阅读说明：本技术 治疗急性肺损伤的组合物和方法 (Compositions and methods for treating acute lung injury ) 是由 A·B·费舍尔 S·I·范斯坦 于 2019-08-15 设计创作，主要内容包括：在各个方面和实施方式中,本发明提供了用于治疗急性肺损伤(ALI)的组合物和方法。(In various aspects and embodiments, the present invention provides compositions and methods for treating Acute Lung Injury (ALI).)

1. A composition comprising a polypeptide consisting of:

SEQ ID NO:4X¹X²X³X⁴X⁵LX⁶X⁷X⁸X⁹HQIL

wherein:

X¹may or may not be present and if present is E;

X²may or may not be present and if present is L;

X³may or may not be present and if present is Q;

X⁴may be present or absent, and if present is a or T;

X⁵may be present or absent, and if present is T or E;

X⁶is H or Y;

X⁷is D or E;

X⁸is F or I; and

X⁹is R or K.

2. The composition of claim 1, wherein the polypeptide is selected from the group consisting of: 5ELQTELYEIKHQIL, 6QTELYEIKHQIL and 7 ELYEIKHQIL.

3. The composition of claim 1, wherein the polypeptide is selected from the group consisting of: 1 SEQ ID NO:1LHDFRHQIL, 2 SEQ ID NO:2LYEIKHQIL or 3 SEQ ID NO:3 LYDIRHQIL.

4. The composition of claim 1, further comprising a pharmaceutically acceptable carrier.

5. The composition of claim 1, wherein the polypeptide is encapsulated in one or more liposomes.

6. The composition of claim 1, wherein the composition is formulated for nebulization inhalation or intratracheal or intravenous injection.

7. A method of treating acute lung injury in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a polypeptide consisting of:

SEQ ID NO:4X¹X²X³X⁴X⁵LX⁶X⁷X⁸X⁹HQIL

wherein:

X¹may or may not be present and if present is E;

X²may or may not be present and if present is L;

X³may or may not be present and if present is Q;

X⁴may be present or absent, and if present is a or T;

X⁵may be T or E;

X⁶is H or Y;

X⁷is D or E;

X⁸is F or I; and

X⁹is R or K

And a pharmaceutically acceptable carrier.

8. The method of claim 7, wherein the polypeptide is selected from the group consisting of: 5ELQTELYEIKHQIL, 6QTELYEIKHQIL and 7 ELYEIKHQIL.

9. The method of claim 7, wherein the polypeptide is selected from the group consisting of: 1 SEQ ID NO:1LHDFRHQIL, 2 SEQ ID NO:2LYEIKHQIL or 3 SEQ ID NO:3 LYDIRHQIL.

10. The method of claim 7, wherein the polypeptide is encapsulated in one or more liposomes.

11. The method of claim 7, wherein the pharmaceutical composition is administered to the subject by nebulized inhalation or by intratracheal or intravenous injection.

12. A method of treating sepsis in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a polypeptide consisting of:

SEQ ID NO:4X¹X²X³X⁴X⁵LX⁶X⁷X⁸X⁹HQIL，

wherein:

X¹may or may not be present and if present is E;

X²may or may not be present and if present is L;

X³may or may not be present and if present is Q;

X⁴may be present or absent, and if present is a or T;

X⁵may be T or E;

X⁶is H or Y;

X⁷is D or E;

X⁸is F or I; and

X⁹is R or K

And a pharmaceutically acceptable carrier.

13. The method of claim 12, wherein the polypeptide is selected from the group consisting of: 5ELQTELYEIKHQIL, 6QTELYEIKHQIL and 7 ELYEIKHQIL.

14. The method of claim 12, wherein the polypeptide is selected from the group consisting of: 1 SEQ ID NO:1LHDFRHQIL, 2 SEQ ID NO:2LYEIKHQIL or 3 SEQ ID NO:3 LYDIRHQIL.

15. The method of claim 12, wherein the polypeptide is encapsulated in one or more liposomes.

16. The method of claim 12, wherein the pharmaceutical composition is administered to the subject by nebulized inhalation or by intratracheal or intravenous injection.

Background

Pulmonary inflammation is an important component of the pathogenesis of Acute Lung Injury (ALI) syndrome due to a variety of etiologies. Pulmonary inflammation associated with Reactive Oxygen Species (ROS) production is an important factor contributing to ALI syndrome. Activation of NADPH oxidase type 2 (NOX2), the major source of ROS in the lung, phospholipase A requiring peroxiredoxin 6(Prdx6)₂(PLA₂) And (4) activity.

Current treatment of ALI is supportive and there are currently no approved drugs specifically for its prevention or treatment. Accordingly, there is a need in the art for methods and compositions for protecting against ALI. The present disclosure addresses this need.

Disclosure of Invention

In one aspect, the invention provides a composition comprising a polypeptide consisting of:

SEQ ID NO:4X¹X²X³X⁴X⁵LX⁶X⁷X⁸X⁹HQIL

wherein:

X¹may or may not be present and if present is E;

X²may or may not be present and if present is L;

X³may or may not be present and if present is Q;

X⁴may be present or absent, and if present is a or T;

X⁵may be present or absent, and if present is T or E;

X⁶is H or Y;

X⁷is D or E;

X⁸is F or I; and

X⁹is R or K.

In various embodiments, the polypeptide is selected from the group consisting of: 5ELQTELYEIKHQIL, 6QTELYEIKHQIL and 7 ELYEIKHQIL.

In various embodiments, the polypeptide is selected from the group consisting of: 1 SEQ ID NO:1LHDFRHQIL, 2 SEQ ID NO:2LYEIKHQIL or 3 SEQ ID NO:3 LYDIRHQIL.

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

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

In various embodiments, the composition is formulated for aerosol inhalation (aerosol inhalation) or for intratracheal or intravenous injection. In various embodiments, the pharmaceutical composition is administered to the subject by intravenous injection.

In another aspect, the present invention provides a method of treating acute lung injury in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a polypeptide consisting of:

SEQ ID NO:4X¹X²X³X⁴X⁵LX⁶X⁷X⁸X⁹HQIL

wherein:

X¹may or may not be present and if present is E;

X²may or may not be present and if present is L;

X³may or may not be present and if present is Q;

X⁴may be present or absent, and if present is a or T;

X⁵may be T or E;

X⁶is H or Y;

X⁷is D or E;

X⁸is F or I; and

X⁹is R or K

And a pharmaceutically acceptable carrier.

In various embodiments, the polypeptide is selected from the group consisting of: 5ELQTELYEIKHQIL, 6QTELYEIKHQIL and 7 ELYEIKHQIL.

In various embodiments, the polypeptide is selected from the group consisting of: 1 SEQ ID NO:1LHDFRHQIL, 2 SEQ ID NO:2LYEIKHQIL or 3 SEQ ID NO:3 LYDIRHQIL.

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

In various embodiments, the pharmaceutical composition is administered to the subject by nebulized inhalation or by intratracheal or intravenous injection.

In another aspect, the invention provides a method of treating sepsis in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a polypeptide consisting of:

SEQ ID NO:4X¹X²X³X⁴X⁵LX⁶X⁷X⁸X⁹HQIL

wherein:

X¹may or may not be present and if present is E;

X²may or may not be present and if present is L;

X³may or may not be present and if present is Q;

X⁴may be present or absent, and if present is a or T;

X⁵may be T or E;

X⁶is H or Y;

X⁷is D or E;

X⁸is F or I; and

X ⁹is R or K

And a pharmaceutically acceptable carrier.

In various embodiments, the polypeptide is selected from the group consisting of: 5ELQTELYEIKHQIL, 6QTELYEIKHQIL and 7 ELYEIKHQIL.

In various embodiments, the polypeptide is selected from the group consisting of: 1 SEQ ID NO:1LHDFRHQIL, 2 SEQ ID NO:2LYEIKHQIL or 3 SEQ ID NO:3 LYDIRHQIL.

