Compositions and methods for the treatment of septic shock

Abstract

This application provides a polypeptide for the treatment of septic shock and a method of using the same.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits and priority of U.S. Provisional Application No. 63 / 500,286, filed on 5 May 2023, which is incorporated herein by reference in its entirety for all purposes.

[0002] Sequence List This application includes a sequence listing which is incorporated herein in its entirety by reference. The XML copy prepared on 3 May 2024 is named PEX-002WO_SL.xml and has a size of 32,768 bytes. [Background technology]

[0003] Septic shock is the final and most severe stage of sepsis, characterized by dangerously low and severe hypotension and / or elevated lactate levels, which cannot be adequately treated with fluid administration alone. Even with vasopressor therapy to maintain blood pressure, the mortality rate from septic shock is high, ranging from 25% to 50% (e.g., Kumar et al, eds., Robbins Basic Pathology (8)). th See Saunders, Elsevier ed., pp.102-3 (2007); Singer et al., "The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3)," JAMA 315(8):801-10 (2016) (these are incorporated herein by reference). Therefore, there is a current need for compositions and methods that can treat septic shock. This embodiment satisfies these needs, among others. [Overview of the project]

[0004] A method for treating septic shock in the necessary population, comprising the following amino acid sequence: X 1 X2 X 3 X 4 X 5 LX 6 X 7 X 8 X 9 HQIL (Sequence number 1) Where: X 1 is E or does not exist, and X 2 is L or does not exist; and X 3 is Q or does not exist, and X 4 is A, T or does not exist, and X 5 is T, E or does not exist, and X 6 is H or Y, and X 7 is D or E, and X 8 is F or I; and X 9A method is provided herein comprising administering to a subject an effective amount of a pharmaceutical composition comprising a polypeptide comprising an amino acid sequence which is R or K, or an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, or 30; and a pharmaceutically acceptable carrier. In some embodiments, the polypeptide comprises an amino acid sequence which is at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In some embodiments, the polypeptide contains an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In some embodiments, the polypeptide comprises SEQ ID NO: 2. In some embodiments, the polypeptide consists of SEQ ID NO: 2. In some embodiments, the polypeptide comprises SEQ ID NO: 3. In some embodiments, the polypeptide consists of SEQ ID NO: 3. In some embodiments, the polypeptide comprises SEQ ID NO: 4. In some embodiments, the polypeptide consists of SEQ ID NO: 4. In some embodiments, the pharmaceutical composition comprises a liposome-encapsulated polypeptide. In some embodiments, the pharmaceutically acceptable carrier is any pharmaceutically acceptable carrier disclosed herein. In some embodiments, the pharmaceutical composition is administered to a subject by any administration method disclosed herein.In some embodiments, the pharmaceutical composition is administered to the subject by aerosol inhalation, intratracheal injection, or intravenous injection. In some embodiments, the subject requiring it is diagnosed with sepsis and has hypotension that is not relieved by intravenous fluid administration alone. In some embodiments, the subject requiring it has a mean arterial pressure (MAP) of less than approximately 65 mmHg. In some embodiments, the subject requiring it is diagnosed with sepsis and has a mean arterial pressure or systolic blood pressure decrease of approximately 44 mmHg or more. In some embodiments, the subject requiring it has a blood lactate level greater than approximately 4 mmol. In some embodiments, the subject requiring it has persistent signs of multi-organ damage or failure. In some embodiments, the subject's blood oxygen level does not decrease after administration of the pharmaceutical composition. In some embodiments, the subject's blood oxygen level does not decrease by more than 50% after administration of the pharmaceutical composition. In some embodiments, the subject's blood oxygen level does not decrease by more than 65% after administration of the pharmaceutical composition. In some embodiments, the subject's blood oxygen level increases after administration of the pharmaceutical composition. In some embodiments, the subject maintains a normal blood oxygen level after administration of the pharmaceutical composition. In some embodiments, the subject requiring it has not been administered a vasopressor, or has previously been administered a smaller amount of a vasopressor, is administered a smaller amount of a vasopressor concurrently, or is subsequently administered a smaller amount of a vasopressor than the amount administered in the absence of the pharmaceutical composition. In some embodiments, the subject requiring it has been diagnosed with septic shock.

[0005] A method for treating subjects with a significant decrease in mean arterial pressure, the following amino acid sequence: X 1 X 2 X 3 X 4 X 5 LX 6 X 7 X 8 X 9 HQIL(Sequence ID 1) In the formula: x 1 is either E, or does not exist, X 2is either L, or does not exist, X 3 is either Q, or does not exist, x 4 is either A, T, or does not exist; X 5 is either T, E, or does not exist; X 6 is H or Y; X 7 is either D or E; X 8 is F or I; and X 9A method is also provided herein, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a polypeptide comprising an amino acid sequence which is R or K; or an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, or 30; and a pharmaceutically acceptable carrier. In some embodiments, the polypeptide comprises an amino acid sequence which is at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In some embodiments, the polypeptide contains an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In some embodiments, the polypeptide comprises SEQ ID NO: 2. In some embodiments, the polypeptide consists of SEQ ID NO: 2. In some embodiments, the polypeptide comprises SEQ ID NO: 3. In some embodiments, the polypeptide consists of SEQ ID NO: 3. In some embodiments, the polypeptide comprises SEQ ID NO: 4. In some embodiments, the polypeptide consists of SEQ ID NO: 4. In some embodiments, the pharmaceutical composition comprises a liposome-encapsulated polypeptide. In some embodiments, the pharmaceutically acceptable carrier is any pharmaceutically acceptable carrier disclosed herein. In some embodiments, the pharmaceutical composition is administered to a subject by any administration method disclosed herein.In some embodiments, the pharmaceutical composition is administered to the subject by aerosol inhalation, intratracheal injection, or intravenous injection. In some embodiments, the subject has a mean arterial pressure or systolic blood pressure that is approximately 44 mmHg or greater, or has been diagnosed with sepsis. In some embodiments, the subject has been diagnosed with sepsis and has hypotension that is not relieved by intravenous fluid administration alone. In some embodiments, the subject has a mean arterial pressure (MAP) of less than approximately 65 mmHg. In some embodiments, the subject requiring it has a blood lactate level greater than approximately 4 mmol. In some embodiments, the subject requiring it has persistent signs of multi-organ damage or failure. In some embodiments, the subject's blood oxygen level does not decrease after administration of the pharmaceutical composition. In some embodiments, the subject's blood oxygen level does not decrease by more than 50% after administration of the pharmaceutical composition. In some embodiments, the subject's blood oxygen level does not decrease by more than 65% after administration of the pharmaceutical composition. In some embodiments, the subject's blood oxygen level increases after administration of the pharmaceutical composition. In some embodiments, the subject maintains a normal blood oxygen level after administration of the pharmaceutical composition. In some embodiments, the subject requiring it has not been administered a vasopressor, or has been previously administered a vasopressor, administered concurrently, or subsequently administered a smaller amount of a vasopressor than the amount administered in the absence of the pharmaceutical composition. In some embodiments, the subject requiring it has been diagnosed with septic shock.

[0006] A method for preventing cardiac injury in a patient diagnosed with septic shock, comprising the following amino acid sequence: X 1 X 2 X 3 X 4 X 5 LX 6 X 7 X 8 X 9 HQIL(Sequence ID 1) In the formula:X 1 is either E, or does not exist, X 2 is either L, or does not exist, X 3is either Q, or does not exist, X 4 is either A, T, or does not exist; X 5 is either T, E, or does not exist; X 6 is H or Y; X 7 is either D or E; X 8 is F or I; and X 9A method is also provided herein, comprising administering to a subject (compared to a subject not treated with the pharmaceutical composition) an effective amount of a polypeptide comprising an amino acid sequence that is R or K; or an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, or 30; and a pharmaceutically acceptable carrier. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In some embodiments, the polypeptide contains an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In some embodiments, the polypeptide comprises SEQ ID NO: 2. In some embodiments, the polypeptide consists of SEQ ID NO: 2. In some embodiments, the polypeptide comprises SEQ ID NO: 3. In some embodiments, the polypeptide consists of SEQ ID NO: 3. In some embodiments, the polypeptide comprises SEQ ID NO: 4. In some embodiments, the polypeptide consists of SEQ ID NO: 4. In some embodiments, the pharmaceutical composition comprises a liposome-encapsulated polypeptide. In some embodiments, the pharmaceutically acceptable carrier is any pharmaceutically acceptable carrier disclosed herein. In some embodiments, the pharmaceutical composition is administered to a subject by any administration method disclosed herein.In some embodiments, the pharmaceutical composition is administered to the subject by aerosol inhalation, intratracheal injection, or intravenous injection. In some embodiments, the subject requiring it has hypotension that is not relieved by intravenous fluid administration alone. In some embodiments, the subject requiring it has a mean arterial pressure (MAP) of less than about 65 mmHg. In some embodiments, the subject requiring it has a mean arterial pressure or systolic blood pressure decrease of about 44 mmHg or more. In some embodiments, the subject requiring it has a blood lactate level greater than about 4 mmol. In some embodiments, the subject requiring it has persistent signs of multi-organ damage or failure. In some embodiments, the subject's blood oxygen level does not decrease after administration of the pharmaceutical composition. In some embodiments, the subject's blood oxygen level does not decrease by more than 50% after administration of the pharmaceutical composition. In some embodiments, the subject's blood oxygen level does not decrease by more than 65% after administration of the pharmaceutical composition. In some embodiments, the subject's blood oxygen level increases after administration of the pharmaceutical composition. In some embodiments, the subject maintains a normal blood oxygen level after administration of the pharmaceutical composition. In some embodiments, the subject requiring it has troponin levels that do not rise after administration of the pharmaceutical composition. In some embodiments, the subject requiring it maintains normal troponin levels after administration of the pharmaceutical composition. In some embodiments, the subject requiring it has not been administered a vasopressor, or the subject has been previously administered a vasopressor, administered concurrently, or subsequently administered a smaller amount of a vasopressor than the amount administered in the absence of the pharmaceutical composition.

[0007] A method for increasing the survival of a patient diagnosed with septic shock, comprising the following amino acid sequence: X 1 X 2 X 3 X 4 X 5 LX 6 X 7 X 8 X 9 HQIL(Sequence ID 1) In the formula:X 1 is either E or does not exist; X2 is either L, or does not exist, X 3 is either Q or does not exist; X 4 is either A, T, or does not exist; X 5 is either T, E, or does not exist; X 6 is H or Y; X 7 is either D or E; X 8 is F or I; and X 9The method comprises administering to a subject (compared to a subject not treated with the pharmaceutical composition) an effective amount of a polypeptide comprising an amino acid sequence that is R or K; or an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, or 30; and a pharmaceutically acceptable carrier. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In some embodiments, the polypeptide includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In some embodiments, the polypeptide includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In some embodiments, the polypeptide includes SEQ ID NO: 2. In some embodiments, the polypeptide comprises SEQ ID NO: 2. In some embodiments, the polypeptide comprises SEQ ID NO: 3. In some embodiments, the polypeptide comprises SEQ ID NO: 3. In some embodiments, the polypeptide comprises SEQ ID NO: 4. In some embodiments, the polypeptide comprises SEQ ID NO: 4. In some embodiments, the pharmaceutical composition comprises a liposome-encapsulated polypeptide. In some embodiments, the pharmaceutically acceptable carrier is any pharmaceutically acceptable carrier disclosed herein. In some embodiments, the pharmaceutical composition is administered to a subject by any administration method disclosed herein.In some embodiments, the pharmaceutical composition is administered to the subject by aerosol inhalation, intratracheal injection, or intravenous injection. In some embodiments, the subject requiring it has been diagnosed with sepsis and has hypotension that is not relieved by intravenous fluid administration alone. In some embodiments, the subject requiring it has a mean arterial pressure (MAP) of less than about 65 mmHg. In some embodiments, the subject requiring it has a mean arterial pressure or systolic blood pressure decrease of about 44 mmHg or more. In some embodiments, the subject requiring it has a blood lactate level greater than about 4 mmol. In some embodiments, the subject requiring it has persistent signs of multi-organ damage or failure. In some embodiments, the subject's blood oxygen level does not decrease after administration of the pharmaceutical composition. In some embodiments, the subject's blood oxygen level does not decrease by more than 50% after administration of the pharmaceutical composition. In some embodiments, the subject's blood oxygen level does not decrease by more than 65% after administration of the pharmaceutical composition. In some embodiments, the subject's blood oxygen level increases after administration of the pharmaceutical composition. In some embodiments, the subject maintains a normal blood oxygen level after administration of the pharmaceutical composition. In some embodiments, the subject requiring it has not been administered a vasopressor, or has been previously administered a vasopressor, administered concurrently, or subsequently administered a smaller amount of a vasopressor than the amount administered in the absence of the pharmaceutical composition. [Brief explanation of the drawing]

