Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/08—Peptides having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/007—Pulmonary tract; Aromatherapy
  • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant

Definitions

• Septic shock is the last and most severe stage of sepsis, where dangerous low and severe drops in blood pressure and/or increased lactate levels and cannot be adequately treated by the administration of fluids alone. Even with the treatment of vasopressors to maintain blood pressure, the mortality rate for septic shock is high, ranging from 25-50% (see, e.g., Kumar et al, eds., Robbins Basic Pathology (8 th ed.), Saunders, Elsevier pp.
• Methods of treating septic shock in a subject in need thereof comprising administering to the subject an effective amount of a pharmaceutical composition comprising a polypeptide comprising an amino acid sequence of:
• X 1 X 2 X 3 X 4 X 5 LX 6 X 7 X 8 X 9 HQIL (SEQ ID NO: 1) wherein: X 1 is E or is absent; X 2 is L or is absent; X 3 is Q or is absent; X 4 is A, T, or is absent; X 5 is T, E, or is absent; X 6 is H or Y; X 7 is D or E; X 8 is F or I; and X 9 is R or K; 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.
• the polypeptide comprises an amino acid sequence that is at least 85% identical to 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 an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 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.
• 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.
• the polypeptide comprises SEQ ID NO: 2.
• the polypeptide consists of SEQ ID NO: 2.
• the polypeptide comprises SEQ ID NO: 3.
• the polypeptide consists of SEQ ID NO: 3.
• the polypeptide comprises SEQ ID NO: 4.
• the polypeptide consists of SEQ ID NO: 4.
• the pharmaceutical composition comprises a liposome encapsulated polypeptide.
• the pharmaceutically acceptable carrier is any pharmaceutically acceptable carrier disclosed herein.
• the pharmaceutical composition is administered to the subject by any administration method disclosed herein.
• the pharmaceutical composition is administered to the subject by aerosol inhalation, intratracheal injection, or intravenous injection.
• the subject in need thereof has been diagnosed with sepsis and has low blood pressure that is not alleviated by the administration of intravenous fluids alone.
• the subject in need thereof has a mean arterial pressure (MAP) less than about 65 mm Hg.
• MAP mean arterial pressure
• the subject in need thereof has been diagnosed with sepsis and has a decrease in mean arterial pressure or systolic blood pressure of about 44 mm Hg or more.
• the subject in need thereof has blood lactate levels that are greater than about 4 mmol. In some embodiments, subject in need thereof has persistent signs of multiple 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 more than 50% after administration of the pharmaceutical composition. In some embodiments, the subject’s blood oxygen level does not decrease 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.
• the subject in need thereof is not administered a vasopressor or the subject has been previously administered a lower amount of a vasopressor, is concurrently administered a lower amount of a vasopressor, or will subsequently be administered a lower amount of a vasopressor than the amount of a vasopressor administered in the absence of the pharmaceutical composition.
• the subject in need thereof has been diagnosed with septic shock.
• Methods of treating subject with a significant drop in mean arterial pressure comprising administering to the subject an effective amount of a pharmaceutical composition comprising a polypeptide comprising an amino acid sequence of:
• X 1 X 2 X 3 X 4 X LX 6 X 7 X 8 X 9 HQIL (SEQ ID NO: 1) wherein: X 1 is E or is absent; X 2 is L or is absent; X 3 is Q or is absent; X 4 is A, T, or is absent; X 5 is T, E, or is absent; X 6 is H or Y;X 7 is D or E; X 8 is F or I; and X 9 is R or K; 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.
• the polypeptide comprises an amino acid sequence that is at least 85% identical to 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 an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 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.
• 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.
• the polypeptide comprises SEQ ID NO: 2.
• the polypeptide consists of SEQ ID NO: 2.
• the polypeptide comprises SEQ ID NO: 3.
• the polypeptide consists of SEQ ID NO: 3.
• the polypeptide comprises SEQ ID NO: 4.
• the polypeptide consists of SEQ ID NO: 4.
• the pharmaceutical composition comprises a liposome encapsulated polypeptide.
• the pharmaceutically acceptable carrier is any pharmaceutically acceptable carrier disclosed herein.
• the pharmaceutical composition is administered to the subject by any administration method disclosed herein.
• the pharmaceutical composition is administered to the subject by aerosol inhalation, intratracheal injection, or intravenous injection.
• the subject has a decrease in mean arterial pressure or systolic blood pressure of about 44 mm Hg or more and has been diagnosed with sepsis.
• the subject has been diagnosed with sepsis and has low blood pressure that is not alleviated by the administration of intravenous fluids alone.
• the subject has a mean arterial pressure (MAP) less than about 65 mm Hg.