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

In various embodiments, the pharmaceutical composition is administered to the subject by nebulized inhalation or by intratracheal or intravenous injection. In various embodiments, the pharmaceutical composition is administered to the subject by intravenous injection.

Drawings

The following detailed description of the preferred embodiments of the present invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIGS. 1A and 1B: wild type C57B1/6 mice, 8-10 weeks old, were injected with 2. mu.g/g body weight PIP-2 via endotracheal (IT) or Intravenous (IV) catheters. The injected peptide was dissolved in saline or incorporated into unilamellar liposomes composed of Dipalmitoylphosphatidylcholine (DPPC), egg Phosphatidylcholine (PC), Phosphatidylglycerol (PG), and cholesterol (molar ratio of lipids 50: 25: 10: 15). We have determined that liposomes containing 75% DPPC or 75% egg PC (plus PG and cholesterol) are as effective as DPPC/PC/PG/cholesterol liposomes for intracellular delivery of PIP-2. Mice were sacrificed after 5min, lungs were perfused until blood was cleared, and then circulating perfusions were performed under temperature-controlled (37 ℃) conditions in the presence of fluorescent indicator Amplex red plus Horse Radish Peroxidase (HRP) to monitor H₂O₂Oxidation of Amplex red. Fluorescence of aliquots of the perfusate was measured at the indicated times and expressed as Arbitrary Fluorescence Units (AFU). Fluorescence increases with perfusion time, indicating H₂O₂The production of (b) reflects the activation of cellular NADPH oxidase (NOX 2). Application of PIP-2 to H in saline₂O₂Has no effect, but PIP-2 in IT (fig. 1A) or IV (fig. 1B) injected liposomes significantly inhibits H₂O₂Is generated.

FIGS. 2A-2F depict lung injury following intratracheal injection of Lipopolysaccharide (LPS) by various markers of tissue oxidation and lung inflammationTime course. Bacteria (e.coli) Lipopolysaccharide (LPS) were administered to wild-type C57B1/6 mice by Intratracheal (IT) injection at 5 μ g/g body weight. As shown, mice were sacrificed 12, 16, 24 or 48h post-LPS. Lungs were removed and lavaged through trachea with saline to obtain BALf; the lungs were then homogenized. Parameters of lung injury are nucleated cells and proteins in BALf and the ratio of lung wet to dry weight (W/D); for W/D, the weight of the upper left lobe of the lung was measured before and after drying to constant weight in an oven. The indices of tissue oxidative stress (lower panel) are thiobarbituric acid reactive species (TBARS), 8-isoprostanes (8-isoprostanes), and protein carbonyl, measured in lung homogenates. The value is the average ±, n ═ 4. P<0.05vs all other values;^§p<0.05vs 12h, 16h and 24 h. Figure 2A depicts a thiobarbituric acid reactive species (TBARS). FIG. 2B depicts 8-isoprostaglandin. Figure 2C depicts protein carbonyl in lung homogenate. Figure 2D depicts the number of cells in bronchoalveolar lavage fluid (BALF). Figure 2E depicts total protein in BALF. Fig. 2F depicts the wet to dry weight ratio of the lungs.

FIGS. 3A-3F produced Acute Lung Injury (ALI) by intratracheal LPS (5mg/g body weight). PIP-2 in liposomes (2 μ g/g body weight) was administered IT with LPS (0h) or Intravenously (IV) 12 or 16h after LPS. IV administration of PIP-2 to avoid a second "attack" on the trachea. Mice were sacrificed at 24h and lung injury and tissue oxidative stress of the lungs were assessed. The result is the mean ± SE, n ═ 4. P <0.05vs all other groups. Figure 3A depicts the number of cells in BALF. Figure 3B depicts total protein in BALF. Figure 3C depicts the wet to dry weight ratio of the lungs. Figure 3D depicts TBARS. FIG. 3E depicts 8-isoprostaglandin. Figure 3F depicts protein carbonyl in lung homogenate.

Fig. 4A and 4B: by the absence of Ca²⁺In the case of (A), phospholipase A of Prdx6 was measured by liberating palmitic acid from dipalmitoylphosphatidylcholine under acidic conditions (pH 4)₂(aiPLA₂). FIG. 4A: AIPLA with increased PIP-2 concentration against recombinant human Prdx6₂The effect of activity. FIG. 4B: AiPLA of mice Lung treated with LPS and PIP-2₂The effect of activity. PI in the absence or presence of liposomesIn the case of P-2(2 μ g/g body weight), mice were treated with intratracheal LPS (2 μ g/g body weight) (n ═ 3 in each case). Animals were sacrificed 6, 12 or 24h after receiving LPS. The lungs are cleared of blood and homogenized. Control lungs were from mice that did not receive LPS. P<0.05vs corresponding control and vs LPS + PIP-2; p § P<0.05vs LPS at 12 and 24 h.

Fig. 5A and 5B: prior to lung isolation, intratracheal instillation of PIP-2 incorporated into liposomes was performed. FIG. 5A: the isolated lung is perfused with artificial media in a recirculation system. NOX2 activity was stimulated by the addition of angiotensin II (Ang II). Amplex red was added to the perfusate along with horseradish peroxidase to detect ROS production. Aliquots of the perfusate were analyzed at intervals by spectrophotometry to determine the oxidation of amplex red, indicating the production of ROS. The results are mean ± SE, N ═ 3-4. FIG. 5B: mice were sacrificed 6, 12, or 24h after LPS administration (5mg/g body weight) and lungs were perfused in situ with saline solution containing the fluorophore (difluorofluorogenic diacetate), DFFDA for 15 min. The lungs were then homogenized and the fluorescence of the lung homogenate was determined as an index of ROS production. The results in fig. 5A and 5B are the mean ± SE, N ═ 4. P <0.05vs corresponding LPS and LPS + PIP-2; p <0.05vs 12h and 24h LPS; Δ P <0.05vs corresponding LPS.

FIG. 6: PIP-2 in liposomes was administered Intratracheally (IT) or Intravenously (IV) to mice at time 0. Lungs were harvested in the interval between 4 and 72h after PIP-2 administration, homogenized and analyzed for Prdx6-PLA2 activity. PIP-2 was effective via either the IT or IV routes with a calculated 1/2 recovery time of about 50 h. The result is an average value + SE, N-3-4.

Fig. 7A and 7B: Kaplan-Meier survival curves. LPS (15mg/g body weight) was administered to all mice by the following method: FIG. 7A: intratracheal (IT) or fig. 7B: intraperitoneal (IP) injection. PIP-2 in liposomes or placebo (liposomes only) was administered Intravenously (IV) 12h after LPS (this is treatment time 0) and then as indicated by the arrow at 12 or 24h intervals for a total of 5 doses. FIG. 7A: PIP-2 at 2. mu.g/g body weight; each group N is 14. FIG. 7B: PIP-2 at 2 or 20 μ g/g body weight; placebo, n-8; PIP-22 mg group, n ═ 7; PIP-220 mg group, n-10.

FIG. 8: PLA2 inhibitory peptide (PIP-2) inhibits ROS production stimulated by angiotensin ii (ang ii) in isolated perfused mouse lungs. PIP-2(2 μ g/g body weight) was administered to intact Wild Type (WT) mice by the IV route. WT-based, WT control and NOX2 knock-out (NOX2 null) did not receive peptides. After 30min, lungs were isolated from anesthetized mice and perfused in the recirculation system with Ang II (50 μ M) and Amplex red plus horseradish peroxidase added as Nox2 activators to detect perfusate ROS. Ang II does not stimulate WT basal lung. After an equilibration period of 15min (referred to as 0 time), aliquots were taken at 15min intervals for fluorescence analysis. Each plot represents the mean ± SE, n ═ 3. The lines are drawn by a least mean square method. The average rate of ROS reduction calculated from the slope of each line is indicated in parentheses. P <0.05vs other 3 slopes.