[0008] [Figure 1] The measured troponin I levels over time in the subjects are shown. Negative controls and subjects treated with PIP-2 did not show an increase in blood troponin protein levels, while subjects not treated with PIP-2 showed an increase in troponin protein levels starting at 1–4 hours. [Figure 2] The measured c-reactive protein (CRP) levels over time in the subjects are shown. Negative controls and subjects treated with PIP-2 did not show an increase in blood CRP protein levels, while subjects not treated with PIP-2 showed an increase in CRP protein levels starting at 1–4 hours. [Figure 3] This shows the average use of vasopressors per animal for negative controls, subjects treated with PIP-2, and subjects not treated with PIP-2. Subjects treated with PIP-2 required approximately 66% less vasopressor treatment to maintain appropriate MAP levels. [Figure 4A] This shows the effect of PIP-2 on mean arterial pressure (MAP). A is a bar graph showing the percentage change in mean arterial pressure (MAP) from baseline in pigs treated with LPS and LPS + PIP-2. [Figure 4B] The effect of PIP-2 on mean arterial pressure (MAP) is shown. Column B shows graphs of MAP values ​​over 8 hours for pigs treated with control, LPS, and LPS+PIP-2. [Figure 5] The survival curves for pigs treated with LPS and LPS + PIP-2 are shown. Pigs were treated with LPS delivered by IV infusion over a period of 1 hour. In addition to LPS, at the end of the LPS infusion, either liposome-containing PIP-2 (n=12) or liposomes alone (n=6) were administered to the pigs as a sustained-release bolus injection IV. Pig mortality by humane slaughter was recorded at 1-hour intervals. All surviving pigs were slaughtered 8 hours after the start of LPS infusion. The addition of PIP-2 increased survival rates compared to the control group. [Figure 6] Figure 6 illustrates that PIP-2 treatment reduced the mortality rate of pigs euthanized by humane slaughter by 50% and the incidence of moderate to severe ARDS by 67%. Figure 6 shows the severity of acute lung injury, as indicated by arterial blood PO2 (aPO2) values ​​during ventilation at 100% O2 (FiO2), measured 8 hours after the start of LPS infusion in three groups of pigs (Group 1: liposomes alone, no LPS, Group 2: LPS + liposomes, and Group 3: LPS + LPS + PIP-2 in liposomes). The percentage of pigs in each group that remained within the normal, mild, moderate, or severe ARDS range, as defined by aPO2 / FiO2 (Horowitz index), is shown for pigs that survived for 8 hours. The percentage of pigs euthanized for humane reasons before the end of the 8-hour study is also shown. Total number of pigs: 2 control, 6 LPS, and 12 LPS + PIP-2. [Figure 7] This figure illustrates that PIP-2 leads to a statistically significantly smaller decrease in lung compliance in subjects treated with LPS to induce septic shock. Specifically, Figure 7 illustrates the static lung compliance of pigs in Group 2 (LPS treatment) and Group 3 (LPS treatment + PIP-2 treatment). Static lung compliance of mechanically ventilated pigs was measured at 1-hour intervals from 0:00 to 8:00 after the start of LPS infusion. Points are connected by continuous lines. Each line represents one pig. Early termination of a line indicates euthanasia of a pig for humane reasons. The thick lines in each graph represent the mean slope of the set of lines. The slopes show the percentage decrease in compliance in pigs in each group: -1.35 + 0.36 ml / cmH2O for the LPS group and -0.56 + 0.41 L / cmH2O for the LPS + PIP-2 pigs. A t-test comparing the two groups with N=16 showed P<0.001. [Modes for carrying out the invention]

[0009] Unless otherwise defined, all technical and scientific terms have the same meaning as they are commonly understood by those skilled in the art in the field to which the disclosed embodiments belong.

[0010] As used herein, the terms "a" or "an" mean "at least one" or "one or more" unless the context clearly indicates otherwise.

[0011] Where used herein, the term “about” means that the numerical value is an approximation and that small variations will not significantly affect the implementation of the disclosed embodiment. Where numerical limitations are used, unless otherwise indicated by the context, “about” means that a ±10% variation in the numerical value will remain within the scope of the disclosed embodiment.

[0012] As used herein, the terms “individual,” “subject,” or “patient” are used synonymously and mean any animal, for example, mammals such as mice, rats, and other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, or primates such as humans.

[0013] As used herein, the terms “comprising” (and any conjugations of “comprising,” such as “comprise,” “comprises,” and “comprised,” “having” (and any conjugations of “having,” such as “have,” and “has,” “has,” “having,” “having,” “having,” “having,” “having,” “having,” “having,” “having,” and any conjugations of “having,” such as “havings,” and “having,” “having,” or “having,” “having,” are open or non-restrictive and do not exclude any additional elements or processes of methods not described herein. Any process or composition using the transitional phrase “comprise” or “comprising” may also be said to describe the same thing as using the transitional phrase “consisting of” or “consists.”

[0014] As used herein, the terms “substitute,” “substituted,” “mutate,” or “mutated” refer to altering, deleting, or inserting one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to create a variant of that sequence.

[0015] The terms "polynucleotide" or "nucleic acid molecule" refer to molecules containing chains of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemical reactions. Double-stranded and single-stranded DNA and RNA are typical examples of polynucleotides.

[0016] The terms “polypeptide” or “protein” refer to a molecule comprising at least two amino acid residues linked by a peptide bond to form a polypeptide. In some embodiments, the term “peptide” may also be used.

[0017] As used herein, the term “sepsis” means a potentially life-threatening condition caused by the body’s response to an infection. In some embodiments, sepsis can lead to failure of one or more organs. While we do not wish to be bound by theory, sepsis has been classified into several stages, with increasing severity. As used herein, the term “septic shock” refers to the final and most severe stage of sepsis, defined as sepsis usually accompanied by organ failure, persistent signs of hypotension, and possibly including cases where lactate levels exceed 2 mmol or 4 mmol and are not relieved by intravenous fluid administration, and / or where vasopressors are required to maintain mean arterial pressure (MAP) above 65 mmHg.

[0018] composition A composition comprising a polypeptide, wherein the polypeptide has the following formula: X 1 X 2 X 3 X 4 X 5 LX 6 X 7 X 8 X 9 A polypeptide having HQIL (Sequence ID 1) During the ceremony: X 1 However, it is either E or does not exist; X 2 However, it is either L or does not exist; X3 is Q or does not exist; X 4 is A, T or does not exist; X 5 is T, E or does not exist; X 6 is H or Y; X 7 is D or E; X 8 is F or I; and X 9 is a polypeptide that is R or K; or a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, or 30, a composition comprising is provided. In some embodiments, this polypeptide is the polypeptide listed in Table 1. [Table 1]

[0019] In some embodiments, this polypeptide is at least 85%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

[0020] As used herein, the term "PIP-2" is a polypeptide having the sequence of SEQ ID NO: 2. As used herein, the term "PIP-4" is a polypeptide having the sequence of SEQ ID NO: 3.

[0021] As used herein, the term "PIP-5" refers to a polypeptide having the sequence of SEQ ID NO: 4. In some embodiments, this polypeptide comprises SEQ ID NOs: 2, 3, or 4. In some embodiments, this polypeptide consists of SEQ ID NOs: 2, 3, or 4.

[0022] Pharmaceutical composition In another embodiment, this embodiment provides a composition, for example, a pharmaceutically acceptable composition, which comprises a polypeptide provided herein, which can be formulated, for example, with one or more excipients. In some embodiments, suitable excipients include, but are not limited to, purified water, calcium carbonate, calcium phosphate, various sugars, starch, cellulose derivatives, gelatin, polymers, such as polyethylene glycol, propylene glycol, PEG400, glycerin, DMA, ethanol, benzyl alcohol, citrate / sodium citrate (pH 3), citrate / sodium citrate (pH 5), tris(hydroxymethyl)aminomethane HCl (pH 7.0), 0.9% physiological saline, and 1.2% physiological saline, as well as any combination thereof.

[0023] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, suitable pharmaceutically acceptable carriers include, but are not limited to, water, silicone, wax, petrolatum, polyethylene glycol, propylene glycol, liposomes, lipids such as cholesterol, cationic lipids such as 1,2,-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2,-dioleoyl-sn-glycero-3-phosphoquiline (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sugars such as mannitol and lactose, and other materials depending on the specific type of formulation used. In some embodiments, suitable pharmaceutically acceptable carriers include, but are not limited to, nanoparticles such as gold or metal nanoparticles.

[0024] In some embodiments, the polypeptide is encapsulated in or formulated with one or more lipids and liposomes. In some embodiments, the lipids and liposomes are cationic lipids, for example, but not limited to, 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoquiline (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 5-carboxyspermylglycine dioctadecyl Luamide (DOGS), 2,3-dioleyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneaminium (DOSPA), 1,2-dioleoyl-3-dimethylammoniumpropane (DODAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), Ptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoic acid (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2 -Dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',1--2'-octadecadienoxy)propane (CpLinDMA), N,The lipids and liposomes include N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), or C12-200. In some embodiments, the lipids and liposomes include, but are not limited to, dipalmitoylphosphatidylcholine (DPPC), egg phosphate, phosphatidylglycerol (PG), egg phosphate, or cholesterol. In some embodiments, the lipids and liposomes contain dipalmitoylphosphatidylcholine (DPPC), egg phosphate (PC), phosphatidylglycerol (PG), and cholesterol in molar ratios of 0.5, 0.25, 0.10, and 0.15.

[0025] In some embodiments, the pharmaceutical composition includes an encapsulated polypeptide, such as the polypeptides provided herein.

[0026] In some embodiments, pharmaceutically acceptable carriers may be suitable for intravenous, intramuscular, intratracheal, subcutaneous, parenteral, rectal, local, topical, spinal, or epithelial administration (e.g., by injection or inhalation). In some embodiments, pharmaceutically acceptable carriers may be suitable for aerosol inhalation.

[0027] The composition may be in various forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., solutions for injection and infusion), dispersions or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic use. Typical compositions are in the form of solutions for injection or infusion. In some embodiments, the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intradermal, intramuscular, intravesical). In some embodiments, the composition is administered by intravenous infusion or injection. In some embodiments, the composition is administered by intramuscular or subcutaneous injection. In some embodiments, the composition is administered by intestinal, sublingual, inhalation, or intranasal administration. In some embodiments, the composition is administered topically, for example, by injection or topical application, to a target site. For example, the pharmaceutical composition may be lyophilized and reconstituted for use before administration to the patient.

[0028] As used herein, the terms “parenteral administration” and “administered parenterally” mean, but are not limited to, conventional methods of administration by injection other than enteral and local administration, including, but are not limited to, intravenous, intramuscular, intra-arterial, subarachnoid, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0029] Compositions such as pharmaceutical compositions are typically sterile and stable under manufacturing and storage conditions. These compositions may be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high concentrations of active ingredients. Sterile injectable solutions may be prepared by incorporating the required amount of active compound (i.e., therapeutic molecules, nucleic acid molecules, cells, polypeptides, vectors, etc.) into a suitable solvent having, if necessary, one or a combination of the components listed above, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying, resulting in a powder with the active ingredient plus any additional desired components from its pre-sterile filtered solution. Appropriate fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Sustained absorption of an injectable composition can be achieved by including absorption-delaying agents in the composition, such as monostearate and gelatin.