• the subject in need thereof has blood lactate levels that are greater than about 4 mmol. In some embodiments, the subject in need thereof has persistent signs of multiple 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 more than 50% after administration of the pharmaceutical composition. In some embodiments, the subject’s blood oxygen level does not decrease 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.
• the subject in need thereof is not administered a vasopressor or the subject has been previously administered, is concurrently administered, or will subsequently be administered a lower amount of a vasopressor than the amount of a vasopressor administered in the absence of the pharmaceutical composition.
• the subject in need thereof has been diagnosed with septic shock.
• Methods of preventing heart damage in a subject with diagnosed with septic shock are also provided for herein, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising a polypeptide comprising an amino acid sequence of:
• X 1 X 2 X 3 X 4 X 5 LX 6 X 7 X 8 X 9 HQIL (SEQ ID NO: 1) wherein: X 1 is E or is absent; X 2 is L or is absent; X 3 is Q or is absent; X 4 is A, T, or is absent; X 5 is T, E, or is absent; X 6 is H or Y; X 7 is D or E; X 8 is F or I; and X 9 is R or K; 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, as compared to a subject not treated with the pharmaceutical composition.
• the polypeptide comprises an amino acid sequence that is at least 85% identical to 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 an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 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.
• 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.
• the polypeptide comprises SEQ ID NO: 2.
• the polypeptide consists of SEQ ID NO: 2.
• the polypeptide comprises SEQ ID NO: 3.
• the polypeptide consists of SEQ ID NO: 3.
• the polypeptide comprises SEQ ID NO: 4.
• the polypeptide consists of SEQ ID NO: 4.
• the pharmaceutical composition comprises a liposome encapsulated polypeptide.
• the pharmaceutically acceptable carrier is any pharmaceutically acceptable carrier disclosed herein.
• the pharmaceutical composition is administered to the subject by any administration method disclosed herein.
• the pharmaceutical composition is administered to the subject by aerosol inhalation, intratracheal injection, or intravenous injection.
• the subject in need thereof has low blood pressure that is not alleviated by the administration of intravenous fluids alone.
• the subject in need thereof has a mean arterial pressure (MAP) less than about 65 mm Hg.
• MAP mean arterial pressure
• the subject in need thereof has a decrease in mean arterial pressure or systolic blood pressure of about 44 mm Hg or more.
• the subject in need thereof has blood lactate levels that are greater than about 4 mmol. In some embodiments, subject in need thereof has persistent signs of multiple organ damage or failure. In some embodiments, 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 more than 50% after administration of the pharmaceutical composition. In some embodiments, the subject’s blood oxygen level does not decrease 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.
• the subject in need thereof has a troponin level that that does not increase after administration of the pharmaceutical composition In some embodiments, the subject in need thereof maintains normal troponin levels after administration of the pharmaceutical composition. In some embodiments, the subject in need thereof is not administered a vasopressor or the subject has been previously administered, is concurrently administered, or will subsequently be administered a lower amount of a vasopressor than the amount of a vasopressor administered in the absence of the pharmaceutical composition.
• Methods of increasing survival of a subject with diagnosed with septic shock comprising administering to the subject an effective amount of a pharmaceutical composition comprising a polypeptide comprising an amino acid sequence of:
• X 1 X 2 X 3 X 4 X LX 6 X 7 X 8 X 9 HQIL (SEQ ID NO: 1) wherein: X 1 is E or is absent; X 2 is L or is absent; X 3 is Q or is absent; X 4 is A, T, or is absent; X 5 is T, E, or is absent; X 6 is H or Y; X 7 is D or E; X 8 is F or I; and X 9 is R or K; or an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 13,
• the polypeptide comprises an amino acid sequence that is at least 85% identical to 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.
• polypeptide comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 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,
• 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.
• the polypeptide comprises SEQ ID NO: 2.
• polypeptide consists of SEQ ID NO: 2.
• polypeptide comprises SEQ ID NO: 3.
• the polypeptide consists of SEQ ID NO: 3.
• the polypeptide comprises SEQ ID NO: 4.
• the polypeptide consists of SEQ ID NO: 4.
• the pharmaceutical composition comprises a liposome encapsulated polypeptide.
• the pharmaceutically acceptable carrier is any pharmaceutically acceptable carrier disclosed herein.
• the pharmaceutical composition is administered to the subject by any administration method disclosed herein.
• the pharmaceutical composition is administered to the subject by aerosol inhalation, intratracheal injection, or intravenous injection.
• the subject in need thereof has been diagnosed with sepsis and has low blood pressure that is not alleviated by the administration of intravenous fluids alone.
• the subject in need thereof has a mean arterial pressure (MAP) less than about 65 mm Hg.