Fig. 9A and 9B: PIP-2 inhibits increased activity of pulmonary aiPLA2 and increased ROS production following LPS administration. LPS (5. mu.g/g body weight) was administered by Intratracheal (IT) instillation together with liposomes alone (labeled LPS) or PIP-2 in liposomes (labeled + PIP-2). The control was liposomes alone without LPS (labeled control). Mice were sacrificed at 6, 12 or 24h post-LPS and lungs were perfused in situ with saline solution containing the fluorophore difluorofluorescein diacetate (DFF-DA) for 15 min. The lungs were then homogenized and analyzed: FIG. 9A: aiPLA2 activity; FIG. 9B: fluorescence of lung homogenates was used as an index of ROS production. The results are the mean ± SE, N-3 for a and 4 for B. P <0.05vs. control and + PIP-2 at the same time point; Δ P <0.05vs. corresponding values at 6 h.

FIG. 10: effect of liposome composition on delivery of PIP-2 to the lung following IV administration. The protocol for liposome-mediated PIP-2 delivery by intravenous infusion is the same as in fig. 5A-5B. % of total lipids; all liposomes also contained 15% cholesterol.Average ± SE, n-3 or n-2. PC or DPPC within liposomes have similar effectiveness for intracellular delivery of PIP-2. The absence of PG resulted in a decrease in efficacy of about 10%.

FIG. 11: "protection" (%) of PIP-2 against lung injury assessed 24hr after IT LPS. Values for PIP-2 effect administered 0, 12 or 16h after LPS. Protection against lung injury (%) was calculated as [1- (injury with PIP-2-control)/(LPS alone-control) ]. By "guard" of PIP-2 > 75%.

FIG. 12: index of lung injury in PIP-2 treated mice surviving high dose LPS. Mice were injected with LPS (15. mu.g/g wt): row B, Intratracheal (IT); or row C, Intraperitoneal (IP). PIP-2 was injected at the time shown in figure 7 at 2. mu.g/g or 20. mu.g/g body weight in liposomes (IV). 5 surviving mice were sacrificed 108h after the start of treatment (120 h after LPS administration). The results were compared to the values of historical control mice (no LPS) (row a). BALf, bronchoalveolar lavage fluid; TBARS, thiobarbituric acid reactive species. Values are mean ± SE, n-4 for control and n-5 for LPS + PIP-2. The average value of LPS + PIP-2 was not statistically different from the corresponding control (p > 0.05). PIP-2 treated mice that survived 5 days post LPS had normal lungs.

FIG. 13: effects of PIP-2 on Ventilation Induced Lung Injury (VILI). Anesthetized mice were challenged with a tidal volume of 12ml/Kg body weight, a respiratory rate of 120/min and 2cm H₂Positive end-expiratory pressure (PEEP) mechanical ventilation of O for 6 h. PIP-2 in liposomes (2 μ g/g body weight) was administered by IT injection at the beginning of mechanical ventilation and mice were sacrificed after 6 h. Controls represent values for normal (non-ventilated) lungs. The% protection was calculated as in table 1. The result is the mean ± SE, n being 4. For VILI + PIP vs VILI, P<0.05. PIP-2 protects against lung injury associated with mechanical ventilation.

Detailed Description

Definition of

Unless defined otherwise, 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 belongs. Although any methods and materials similar or equivalent to those described herein can be used in practice to test the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

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

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

As used herein, "about" when referring to measurable values such as amounts, time durations, and the like, is meant to encompass variations from the specified values of ± 20% or ± 10%, more preferably ± 5%, even more preferably ± 1%, still more preferably ± 0.1%, as such variations are suitable for carrying out the methods of the present disclosure.

As used herein, "acute lung injury" or "ALI" refers to a syndrome not associated with heart failure characterized by acute episodes of bilateral pulmonary infiltration due to hypoxemia.

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

As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of at least one compound useful within the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. There are a variety of techniques in the art for administering compounds including, but not limited to, intravenous, oral, aerosol, parenteral, ocular, pulmonary, and topical administration.

An "effective amount" or "therapeutically effective amount" of a compound is an amount of the compound sufficient to provide a beneficial effect to the subject to which the compound is administered. An "effective amount" of a delivery vehicle is an amount sufficient to effectively bind or deliver a compound.

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

As used herein, the term "pharmaceutically acceptable" refers to a material (e.g., carrier or diluent) that does not abrogate the biological activity or properties of the compound and is relatively non-toxic, i.e., the material can be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of a composition in which it is contained.

As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle (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 invention within or to a patient such that it 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 compounds useful within the invention, and not injurious to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars (such as lactose, glucose and sucrose); starches (e.g., corn starch and potato starch); cellulose and its derivatives (such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate); powdered tragacanth; 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 glycerol, sorbitol, mannitol, and polyethylene glycol); esters (e.g., ethyl oleate and ethyl laurate); agar; buffering agents (such as magnesium hydroxide and aluminum hydroxide); a surfactant; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol; a phosphate buffer solution; and other non-toxic compatible materials employed in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents and the like that are compatible with the activity of the compounds useful within the present invention and physiologically acceptable to a patient. Supplementary active compounds may also be incorporated into the compositions. The "pharmaceutically acceptable carrier" may further include pharmaceutically acceptable salts of the compounds useful within the present invention. Other additional ingredients that may be included in the Pharmaceutical compositions used in the practice of 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.

As used herein, "PIP-2" means a peptide having SEQ ID NO 1 LHDFRHQIL.

As used herein, "PIP-4" means the peptide having SEQ ID NO 2 LYEIKHQIL.

As used herein, "PIP-5" means a peptide having SEQ ID NO 3 LYDIRHQIL.

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

As used herein, "sepsis" is a potentially life-threatening condition caused by the body's response to an infection and can lead to multiple organ failure.

As used herein, the term "treatment" encompasses prophylaxis and/or therapy. Thus, the compositions and methods of the present invention are not limited to therapeutic applications and may be used for prophylactic applications. Thus, "treating" a state, disorder or condition includes: (i) preventing or delaying the occurrence of clinical symptoms of a state, disorder or condition developing in a subject who may be suffering from or predisposed to a state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (ii) inhibiting a state, disorder or condition, i.e., arresting or reducing the development of a disease or at least one clinical or subclinical symptom thereof; or (iii) alleviating the disease, i.e., causing regression of at least one of the states, disorders or conditions or clinical or subclinical symptoms thereof.

The range is as follows: throughout this disclosure, various aspects of the present invention may be presented in a range format. It is to be understood that the description of the range format is merely for convenience and brevity and should not 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 the possible sub-ranges as well as individual numerical values within that range. For example, the description of a range (e.g., 1 to 6) should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6,3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description of the invention

Composition comprising a metal oxide and a metal oxide

The present invention is based in part on aiPLA useful for treating ALI₂Engineering of specific peptide inhibitors of (a). AiPLA of several peptides of the invention₂The inhibitory activity is shown in table 1 below.

Table 1: effect of peptides on AiPLA2 Activity of recombinant hPrdx6

Table 2: by aiPLA of human recombinant proteins₂Effect of Activity to optimize the size of inhibitory peptides

Table 3: substitutions in PIP-2: effect on inhibition of aiPLA2 Activity of human recombinant Prdx6

Thus, in one aspect, the invention provides a composition comprising a polypeptide consisting of:

SEQ ID NO:4X¹X²X³X⁴X⁵LX⁶X⁷X⁸X⁹HQIL

wherein:

X¹may or may not be present and if present is E;

X²may or may not be present and if present is L;

X³may or may not be present and if present is Q;

X⁴may be present or absent, and if present is a or T;

X⁵may be present or absent, and if present is T or E;

X⁶is H or Y;

X⁷is D or E;

X⁸is F or I; and

X⁹is R or K.

In various embodiments, the composition comprises a polypeptide consisting of SEQ ID NO:1LHDFRHQIL (PIP-2), SEQ ID NO:2LYEIKHQIL (PIP-4), or SEQ ID NO:3LYDIRHQIL (PIP-5). The compositions of the present invention may be provided to a subject as a pharmaceutical composition. Thus, in various embodiments, the composition further comprises 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 composition is formulated for aerosol inhalation or intratracheal or intravenous injection. Suitable pharmaceutically acceptable carriers and inhalable or injectable formulations are described elsewhere herein.