[0030] As those skilled in the art will understand, the route and / or mode of administration varies depending on the desired outcome. In certain embodiments, the active compound may be prepared with a carrier that protects the compound from rapid release, such as a controlled-release formulation including implants, transdermal patches, and microcapsule-encapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Many methods for preparing such formulations are patented or known to those skilled in the art. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0031] In certain embodiments, the pharmaceutical composition may be administered orally, for example, with an inert diluent or an assimilated food carrier. The compound (and optionally other components) may also be encapsulated in hard-shell or soft-shell gelatin capsules, compressed into tablets, or directly incorporated into the target meal. For oral therapeutic administration, the compound may be mixed with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, warphers, etc. To administer compositions such as those provided herein by means other than parenteral administration, it may be necessary to coat the composition with a material that prevents its inactivation, or to administer the composition together with such material. The composition may also be administered using medical devices known in the art.

[0032] The drug regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgent requirements of the treatment situation. For ease of administration and dose uniformity, it is particularly beneficial to formulate parenteral compositions in unit dosage forms. As used herein, unit dosage forms refer to physically separate units suitable as dose units for treating a subject; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier.

[0033] An exemplary, non-limiting range of therapeutically or prophylactically effective amounts of the therapeutic compound is 0.1 to 30 mg / kg, more preferably 1 to 25 mg / kg. The dose and treatment regimen of the therapeutic compound can be determined by those skilled in the art. In certain embodiments, the therapeutic compound is administered by injection (e.g., subcutaneously or intravenously) in doses of about 1 to 40 mg / kg, for example, 1 to 30 mg / kg, for example, about 5 to 25 mg / kg, about 10 to 20 mg / kg, about 1 to 5 mg / kg, 1 to 10 mg / kg, 5 to 15 mg / kg, 10 to 20 mg / kg, 15 to 25 mg / kg, or about 3 mg / kg. The dosing schedule may vary, for example, between once a week and once every 2, 3, or 4 weeks, or in some embodiments, the dosing schedule may be once a month, every 2 months, every 3 months, or every 6 months. In one embodiment, the therapeutic compound is administered every other week at a dose of about 10–20 mg / kg. The therapeutic compound may be administered by intravenous infusion at a rate greater than 20 mg / min, e.g., 20–40 mg / min, typically 40 mg / min or higher, until a dose of about 35–440 mg / m2, typically about 70–310 mg / m2, more typically about 110–130 mg / m2 is reached. In one embodiment, an infusion rate of about 110–130 mg / m2 achieves a level of about 3 mg / kg. In another embodiment, the therapeutic compound may be administered by intravenous infusion at a rate less than 10 mg / min, e.g., 5 mg / min or less, until a dose of about 1–100 mg / m2, e.g., about 5–50 mg / m2, about 7–25 mg / m2, or about 10 mg / m2 is reached. In some embodiments, the therapeutic compound is infused over a period of about 30 minutes. It should be noted that dosage values ​​may vary depending on the type and severity of the condition being alleviated. Furthermore, it should be understood that for any particular subject, a specific medication regimen should be adjusted over time according to the individual's needs and the professional judgment of the person administering or supervising the administration of the composition, and that the dosage ranges described herein are illustrative only and are not intended to limit the scope or implementation of the claimed composition.

[0034] A pharmaceutical composition may contain a "therapeutic dose" or a "preventive dose" of a composition, vector, cell, polypeptide, or nucleic acid molecule encoding them. "Therapeutic dose" refers to the amount effective in the dose and duration necessary to achieve the desired therapeutic outcome. The therapeutically effective amount of the active ingredient or molecule may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic compound to induce the desired response in that individual. A "therapeutic dose" may, for example, inhibit measurable parameters, such as tumor growth by at least about 20%, at least about 40%, at least about 60%, and at least about 80% compared to an untreated subject. Measurable parameters, such as the ability of a compound to inhibit tumor growth, can be evaluated in animal model systems that predict efficacy in tumor growth. Alternatively, this property of a composition can be evaluated by testing the compound's ability to inhibit such inhibition in vitro using assays known to those skilled in the art.

[0035] The "prophylactic effective dose" refers to the amount effective in achieving the desired prophylactic outcome at the required dosage and duration of administration. Typically, since prophylactic doses are used in patients before or in the early stages of disease, the prophylactic effective dose may be less than the therapeutic effective dose, but this is not always the case.

[0036] Similarly, this specification also provides kits comprising compositions, cells, vectors, nucleic acid molecules, or polypeptides described herein. The kit may comprise one or more other elements, e.g., instructions for use; other reagents, e.g., labels, therapeutic agents, or factors useful for chelating or otherwise binding a molecule to a label, other therapeutic agent, or radioactive composition; apparatus or other materials for preparing molecules for administration; pharmaceutically acceptable carriers; and apparatus or other materials for administration to a subject.

[0037] Vasopressor In some embodiments, the pharmaceutical composition comprising the polypeptide is administered after, before, or in combination with a vasopressor. In some embodiments, the subject requiring it has not been administered a vasopressor, or has previously been administered a smaller amount of a vasopressor, is administered a smaller amount of a vasopressor concurrently, or is subsequently administered a smaller amount of a vasopressor than the amount administered in the absence of the pharmaceutical composition. In some embodiments, the subject requiring treatment receives about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% less of the vasopressor than the subject not receiving the polypeptide-containing pharmaceutical composition. In some embodiments, the subject requiring treatment receives 1 / 1, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, or 1 / 10 less of the vasopressor than the subject not receiving the polypeptide-containing pharmaceutical composition.

[0038] Examples of vasopressors include, but are not limited to, Vasopressin (Pitressin® or Vasostrict®), Phenylephrine (Biorphen® or Vazculep®), epinephrine, norepinephrine, droxidopa, phenylephrine, ephedrine, dobutamine, dopamine, angiotensin-II, and terlipressin.

[0039] method "Treatment, therapy" of any disease referred to in this specification includes alleviation of at least one symptom of the disease, reduction of the severity of the disease, or in some cases, delay or prevention of progression of the disease to more severe symptoms, which may be accompanied by the disease or at least one other disease. Treatment (therapy) does not necessarily mean complete cure of the disease. A useful therapeutic agent only needs to reduce the severity of the disease, reduce the severity of the disease or the symptom(s) associated with the disease or its treatment, or delay the onset of more severe symptoms or more severe diseases that may occur at a certain frequency after the treated condition. For example, if the disease is a tumor, the composition may reduce the growth or spread of the tumor or the effect of the tumor on the tissue in which the tumor is present. The patient's condition can be evaluated by standard techniques. Appropriate procedures vary depending on the patient's condition and symptoms.

[0040] In some embodiments, the compositions provided herein can be used to treat septic shock. In some embodiments, the method includes administering to a patient a polypeptide provided herein. In some embodiments, the method includes administering to a patient an effective amount of a pharmaceutical composition comprising a polypeptide provided herein.

[0041] In some embodiments, a method of treating septic shock in a subject in need thereof, the polypeptide comprising the following amino acid sequence: X 1 X 2 X 3 X 4 X 5 LX 6 X 7 X 8 X 9 HQIL (SEQ ID NO: 1) Where: X 1 is E or does not exist; X 2 is L or does not exist; X 3 is Q or does not exist; X4 However, it is either A, T, or does not exist; X 5 However, it is either T, E, or does not exist; X 6 However, it is either H or Y; X 7 However, it is either D or E; X 8 However, it is F or I; and X 9 However, a polypeptide containing an amino acid sequence that is R or K; or Polypeptides containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, or 30; A method is provided which involves administering an effective amount of a pharmaceutical composition, comprising a pharmaceutically acceptable carrier, to the subject.

[0042] In some embodiments, the polypeptide is at least 85%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. In some embodiments, this polypeptide comprises SEQ ID NOs: 2, 3, or 4.

[0043] In some embodiments, the subject requiring it has been diagnosed with sepsis and has hypotension that is not relieved by intravenous fluid administration alone. In some embodiments, the subject has a mean arterial pressure (MAP) of less than approximately 65 mmHg. In some embodiments, the subject has a mean arterial pressure (MAP) of less than approximately 60 mmHg. In some embodiments, the subject has a mean arterial pressure (MAP) of less than approximately 55 mmHg. In some embodiments, the subject has a mean arterial pressure or systolic blood pressure that is lower than approximately 44 mmHg. In some embodiments, the subject has a blood lactate level greater than approximately 2 mmol. In some embodiments, the subject has a blood lactate level greater than approximately 4 mmol. In some embodiments, the subject has a blood lactate level greater than approximately 6 mmol. In some embodiments, the subject has a blood lactate level greater than approximately 8 mmol. In some embodiments, the subject has a blood lactate level greater than approximately 10 mmol. In some embodiments, the subject has persistent signs of multi-organ damage or failure. In some embodiments, the subject has been diagnosed with septic shock.

[0044] In some embodiments, methods are also provided for treating a subject with significantly reduced MAP, comprising administering to the subject an effective amount of any pharmaceutical composition described herein. In some embodiments, the subject has a MAP or systolic blood pressure reduction of about 44 mmHg or greater, or has been diagnosed with sepsis. In some embodiments, the subject has been diagnosed with sepsis and has hypotension that is not relieved by intravenous fluid administration alone. In some embodiments, the subject has a MAP of less than about 65 mmHg. In some embodiments, the subject has a blood lactate level greater than about 4 mmol. In some embodiments, the subject has persistent signs of multi-organ damage or failure. In some embodiments, the subject has been diagnosed with septic shock.

[0045] In some embodiments, the subject maintains normal blood oxygen levels after administration of the pharmaceutical composition. Normal blood oxygen levels are generally 95% to 100%. In some embodiments, the subject maintains blood oxygen levels of 95% to 100% after administration of the pharmaceutical composition. In some embodiments, the subject's blood oxygen level does not decrease by more than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% after administration of the pharmaceutical composition. In some embodiments, the subject's blood oxygen level increases after administration of the pharmaceutical composition. In some embodiments, the subject's blood oxygen level increases by more than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% after administration of the pharmaceutical composition.

[0046] In some embodiments, the pharmaceutically acceptable carrier is any pharmaceutically acceptable carrier disclosed herein. In some embodiments, the pharmaceutically acceptable carrier is water.

[0047] To treat the disease of interest, the compositions and polypeptides described herein may be administered by any suitable method, including but not limited to parenteral, topical, oral, nasal, vaginal, rectal, or pulmonary (by inhalation) administration. When administered by injection, the composition(s) may be administered intra-articular, intravenous, intra-arterial, intramuscular, intravesical, intra-bladder, intraperitoneal, intratracheal, or subcutaneously by bolus injection or continuous infusion. Topical administration, i.e., administration at the site of disease, as well as transdermal delivery and sustained release from implants, skin patches, or suppositories, are intended. Delivery by inhalation includes, for example, nasal or oral inhalation, use of a nebulizer, and inhalation in aerosol form. Administration via suppositories inserted into a body cavity may be achieved, for example, by inserting the solid form of the composition into a selected body cavity and allowing it to dissolve. Other alternatives include eye drops, oral preparations, e.g., pills, lozenges, syrups, and chewing gums, and topical preparations, e.g., lotions, gels, sprays, and ointments.

[0048] In the administration of a treatment method, the compositions described herein may be administered as described herein and above. For example, the compositions may be administered in any dose, frequency, and duration that may be effective in treating the condition being treated. The dosage depends on the molecular nature of the active ingredient and the nature of the disorder being treated. Treatment may be continued for as long as necessary to achieve the desired outcome. The compositions provided herein may be administered as a single dose or as a series of doses given regularly, including multiple doses per day, daily, every other day, twice a week, three times a week, weekly, every other week, and monthly, among other possible administration regimens. The treatment cycle may or may not be constant throughout the entire period of treatment. For example, treatment may be initially given at weekly intervals and later every other week. Treatments having durations of days, weeks, months, or years are encompassed by the embodiments provided herein. Treatment may be discontinued and then resumed. A maintenance dose may or may not be administered after the initial treatment.

[0049] The dosage is given as milligrams per kilogram of body weight (mg / kg) or as milligrams per square meter of skin surface (mg / m²). 2 ) may be measured as a fixed dose regardless of height or weight. These are all standard dosage units in the field. A person's skin surface area is calculated from their height and weight using a standard formula.