• the subject in need thereof has a decrease in mean arterial pressure or systolic blood pressure of about 44 mm Hg or more. In some embodiments, the subject in need thereof has blood lactate levels that are greater than about 4 mmol. In some embodiments, the subject in need thereof has persistent signs of multiple 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 more than 50% after administration of the pharmaceutical composition. In some embodiments, the subject’s blood oxygen level does not decrease 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.
• the subject maintains a normal blood oxygen level after administration of the pharmaceutical composition.
• the subject in need thereof is not administered a vasopressor or the subject has been previously administered, is concurrently administered, or will subsequently be administered a lower amount of a vasopressor than the amount of a vasopressor administered in the absence of the pharmaceutical composition
• FIG. 1 illustrates a measurement of troponin I levels in subjects over time.
• the negative control and subjects treated with PIP-2 treated showed no increase in troponin protein levels in the blood, while the subjects were not treated with PIP-2 showed an increase in troponin protein levels starting between 1 and 4 hours.
• FIG. 2 illustrates a measurement of c-reactive protein (CRP) levels in subjects over time.
• the negative control and subjects treated with PIP-2 treated showed no increase in CRP protein levels in the blood, while the subjects not treated with PIP-2 showed an increase in CRP protein levels starting between 1 and 4 hours.
• FIG. 3 shows the mean use of vasopressors per animal for negative control, PIP-2 treated, and subjects not treated with PIP-2.
• the PIP-2 treated subjects required about 66% less vasopressor treatment to maintain appropriate MAP levels.
• FIG. 4A shows a bar graph of the percent change in mean arterial pressure (MAP) vs. baseline for pigs treated with LPS and LPS + PIP-2.
• FIG. 4B shows a graph of the MAP values for control-, LPS-, and LPS + PIP-2-treated pigs over 8 hours.
• FIG. 5 shows a survival curve for pigs treated with LPS and LPS + PIP-2.
• Pigs were treated with LPS delivered by IV infusion over a 1 hour period.
• Death of pigs due to sacrifice for humane considerations was recorded at hourly intervals. All surviving pigs were sacrificed at 8 hours after the start of LPS infusion. The addition of PIP-2 led to an increase in survival as compared to the control.
• FIG. 6 illustrates that PIP-2 treatment resulted in a 50% decrease in pig mortality by humane sacrifice and a 67% decrease in the incidence of moderate to severe ARDS.
• FIG. 6 illustrates the severity of acute lung injury as indicated by arterial blood PO2 (aPCh) values during ventilation with 100% O2 (FIO2) for 3 groups of pigs (Group 1 : liposomes alone, no LPS; Group 2: LPS + liposomes; and Group 3: LPS + PIP-2 in liposomes) measured at 8 hours after the start of LPS infusion.
• aPCh arterial blood PO2
• FIO2 O2
• FIG. 7 illustrates that PIP-2 led to a statistically significant lesser decrease in lung compliance in subject treated with LPS to induce septic shock.
• FIG. 7 illustrates static lung compliance for pigs in group 2 (LPS treated) and group 3 (LPS treated + PIP-2 treated).
• Static lung compliance in mechanically ventilated pigs was measured at hourly intervals between zero time and 8 hours after the start of LPS infusion. The points were connected by a continuous line. Each line represents one pig. Early termination of a line indicates euthanasia of the pig for humane considerations.
• the heavy-weighted line in each graph represents the mean slope for each set of lines.
• the term “individual” or “subject,” or “patient” used interchangeably, means any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, such as humans.
• the terms “comprising” (and any form of comprising, such as “comprise”, “comprises”, and “comprised”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”), are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Any step or composition that uses the transitional phrase of “comprise” or “comprising” can also be said to describe the same with the transitional phase of “consisting of” or “consists.”
• polynucleotide or “nucleic acid molecule” means a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. Double and single- stranded DNAs and RNAs are typical example of polynucleotides.
• polypeptide or “protein” means a molecule that comprises at least two amino acid resides linked by a peptide bond to form a polypeptide. In some embodiments, the term “peptide” can also be used.
• sepsis as used herein means a potentially life-threatening condition caused by the body’s response to an infection. In some embodiments, sepsis can lead to the failure of one or more organs. Without wishing to be bound by theory, sepsis is categorized into multiple stages, which increased in severity.
• septic shock means the last and most severe stage of sepsis, usually defined by sepsis with persistent signs of organ damage, low blood pressure and, in some instance, lactate levels that measure greater than 2 mmol or 4 mmol that is not alleviated by the administration of intravenous fluids and/or where vasopressors are required to maintain mean arterial pressure (MAP) of greater than or equal to 65 mm Hg.