Method of treating acute lung injury

In another aspect, the present invention provides a method of treating acute lung injury in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a polypeptide consisting of:

SEQ ID NO:4X¹X²X³X⁴X⁵LX⁶X⁷X⁸X⁹HQIL

wherein:

X¹may or may not be present and if present is E;

X²may or may not be present and if present is L;

X³may or may not be present and if present is Q;

X⁴may be present or absent, and if present is a or T;

X⁵can be T or E;

X⁶is H or Y;

X⁷is D or E;

X⁸is F or I; and

X⁹is R or K;

and a pharmaceutically acceptable carrier. In various embodiments, the polypeptide can be a polypeptide consisting of SEQ ID NO 1LHDFRHQIL, SEQ ID NO 2LYEIKHQIL, or SEQ ID NO 3 LYDIRHQIL. 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 nebulized inhalation or by intratracheal or intravenous injection.

Methods of treating sepsis

In another aspect, the invention provides a method of treating sepsis in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a polypeptide consisting of:

SEQ ID NO:4X¹X²X³X⁴X⁵LX⁶X⁷X⁸X⁹HQIL

wherein:

X¹may or may not be present and if present is E;

X²may or may not be present and if present is L;

X³may or may not be present and if present is Q;

X⁴may be present or absent, and if present is a or T;

X⁵can be T or E;

X⁶is H or Y;

X⁷is D or E;

X⁸is F or I; and

X⁹is R or K;

and a pharmaceutically acceptable carrier. In various embodiments, the polypeptide can be a polypeptide consisting of SEQ ID NO 1LHDFRHQIL, SEQ ID NO 2LYEIKHQIL, or SEQ ID NO 3 LYDIRHQIL. 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 nebulized inhalation or by intratracheal or intravenous injection. In various embodiments, the pharmaceutical composition is administered to the subject by intravenous injection.

Administration/dose/formulation

The regimen of administration may affect the constitution of the effective amount. The therapeutic formulation can be administered to the subject before or after the onset of the injury. Further, several divided doses (divided doses) and staggered doses may be administered daily or sequentially, or the doses may be continuously infused, or may be bolus injections. Further, the dosage of the therapeutic agent may be increased or decreased in proportion to the urgency of the therapeutic or prophylactic condition.

The compositions of the present invention can be administered to a patient, preferably a mammal, more preferably a human, using known procedures at dosages and for periods of time effective to treat lung injury in the patient. The effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary depending on factors such as the state of the disease or disorder in the patient, the age, sex, and weight of the patient, and the ability of the therapeutic compound to treat or prevent acute lung injury in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be reduced proportionally to the urgency of the treatment situation. A non-limiting example of an effective dosage range of a therapeutic compound of the invention is about 1 to 5,000mg/kg body weight per day. One of ordinary skill in the art will be able to study the relevant factors and determine an effective amount of a therapeutic compound without undue experimentation.

The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

In particular, the selected dosage level will depend upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion or decomposition of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and past medical history of the patient being treated, and like factors well known in the medical arts.

A physician, such as 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, a physician or veterinarian can start a dose of a compound of the invention employed in a pharmaceutical composition at a level below that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

In certain embodiments, it is particularly advantageous to formulate the compounds in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of a therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The dosage unit forms of the invention are specified and directly dependent on: (a) the unique characteristics of a therapeutic compound and the particular therapeutic effect to be achieved, and (b) limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of lung injury in a patient.

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.

In certain embodiments, the compositions of the present invention are administered to a patient in a dosage ranging from 1 to 5 or more times per day. In other embodiments, the compositions of the present invention are administered to a patient in 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 apparent to those skilled in the art that the frequency of administration of the various combination compositions of the invention will vary from individual to individual, depending on a variety of factors including, but not limited to, age, the disease or disorder being treated, sex, general health, and other factors. Thus, the invention should not be construed as limited to any particular dosage regimen, and the precise dosages and compositions to be administered to any patient will be determined by the attending physician taking into account all other factors associated with the patient.

The compounds of the invention for administration may be in the range of about 1 μ g to about 10,000mg, about 20 μ g to about 9,500mg, about 40 μ g to about 9,000mg, about 75 μ g to about 8,500mg, about 150 μ g to about 7,500mg, about 200 μ g to about 7,000mg, about 350 μ g to about 6,000mg, about 500 μ g to about 5,000mg, about 750 μ g to about 4,000mg, about 1mg to about 3,000mg, about 10mg to about 2,500mg, about 20mg to about 2,000mg, about 25mg to about 1,500mg, about 30mg to about 1,000mg, about 40mg to about 900mg, about 50mg to about 800mg, about 60mg to about 750mg, about 70mg to about 600mg, about 80mg to about 500mg, and any and all or some increment therebetween.

In some embodiments, the compound of the invention is administered in a dose of about 1mg to about 2,500 mg. In some embodiments, the dose of a compound of the invention for use in the compositions described herein is less than about 10,000mg, or less than about 8,000mg, or less than about 6,000mg, or less than about 5,000mg, or less than about 3,000mg, or less than about 2,000mg, or less than about 1,000mg, or less than about 500mg, or less than about 200mg, or less than about 50 mg. Similarly, in some embodiments, the dose of the second compound described herein is less than about 1,000mg, or less than about 800mg, or less than about 600mg, or less than about 500mg, or less than about 400mg, or less than about 300mg, or less than about 200mg, or less than about 100mg, or less than about 50mg, or less than about 40mg, or less than about 30mg, or less than about 25mg, or less than about 20mg, or less than about 15mg, or less than about 10mg, or less than about 5mg, or less than about 2mg, or less than about 1mg, or less than about 0.5mg, and any and all or part increments thereof.

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

The formulations may be employed in admixture with conventional excipients, i.e. pharmaceutically acceptable organic or inorganic carrier materials suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral or any other suitable mode of administration known in the art. The pharmaceutical formulations can be sterilized and, if desired, mixed with adjuvants (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like). They may also be combined with other active agents (e.g., other analgesics) as desired.

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 may be formulated for administration by any suitable route, such as oral or parenteral, e.g., transdermal, transmucosal (e.g., sublingual, lingual, (buccal), (transurethral), vaginal (e.g., transvaginal and perivaginal), nasal (intra) and (transrectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets, capsules, caplets (caplets), pills, gelcaps (gel caps), lozenges, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, granules, creams, lozenges, creams, pastes, ointments, emulsions, wafers, suppositories, liquid sprays for nasal or oral administration, dry or aerosolized formulations for inhalation, compositions and formulations for intravesical administration, and the like. It is to be understood that the formulations and compositions that will be useful in the present invention are not limited to the specific formulations and compositions described herein.

Oral administration

For oral use, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. Compositions intended for oral use may be prepared according to any method known to the art, and such compositions may contain one or more agents selected from inert, non-toxic pharmaceutical excipients suitable for the manufacture of tablets. Such excipients include, for example, inert diluents (e.g., lactose); granulating and disintegrating agents (e.g., corn starch); binders (e.g., starch); and lubricating agents (such as magnesium stearate). The tablets may be uncoated or they may be coated by known techniques for aesthetics or to delay release of the active ingredient. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.

The present invention also includes a multilayer tablet comprising a layer providing delayed release of one or more compounds of the present invention and another layer providing immediate release of a drug for the treatment of certain diseases or disorders. Using a wax/pH sensitive polymer mixture, a gastro-insoluble composition can be obtained in which the active ingredient is entrapped, thus ensuring its delayed release.

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 for administration in large doses and/or continuous infusion. Suspensions, solutions or emulsions in oily or aqueous vehicles, optionally containing other formulating agents (such as suspending, stabilizing and/or dispersing agents), may be used.

In addition administration forms

Additional dosage forms of the present invention include those described in U.S. Pat. nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and a dosage form as described in 5,007,790. Additional dosage forms of the invention also include U.S. patent application nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of the invention also include 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 dosage forms as described in WO 90/11757.