[0050] As used herein, the phrase “needs it” means that the subject (animal or mammal) is identified as having a need for a particular method or treatment. In some embodiments, identification may be by any diagnostic means. In any of the methods and treatments described herein, the animal or mammal may need it. In some embodiments, the animal or mammal is in or will go into an environment where a particular disease, disorder, or condition is prevalent.

[0051] Embodiment In some embodiments, the embodiments provided herein include, but are not limited to, the following:

[0052] 1. A method for treating septic shock in the necessary population, comprising the following amino acid sequence: X1X2X3X4X5LX6X7X8X9HQIL(Sequence ID 1) During the ceremony: X1 is either E or does not exist; X2 is either L or does not exist; X3 is either Q or does not exist; X4 is either A, T, or does not exist; X5 is either T, E, or does not exist; X6 is either H or Y; X7 is either D or E; X8 is either F or I; and An amino acid sequence in which X9 is R or K; or Amino acid sequences selected from the group consisting of sequence numbers 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, or 30; A method comprising administering to a subject an effective amount of a pharmaceutical composition containing a polypeptide containing [a specific substance].

[0053] 2. The method according to Embodiment 1, wherein the polypeptide contains an amino acid sequence that is at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

[0054] 3. The method according to Embodiment 1, wherein the polypeptide includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

[0055] 4. The method according to Embodiment 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

[0056] 5. The method according to Embodiment 1, wherein the polypeptide includes SEQ ID NO: 2.

[0057] 6. The method according to Embodiment 1, wherein the polypeptide is comprised of Sequence ID No. 2.

[0058] 7. The method according to Embodiment 1, wherein the polypeptide includes SEQ ID NO: 3.

[0059] 8. The method according to Embodiment 1, wherein the polypeptide is comprised of Sequence ID No. 3.

[0060] 9. The method according to Embodiment 1, wherein the polypeptide comprises SEQ ID NO: 4.

[0061] 10. The method according to Embodiment 1, wherein the polypeptide comprises Sequence ID No. 4.

[0062] 11. The method according to any one of the preceding embodiments, wherein the pharmaceutical composition comprises a liposome-encapsulated polypeptide.

[0063] 12. The method according to any one of the prior embodiments, wherein the pharmaceutically acceptable carrier is any pharmaceutically acceptable carrier disclosed herein.

[0064] 13. The method according to any one of the preceding embodiments, wherein the pharmaceutical composition is administered to the subject by any administration method disclosed herein.

[0065] 14. The method according to any one of the preceding embodiments, wherein the pharmaceutical composition is administered to the subject by aerosol inhalation, intratracheal injection, or intravenous injection.

[0066] 15. The method according to any one of the preceding embodiments, wherein the subject requiring the method is diagnosed with sepsis and has hypotension that is not relieved by intravenous fluid administration alone.

[0067] 16. The method according to Embodiment 15, wherein the subject requiring it has a mean arterial pressure (MAP) of less than approximately 65 mmHg.

[0068] 17. The method according to any one of the preceding embodiments, wherein the subject requiring the method is diagnosed with sepsis and has a mean arterial pressure or systolic blood pressure that has decreased by approximately 44 mmHg or more.

[0069] 18. The method according to any one of Embodiments 15 to 17, wherein the subject requiring the method has a blood lactate level greater than approximately 4 mmol.

[0070] 19. The method according to any one of claims 15 to 17, wherein the subject requiring the method has persistent signs of multiple organ damage or failure.

[0071] 20. The method according to any one of the preceding embodiments, wherein the blood oxygen level of the subject does not decrease after administration of the pharmaceutical composition.

[0072] 21. The method according to Embodiment 20, wherein the blood oxygen level of the subject does not decrease by more than 50% after administration of the pharmaceutical composition.

[0073] 22. The method according to Embodiment 20, wherein the blood oxygen level of the subject does not decrease by more than 65% after administration of the pharmaceutical composition.

[0074] 23. The method according to any one of the preceding embodiments, wherein the blood oxygen level of the subject increases after administration of the pharmaceutical composition.

[0075] 24. The method according to any one of the preceding embodiments, wherein the subject maintains a normal blood oxygen level after administration of the pharmaceutical composition.

[0076] 25. The method according to any one of the preceding embodiments, wherein the subject requiring it has not been administered a vasopressor, or has been previously administered a smaller amount of a vasopressor, is administered a smaller amount of a vasopressor simultaneously, or is subsequently administered a smaller amount of a vasopressor than the amount of a vasopressor administered in the absence of the pharmaceutical composition.

[0077] 26. The method according to any one of the preceding embodiments, wherein the subject requiring it has been diagnosed with septic shock.

[0078] 27. A method for treating a subject with a significant decrease in mean arterial pressure, the following amino acid sequence: X1X2X3X4X5LX6X7X8X9HQIL(Sequence ID 1) During the ceremony: X1 is either E or does not exist; X2 is either L or does not exist; X3 is either Q or does not exist; X4 is either A, T, or does not exist; X5 is either T, E, or does not exist; X6 is either H or Y; X7 is either D or E; X8 is either F or I; and An amino acid sequence in which X9 is R or K; or Amino acid sequences selected from the group consisting of sequence numbers 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, or 30; The method comprising administering to a subject an effective amount of a pharmaceutical composition comprising a polypeptide containing and a pharmaceutically acceptable carrier.

[0079] 28. The method according to Embodiment 27, wherein the polypeptide includes an amino acid sequence that is at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

[0080] 29. The method according to Embodiment 27, wherein the polypeptide includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

[0081] 30. The method according to Embodiment 27, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

[0082] 31. The method according to Embodiment 27, wherein the polypeptide includes Sequence ID No. 2.

[0083] 32. The method according to Embodiment 27, wherein the polypeptide is the polypeptide of Sequence ID No. 2.

[0084] 33. The method according to Embodiment 27, wherein the polypeptide includes Sequence ID No. 3.

[0085] 34. The method according to Embodiment 27, wherein the polypeptide is sequence number 3.

[0086] 35. The method according to Embodiment 27, wherein the polypeptide comprises Sequence ID No. 4.

[0087] 36. The method according to Embodiment 27, wherein the polypeptide is sequence number 4.

[0088] 37. The method according to any one of the preceding embodiments, wherein the pharmaceutical composition comprises a liposome-encapsulated polypeptide.

[0089] 38. The method according to any one of the preceding embodiments, wherein the pharmaceutically acceptable carrier is any pharmaceutically acceptable carrier disclosed herein.

[0090] 39. The method according to any one of the preceding embodiments, wherein the pharmaceutical composition is administered to the subject by any administration method disclosed herein.

[0091] 40. The method according to any one of the preceding embodiments, wherein the pharmaceutical composition is administered to the subject by aerosol inhalation, intratracheal injection, or intravenous injection.

[0092] 41. The method according to any one of the preceding embodiments, wherein the subject has a mean arterial pressure or systolic blood pressure that has decreased by approximately 44 mmHg or more and has been diagnosed with sepsis.

[0093] 42. The method according to any one of the preceding embodiments, wherein the subject is diagnosed with sepsis and has hypotension that is not relieved by intravenous fluid administration alone.

[0094] 43. The method according to Embodiment 42, wherein the subject has a mean arterial pressure (MAP) of less than approximately 65 mmHg.

[0095] 44. The method according to any one of Embodiments 41 to 43, wherein the subject requiring the method has a blood lactate level greater than approximately 4 mmol.

[0096] 45. The method according to any one of claims 41 to 43, wherein the subject requiring the method has persistent signs of multiple organ damage or failure.

[0097] 46. ​​The method according to any one of the preceding embodiments, wherein the blood oxygen level of the subject does not decrease after administration of the pharmaceutical composition.

[0098] 47. The method according to Embodiment 46, wherein the blood oxygen level of the subject does not decrease by more than 50% after administration of the pharmaceutical composition.

[0099] 48. The method according to Embodiment 46, wherein the blood oxygen level of the subject does not decrease by more than 65% after administration of the pharmaceutical composition.

[0100] 49. The method according to any one of the preceding embodiments, wherein the blood oxygen level of the subject increases after administration of the pharmaceutical composition.

[0101] 50. The method according to any one of the preceding embodiments, wherein the subject maintains a normal blood oxygen level after administration of the pharmaceutical composition.

[0102] 51. The method according to any one of the preceding embodiments, wherein the subject requiring it has not been administered a vasopressor, or has been previously administered a vasopressor, is administered concurrently with it, or is subsequently administered a smaller amount of vasopressor than the amount administered in the absence of the pharmaceutical composition.

[0103] 52. The method according to any one of the preceding embodiments, wherein the subject requiring it has been diagnosed with septic shock.

[0104] 53. A method for preventing cardiac injury in a subject diagnosed with septic shock, comprising the following amino acid sequence: X1X2X3X4X5LX6X7X8X9HQIL(Sequence ID 1) During the ceremony: X1 is either E or does not exist; X2 is either L or does not exist; X3 is either Q or does not exist; X4 is either A, T, or does not exist; X5 is either T, E, or does not exist; X6 is either H or Y; X7 is either D or E; X8 is either F or I; and An amino acid sequence in which X9 is R or K; or Amino acid sequences selected from the group consisting of sequence numbers 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, or 30; The method comprising administering to a subject (compared to a subject not treated with the pharmaceutical composition) an effective amount of a pharmaceutical composition comprising a polypeptide containing and a pharmaceutically acceptable carrier.

[0105] 54. The method according to Embodiment 53, wherein the polypeptide includes an amino acid sequence that is at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

[0106] 55. The method according to Embodiment 53, wherein the polypeptide includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

[0107] 56. The method according to Embodiment 53, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

[0108] 57. The method according to Embodiment 53, wherein the polypeptide includes Sequence ID No. 2.

[0109] 58. The method according to Embodiment 53, wherein the polypeptide is sequence number 2.

[0110] 59. The method according to Embodiment 53, wherein the polypeptide comprises Sequence ID No. 3.

[0111] 60. The method according to Embodiment 53, wherein the polypeptide is sequence number 3.

[0112] 61. The method according to Embodiment 53, wherein the polypeptide comprises Sequence ID No. 4.

[0113] 62. The method according to Embodiment 53, wherein the polypeptide is sequence number 4.

[0114] 63. The method according to any one of the preceding embodiments, wherein the pharmaceutical composition comprises a liposome-encapsulated polypeptide.

[0115] 64. The method according to any one of the preceding embodiments, wherein the pharmaceutically acceptable carrier is any pharmaceutically acceptable carrier disclosed herein.

[0116] 65. The method according to any one of the preceding embodiments, wherein the pharmaceutical composition is administered to the subject by any administration method disclosed herein.

[0117] 66. The method according to any one of the preceding embodiments, wherein the pharmaceutical composition is administered to the subject by aerosol inhalation, intratracheal injection, or intravenous injection.

[0118] 67. The method according to any one of the preceding embodiments, wherein the subject requiring the method has hypotension that is not relieved by intravenous fluid administration alone.

[0119] 68. The method according to embodiment 67, wherein the subject requiring it has a mean arterial pressure (MAP) of less than approximately 65 mmHg.

[0120] 69. The method according to any one of the preceding embodiments, wherein the subject requiring it has a mean arterial pressure or systolic blood pressure that has decreased by approximately 44 mmHg or more.

[0121] 70. The method according to any one of embodiments 67 to 69, wherein the subject requiring it has a blood lactate level greater than approximately 4 mmol.

[0122] 71. The method according to any one of claims 67 to 69, wherein the subject requiring the method has persistent signs of multi-organ damage or failure.

[0123] 72. The method according to any one of the preceding embodiments, wherein the blood oxygen level of the subject does not decrease after administration of the pharmaceutical composition.

[0124] 73. The method according to Embodiment 72, wherein the blood oxygen level of the subject does not decrease by more than 50% after administration of the pharmaceutical composition.

[0125] 74. The method according to Embodiment 72, wherein the blood oxygen level of the subject does not decrease by more than 65% after administration of the pharmaceutical composition.

[0126] 75. The method according to any one of the preceding embodiments, wherein the blood oxygen level of the subject increases after administration of the pharmaceutical composition.

[0127] 76. The method according to any one of the preceding embodiments, wherein the subject maintains a normal blood oxygen level after administration of the pharmaceutical composition.

[0128] 77. The method according to any one of the preceding embodiments, wherein the required subject has a troponin level that does not increase after administration of the pharmaceutical composition.