• MAP mean arterial pressure
• composition comprising a polypeptide
• the polypeptide having the formula of:
• X 1 is E or is absent
• X 2 is L or is absent
• X 3 is Q or is absent
• X 4 is A, T, or is absent
• X 5 is T, E, or is absent
• X 6 is H or Y
• X 7 is D or E
• X 8 is F or I
• X 9 is R or K; or a polypeptide comprising 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.
• the polypeptide is a polypeptide listed in Table 1.
• the polypeptide is at least 85%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 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.
• the term “PIP-2” is the polypeptide having the sequence of SEQ ID NO: 2.
• the term “PIP-4” is the polypeptide having the sequence of SEQ ID NO: 3
• the term “PIP-5” is the polypeptide having the sequence of SEQ ID NO: 4.
• the polypeptide comprises SEQ ID NOs: 2, 3, or 4.
• the polypeptide consists of SEQ ID NOs: 2, 3, or 4.
• the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
• suitable pharmaceutically acceptable carries include, but are not limited to, water, silicone, waxes, petroleum jelly, polyethylene glycol, propylene glycol, liposomes, a lipid such as cholesterol, cationic lipids such as 1, 2, -dioleoyl-3-trimethylammonium propane (DOTAP), l,2,-dioleoyl-sn-glycero-3- phosphochiline (DOPC), and l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sugars such as mannitol and lactose, and other materials depending on the specific type of formulation used.
• suitable pharmaceutically acceptable carries include, but are not limited to, nanoparticles such as gold or metallic nanoparticles.
• the polypeptide is encapsulated in or formulated with one or more lipids and liposomes.
• the lipids and liposomes comprises a cationic lipid, such as, but not limited to, l,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP), l,2,-dioleoyl-sn-glycero-3-phosphochiline (DOPC), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 5-carboxyspermylglycinedioctadecylamide (DOGS), 2,3-dioleyloxy-N- [2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-propan
• DOTAP l,2-Di
• the lipids and liposomes comprise, but are not limited to, dipalmitoyl phosphatidylcholine (DPPC), egg PC, phosphatidylglycerol (PG), egg PC, or cholesterol.
• DPPC dipalmitoyl phosphatidylcholine
• PG phosphatidylglycerol
• the lipids and liposomes comprise dipalmitoyl phosphatidylcholine (DPPC), egg PC, phosphatidylglycerol (PG), and cholesterol in the molar ratio 0.5, 0.25, 0.10, 0.15.
• the pharmaceutical composition comprises an encapsulated polypeptide, such as the polypeptides as provided for herein.
• the pharmaceutically acceptable carrier can be suitable for intravenous, intramuscular, intratracheal subcutaneous, parenteral, rectal, local, topical, spinal or epidermal administration (e.g. by injection or infusion). In some embodiments, the pharmaceutically acceptable carrier can be suitable for aerosol inhalation.
• compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories.
• liquid solutions e.g., injectable and infusible solutions
• dispersions or suspensions e.g., dispersions or suspensions, liposomes and suppositories.
• Typical compositions are in the form of injectable or infusible solutions.
• the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intradermal, intramuscular, intra vesicular).
• the composition is administered by intravenous infusion or injection.
• the composition is administered by intramuscular or subcutaneous injection.
• the composition is administered by enteral, sublingual, inhalation, or intranasal. In some embodiments, the composition is administered locally, e.g., by injection, or topical application, to a target site.
• the pharmaceutical compositions can be lyophilized and reconstituted for use prior to administration to the patient.
• parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion.
• compositions such as pharmaceutical compositions, typically are sterile and stable under the conditions of manufacture and storage.
• the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for a high concentration of the active ingredient.
• Sterile injectable solutions can be prepared by incorporating the active compound (i.e., therapeutic molecule, nucleic acid molecule, cell, polypeptide, vector, etc.) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
• the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
• a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
• the pharmaceutical composition can be orally administered, for example, with an inert diluent or an assimilable edible carrier.
• the compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet.
• the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
• To administer a compositions as provided for herein by other than parenteral administration it may be necessary to coat the compositions with, or co-administer the compositions with, a material to prevent its inactivation.
• the compositions can also be administered with medical devices known in the art.
• Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
• Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• An exemplary, non- limiting range for a therapeutically or prophylactically effective amount of a therapeutic compound is 0.1-30 mg/kg, more preferably 1-25 mg/kg. Dosages and therapeutic regimens of the therapeutic compound can be determined by a skilled artisan.
• the therapeutic compound is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 1 to 40 mg/kg, e.g., 1 to 30 mg/kg, e.g., 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 can vary from e.g., once a week to once every 2, 3, or 4 weeks, or, in some embodiments, the dosing schedule can be, once every month, every 2 months, every 3 months, or every 6 months.
• the therapeutic compound is administered at a dose from about 10 to 20 mg/kg every other week.
