related but distinct animal models of ALI, including gram-negative sepsis, endotoxin, severe trauma, hemorrhagic shock, and oleic acid infusion. Perhaps the primary manifestation of oxidant stress associated with NOX2 activation is the oxidation of tissue macromolecules, as shown in this study. However, another important pathophysiological role associated with NOX2-derived ROS is based on evidence that ROS are responsible for signals leading to neutrophil recruitment to the lung and the resulting pulmonary inflammation characteristic of ALI. The significant reduction in nucleated cells in BALf after treatment with PIP-2 suggests that this function of ROS is important for recovery from lung injury. In this regard, a peptide inhibitor of myristoylated alanine-rich C kinase substrate (Marcks) protein also protects against LPS-induced lung injury in mice. Although this latter peptide has not previously been shown to inhibit NOX2 activation, its effect may be mediated by alterations in cell motility that prevent PMN influx into the lung. Thus, inhibitors of NOX2, such as PIP-2, simvastatin, Marcks protein inhibitors, and possibly apocynin, may all prevent PMN influx into the lung after LPS, thereby reversing inflammation and associated lung injury.

[0125] Based on these results, peptide inhibitors of NOX2 activation may be effective as preventative agents for patients at risk for ALI and as treatments for patients already suffering from ALI. While the toxicity of these small peptides is not expected given their normal expression in the lung as components of the SP-A protein, toxicity should still be investigated. The peptides' potential for antigenicity is theoretically low, but this will need to be confirmed in humans. Other potential side effects of the peptides include those related to the inhibition of Rac activation and the loss of ROS signaling and regulatory functions. Notably, no significant effects that could be related to Rac inhibition have been reported so far for the widely used drug simvastatin. A potentially more important "side effect" of treatment with PIP may be the effect that the inhibition of ROS production has on the bactericidal activity of inflammatory cells (PMNs and AMs), which use superoxide anions generated by NOX2 activity to kill bacteria. Furthermore, some antibiotics have been shown to require ROS for maximal efficacy. Despite their theoretical potential to alter responses to infection, inhibitors of NOX2 activation did not reduce PMN bactericidal activity in the LPS model of ALI. This may reflect the ability of non-NOX2 pathways to compensate for the loss of NOX2-derived ROS. While this may highlight the important role of antibiotic protection in patients treated with NOX2 inhibitors, it is important to note that the use of antibiotics alone is not effective in reducing the mortality rate of this disease significantly below 40%.

[0126] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entireties.

[0127] While the present invention has been disclosed with reference to particular embodiments, it will be apparent that other embodiments and variations of the present invention may be devised by others skilled in the art without departing from the true spirit and scope of the present invention, and it is intended that the appended claims be construed to include all such embodiments and equivalent variations.

[0128] Sequence information SEQUENCE LISTING <110> The Trustees of the University of Pennsylvania <120> Compositions and Methods for Treatment of Acute Lung Injury <150> US 62 / 719,217 <151> 2018-08-17 <160> 34 <170> PatentIn version 3.5 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 1 Leu His Asp Phe Arg His Gln Ile Leu 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 2 Leu Tyr Glu Ile Lys His Gln Ile Leu 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 3 Leu Tyr Asp and Arg His Gln and Leu 1 5 <210> 4 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <220> <221> misc_feature <222> (1)..(5) <223> It can be any naturally occurring amino acid. <220> <221> misc_feature <222> (7)..(10) <223> It can be any naturally occurring amino acid. <400> 4 Yes Yes Yes Yes Yes Yes Yes Yes Yes His Gln Isle Leu 1 5 10 <210> 5 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 5 Glu Leu Gln Thr Glu Leu Tyr Glu Ile Lys His Gln Ile Leu 1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 6 Gln Thr Glu Leu Tyr Glu Ile Lys His Gln Ile Leu 1 5 10 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 7 Glu Leu Tyr Glu Ile Lys His Gln Ile Leu 1 5 10 <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 8 Asp Glu Glu Leu Gln Ala Thr Leu His Asp Phe Arg His Gln Ile Leu 1 5 10 15 <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 9 Asp Glu Glu Leu Gln Thr Glu Leu Tyr Glu Ile Lys His Gln Ile Leu 1 5 10 15 <210> 10 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 10 Glu Leu Gln Thr Glu Leu Tyr Glu Ile Lys His Gln Ile Leu 1 5 10 <210> 11 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 11 Gln Thr Glu Leu Tyr Glu Ile Lys His Gln Ile Leu 1 5 10 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 12 Glu Leu Tyr Glu Ile Lys His Gln Ile Leu 1 5 10 <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 13 Tyr Glu Ile Lys His Gln Ile Leu 1 5 <210> 14 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 14 Asp Glu Glu Leu Gln Thr Glu Leu Tyr Glu Ile Lys His Gln 1 5 10 <210> 15 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 15 Asp Glu Glu Leu Gln Thr Glu Leu Tyr Glu Ile Lys 1 5 10 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 16 Asp Glu Glu Leu Gln Thr Glu Leu Tyr Glu Ile 1 5 10 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 17 Asp Glu Glu Leu Gln Thr Glu Leu 1 5 <210> 18 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 18 Thr Leu His Asp Phe Arg His Gln Ile Leu 1 5 10 <210> 19 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 19 Thr Leu His Asp Phe Arg His Gln Ile 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 20 Leu His Asp Phe Arg His Gln Ile 1 5 <210> 21 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 21 Glu Leu Tyr Glu Ile Lys His Gln Ile 1 5 <210> 22 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 22 Leu Tyr Glu Ile Lys His Gln Ile 1 5 <210> 23 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 23 Leu Lys Ile Glu Tyr His Gln Ile Leu 1 5 <210> 24 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 24 Leu Arg Phe Asp His His Gln Ile Leu 1 5 <210> 25 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 25 Leu His Glu Phe Lys His Gln Ile Leu 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 26 Leu Phe Lys Leu Glu His Gln Ile Leu 1 5 <210> 27 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 27 Leu His Asp Phe Arg Asp Gln Ile Leu 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 28 Leu His Asp Phe Arg Pro Gln Ile Leu 1 5 <210> 29 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 29 Leu His Asp Phe Arg His Asn Ile Leu 1 5 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 30 Leu His Asp Phe Arg His Ile Ile Leu 1 5 <210> 31 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 31 Leu His Asp Phe Arg His Gln Leu Leu 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 32 Leu His Asp Phe Arg His Gln Thr Leu 1 5 <210> 33 <211> 10 <212> PRT <213> Homo sapiens <400> 33 Glu Glu Glu Ala Lys Lys Leu Phe Pro Lys 1 5 10 <210> 34 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Inhibitory peptide. <400> 34 Ile Lys His Gln Ile Leu 1 5

Claims

1. A polypeptide consisting of the amino acid sequence of SEQ ID NO:

1.

2. A pharmaceutical composition comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and a pharmaceutically acceptable excipient.

3. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a liposome encapsulating a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

Images