[0129] 78. The method according to any one of the preceding embodiments, wherein the subject concerned maintains normal troponin levels after administration of the pharmaceutical composition.

[0130] 79. The method according to any one of the preceding embodiments, wherein the subject requiring it has not been administered a vasopressor, or has been previously administered a vasopressor, is administered concurrently with it, or is subsequently administered a smaller amount of vasopressor than the amount administered in the absence of the pharmaceutical composition.

[0131] 80. A method for increasing the survival of a subject diagnosed with septic shock, comprising the following amino acid sequence: X1X2X3X4X5LX6X7X8X9HQIL(Sequence ID 1) During the ceremony: X1 is either E or does not exist; X2 is either L or does not exist; X3 is either Q or does not exist; X4 is either A, T, or does not exist; X5 is either T, E, or does not exist; X6 is either H or Y; X7 is either D or E; X8 is either F or I; and An amino acid sequence in which X9 is R or K; or Amino acid sequences selected from the group consisting of sequence numbers 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, or 30; The method comprising administering to a subject (compared to a subject not treated with the pharmaceutical composition) an effective amount of a pharmaceutical composition comprising a polypeptide containing and a pharmaceutically acceptable carrier.

[0132] 81. The method according to Embodiment 80, wherein the polypeptide contains an amino acid sequence that is at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

[0133] 82. The method according to Embodiment 80, wherein the polypeptide includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

[0134] 83. The method according to Embodiment 80, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

[0135] 84. The method according to Embodiment 80, wherein the polypeptide includes Sequence ID No. 2.

[0136] 85. The method according to Embodiment 80, wherein the polypeptide is comprised of Sequence ID No. 2.

[0137] 86. The method according to Embodiment 80, wherein the polypeptide comprises Sequence ID No. 3.

[0138] 87. The method according to Embodiment 80, wherein the polypeptide is SEQ ID NO: 3.

[0139] 88. The method according to Embodiment 80, wherein the polypeptide comprises SEQ ID NO: 4.

[0140] 89. The method according to Embodiment 80, wherein the polypeptide comprises Sequence ID No. 4.

[0141] 90. The method according to any one of the preceding embodiments, wherein the pharmaceutical composition comprises a liposome-encapsulated polypeptide.

[0142] 91. The method according to any one of the prior embodiments, wherein the pharmaceutically acceptable carrier is any pharmaceutically acceptable carrier disclosed herein.

[0143] 92. The method according to any one of the preceding embodiments, wherein the pharmaceutical composition is administered to the subject by any administration method disclosed herein.

[0144] 93. The method according to any one of the preceding embodiments, wherein the pharmaceutical composition is administered to the subject by aerosol inhalation, intratracheal injection, or intravenous injection.

[0145] 94. The method according to any one of the preceding embodiments, wherein the subject requiring the method is diagnosed with sepsis and has hypotension that is not relieved by intravenous fluid administration alone.

[0146] 95. The method according to Embodiment 94, wherein the subject requiring it has a mean arterial pressure (MAP) of less than approximately 65 mmHg.

[0147] 96. The method according to any one of the preceding embodiments, wherein the subject requiring it has a mean arterial pressure or systolic blood pressure that has decreased by approximately 44 mmHg or more.

[0148] 97. The method according to any one of embodiments 94 to 96, wherein the subject requiring it has a blood lactate level greater than approximately 4 mmol.

[0149] 98. The method according to any one of claims 94 to 96, wherein the subject requiring the method has persistent signs of multi-organ damage or failure.

[0150] 99. The method according to any one of the preceding embodiments, wherein the blood oxygen level of the subject does not decrease after administration of the pharmaceutical composition.

[0151] 100. The method according to Embodiment 99, wherein the blood oxygen level of the subject does not decrease by more than 50% after administration of the pharmaceutical composition.

[0152] 101. The method according to Embodiment 99, wherein the blood oxygen level of the subject does not decrease by more than 65% after administration of the pharmaceutical composition.

[0153] 102. The method according to any one of the preceding embodiments, wherein the blood oxygen level of the subject increases after administration of the pharmaceutical composition.

[0154] 103. The method according to any one of the preceding embodiments, wherein the subject maintains a normal blood oxygen level after administration of the pharmaceutical composition.

[0155] 104. The method according to any one of the preceding embodiments, wherein the subject requiring it has not been administered a vasopressor, or has been previously administered a vasopressor, is administered concurrently with it, or is subsequently administered a smaller amount of vasopressor than the amount administered in the absence of the pharmaceutical composition. [Examples]

[0156] The following examples illustrate, but are not limiting, the compounds, compositions, and methods described herein. Other suitable modifications and alterations known to those skilled in the art are within the scope of the embodiments shown herein.

[0157] Example 1: Inhibition of human Prdx6 aiPLA2 activity by a selected polypeptide. The polypeptides disclosed herein are based in part on the engineering of specific peptide inhibitors of aiPLA2. Tables 2, 3, and 4 below show the effects of selected polypeptides on the aiPLA2 activity of recombinant human Prdx6 (Table 2), the size optimization of inhibitory polypeptides by the effect of human recombinant proteins on aiPLA2 activity (Table 3), and the effect of selective substitution in PIP-2 inhibition of aiPLA2 activity of human recombinant Prdx6 (Table 4). [Table 2] [Table 3] [Table 4]

[0158] Example 2: Treatment of septic shock-induced organ injury To determine the efficacy of PIP-2 in treating acute lung and other organ injuries resulting from septic shock, healthy growing Yorkshire pig subjects were divided into three groups: (Group 1) healthy controls + standard treatment with mechanical ventilation (negative control); (Group 2) placebo + standard treatment with mechanical ventilation, fluids, glucose, and vasopressors (positive control); and (Group 3) PIP-2 treatment + the same standard treatment as Group (2). Subjects were administered via bolus intravenous push in both arms, and the boluses in Groups (2) and (3) contained bacterial lipopolysaccharide (LPS) to induce septic shock. The entire study period was 8 hours from injection.

[0159] LPS induced severe and rapid septic shock in subjects, with the worst-case scenario being death within 4–7 hours of LPS administration. Furthermore, septic shock resulted from protein accumulation in the lung lavage fluid as a result of damage to the alveolar-capillary barrier; myeloperoxidase (MPO) in the lung lavage fluid reflecting an increase in white blood cell count; a "cytokine storm" resulting from excessive immune activation and damage; and elevated troponin levels as a result of cardiac damage.

[0160] The results, as shown in Table 5, indicated that treatment with PIP-2 reduced the amount of protein located in the lung lavage fluid compared to the negative control group. [Table 5]

[0161] The results also showed that treatment with PIP-2 protected against inflammation and leukocyte activation by lowering MPO levels, an indicator of the immune response to pneumonia and injury, as shown in Table 6. [Table 6]

[0162] Furthermore, PIP-2 treatment also prevented a "cytokine storm" in the measurement of three different cytokine levels in bronchoalveolar lavage fluid (BALf), as shown in Table 7. High cytokine levels in BALf indicated activation of the lung immune response to injury. [Table 7]

[0163] Furthermore, animals treated with PIP-2 showed dramatic improvement in Horowitz Index performance, a measure used to assess lung function and damage in patients, particularly those using mechanical ventilation. In the Horowitz Index, a score of 300 or higher indicates normal lung function, a score of 201–300 indicates mild lung damage, a score of 100–200 indicates moderate lung damage, and a score below 100 indicates severe lung damage. Table 8 shows the Horowitz Index scores for the placebo (LPS control) and PIP-2 treated groups. The PIP-2 treated group was more than twice as likely to have normal lung function and half as likely to die. [Table 8]

[0164] In addition to positive results in the lungs, PIP-2 treatment also produced positive results in the subjects' cardiac and vascular systems. Firstly, blood troponin is an indicator of cardiac injury because this protein is not normally present in the blood, but when the myocardium is damaged, it leaks into the bloodstream, potentially leading to septic shock. As shown in Figure 1, troponin levels in the placebo-LPS control group began to rise rapidly from 1 to 8 hours into the study, with a significant difference observed at 4 hours. However, the PIP-2 treatment group did not show such an increase and remained stable with the control group.

[0165] Secondly, treatment with PIP-2 also protected blood creatine kinase (CK) protein levels. Similar to troponin, CK protein leaks into the bloodstream when the myocardium and other skeletal muscles are damaged. At a single time point, control subjects had a mean CK level of 19% (U / mL), while placebo-treated LPS controls had a much higher CK level of 89%. However, in subjects treated with PIP-2, CK levels decreased to approximately 15%. Therefore, PIP-2 protected animals from LPS-induced septic shock.

[0166] Similarly, treatment with PIP-2 also prevented an increase in c-reactive protein (CRP), an indicator of inflammation. As shown in Figure 2, CRP levels in the placebo-LPS control group began to rise sharply between 1 and 8 hours, similar to the rise observed with troponin levels. The PIP-2 treated group did not show such an increase, and CRP levels remained low. A statistically significant difference in blood c-reactive protein levels between the PIP-2 treated group and the placebo group was observed at 8 hours.

[0167] Septic shock caused a fatal decrease in mean arterial pressure (MAP), which is one of the leading causes of death in this condition. Since IV fluid resuscitation alone does not produce a response with increased MAP in patients with septic shock, vasopressor therapy is a primary tool for increasing MAP and stabilizing blood flow. As shown in Figure 3, subjects in the placebo-LPS control group required salvage vasopressor therapy and continuous infusion to attempt to stabilize MAP. In comparison, subjects treated with PIP-2 required approximately 66% less vasopressor therapy and little to no salvage therapy. Furthermore, subjects treated with PIP-2 had better overall MAP even without the use of vasopressors. Measuring the mean change in MAP from baseline to the end of the 8-hour study, the placebo-LPS control group experienced a mean decrease of -32% in MAP, compared to only -25% in subjects treated with PIP-2 (Figures 4A and 4B).

[0168] In summary, in this 8-hour LPS-induced septic shock model, PIP-2 treatment demonstrated superior outcomes across several key measurements. PIP-2 treatment halved the septic shock mortality rate, while 50% of treated subjects showed complete protection. Unexpectedly, further PIP-2 treatment showed several benefits, including decreased intrapulmonary protein levels, reduced serum troponin, creatine kinase, and CRP levels, reduced use of vasopressors to stabilize MAP, and overall improvement in cardiovascular capacity.

[0169] Example 3: Reduction of lipopolysaccharide (LPS)-induced acute lung injury in pigs by treatment with a nonapeptide (PIP-2) that inhibits the phospholipase A2 activity of peroxiredoxin 6. The efficacy of liposomal-encapsulated 9-amino acid peptide (PIP-2) for the prevention / treatment of lipopolysaccharide (LPS)-induced sepsis and acute lung injury (ALI) was investigated in pigs. PIP-2 inhibits the PLA2 activity of peroxiredoxin 6, thereby preventing the activation of NADPH oxidases type 1 and type 2. Twenty Yorkshire female pigs were intravenously administered liposomes alone (control, n=2), LPS + liposomes (untreated, n=6), and LPS + PIP-2 in liposomes (treated, n=12). Animals were mechanically ventilated and euthanized within 8 hours if pre-defined humane endpoints were reached. Control pigs remained essentially unchanged over the 8-hour trial. LPS administration resulted in systemic inflammation with signs of clinical sepsis, decreased lung function with a marked decrease in arterial PO2, and vascular instability, and 50% of untreated animals were euthanized early. PIP-2 treatment reduced the need for adjunctive vasopressors and signs of lung injury, resulting in only 25% of animals requiring early euthanasia. Bronchoalveolar lavage fluid showed significantly lower total protein, cytokine (TNF-α, IL-6, and IL-1β), and myeloperoxidase levels in PIP-2-treated pigs compared to untreated pigs. Thus, the porcine LPS-induced sepsis model was associated with moderate to severe lung pathophysiology consistent with ALI. PIP-2 treatment significantly reduced lung injury, cardiovascular instability, and early euthanasia of pigs. These results indicate that inhibition of ROS production via NOX1 / 2 has a beneficial effect in the treatment of pigs with LPS-induced sepsis and ALI. Therefore, PIP-2 and the peptides provided herein may be used to treat septic shock and associated injuries, such as ALI.