• the therapeutic compound can be administered by intravenous infusion at a rate of more than 20 mg/min, e.g., 20-40 mg/min, and typically greater than or equal to 40 mg/min to reach a dose of about 35 to 440 mg/m2, typically about 70 to 310 mg/m2, and more typically, about 110 to 130 mg/m2.
• the infusion rate of about 110 to 130 mg/m2 achieves a level of about 3 mg/kg.
• the therapeutic compound can be administered by intravenous infusion at a rate of less than 10 mg/min, e.g., less than or equal to 5 mg/min to reach a dose of about 1 to 100 mg/m2, e.g., about 5 to 50 mg/m2, about 7 to 25 mg/m2, or, about 10 mg/m2.
• the therapeutic compound is infused over a period of about 30 min. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated.
• the pharmaceutical compositions may include a “therapeutically effective amount” or a “prophylactically effective amount” of the compositions, vectors, cells, polypeptides, or nucleic acid molecules encoding the same.
• a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
• a therapeutically effective amount of an active ingredient or molecule may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic compound to elicit a desired response in the individual.
• a “therapeutically effective dosage” can, for example, inhibit a measurable parameter, e.g., tumor growth, by at least about 20%, by at least about 40%, by at least about 60%, and by at least about 80% relative to untreated subjects.
• a measurable parameter e.g., tumor growth
• the ability of a compound to inhibit a measurable parameter, e.g., tumor growth can be evaluated in an animal model system predictive of efficacy in tumor growth.
• this property of a composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner.
• a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount can be, but is not necessarily, less than the therapeutically effective amount.
• kits comprising compositions, cells, vectors, nucleic acid molecules, or polypeptides as described herein.
• the kit can include one or more other elements including: instructions for use; other reagents, e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, a molecule to a label or other therapeutic agent, or a radioprotective composition; devices or other materials for preparing the molecule for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.
• other reagents e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, a molecule to a label or other therapeutic agent, or a radioprotective composition
• devices or other materials for preparing the molecule for administration e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, a molecule to a label or other therapeutic agent, or a radioprotective composition
• devices or other materials for preparing the molecule for administration e.g., a label, a therapeutic agent, or an agent useful for
• the pharmaceutical composition comprising the polypeptide is administered subsequent to, prior to, or in combination with a vasopressor.
• the subject in need thereof is not administered a vasopressor or the subject has been previously administered a lower amount of a vasopressor, is concurrently administered a lower amount of a vasopressor, or will subsequently be administered a lower amount of a vasopressor than the amount of a vasopressor administered in the absence of the pharmaceutical composition.
• the subject in need is administered about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or between about 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50- 60%, 60-70%, 70-80%, 80-90%, or 90-100% less vasopressor than a subject not administered the pharmaceutical composition comprising the polypeptide.
• the subject in need is administered 1-fold less, 2-fold less, 3-fold less , 4-fold less, 5-fold less, 6- fold less, 7-fold less, 8-fold less, 9-fold less, or 10-fold less vasopressor than a subject not administered the pharmaceutical composition comprising the polypeptide.
• vasopressors include, but are not limited to, Vasopressin (Pi tressin® or Vasostrict®), Phenylephrine (Biorphen® or Vazculep®), epinephrine, norepinephrine, droxidopa, phenylephrine, ephedrine, dobutamine, Dopamine, Angiotensin-II, and Terlipressin.
• Treatment of any disease mentioned herein encompasses an alleviation of at least one symptom of the disease, a reduction in the severity of the disease, or the delay or prevention of disease progression to more serious symptoms that may, in some cases, accompany the disease or to at least one other disease. Treatment need not mean that the disease is totally cured.
• a useful therapeutic agent needs only to reduce the severity of a disease, reduce the severity of symptom(s) associated with the disease or its treatment, or delay the onset of more serious symptoms or a more serious disease that can occur with some frequency following the treated condition.
• the composition may reduce the growth or spread of the tumor, or the tumors effect on the tissue in which it is present.
• a patient's condition can be assessed by standard techniques. Suitable procedures vary according to the patient's condition and symptoms.
• compositions provided for herein can be used to treat septic shock.
• the methods comprise administering to the patient a polypeptide as provided for herein.
• the methods comprise administering to the patient an effective amount of a pharmaceutical composition comprising a polypeptide as provided for herein.
• a method of treating septic shock in a subject in need thereof comprising administering to the subject an effective amount of a pharmaceutical composition comprising a polypeptide comprising an amino acid sequence of: X 1 X 2 X 3 X 4 X 5 LX 6 X 7 X 8 X 9 HQIL (SEQ ID NO: 1) wherein:
• X 1 is E or is absent
• X 2 is L or is absent
• X 3 is Q or is absent
• X 4 is A, T, or is absent
• X 5 is T, E, or is absent
• X 6 is H or Y
• X 7 is D or E
• X 8 is F or I
• X 9 is R or K; or a polypeptide comprising 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.