Controlled release formulations and drug delivery systems

In certain embodiments, the formulations of the present invention may be, but are not limited to, short-term, rapid-offset (rapid-offset) and controlled, e.g., sustained-release, delayed-release, and pulsatile-release formulations.

The term sustained release is used in its conventional sense to refer to a drug formulation that provides a gradual release of the drug over an extended period of time and, although not necessarily, results in a substantially constant blood level of the drug over an extended period of time. The time period may be up to a month or more and should be a longer release than an equivalent amount of the agent administered in a high dose form.

For sustained release, the compound can be formulated with a suitable polymeric or hydrophobic material that provides the compound with sustained release properties. Thus, the compounds for use in the methods of the invention may be administered in the form of microparticles, for example by injection, or by implantation in the form of a wafer or disc.

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

The term delayed release is used herein in its conventional sense to refer to a pharmaceutical formulation that provides for the initial release of the drug after some delay following administration of the drug, and although may not necessarily include a delay of about 10 minutes up to about 12 hours.

The term pulsatile release is used herein in its conventional sense to refer to a pharmaceutical formulation that provides release of a drug in a manner that produces a pulsatile plasma distribution of the drug after administration of the drug.

The term immediate release is used in its conventional sense to refer to a pharmaceutical formulation that provides immediate release of the drug after administration of the drug.

As used herein, short-term refers to any period of 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 or all whole or partial increments thereof.

As used herein, rapid compensation refers to any period of 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.

Administration of drugs

The therapeutically effective amount or dose of the compound of the present invention depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of acute lung injury in the patient being treated. The skilled person will be able to determine the appropriate dosage in view of these and other factors.

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

It is understood that in non-limiting examples, the amount of compound administered daily can be administered daily, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, for administration every other day, a dose of 5mg per day may begin on Monday, with the first subsequent dose of 5mg per day administered on Wednesday, the second subsequent dose of 5mg per day administered on Friday, and so on.

In the event that the patient's condition does improve, administration of the inhibitor of the invention is optionally continued, at the discretion of the physician; optionally, the dose of drug being administered is temporarily reduced or temporarily terminated for a period of time (i.e., a "drug holiday"). The length of the drug-deprivation period optionally varies from 2 days to 1 year, including, 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 reductions during the drug-off period 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%.

Once the patient's condition has improved, a maintenance dose is administered as necessary. Subsequently, depending on the viral load, either the dose or the frequency of administration or both are reduced to a level at which the improved disease is retained. In certain embodiments, the patient requires long-term intermittent treatment upon any recurrence of symptoms and/or infection.

The compounds for use in the methods of the invention may be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for patients undergoing therapy, wherein each unit contains a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form can be one of a single daily dose or multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are employed, the unit dosage form for each dose may be the same or different.

Toxicity and therapeutic efficacy of such treatment regimens are optionally determined in cell cultures or experimental animals, including but not limited to determining LD₅₀(dose lethal to 50% of the population) and ED₅₀(a dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, expressed as LD₅₀With ED₅₀The ratio of (a) to (b). The data obtained from cell culture assays and animal studies can optionally be used to formulate dosage ranges for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that include the ED with minimal toxicity₅₀. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.

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 this invention and are covered by the following claims. For example, it is understood that reaction conditions, including but not limited to, modifications in reaction times, reaction sizes/volumes, and experimental reagents (such as solvents, catalysts, pressures, atmospheric conditions (e.g., nitrogen atmosphere) and reducing/oxidizing agents), as well as art-recognized alternatives and use of only routine experimentation, are within the scope of the present application.

It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed within that value and range are intended to be encompassed within the scope of the present invention. Moreover, all values falling within these ranges, as well as upper and lower limits of the ranges of values, are also contemplated by this application.

Experimental examples

The present invention is described in further detail with reference to the following experimental examples. These embodiments are provided for the purpose of example only and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should in no way be construed as limited to the following examples, but rather should be construed to cover any and all variations which become evident as a result of the teachings provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and use the compounds of the present invention and practice the claimed methods. The following working examples therefore particularly point out preferred embodiments of the invention and should not be construed as limiting the remainder of the disclosure in any way.

Example 1: activity of PIP peptides in vivo

Injecting PIP-2(2 μ g/g body weight) (see fig. 1) IT (fig. 1A) or IV (fig. 1B) in liposomes into mice; these liposomes are found in DPPThe 9 and 10 positions of the sn-2 palmitate of C contain a tracer [ 2 ]³H]. Lungs were removed from mice and studied in a separate system. The slope of the line indicates the oxidant (H)₂O₂) Is generated.

PIP-2 in IV or IT injected liposomes inhibited Prdx6 activity in lung homogenates. Maximum inhibition was observed within 4 hours after PIP-2 administration. Recovery from inhibition began at about 36 hours and was completed by 48 hours. The results for PIP-1, 2, 4 were similar (PIP-3 and-5 were not tested). Based on the results in mice, PIP-2 or PIP-4 may be administered once every 24 to 36 hours to maintain maximum inhibition of Prdx6 activity and NOX2 activation. Effectiveness requires liposomes for peptide delivery. Inhibition after IT or IV administration was similar. The data are presented in tables 4-6 below.

Table 4: AiPLA2 Activity in mouse Lung 24 hours after IV injection of PIP-4 with and without liposomes for delivery

PIP-4 ═ 2 μ g/g wt of mice. Mean +/-SE; n is 3.

Table 5: aiPLA2 activity of mouse lung homogenate at increased time after IT or IV injection of PIP-2: persistence in vivo

Table 6: prdx6-PLA2 activity of mouse lung homogenates at increased time after IT or IV injection of PIP

Lung lavage was performed before the assay to avoid possible effects of non-internalized SP- ｃA peptide.

Example 2: time course of post-IT LPS injury.

Bacterial (E.coli) Lipopolysaccharide (LPS) was administered to wild type C57Bl/6 mice by Intratracheal (IT) injection at 5. mu.g/g body weight. As shown, mice were sacrificed 12, 16, 24 or 48 hours post-LPS. Lungs were removed and lavaged through trachea with saline to obtain BALF; the lungs were then homogenized. The parameters measured were nucleated cells and proteins in BALF, thiobarbituric acid reactive substance (TBARS) in lung homogenate, 8-isoprostane and protein carbonyl, and the ratio of wet to dry weight of lung (W/D). The value is the average ±, n ═ 4.

As shown in figure 2, the increase in cells in BALF reflects inflammation, the increase in proteins and wet-to-dry weight ratio in BALF reflects changes in alveolar permeability, TBARS and 8-isoprostane reflect cellular membrane lipid peroxidation, and protein carbonyls reflect tissue protein oxidation. These effects are all characteristic of ALI syndrome. There was significant lung injury after a single dose of LPS, which was essentially unchanged between 12 and 24 hours post-LPS. Partial recovery was observed at 48 hours. LPS damage to the lung was relatively stable between 12 and 24 hours; presumably, this reflects a balance between sustained lung injury and the recovery process. The data are presented in table 7 below.

Table 7: time course of post-IT LPS injury

LPS 5μg/g

Example 3: PIP-2 for preventing lung injury

The IT model for Acute Lung Injury (ALI) shown in table 9 was used to test the effect of PIP-2. PIP-2 in liposomes (2 μ g/g body weight) was administered IT with LPS (0 hours) or Intravenously (IV) 12 or 16 hours after LPS. IV administration using PIP-2 to avoid a second "attack" on the trachea. Mice were sacrificed at 24 hours and lung injury was assessed as described in table 9. Protection against lung injury (%) was calculated as [1- (injury with PIP-2-control)/(LPS alone-control)]. The result is thatMean ± SE, n ═ 4. P<0.01vs control.vs PIP free, 24 hours.