[0170] Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) can be associated with various pulmonary (e.g., aspiration, pneumonia) or non-pulmonary (e.g., sepsis, trauma) etiologies. Physiological effects associated with ARDS include pulmonary rigidity (reduced lung compliance), pulmonary edema, and progressive hypoxemia. Current standard treatment is based on treating the underlying disease and respiratory support using controlled ventilation to avoid mechanical damage to lung tissue. However, despite optimal respiratory support, ARDS currently has a mortality rate of approximately 40%.

[0171] While the cause of ALI is multifactorial, it is now recognized that excessive release of reactive oxygen species (ROS), including oxygen-derived radicals, can play a significant role in lung damage associated with many different etiologies. NADPH oxidase (NOX) is the primary enzyme source of ROS in the lung. ROS produced by the NOX enzyme are essential for regulating important cellular functions such as host defense, cell signaling, cell migration, cell differentiation, and post-translational protein processing. However, excessive production of ROS can lead to the oxidation of tissue macromolecules (lipids, proteins, and DNA), potentially resulting in widespread cellular damage. ROS can be produced in the lung by inflammatory cells, including polymorphonuclear leukocytes (PMNs) and alveolar macrophages (AMs), as well as by lung parenchymal cells, including both epithelial and endothelial cells. Therefore, ROS release may play a crucial pathophysiological role in the signs of ALI / ARDS.

[0172] The NOX family consists of seven different proteins. NOX2 is the major NOX enzyme in phagocytes and the lungs, while NOX1 and NOX2 are dominant in the cardiovascular system. NOX2 has two cell membrane-bound protein components, gp91 phox and p22 phox It is a complex and is inactive in its resting state. Activation of intracellular enzymes is due to gp91 phox Phosphorylation of three additional cytoplasmic proteins (p67) phox p47 phox p40 phoxThis process involves translocation of rac to the membrane and activation of the cytoplasmic rac protein (either rac1 or rac2). Rac is a member of the Rho family of GTPases; rac1 is an activation cofactor in lung parenchymal cells, while rac2 is required in PMNs. The protein peroxiredoxin 6 (Prdx6) is required for rac activation and therefore plays a crucial role in NOX2-mediated ROS production. Thus, the activation process requires the phospholipase A2 activity (aiPLA2) of Prdx6, which regulates rac release via lysophosphatidic acid receptor signaling. NOX1 also requires the rac protein to activate ROS production, although the other five NOX enzymes are independent of rac.

[0173] The lipid compound MJ33 and surfactant protein A (SP-A) are two agents that bind to Prdx6, inhibiting aiPLA2 activity and consequently leading to rac activation failure. The nine-amino acid sequence of SP-A was identified as the cause of the inhibition of NOX2 activation. This nine-amino acid peptide sequence is generally conserved in mammals, and some minor mutations are called peroxiredoxin 6 PLA2 inhibitory peptides (PIPs); the peptide corresponding to the human amino acid peptide sequence of SP-A is called PIP-2.

[0174] Numerous models of experimental ALI are described, including commonly used models associated with the administration of bacterial lipopolysaccharide (LPS). LPS can be administered either intratracheally (leading to primary pneumonia) or intraperitoneally (IP) or intravenously (IV) (leading to systemic sepsis with pneumonia). In various mouse models of ALI, inhibition of NOX2 activation or genetic inactivation of aiPLA2 activity by treatment with MJ33 or PIP-2 can significantly improve lung injury. The studies described herein are designed to evaluate the possible protective role of PIP-2 in animal models with lungs that more closely resemble the anatomical structure and physiological function of the human lung.

[0175] method medication A 9-amino acid peptide called PIP-2 was synthesized by APeptide, Shigh, China using a C-terminal HCl group. The peptide was encapsulated in liposomes for IV delivery. The liposomes consisted of dipalmitoylphosphatidylcholine (DPPC), egg polyphosphate (PC), phosphatidylglycerol (PG), and cholesterol in molar ratios of 0.5, 0.25, 0.10, and 0.15; this composition reflects the lipid components of lung surfactant. The liposomes were stored at -20°C before use. Samples were warmed to room temperature and used within 3 hours. The PIP-2 encapsulation efficiency of the liposomes was approximately 15-20%. LPS, Escherichiacoli O55:B5, was obtained from Sigma (product code: L2637). Source batch: 12181107, sub-batches: 0000102731 and 0000119457. LPS was stored at 3-8°C. @@Remove@@

[0176] animal The model used was based on a previously reported study on LPS-induced injury in pigs (Hochhausen N, Orschulik J, Follmann A, Santos SA, Dohmeier H, Leonhardt S, Rossaint R, Czaplik M. Comparison of two experimental ARDS models in pigs using electrical impedance tomography. PLoS One. 2019 Nov 13;14(11):e0225218.). Twenty nulliparous female Yorkshire pigs (Sus scrofa domesticus), 2-3 months old, weighing 20.2-29.4 kg (average 24.9 ± 2.1 kg), were purchased from Animal Biotech Industries, Danboro, PA. All animals were housed in quarantine before being assigned to the study. The animals were housed under conditions that met or exceeded the requirements listed in the USDA EVA / AWR and the guidelines. Environmental conditions within the animal facility (temperature, relative humidity, and light) were monitored and maintained within acceptable limits. Drinking water was freely available to all animals. Pigs were fed Purina Lab Diet (#5084 Laboratory Porcine Diet Grower) once daily; no other feed was given on experimental days.

[0177] The animals were prepared for surgery using approved standard veterinary care. Tiletamine-zolazepam (4–6 mg / kg, IM) or buprenorphine (0.01 mg / kg, IM) was administered as a preoperative anesthetic. Isoflurane (delivered with 100% oxygen) was administered via mask / nose cone until the animals were anesthetically tuned to facilitate endotracheal intubation. Once sufficiently anesthetized, the animals were intubated, and two IV catheters were connected to peripheral veins to allow for the administration of escaping IV fluids and propofol, and to collect blood samples to monitor blood gases and electrolytes; catheters were inserted into the carotid or femoral artery to directly monitor arterial blood pressure and obtain arterial blood samples. An "induction sheath" was inserted into each container to provide access and advanced. Propofol was administered as a continuous infusion, initially as a bolus of 4–8 mg / kg IV, followed by approximately 0.2–0.4 mg / kg / min IV; the animals were maintained under propofol anesthesia for the remainder of the procedure. An ophthalmic lubricant was applied to the eyes. If necessary, a Foley catheter was inserted into the bladder under cystoscopy guidance to allow for urine collection throughout the experiment. A heated water pad was used to help maintain an appropriate body temperature while under anesthesia; animals with a body temperature below 96°F were provided with a warm blanket and warm liquid. Lactic acidosis (serum lactate >10 mM) was treated with sodium bicarbonate, and hypoglycemia was treated with IV dextrose. Hypotension during the experiment was treated with IV administration of norepinephrine and / or phenylephrine.

[0178] Experimental protocol Prior to the start of the experiment, a series of systemic, cardiovascular, and respiratory tests were performed. These are referred to as baseline measurements. Pigs were infused IV (0 hours) with the following: Group 1, liposomes alone, no LPS (control, n=2); Group 2, LPS + liposomes (ALI untreated, n=6); and Group 3, LPS + PIP-2 in liposomes (ALI treated, n=12). Technicians responsible for animal management were "blinded" to the treatment conditions. LPS was administered as an IV infusion of 50 ug / kg body weight over the first hour of the experiment. Several pigs were IV treated with 100 ug / kg LPS before becoming immobilized on the 50 ug / kg dose of LPS, resulting in severe cardiovascular instability and premature euthanasia. Either liposomes alone (Group 2) or PIP-2 in liposomes (Group 3) were infused as a "sustained-release" bolus at the end of the LPS infusion. Liposomes were administered at 2.22 mg / kg body weight. PIP-2 was administered at 20 mg / kg body weight. Based on a measured encapsulation efficiency of approximately 15–20%, IV-administered PIP-2 amounted to approximately 3–4 mg / kg body weight in liposomes, with the remainder in aqueous solution; presumably, encapsulated PIP-2 was internalized by cells, while unencapsulated PIP-2 remained extracellular.

[0179] Animals were mechanically ventilated until 8am in volume-controlled mode with a tidal volume of 8 mL / kg body weight, an inspiration-to-expiration ratio of 1:1, an inspired oxygen fraction (FiO2) of 1.0, and a positive end-expiratory pressure (PEEP) of 5 cmH2O. After baseline measurements, respiratory, hemodynamic, blood gas, and other blood parameters were assessed at 1-hour intervals. Arterial blood O2 (aPO2) was measured using an O2 electrode. Static lung compliance was measured as the pressure change at the end of a "breath-hold" at a given tidal volume. These hourly measurements were used to assess the animals' condition and provide predefined supportive care as needed. Blood samples were collected from peripheral veins and hematological analysis was performed in-house; serological chemistry was performed at private clinical laboratories (cardiac markers at Antech Diagnostics, Levittown, PA; other assays at Idexx Laboratories, Westbrook, ME).

[0180] Animals were euthanized before the 8-hour study period if they did not respond to basic treatment and met pre-established humane endpoints. These endpoints included: arterial pO2 < 50 mmHg (Horowitz index < 50) during 100% O2 respiration, marked fever (temperature > 105°C) or hypothermia (temperature < 96°C), severe hypotension (blood pressure < 50 mmHg) (despite taking intravenous vasopressor drugs), or severe lactic acidosis (whey lactate persistently > 10 mM). Unless otherwise specified, the physiological parameter values ​​presented in this report are the final measurements taken before slaughter.

[0181] Animals that survived for 8 hours were subjected to bronchoalveolar lavage (BAL) after 8 hours of cardiopulmonary monitoring. FITC-dextran was intravenously administered over 15 minutes, followed by the insertion of a bronchoscope into the airway and guidance into the subsegmental bronchi. One end of a tube was attached to the working channel of the bronchoscope, and the other end was attached to a syringe containing saline via a three-way stopcock. The third port of the three-way stopcock was attached to a trap, and BAL fluid (BALF) was collected. Fluid was collected when the three-way stopcock to the syringe was turned off, causing aspiration through the trap. The porcine lung lavage fluid (BALf) was centrifuged at 1000xg for 10 minutes and used for biochemical assays. Protein content was measured using a Bio-Rad assay kit containing gamma globulin as a standard. Myeloperoxidase (MPO) activity was measured by an ELISA assay using a commercially available kit (Biomatik, Wilmington DE). Cytokines (IL-6, IL-1β, TNF-α) were also measured using ELISA assays with commercially available assay kits (Invitrogen, ThermoFisher Scientific, Federic MD).

[0182] At the end of each experiment, animals were euthanized by overdose of euthanasia solution (IV) in accordance with acceptable AVMA guidelines. For all animals, the lungs, liver, kidneys, and heart were collected post-sacrificially, fixed in 10% formalin, paraffin-treated, and stained with hematoxylin and eosin (H&E) for histopathological evaluation.

[0183] statistical analysis Results are expressed as mean ± standard deviation (SD). Between-group differences were assessed using two-sided t-tests or one-way ANOVA, followed by post-hoc t-tests, and Bonferroni correction as needed. For some studies, results were plotted against the time elapsed since the start of the experiment, and the slope of the line was calculated using the least mean squares method. For statistical analysis, the results of the pig groups administered LPS+ / -PIP-2 were compared with baseline pigs (i.e., pigs not subjected to mechanical ventilation and not administered LPS); control pigs (i.e., those with mechanical ventilation but no LPS) were not included in the statistical evaluation due to insufficient numbers (n=2). Statistical significance was assessed using SigmaStat software (Jandel Scientific, San Jose, CA). Statistical significance was accepted at p<0.05 for all studies.

[0184] result overview Untreated control pigs (no LPS, n=2) showed only slight changes in cardiovascular and pulmonary parameters compared to baseline measurements and were able to tolerate 8 hours of mechanical ventilation under anesthesia well (see data below). Based on the above humanitarian considerations, 50% of the pigs given LPS alone (3 out of 6) were euthanized before the planned 8-hour point for the study, while only 25% of the pigs given LPS and treated with PIP-2 (3 out of 12) were euthanized for similar considerations (Figure 5).