• the subject in need thereof has been diagnosed with sepsis and has a low blood pressure that is not alleviated by the administration of intravenous fluids alone.
• the subject has a mean arterial pressure (MAP) of less than 65 mm Hg.
• the subject has a mean arterial pressure (MAP) of less than 60 mm Hg.
• the subject has a mean arterial pressure (MAP) of less than 55 mm Hg.
• the subject has a decrease in mean arterial pressure or systolic blood pressure of about 44 mm Hg or more.
• the subject has blood lactate levels that are greater than about 2 mmol.
• MAP mean arterial pressure
• a method of preventing heart damage in a subject with diagnosed with septic shock comprising administering to the subject an effective amount of a pharmaceutical composition comprising a polypeptide comprising an amino acid sequence of: X1X2X3X4X5LX6X7X8X9HQIL (SEQ ID NO: 1) wherein:
• XI is E or is absent
• X2 is L or is absent
• X3 is Q or is absent
• X4 is A, T, or is absent
• X5 is T, E, or is absent
• X6 is H or Y
• X7 is D or E
• X8 is F or I
• X9 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, as compared to a subject not treated with the pharmaceutical composition.
• the polypeptide comprises an amino acid sequence that is at least 85% identical to 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.
• polypeptide comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to 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.
• 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.
• polypeptide comprises SEQ ID NO: 2.
• MAP mean arterial pressure
• 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.
• Results showed that treatment with PIP-2 lowered the protein located in the lung lavage to that of the negative control group, as shown in Table 5.
• Results also showed that treatment with PIP-2 protects from inflammation and white cell activation by lowering MPO levels, which is an indicator of lung inflammation and immune response to injury, as shown in Table 6.
• troponin in the blood is an indication of heart damage, because the protein is normally not present in the blood but leaks into the bloodstream when heart muscles are damaged, which can occur in septic shock. As shown in FIG. 1 , troponin levels in the placebo LPS control group began to sharply rise from
• treatment with PIP-2 also provided protection against creatine kinase (CK) protein levels in the blood.
• CK protein leaks in to the bloodstream when heart muscles and other skeletal muscles are damaged.
• control subjects had a mean CK level of 19% (U/mL), while placebo treated LPS controls had a much higher 89% CK level.
• Subjects treated with PIP-2 however had the CK level drop to about 15%. Therefore, PIP-2 protected the animals from the LPS induced septic shock.
• treatment with PIP-2 also prevented the increase of c-reactive protein (CRP) an indicator of inflammation. As shown in FIG.
• CRP c-reactive protein
• MAP mean arterial pressure
• vasopressor treatment is a primary tool to increase MAP and stabilize blood flow.
• placebo LPS control group subjects needed treatment with rescue vasopressors as well as continuous infusion in an attempt to stabilize MAP.
• PIP-2 treated subjects required about 66% less vasopressor treatment and little to no rescue treatment. Additionally, PIP-2 treated subjects had better overall MAP even without the use of vasopressors.
• PIP-2 liposome-encapsulated 9 amino acid peptide
• LPS lipopolysaccharide
• ALI acute lung injury
• Broncho-alveolar lavage fluid showed markedly lower total protein, cytokines (TNF-a, IL-6, and IL- 1 [ ), and myeloperoxidase levels in PIP-2- treated as compared to untreated pigs.
• cytokines TNF-a, IL-6, and IL- 1 [
• myeloperoxidase levels in PIP-2- treated as compared to untreated pigs.
• the porcine LPS-induced sepsis model was associated with moderate to severe lung pathophysiology compatible with ALL Treatment with PIP-2 markedly decreased lung injury, cardiovascular instability, and early sacrifice of pigs.
• Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) can be associated with a variety of pulmonary (e.g., aspiration, pneumonia) or non-pulmonary (e.g., sepsis, trauma) etiologies.
• pulmonary e.g., aspiration, pneumonia
• non-pulmonary e.g., sepsis, trauma
• the physiological effects associated with ARDS include stiff lungs (decreased lung compliance), lung edema, and progressive hypoxemia.
• the current standard of care is based on treatment of the underlying pathology and respiratory support using controlled ventilation that avoids mechanical injury to lung tissue. But, despite optimal respiratory support, ARDS currently has a mortality rate of -40%.
• ROS reactive oxygen species
• NOX NADPH oxidases
• ROS generated by NOX enzymes are crucial for the regulation of important cellular functions such as host defense, cellular signaling, cell migration, cell differentiation, and post-translational protein processing.
• excessive production of ROS can lead to oxidation of tissue macromolecules (lipids, proteins, DNA) resulting in widespread cellular injury.