As shown in figure 3, PIP-2 administered with LPS completely prevented lung injury when assessed 24 hours after LPS administration. PIP-2 administered 12 or 16 hours post-LPS provides about 85-95% protection against lung injury (assessed 24 hours post-LPS). The effect of PIP-2 is very significant. PIP-2 or PIP-4 prevented lung injury when administered at time 0, further injury when administered at 12 to 16 hours, allowing the injured lung to heal during the interval between 12 to 16 hours post-LPS and 24 hours (h) sacrifice. Therefore, PIP-2 and PIP-4 can prevent lung injury as well as treat lung injury. Data for various markers of lung injury are presented in tables 8-11.

Table 8: effect of PIP-2 on inflammation and edema after LPS

n-4, PIP-2 concentration (2 μ g/g wt in mice), LPS 5 μ g/g

Table 9: effect of PIP-2 on Lung tissue Oxidation after LPS

n-4, PIP concentration (2 μ g/g wt in mice), LPS 5 μ g/g

Table 10: effect of PIP-4 on inflammation and edema after LPS

n is 4; PIP concentration, 2. mu.g/g wt of mice, 5. mu.g/g LPS

Table 11: effect of PIP-4 on Lung tissue Oxidation after LPS

n-4, PIP concentration (2 μ g/g wt in mice), LPS 5 μ g/g

Example 4: PIP-2 is stable as a dry powder.

AiPLA was measured at intervals₂Activity to confirm that the peptide can maintain it as aiPLA₂The time of efficacy of the active inhibitor. The peptide was stable in 4 months of observation.

Table 12: the activity of PIP-2 as a dry powder during storage at room temperature for 4 months indicates stability.

Example 5:

the materials and methods used in the following examples are described herein.

Animal(s) production

C57Bl/6J or NADPH oxidase (Nox2) knockout mice were obtained from Jackson laboratories (Bar Harbor, ME) and maintained in HEPA filtered air with a 12h light/dark cycle in the facilities of the University of Pennsylvania chemical Animal Resources center (University of Pennsylvania Laboratory Resources) (ULAR).

Reagent

The estimated purity of the peptide assessed by mass spectrometry was > 89%. Lipopolysaccharide (LPS) derived from the cell membrane of Escherichia coli 0111: B4 and purified by gel filtration chromatography was obtained from Sigma-Aldrich (St. Louis, Mo, USA, Cat. No. L3012). The carboxyl adduct of amplex red/horseradish peroxidase (HRP) assay kit (Cat. A22188) and reduced difluorofluorescein diacetate (DFF-DA, Cat. 13293) (by Thermo-Fisher Scientific) was purchased from Life Technologies, Grand Island, NY, USA. Angiotensin II (Ang II) was obtained from Bachem, Torrance, CA, USA (Cat. No. 4095850.0005). Real lipids (Authentic lipids) were purchased from Sigma-Aldrich, st.louis, MO, USA, and liposomes were prepared by evaporation to dryness and then reconstitution in saline as described previously to reflect the composition of the lung surfactant; the composition of the liposome is: on a molar fraction basis, 0.5 Dipalmitoylphosphatidylcholine (DPPC), 0.25 egg Phosphatidylcholine (PC), 0.10 Phosphatidylglycerol (PG), and 0.15 cholesterol. When PIP-2 was added, the concentration was 0.15. mu.g PIP-2/. mu.g lipid.

Administration of LPS and PIP-2

Anesthetized mice were administered LPS (5 or 15 μ g/g body weight) in 20 μ L saline, which was instilled into the lungs through an endotracheal tube placed on the level of the tracheal bulge (tracheal carina). We have previously shown that PIP-2 is ineffective if injected alone, and if encapsulated in liposomes, it inhibits aiPLA2 activity for about 50h of 1/2 time. PIP-2 in liposomes was suspended in 20 μ L saline for IV or IT injection. For studies to evaluate the effect of PIP-2 at time 0, liposomes ± PIP-2 were also administered by IT instillation after LPS administration. For studies to evaluate the effect of administering PIP-2 at a later time after LPS, liposomes ± PIP-2 were administered by injection into the retinal artery. This change in route of administration is used to minimize damage to the mouse trachea that may occur in a repeated tracheostomy and result in adverse effects on the lungs. The dose of PIP-2 for treatment after intratracheal LPS was 2. mu.g/g mouse body weight; in control mice, this dose of PIP-2 has been shown to maximally inhibit pulmonary aiPLA₂The activity is at least 24 h. We given a second dose of PIP-2 at 12h to determine maximum coverage, and then switched to administering PIP-2 every 24 h. For a model of sepsis, mice were treated intraperitoneally with LPS in 20 μ L saline (15 μ g/g body weight) and with IV PIP-2 at 2 or 20 μ g/g body weight; note that is used forThe initial dose of PIP-2 for this sepsis model was IV, not IT. We used the same number of PIP-2 administrations in the sepsis model as in the IT LPS model. After recovery from anesthesia, all mice were maintained in a rearing chamber (vivarium), with free (ad lib) access to food and water.

Assessment of Lung injury

At the end of each experiment with IT LPS (24h or 120h), surviving mice were sacrificed by exsanguination under anesthesia. The lungs are cleared of blood in situ by pulmonary arterial perfusion, followed by tracheal lavage with saline. The lungs were then removed from the chest cavity for histological examination. We assessed the effect of LPS on lung injury by measuring the number and protein content of nucleated cells in lung bronchoalveolar lavage fluid (BALf), the wet-to-dry weight ratio of the lung using the upper left lobe of the lung, and the oxidation of lipid and protein components of lung tissue by thiobarbituric acid-reactive products (TBARS), 8-isoprostane and protein carbonyl in lung homogenates. To study the mortality of mice, survival curves were constructed using Kaplan-Meier estimation.

Measuring pulmonary ROS production and aiPLA₂Activity of

The effect of PIP on ROS production in control (untreated) lungs was determined in vitro using isolated perfused lungs. PIP-2 in liposomes was administered by the IV route at 2 μ g/g mouse weight. After 30min, mice were anesthetized and lungs were isolated, blood was removed and perfused into the recirculation system with perfusate containing Ang II (50 μ M) as Nox2 activator and Amplex red plus horseradish peroxidase to detect ROS. Lungs from wild type mice and lungs from NOX2 knock-out mice that were not treated with PIP-2 were used as controls. Basal rates of ROS production were assessed with WT lungs perfused in the absence of AngII. The perfusion protocol included a 15min equilibration period followed by a 60min experimental period. Aliquots of the perfusate were removed at 15min intervals and analyzed fluorometrically for the Amplex red oxidation product resorufin (resorufin) (. lambda.) (Amersham biosciences)_Excitation568nm、λ_Launching581 nm). The rate of Amplex red oxidation was calculated and expressed as Arbitrary Fluorescence Units (AFU) and normalized to mouse body weight. In the absence of HRP in the perfusate, the rate of Amplex red oxidation was low (about AngII-excited fluorescence)7%) indicating non-ROS mediated oxidation of the fluorophore; this value is subtracted to obtain the reported value.

To determine the production of lung ROS following LPS treatment, whole mice were treated with LPS (5. mu.g/g). + -. PIP-2 (2. mu.g/g). Mice were anesthetized 6, 12, or 24h after LPS treatment, and the lungs were cleared of blood in situ, and then perfused with saline solution containing the fluorophore DFF-DA (which is hydrolyzed intracellularly to DFF) for 10 min. The lungs were then homogenized and the fluorescence of the homogenate was measured at Ex495nm, Em 525 nm. Lung fluorescence was expressed as AFU per minute of perfusion and normalized to mouse body weight.

Statistical analysis

Data are presented as mean ± Standard Error (SE). The slope of the linear plot was calculated by the least mean square error method. Statistical significance was assessed using SigmaStat software (Jandel Scientific, San Jose, Calif.). The mean of the differences between the groups was assessed by one-way anova, then by Bonferroni pairwise comparison analysis (Bonferroni post hoc test). For the comparison of both groups, the mean values were compared by t-test. The difference between the mean values was considered statistically significant (P < 0.05).