[0185] Lung parameters aPO2 was measured at time intervals and used to calculate arterial blood PO2 divided by the inspiratory O2 fraction (FIO2); this parameter (aPO2 / FIO2) is called the Horowitz index. Since the proportion of O2 used for ventilation in this study was 1.0, the Horowitz index in these studies is the same as aPO2. At both 4 and 8 hours of assisted ventilation, the aPO2 (Horowitz index) of the two control animals did not change from baseline (Table 9). In LPS-treated pigs (Group 2), aPO2 decreased significantly by approximately 65% ​​at 4 hours (n=6) after the start of LPS infusion and remained almost the same at 8 hours (n=3) (Table 9). In contrast, the mean aPO2 decreased by only 50% at 4 hours (n=12) and 37% at 8 hours (n=9) after the start of LPS infusion (Table 9). [Table 9]

[0186] aPO2 / FiO2 (Horowitz index) is the primary parameter used to classify the severity of ALI. When assessed at the end of the 8-hour study period, moderate to severe ALI was observed in 2 out of the remaining 3 untreated LPS pigs (67%), compared to only 2 out of the remaining 9 PIP-2 treated pigs (22%) (Figure 6). Thus, 58% of the PIP-2 treated pigs had either normal lungs or mild ARDS, compared to only 17% of the untreated pigs. As a result, PIP-2 treatment reduced the mortality rate of pigs by 50% due to humane slaughter, and among pigs that survived the entire 8-hour study period, the incidence of moderate to severe ARDS was reduced by 67%. Eight hours after the start of LPS infusion, 50% of the PIP-2 treated pigs (Group 3), while only 17% of the LPS-only treated pigs (Group 2), had aPO2 levels within the normal range (Figure 2).

[0187] Blood assay All pigs in Group 2 (LPS + liposomes) and Group 3 (LPS + PIP-2 in liposomes) developed lactic acidosis within 30–60 minutes of LPS infusion. Lactate levels remained stable but slightly elevated in both groups throughout the study period, with no substantial difference between the two groups. Similarly, there were no significant differences between PIP-2 treated and untreated pigs in serum BUN, creatinine, alkaline phosphatase, and aspartate aminotransferase levels. On the other hand, the mean C-reactive protein, an indicator of systemic inflammation, increased approximately fivefold with LPS administration, and this increase was completely suppressed in PIP-2 treated pigs (p<0.05) (Table 10). Thus, PIP-2 treatment of LPS-induced sepsis resulted in significantly lower serum C-reactive protein levels compared to untreated pigs. [Table 10] N=20 (relative to baseline); N=2 (4-hour and 8-hour control); N=6 and 3 (for 4-hour and 8-hour LPS, respectively); N=12 and 9 (for 4-hour and 8-hour LPS + PIP-2, respectively). Results are given as mean + SD or mean + range for n=2. *P<0.05 compared to baseline; ‡P<0.05 (compared to LPS).

[0188] Pigs administered with LPS showed a significant decrease in white blood cell (WBC) and platelet counts (Table 10), consistent with the response to sepsis. The mean PMN white blood cell count after LPS (group 2) decreased by approximately 80–90% compared to controls at both 4 and 8 hours in animals treated with PIP, but only by approximately 60% and 40% at 4 and 8 hours, respectively (Table 11). However, these positive or negative differences in PIP-2 were not statistically significant. PIP-2 treatment did not affect the decrease in platelets associated with LPS.

[0189] Cardiovascular parameters Animals in Group 2 (LPS) developed elevated plasma levels of cardiac troponin and creatine kinase during the 4-hour and 8-hour observation periods (Table 12). These enzymes are considered indicators of myocardial damage. There was no change from baseline in the levels of these two enzymes in animals in Group 3 (LPS + PIP-2) (Table 12). Therefore, the increase in serum troponin and creatine kinase in pigs in Group 2 was statistically significant compared to pigs treated in Group 3 (PIP-2). [Table 11] N=20 (relative to baseline); N=2 (4-hour and 8-hour control); N=6 and 3 (for 4-hour and 8-hour LPS, respectively); N=12 and 9 (for 4-hour and 8-hour LPS + PIP-2, respectively). Results are given as mean + SD or mean + range for n=2. *P<0.05 compared to baseline.

[0190] Pigs treated with LPS exhibited significant cardiovascular instability, with a significantly increased heart rate and decreased arterial blood pressure (Table 13). The latter required administration of vasopressors (i.e., norepinephrine, epinephrine, and / or phenylephrine) to maintain systemic arterial blood pressure within the normal range. Pigs treated with PIP-2 (Group 3) required considerably less supportive care to maintain acceptable arterial blood pressure levels compared to untreated pigs (Group 2) (80-90%) (Table 13). [Table 12] Total vasopressor is the sum of epinephrine, norepinephrine, and phenylephrine administered to maintain adequate arterial blood pressure during the first 4 hours of the experiment and the following 4-8 hours. N=20 (relative to baseline); N=2 (4-hour and 8-hour control); N=6 and 3 (for LPS at 4-hour and 8-hour, respectively); N=12 and 9 (for LPS + PIP-2 at 4-hour and 8-hour, respectively). Results are given as mean + SD or mean + range for n=2. *P<0.05 relative to baseline.

[0191] Lung parameters - Lung compliance In addition to PaO2 measurements compatible with ALI, lung injury was assessed by measuring lung compliance and analyzing bronchopulmonary lavage fluid. Decreased lung compliance, indicating lung rigidity, is a common feature of ALI. The mean static lung compliance (CL) decreased from 20.3 ml / cmH2O in pigs at baseline to 11.0 ml / cmH2O in pigs in group 2 4 hours after the start of LPS infusion (a 436% reduction), and there was essentially no further change in the mean value at the 8-hour study (Table 14). Treatment of pigs with PIP-2 (group 3) resulted in less reduction in lung compliance compared to untreated pigs (group 2, no PIP-2) (40% at 4 hours, 30% at 8 hours). In both pigs in group 2 and group 3, the significant reduction in lung compliance occurred during the first 4 hours of the experiment. [Table 13] N=20 (relative to baseline); N=2 (4-hour and 8-hour control); N=6 and 3 (for 4-hour and 8-hour LPS, respectively); N=12 and 9 (for 4-hour and 8-hour LPS + PIP-2, respectively). Results are given as mean + SD or mean + range for n=2. *P<0.05 compared to baseline.

[0192] Lung compliance data was further analyzed by plotting values ​​obtained at 1-hour intervals for pigs in groups 2 and 3 (Figure 7). The slopes of the lines under the two conditions indicated that the decrease in lung compliance was statistically significantly smaller in pigs treated with PIP-2 (P<0.001).

[0193] Lung parameters, bronchoalveolar lavage: At the end of the experiment, 20 pigs were subjected to lung lavage; the lavage fluid of 1 pig was lost and not analyzed. The lavage fluids of the 19 pigs analyzed included control (n = 2), LPS (n = 5), and LPS+PIP-2 (n = 12) groups. In the LPS group, 3 of the lavages were performed at the end of 8 hours of the experiment, and 2 were performed on pigs that were euthanized early 5 - 6 hours after the start of LPS injection. In the LPS+PIP-2 group, 3 pigs were euthanized early and then lavaged, but bloody lavage fluids were obtained from all 3, and these samples were discarded without further analysis. Therefore, the analysis of lavage fluids was recorded for the BALF of 16 pigs.

[0194] The mean total protein in BALF significantly increased (about 3.5 - fold) in pigs administered LPS alone (group 2); the increase in protein with the administration of PIP-2 (group 3) was 50% less (Table 15), indicating that the alveolar - capillary barrier was better preserved. Lung inflammation was evaluated by the expression of MPO and cytokines. Despite systemic leukopenia, the myeloperoxidase (MPO) content in the lung lavage fluid of pigs given LPS alone increased nearly 6 - fold (Table 15); MPO is generally accepted as reflecting the presence of PMN. The increase in MPO by LPS was nullified by treatment with PIP-2. Similarly, the lung lavage cytokines (IL - 6, IL - 1β, TNFα) increased 2 - 3 - fold by LPS treatment, and this increase was blocked by treatment with PIP-2 (Table 15). Therefore, treatment with PIP-2 in LPS - exposed animals resulted in a protective effect on the permeability of alveolar capillary proteins in the lung and reduced lung inflammation, as indicated by BALF MPO and cytokine values.

Table 14

[0195] Histology: Control animals showed relatively unremarkable evidence of mechanically ventilator-tolerant lung injury under control conditions for 8 hours. Lungs of LPS-treated animals showed evidence of mild to moderate inflammation, with no significant difference between PIP-2-treated and non-PIP-2-treated pigs. Notably, the lungs were not inflation-fixed, which limited the usefulness of morphological examination. Similarly, examination of the heart, liver, and kidneys showed relatively mild inflammation and hemorrhage, with no significant difference between PIP-2-treated and untreated animals. The relatively minor changes at the time of examination probably reflect the relatively short duration of LPS exposure (less than 8 hours).

[0196] Summary of Results The cardio-pulmonary results shown in Tables 9 - 15 and Figures 4 - 7 are summarized. Treatment with PIP-2 resulted in: 1) a 50% reduction in mortality; 2) a three-fold increase in pigs maintaining normal arterial PO2; 3) prevention of the increase in serum troponin and creatine kinase after LPS, consistent with a decrease in myocardial injury; 4) a marked reduction in the need for vasopressors to maintain arterial blood pressure; 5) a significant reduction in lung stiffness (improvement in lung compliance); 6) reduction of LPS-mediated changes in alveolar-capillary protein permeability; and 7) reduction of lung inflammation, shown by both a reduction in cytokine release and a decrease in MPO content in the lung lavage fluid.

[0197] Discussion The main characteristics of ALI are lung inflammation and the release of potentially damaging substances from inflammatory cells. These damaging agents include reactive oxygen species (ROS), which, when produced in excess, can oxidize tissue macromolecules and lead to widespread cellular destruction. ROS can be produced in the lung through several mechanisms, the primary cause being the enzymatic activity of NADPH oxidase (NOX) enzymes, specifically NOX1 and NOX2. Recent comprehensive reviews have described how oxidative stress plays a crucial role in many acute and chronic disease states (Forman HJ, Zhang H. Targeting oxidative stress in disease: promise and limitations of antioxidant therapy. Nat Rev Drug Discov. 2021 Sep;20(9):689-709. doi:10.1038 / s41573-021-00233-1. Epub 2021 Jun 30. PMID:34194012). The review states that small peptides that inhibit NOX oxidase assembly have therapeutic potential, but notes that none of the proposed inhibitors have progressed to clinical trials. Although ALI / ARDS is not specifically discussed in this review, the clearly excessive ROS production that leads to oxidative stress plays a significant role in lung damage in this syndrome.

[0198] The studies presented herein utilize a small molecule (9 amino acid) peptide that inhibits rac release from the cell membrane, thereby inhibiting oxidase assembly and thus inhibiting the activation of NOX1 and NOX2. Inhibition of rac release results from the inhibition of aiPLA2 activity in Prdx6. This role of aiPLA2 activity in NOX1 and NOX2 activation was demonstrated by using either MJ33 or PIP-2 to inhibit aiPLA2 activity in Prdx6, and by mutations in the Prdx6 molecular site required for aiPLA2 activity expression. Since MJ33 is a lipid molecule that inhibits various PLA2 enzymes, including pancreatic PLA2, and the aiPLA2 activity of Prdx6, it was demonstrated that inhibition of NOX2 activation is associated with the inhibition of aiPLA2 (PLA2 activity of Prdx6). The surfactant-related protein SP-A binds to Prdx6 and inhibits its aiPLA2 activity. Using protein cleavage, the minimum effective 246-amino acid sequence of human SP-A for inhibiting aiPLA2 was determined. A 9-amino acid sequence, called a phospholipase A2 inhibitory peptide (PIP), was determined to have complete inhibitory activity. The human SP-A PIP sequence was named PIP-2.