• ROS can be produced in the lung by inflammatory cells including polymorphonuclear leukocytes (PMN) and alveolar macrophages (AM) as well as by parenchymal lung cells including both epithelial and endothelial cells.
• PMN polymorphonuclear leukocytes
• AM alveolar macrophages
• parenchymal lung cells including both epithelial and endothelial cells.
• N0X2 is the major NOX enzyme in phagocytic cells and in the lung while NOXes 1 and 2 are predominant in the cardiovascular system.
• N0X2 is a complex of 2 cell membrane-associated protein components, gp91 phox and p22 phox , that is inactive in the resting state. Activation of the enzyme in cells involves phosphorylation of gp91 phox , the translocation to the membrane of 3 additional cytosolic proteins (p67 pllox , p47 ptlox , p40 ptlox ), and the activation of cytosolic rac protein (either racl or rac2).
• Rho is a member of the Rho family of GTPases; racl is the activating co-factor in parenchymal lung cells while rac2 is required in PMN.
• the protein peroxiredoxin 6 (Prdx6) has an important role in ROS production via N0X2 since it is required for activation of rac.
• the activation process requires the phospholipase A2 activity (aiPLA2) of Prdx6 that modulates rac release through lysophosphatidic acid receptor signaling.
• N0X1 also requires rac protein for activation of ROS production but the other 5 NOX enzymes are independent of rac.
• the lipid compound termed MJ33 and the surfactant protein A (SP-A) are 2 agents that bind to Prdx6 preventing aiPLA2 activity, in turn resulting in failure of rac activation.
• SP-A surfactant protein A
• a 9 amino acid sequence of SP-A was identified as being responsible for inhibition of N0X2 activation. This 9 amino acid peptide sequence is generally conserved in mammals with some minor variation is termed peroxiredoxin 6 PLA2 inhibitory peptide (PIP); the peptide corresponding to the human amino acid peptide sequence in SP-A is termed PIP-2.
• EPS bacterial lipopolysaccharide
• IP intraperitoneal
• IV intravenous
• NOX2 activation by treatment with MJ33 or PIP-2 or genetic inactivation of aiPLA2 activity can significantly ameliorate lung injury.
• the study described herein was designed to evaluate the possible protective role for PIP-2 in an animal model with lungs more closely resembling the anatomy and physiology of human lungs.
• the 9 amino acid peptide called PIP-2 was synthesized with a C-terminal HC1 group by APeptide, Shanghai, China.
• the peptide was encapsulated in liposomes for IV delivery.
• Liposomes were composed of dipalmitoyl phosphatidylcholine (DPPC), egg PC, phosphatidylglycerol (PG), and cholesterol in the molar ratio 0.5, 0.25, 0.10, 0.15; this composition reflects the lipid composition of lung surfactant.
• DPPC dipalmitoyl phosphatidylcholine
• PG phosphatidylglycerol
• cholesterol in the molar ratio 0.5, 0.25, 0.10, 0.15; this composition reflects the lipid composition of lung surfactant.
• Liposomes were stored at -20 degrees C prior to use. Samples were warmed to room temperature and were used within 3 hours.
• the PIP-2 encapsulation efficiency for the liposomes was about 15-20%
• LPS Escherichia coli O55:B5
• Sigma product Code: L2637
• source batch 12181107 with sub-batches: 0000102731 and 0000119457.
• LPS was stored at 3-8 deg. C.
• the animals were intubated and two IV catheters were placed in peripheral veins for administering supportive IV fluids and propofol and to allow collection of blood samples for monitoring blood gases and electrolytes; a carotid or femoral artery was catheterized to directly monitor arterial blood pressure and to obtain arterial blood samples.
• An “introducer sheath” was inserted and advanced into each vessel for providing access.
• Propofol was administered, initially 4-8 mg/kg IV as a bolus followed by -0.2-0.4 mg/kg/min IV as a continuous infusion; animals were maintained on propofol anesthesia for the remainder of the procedure.
• An ophthalmic lubricant was applied to the eyes.
• a Foley catheter was placed in the bladder, under cystoscopic guidance, if necessary, to allow urine collection throughout the experiment.
• Warm water heating pads were used to help maintain adequate body temperature while under anesthesia; animals with body temperatures below 96°F were provided warming blankets and warm fluids.
• Lactic acidosis serum lactate >10 mM
• hypoglycemia was treated with IV dextrose.
• Hypotension during the experiment was treated with IV administration of norepinephrine and/or phenylephrine.
• the IV administered PIP-2 was -3-4 mg/kg body weight in liposomes and the remainder was in aqueous solution; presumably, the encapsulated PIP-2 was internalized by cells while the unencapsulated PIP-2 remained extracellular.