As a result:

PIP inhibits pulmonary ROS production.

To confirm the inhibitory effect of PIP compounds on NOX2 activation, we investigated ROS produced by isolated perfused lungs in the presence of the known NOX2 activator Ang II. Amplex red oxidation was used as an index for ROS production. Under control conditions, i.e., no added stimulant of NOX2 activity (fig. 8, WT basis), the baseline rate of ROS production in the perfused lungs was very low. By adding Ang II to the perfusate to activate NOX2, ROS production was significantly increased (fig. 8, WT control). The reduction in ROS production in NOX2 knockdown compared to WT lungs was 76%, indicating that NOX2 is the major source of ROS entering the perfusate following Ang ii stimulation. As shown previously, addition of PIP-2 (in liposomes) to WT lungs inhibited ROS production (about 75%), which is similar to NOX2 knock-out. Thus, PIP-2 results in a substantially complete inhibition of NOX 2-mediated ROS production.

Next, we determined that PIP-2 (in liposomes) after LPS is directed to aiPLA in the lung₂Activity and ROS production. 6, 12, after IT LPS administration,These parameters are determined 24 h. The activity of aiPLA2 in lung homogenates increased by about 50% at 6h after treatment with LPS and by 50% again at 12 and 24h compared to the control (fig. 9A). We used intracellular fluorophore (DFF-DA) to determine lung ROS generation. ROS-induced fluorescence was very low in control lungs not treated with LPS, but ROS-induced fluorescence in mice lungs treated with LPS increased about 10-fold at 6h and about 20-fold at 12h and 24h (fig. 9B). This increase in lung DFF fluorescence after LPS may be slightly underestimated due to signal dilution caused by the presence of edema in these lungs (see below). Pre-treatment of mice with PIP-2 prior to LPS administration resulted in a significant decrease in the fluorescence of aiPLA2 activity and ROS generation over all 3 time periods to values similar to those of controls not treated with LPS. These results indicate that intratracheal administration of LPS results in increased ROS production in the lungs, which can be maintained for at least 24h and can be almost completely inhibited by lungs pre-treated with PIP-2.

Time course of LPS-mediated lung injury

The sensitivity of LPS-mediated damage varies significantly in mouse strains. For this study, we determined the course of lung injury in C57Bl/6J mice given LPS at 5. mu.g/g body weight IT (FIGS. 2A to 2F). When evaluated 12h post-LPS, the lungs showed considerable damage as indicated by an increase in nucleated cells in BALf, an increase in BALf protein, and an increase in the wet to dry weight ratio of the lungs (p < 0.05). These results are consistent with lung inflammation (cells in BALf), alterations in the alveolar-capillary permeability barrier (BALf protein), and lung fluid accumulation (wet/dry weight of lung). An increase in lung tissue TBARS, 8-isoprostane, and protein carbonyl indicate that oxidative stress is accompanied by oxidation of lung tissue lipid and protein components. These lung injury indices showed similar values at 12, 16 or 24h post-LPS (fig. 2A to 2F), indicating that the degree of lung injury was substantially stable at 12 to 24h post-non-lethal dose of LPS. Partial recovery was seen at 48h for the lung injury index (approximately 50%, P <0.05), although it was still elevated compared to the control (P < 0.05).

Effect of PIP-2 on LPS-mediated Lung injury

To investigate the effect of PIP-2 administration on lung injury, mice were treated with IT-administered LPS (5 μ g/g body weight). The dose of LPS was selected based on our previous study with the same batch of LPS, which showed relatively low levels of lung injury (1 μ g/g body weight), and larger injury with insignificant mortality (5 μ g/g body weight was used). PIP-2 (2. mu.g/g body weight in liposomes) was administered 0, 12 or 16h after LPS. We have previously shown that this dose of PIP-2 inhibits pulmonary aiPLA2 activity by about 90% for at least 24 h. PIP-2 was given at time IT at 0 and at 12 or 16h IV to avoid excessive damage to the trachea. Animals were sacrificed 24h post LPS and lungs were examined. All lung injury indices (reflecting lung inflammation, alveolar-capillary barrier dysfunction, lung fluid accumulation, and tissue oxidative stress) were elevated in LPS-treated mice compared to controls (p < 0.05). PIP-2 administered at time 0 completely prevented lung injury when assessed 24h post-LPS (fig. 3A to 3F). The index of tissue damage was also significantly reduced in the lungs of mice treated with PIP-2 at 12 and 16h compared to LPS alone, and the values were not significantly different from the control values (fig. 3A to 3F). Due to the presence of lung lesions in the lungs 12 and 16h after LPS, normal values at 24h in the lungs of LPS-treated mice given PIP-2 at 12 or 16h could only indicate that the lungs were able to recover completely from their lesions during the 8 to 12h interval between administration of PIP-2 and examination of the lungs.

PIP-2 treatment prevented mouse death from high doses of LPS.

Although mice treated with low doses of LPS (5 μ g/g body weight) suffered significant lung injury, it was transient and essentially all mice recovered from the injury (not shown). To test the effect of PIP-2 treatment with a more severe injury model, a higher dose of LPS (15 μ g/g body weight) was administered to the mice. Plotting survival data with initial PIP-2 treatment at time 0; LPS was administered 12h before PIP-2 (fig. 7A and fig. 7B, panel-12 h). At this higher dose of LPS, mice treated with placebo (liposomes alone) showed 73% mortality during 24hr post-LPS and 100% mortality by 48 h. For the treatment group, mice were administered PIP-2 12, 24, 48, 72 and 96h post-LPS and mice were sacrificed at 120 h; PIP-2 treated mice showed only 17% mortality (83% survival) 36h after PIP-2 treatment was initiated and no further mortality during the observation period. In addition to the effect on mortality, a significant difference was observed in the behaviour of mice receiving PIP-2 after LPS, with most mice reverting to normal physical activity 12h after receiving PIP-2. Lung injury index of treated mice sacrificed 120h post-LPS showed no abnormality (table 13).

Table 13 lung injury was repaired in mice surviving high dose LPS.

Mice were instilled with LPS (15. mu.g/g wt) IT; PIP-2 in liposomes (2 μ g/g body weight) was Injected (IV) at the times shown in figure 7A. 5 surviving mice were sacrificed 120h post LPS; control mice were given liposomes, but no LPS. BALf, bronchoalveolar lavage fluid; TBARS, thiobarbituric acid reactive species. Values are mean ± SE, N-4 for control, and N-5 for LPS + PIP-2. The average value of LPS + PIP-2 was not statistically different from the corresponding control (p > 0.05).

Next, we evaluated the effect of PIP-2 in mice given LPS by the intraperitoneal route (15 μ g LPS/g body weight) as a model of ALI associated with systemic sepsis. We selected the dose of LPS based on our previous study, which showed a mortality rate of 60% at 10 μ g LPS/g body weight; our goal was to produce 100% mortality in placebo-treated mice, similar to that seen by the high-dose IT LPS study. The survival of placebo (liposome only) treated mice was less than 40% 24h post-LPS, and 100% of the mice died by 48h (fig. 7B). In contrast, the survival of mice treated with PIP-2(2 μ g/g body weight) after LPS increased to 86% at 36h and 43% of mice were fully recovered. With higher dose of PIP-2(20 μ g/g body weight), long-term survival rates were significantly improved, reaching 70%. Therefore, PIP-2 significantly increased the survival of mice in this model of ALI associated with systemic sepsis.