[0199] Mouse studies using several different ALI models have shown that inhibition of rac1,2 activation by either MJ33 or PIP-2 has a significant protective effect against lung injury. Mouse models of ALI showed protection against lung injury upon treatment with one of the rac inhibitors. In the studies presented herein, inhibition of ROS production by inhibiting NOX2 in LPS-induced sepsis plus ALI has also been shown to be protective against signs of lung and cardiovascular injury in pigs, an animal species with lungs more similar in anatomical and physiological function to human lungs.

[0200] As evidence of protection by PIP-2, mortality (based on humanely slaughtered animals as indicated by IACUC) was reduced by 50% in PIP-2 treated pigs. Secondly, 50% of PIP-2 treated pigs, compared to only 17% of untreated pigs, maintained normal arterial oxygenation throughout the experimental period. Additional observed beneficial effects of PIP-2 treatment included: a significant reduction in increased indicators of myocardial damage, indicated by serum troponin 1 and creatine kinase levels; a significant reduction in vasopressor requirements for arterial blood pressure control; reduced changes in lung gas exchange, indicated by a decrease in abnormal alveolar-to-arterial (Aa)PO2 gradients; protection of the alveolar-capillary barrier of the lung, indicated by a significant reduction in protein leakage into the lung alveolar space; less decrease in serum WBCs, consistent with reduced signs of sepsis; a significant reduction in elevated serum C-reactive protein, also consistent with reduced sepsis; and reduced pneumonia, indicated by a decrease in lung lavage fluid WBCs (estimated by MPO content). Notably, for all parameters studied, treatment with PIP-2 did not affect or had a positive effect on the signs of LPS-induced injury and sepsis; that is, none of the measured parameters were significantly worsened by treatment with PIP-2. Therefore, the use of PIP-2 was prophylactic in this model of ALI, and there was no evidence of adverse side effects.

[0201] The efficacy of PIP-2 as a potential agent for the treatment of clinical ALI and / or sepsis is based on the crucial role of NOX-generating ROS in the pathophysiology of lung injury. Excessive ROS generation is considered paramount in the pathophysiology of acute lung injury, and inhibition of ROS generated by NOX1 / 2 is expected to significantly reduce oxidative stress associated with this syndrome. Therefore, as a non-toxic inhibitor with a relatively good tissue half-life, PIP-2 may be considered a strong candidate to be tested as an adjunctive therapy for the prevention or treatment of sepsis and / or ALI.

[0202] In summary, PIP-2 reduced animal mortality in septic shock by 50%, a result not observed in other studies using this model. Furthermore, PIP-2 biomarkers were nearly normal in treated animals, while placebo animals showed significant damage. While we do not wish to be bound by theory, this result, due to the nature of sepsis and the observed decrease in biomarkers, suggests that there may have been further deaths in the placebo animals. However, some of the mildly and moderately affected animals in the PIP-2 treatment group may have fully recovered, as their biomarkers were nearly normal after PIP-2 treatment.

[0203] Furthermore, this study demonstrated several unexpected benefits of PIP-2 treatment. Firstly, troponin levels in the placebo arm showed the expected rapid increase in observed septic shock injury. However, PIP-2 treated animals showed nearly normal troponin levels. While we do not wish to be bound by theory, PIP-2 treatment resulted in cardioprotection. The ability of PIP-2 to protect cardiac function in these studies was unexpected. No other compound tested to date has shown such a rapid and significant cardioprotective effect. Secondly, the use of vasopressors is the standard treatment for septic shock injury. One of the effects in septic shock is loss of perfusion due to endothelial dysfunction, and the most common treatment is the use of vasopressors. Loss of perfusion is a common cause of death in septic shock injury. In this study, PIP-2 treated animals required one-third (1 / 3) the total mg of vasopressors compared to placebo animals, while achieving higher mean arterial pressure. It was also unexpected that this type of mean arterial pressure was achieved in septic shock injury by a novel mechanism, and since the use of vasopressors has many negative side effects that PIP-2 did not show, it may have beneficial effects in patients. Therefore, the use of PIP-2 and related peptides offers significant advantages over current standard treatment for septic shock.

[0204] This specification contains numerous citations to patents, patent applications, accession numbers, and / or publications, each of which is incorporated herein by reference for any purpose.

Claims

1. A method for treating septic shock in the target population, comprising the following amino acid sequence: X １ X ２ X ３ X ４ X ５ LX ６ X ７ X ８ X ９ HQIL (SEQ ID NO: 1) During the ceremony: X １ However, it is either E or does not exist; X ２ However, it is either L or does not exist; X ３ However, it is either Q or does not exist; X ４ However, it is either A, T, or does not exist; X ５ However, it is either T, E, or does not exist; X ６ However, it is either H or Y; X ７ However, it is either D or E; X ８ However, it is F or I; and X ９ However, an amino acid sequence in which R or K is present; or Amino acid sequences selected from the group consisting of sequence numbers 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, or 30; The method comprising administering to a subject an effective amount of a pharmaceutical composition comprising a polypeptide containing and a pharmaceutically acceptable carrier.

2. The method according to claim 1, wherein the polypeptide includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

3. The method according to claim 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

4. The method according to claim 1, wherein the polypeptide comprises SEQ ID NO:

2.

5. The method according to claim 1, wherein the polypeptide comprises SEQ ID NO:

2.

6. The method according to claim 1, wherein the polypeptide comprises SEQ ID NO:

3.

7. The method according to claim 1, wherein the polypeptide comprises sequence number 3.

8. The method according to claim 1, wherein the polypeptide comprises SEQ ID NO:

4.

9. The method according to claim 1, wherein the polypeptide comprises sequence number 4.

10. The method according to claim 1, wherein the pharmaceutical composition comprises a liposome-encapsulated polypeptide.

11. The method according to claim 1, wherein the pharmaceutically acceptable carrier is any pharmaceutically acceptable carrier disclosed herein.

12. The method according to claim 1, wherein the pharmaceutical composition is administered to the subject by aerosol inhalation, intratracheal injection, or intravenous injection.

13. The method according to claim 1, wherein the subject requiring the same is diagnosed with sepsis and has hypotension that is not relieved by intravenous fluid administration alone.

14. The method according to claim 13, wherein the subject requiring the same has a mean arterial pressure (MAP) of less than approximately 65 mmHg.

15. The method according to claim 1, wherein the subject requiring the same has been diagnosed with sepsis and has a mean arterial pressure or systolic blood pressure that has decreased by approximately 44 mmHg or more.

16. The method according to claim 1, wherein the subject requiring the same has a blood lactate level greater than approximately 4 mmol.

17. The method according to claim 1, wherein the subject requiring the same has persistent signs of multiple organ damage or failure.

18. The method according to claim 1, wherein the blood oxygen level of the subject does not decrease after administration of the pharmaceutical composition.

19. The method according to claim 18, wherein the blood oxygen level of the subject does not decrease by more than 50% or 65% after administration of the pharmaceutical composition.

20. The method according to claim 1, wherein the blood oxygen level of the subject increases after administration of the pharmaceutical composition.

21. The method according to claim 1, wherein the subject maintains a normal blood oxygen level after administration of the pharmaceutical composition.

22. The method according to claim 1, wherein the subject requiring it has not been administered a vasopressor, or has been previously administered a smaller amount of a vasopressor, is administered a smaller amount of a vasopressor simultaneously, or is subsequently administered a smaller amount of a vasopressor than the amount administered in the absence of the pharmaceutical composition.

23. A method for treating subjects with a significant decrease in mean arterial pressure, the following amino acid sequence: X １ X ２ X ３ X ４ X ５ LX ６ X ７ X ８ X ９ HQIL (Sequence ID 1) During the ceremony: X １ However, it is either E or does not exist; X ２ However, it is either L or does not exist; X ３ However, it is either Q or does not exist; X ４ However, it is either A, T, or does not exist; X ５ However, it is either T, E, or does not exist; X ６ However, it is either H or Y; X ７ However, it is either D or E; X ８ However, it is F or I; and X ９ However, an amino acid sequence in which R or K is present; or Amino acid sequences selected from the group consisting of sequence numbers 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, or 30; The method comprising administering to a subject an effective amount of a pharmaceutical composition comprising a polypeptide containing and a pharmaceutically acceptable carrier.

24. The method according to claim 23, wherein the polypeptide includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

25. The method according to claim 23, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

26. The method according to claim 23, wherein the polypeptide includes SEQ ID NO:

2.

27. The method according to claim 23, wherein the polypeptide is sequence number 2.

28. The method according to claim 23, wherein the polypeptide includes SEQ ID NO:

3.

29. The method according to claim 23, wherein the polypeptide comprises sequence number 3.

30. The method according to claim 23, wherein the polypeptide includes sequence number 4.

31. The method according to claim 23, wherein the polypeptide comprises sequence number 4.

32. The method according to claim 23, wherein the pharmaceutical composition comprises a liposome-encapsulated polypeptide.

33. The method of claim 23, wherein the pharmaceutically acceptable carrier is any pharmaceutically acceptable carrier disclosed herein.

34. The method according to claim 23, wherein the pharmaceutical composition is administered to the subject by aerosol inhalation, intratracheal injection, or intravenous injection.

35. The method according to claim 23, wherein the subject has a mean arterial pressure or systolic blood pressure that has decreased by approximately 44 mmHg or more, and has been diagnosed with sepsis.

36. The method according to claim 23, wherein the subject has been diagnosed with sepsis and has hypotension that is not relieved by intravenous fluid administration alone.

37. The method according to claim 36, wherein the subject has a mean arterial pressure (MAP) of less than approximately 65 mmHg.

38. The method according to claim 23, wherein the subject has a blood lactate level of more than approximately 4 mmol.

39. The method according to claim 23, wherein the subject requiring the same has persistent signs of multi-organ damage or failure.

40. The method according to claim 23, wherein the blood oxygen level of the subject does not decrease after administration of the pharmaceutical composition.

41. The method according to claim 40, wherein the blood oxygen level of the subject does not decrease by more than 50% or 65% after administration of the pharmaceutical composition.

42. The method according to claim 23, wherein the blood oxygen level of the subject increases after administration of the pharmaceutical composition.

43. The method according to claim 23, wherein the subject maintains a normal blood oxygen level after administration of the pharmaceutical composition.

44. The method according to claim 23, wherein the subject requiring the same has not been administered a vasopressor, or has been previously administered a vasopressor, is administered concurrently with it, or is subsequently administered a smaller amount of a vasopressor than the amount administered in the absence of the pharmaceutical composition.

45. The method according to claim 23, wherein the subject requiring the same has been diagnosed with septic shock.

46. A method for preventing cardiac injury in a patient diagnosed with septic shock, comprising the following amino acid sequence: X １ X ２ X ３ X ４ X ５ LX ６ X ７ X ８ X ９ HQIL (Sequence ID 1) During the ceremony: X １ However, it is either E or does not exist; X ２ However, it is either L or does not exist; X ３ However, it is either Q or does not exist; X ４ However, it is either A, T, or does not exist; X ５ However, it is either T, E, or does not exist; X ６ However, it is either H or Y; X ７ However, it is either D or E; X ８ However, it is F or I; and X ９ However, an amino acid sequence in which R or K is present; or Amino acid sequences selected from the group consisting of sequence numbers 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, or 30; The method comprising administering to a subject (compared to a subject not treated with the pharmaceutical composition) an effective amount of a pharmaceutical composition comprising a polypeptide containing and a pharmaceutically acceptable carrier.

47. The method according to claim 46, wherein the polypeptide includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

48. A method for increasing the survival of a patient diagnosed with septic shock, comprising the following amino acid sequence: X １ X ２ X ３ X ４ X ５ LX ６ X ７ X ８ X ９ HQIL (Sequence ID 1) During the ceremony: X １ However, it is either E or does not exist; X ２ However, it is either L or does not exist; X ３ However, it is either Q or does not exist; X ４ However, it is either A, T, or does not exist; X ５ However, it is either T, E, or does not exist; X ６ However, it is either H or Y; X ７ However, it is either D or E; X ８ However, it is F or I; and X ９ However, an amino acid sequence in which R or K is present; or Amino acid sequences selected from the group consisting of sequence numbers 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, or 30; The method comprising administering to a subject (compared to a subject not treated with the pharmaceutical composition) an effective amount of a pharmaceutical composition comprising a polypeptide containing and a pharmaceutically acceptable carrier.

49. The method according to claim 48, wherein the polypeptide includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

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