ALI is a severe disease syndrome with a mortality rate of about 40%. Inflammation is an important factor that can amplify lung injury associated with primary injury. To date, there is no approved drug treatment for the inflammatory component of the syndrome. Inflammation of lungThe mechanism of lung injury during the disease is complex, but excessive ROS production appears to play a major role. We have previously shown that aiPLA2 activity of Prdx6 is essential for activating ROS production by NOX2, and have described several nonapeptides derived from the lung surfactant protein ｃA (SP- ｃA) sequence that inhibit aiPLA₂Activity, thereby inhibiting the activation of NOX2 in lung cells. This study demonstrated that these are termed PLA₂Peptides that inhibit peptides (PIP-2, PIP-4 and PIP-5) inhibit ANGII-activated NOX2 from producing ROS in isolated mouse lungs. Although PIP-2 appears to be slightly more active than the other 2, all 3 PIP compounds are effective as inhibitors, presumably reflecting, in part, the high degree of conservation of Prdx6 amino acid sequence across species. We have demonstrated that the binding site for the 16 amino acid precursor of PIP is an amino acid sequence comprising amino acids 195 to 204 of Prdx 6. The sequence of such a fragment of human Prdx6 is: 34195-EEEAKKLFPK-204; for 8 of 10 amino acids, the corresponding mouse sequences are identical, with Q instead of K at position 200 and C instead of L at position 201. We selected PIP-2 (which is derived from the relevant sequence of human SP- ｃA) for subsequent investigation. The PIP-2 amino acid sequence is: 1LHDFRHQIL SEQ ID NO.

The main objective 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 inhibits AngII-mediated ROS generation; as we have shown previously, AngII is a known activator of NOX2, activation requiring aiPLA₂And (4) activity. Then, we show that treatment with LPS results in a significant increase in lung aiPLA2 activity, and also a significant increase in ROS production through activation of NOX 2; LPS-mediated increase in aiPLA2 activity and ROS production was also inhibited by PIP-2.

The first study of the effectiveness of PIP-2 in a lung injury model was the simultaneous administration of PIP-2 and LPS, which significantly protected against subsequent lung injury. Measures to assess acute lung injury post-LPS include: a) nucleated cells in BALf (inflammation); b) proteins in BALf (alveolar-capillary permeability); c) wet to dry weight ratio of lungs (pulmonary edema); and d) lung TBARS, 8-isoprostaglandin, and protein carbonyl (oxidation of tissue lipids and proteins). All these lesion indices evaluated 12 to 24h after LPS administration were significantly elevated in the lungs. However, neither of these indices of tissue damage in the lung was altered when PIP-2 was administered concurrently with LPS. Thus, PIP-2 can prevent ALI associated with LPS administration in mice.

The next study investigated the effect of administering PIP-2 as a means of treatment (versus prophylaxis) 12 or 16h after LPS administration. As shown in figures 3A to 3F, tissue damage associated with non-lethal LPS was greatest at this time. PIP-2 was administered 12 or 16h after LPS and the parameters of lung injury had returned to essentially normal values at 24h post LPS examination. We conclude from this study that: PIP-2 prevented the persistent lung injury associated with LPS during 8-12h between PIP-2 administration and sacrifice of animals and allowed the lung to self-repair.

Our final study was to evaluate the effect of PIP-2 on mouse lung function and survival after administration of a lethal dose of LPS. Administration of PIP-2 every 12 to 24h after LPS administration resulted in significantly improved mouse behavior, significantly reduced mouse mortality, and resulted in a return of the index of lung injury to normal values. Thus, Prdx6 PLA₂Active nonapeptide inhibitors prevent ROS production following NOX2 activation and prevent mortality associated with administration of lethal doses of LPS. These results indicate that PIP-2 can prevent and treat a mouse model of LPS-induced ALI.

The current results of PIP-2 gave similar conclusions to our previous study, showing the use of several different approaches to inhibit aiPLA₂Activity and subsequent NOX2 activation protected against LPS-induced ALI. These include: a) administration of aiPLA₂Active lipid inhibitors, MJ 33; b) knockout mice were used with Prdx6 (not a perfect model since the peroxidase activity of Prdx6 was also lost); and c) at Prdx6 (aiPLA)₂An important component of the active site) of the mouse with the amino acid D140 mutation. MJ 33-inhibited mice, D140A-mutated mice, and PIP-2-treated mice all retained the peroxidase activity of Prdx6, whereas this activity was abolished in Prdx6 knockout mice. In these previous studies, LPS was administered by the IT route in a) and b) as a model for direct lung injury, and LPS was administered by the intraperitoneal route in c) as a model for non-infectious sepsis. We have alreadyIt is proposed that the protection mechanism provided by PIP-2 is its inhibition of aiPLA of Prdx6 due to allosteric effects caused by peptide binding to Prdx6₂And (4) activity. PIP peptide does not inhibit other lung PLA₂Enzymes, as experimentally demonstrated and expected from differences in potential binding sites on different proteins. ai PLA₂Inhibition of activity prevents the production of lysoPC and its downstream products, thereby preventing the activation of Rac, an essential cofactor for Nox2 activation. Interestingly, the cholesterol lowering drug simvastatin also inhibited Rac activation and has been shown to inhibit the production of ROS by endothelial cells and has a protective effect in a mouse model of LPS-induced ALI. Although there is no definitive evidence at this time, inhibition of Rac activation may have a beneficial effect on non-ROS mediated ALI expression in addition to its effect on NOX2 activation.

Current and previous studies have shown that NOX2 is the major source of ROS in the lungs, and the enzyme is activated in the presence of LPS. In addition to the LPS model, ROS generation by NOX2 has been shown to play an important role in several other related and different ALI animal models, including gram-negative sepsis, endotoxin, severe trauma, hemorrhagic shock, and oleic acid instillation. It is speculated that the major manifestation of oxidative stress associated with NOX2 activation is oxidation of tissue macromolecules, as shown in this study. However, another important pathophysiological role associated with NOX 2-derived ROS is based on the following evidence: ROS are responsible for the signals leading to the recruitment of neutrophils to the lung and the resulting pulmonary inflammation (which is characteristic of ALI). The significant reduction of nucleated cells in BALf after treatment with PIP-2 indicates that this function of ROS is important for the recovery of lung injury. In this regard, peptide inhibitors of C kinase substrate (Marcks) protein rich in myristoylated alanine also protected mice from LPS-induced lung injury. Although the latter peptide has not been shown to inhibit NOX2 activation, its effect may be mediated by altered cell motility, thereby preventing PMNs from flowing into the lungs. Thus, PIP-2, simvastatin, inhibitors of the Marcks protein, and possibly inhibitors of NOX2 (such as acetovanillone) all prevent post-LPS PMN flow into the lungs, thereby reversing inflammation and associated lung injury.

Based on the current results, peptide inhibitors of NOX2 activation may be effective prophylactic agents for patients at risk of ALI, as well as for the treatment of patients with ALI that have been diagnosed. Although the toxicity of these small peptides was not expected based on their normal expression in the lung as ｃA component of the SP- ｃA protein, it still had to be studied. The antigenic potential of the peptide is theoretically low, but this would need to be demonstrated in humans. Other possible side effects of peptides include those associated with inhibition of Rac activation and loss of signaling and regulatory functions of ROS. Notably, no major effects have been reported that may be associated with the inhibition of Rac with the widely used drug simvastatin. A potentially more important "side effect" of treatment with PIP may be the inhibition of the effect of ROS production on the bactericidal activity of inflammatory cells (PMNs and AMs) that utilize superoxide anions generated by the activity of NOX2 to kill bacteria. Furthermore, it has been shown that some antibiotics require ROS for maximum efficacy. Although it is theoretically possible to alter the response to infection, inhibitors of NOX2 activation do not reduce the bactericidal activity of PMNs in the LPS model of ALI. This may reflect the ability of the non-NOX 2 pathway to compensate for the loss of NOX 2-derived ROS. Although this would emphasize the important role of antibiotic coverage in patients treated with NOX2 inhibitors, it is important to note that antibiotics alone are not effective in reducing the mortality rate of this disease to values significantly below 40%.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety.

Although the present invention has been disclosed with reference to specific embodiments, it is 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. It is intended that the following claims be interpreted to embrace all such embodiments and equivalent variations.

Sequence listing

<110> board of the university of pennsylvania

Fisher, Aron B.

Feinstein, Sheldon I.

<120> compositions and methods for treating acute lung injury

<130> 046483-7211WO1(02047)

<150> US 62/719,217

<151> 2018-08-17

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