• Animals were mechanically ventilated for up to 8 hours in a volume-controlled mode using a tidal volume of 8 mL/kg body weight, an inspiratory-expiratory ratio of 1 : 1 , a fraction of inspired oxygen (FiO2) of 1.0, and a positive end-expiratory pressure (PEEP) of 5 cm H2O.
• aPO2 Arterial blood O2
• Static lung compliance was measured as the change in pressure at the end of a “breath hold” with a given tidal volume.
• FITC-dextran was infused IV over 15 minutes and then a bronchoscope was inserted into the airway and navigated into a subsegmental bronchus.
• Tubing was attached to the working channel of the bronchoscope on one end and to a syringe containing saline through a three-way stopcock on the other end.
• the third port of the stopcock was attached to a trap to collect the BAL fluid (BALF). The effluent was collected when the stopcock was turned off to the syringe causing suction through the trap.
• BALF BAL fluid
• Pig lung lavage (BALf) was centrifuged at lOOOxg for 10 min and used for biochemical assays. Protein content was measured using a Bio-Rad assay kit with gamma globulin as standard. Myeloperoxidase (MPO) activity was measured by ELISA assay using a commercial kit (Biomatik, Wilmington DE). Cytokines (IL-6, IL-ip, TNF-a) also were measured by ELISA assay using a commercial assay kit (Invitrogen, ThermoFisher Scientific, Federic MD).
• the aPCh was measured at hourly intervals and used to calculate the arterial blood PO2 divided by the fractional inspired O2 (FIO2); this parameter (aPO2/FIO2) has been called the Horowitz index. Since the fractional O2 used in this study for ventilation was 1 .0, the Horowitz index in these studies is identical to the aPCh.
• the aPCh Horowitz index
• the aPCh/FiCh has been the major parameter used to grade the severity of ALL
• moderate to severe ALI was noted in 2 of the remaining 3 (67%) untreated LPS pigs but in only 2 of the 9 remaining PIP-2 treated pigs (22%) (FIG. 6).
• PIP-2 treatment resulted in a 50% decrease in pig mortality by humane sacrifice and a 67% decrease in the incidence of moderate to severe ARDS in the pigs that survived for the entire 8 hour study duration.
• Pigs given LPS showed significant depression of the blood white cell (WBC) and platelet counts (Table 10) compatible with the response to sepsis.
• the mean PMN leukocyte count after LPS (group 2) was decreased by ⁇ 80-90% vs control at both 4 and 8 hours but by only ⁇ 60% and 40% at 4 and 8 hours, respectively, in animals treated with PIP-2 (Table 11), although these differences between plus or minus PIP-2 were not statistically significant.
• PIP- 2 treatment had no effect on the decrease in blood platelets associated with LPS.
• Group 2 (LPS) animals developed elevation in the plasma levels of cardiac troponin and of creatine kinase at the 4 and 8 hour observation periods (Table 12). These enzymes are considered to be indicators of cardiac muscle injury. There was no change from baseline in levels of these 2 enzymes in the group 3 (LPS + PIP-2) animals (Table 12). Thus, the increases in serum troponin and creatine kinase in the group 2 pigs were statistically significant as compared to the group 3 (PIP-2) treated pigs.
• Total pressor is the total amount of epinephrine, norepinephrine, and phenylephrine given to maintain adequate arterial blood pressure during the initial 4 hrs and during the subsequent 4 -8 hrs of the experiment.
• lung injury was assessed by measurement of lung compliance and analysis of broncho-pulmonary lavage fluid. Decreased lung compliance indicating stiff lungs is a common feature of ALL
• the mean value for static lung compliance (CL) was reduced from 20.3 ml/cm H2O for pigs at baseline to 11.0 ml/cm H2O (436% decrease) for group 2 pigs at 4 hours after the start of LPS infusion, with essentially no further change in the mean value at 8 hours of study (Table 14).
• Treatment of pigs with PIP-2 (group 3) resulted in a lesser decrease (40% at 4 hours and 30% at 8 hours) in lung compliance compared with untreated (group 2, no PIP- 2) pigs. For both Group 2 and group 3 pigs, the major decrease in lung compliance occurred during the initial 4 hours of the experiment.
• lung lavage cytokines (IL-6, IL-1 p, TNFa) were increased 2-3 fold with LPS treatment and this increase was blocked by treatment with PIP-2 (Table 15).
• PIP-2 treatment of LPS-exposed animals had a protective effect on lung alveolar capillary protein permeability and decreased lung inflammation as indicated by BALF MPO and cytokine levels.
• the control animals showed relatively trivial evidence of lung injury compatible with tolerance to mechanical ventilation for 8 hours under control conditions.
• Lungs from LPS- treated animals demonstrated evidence of mild to moderate inflammation with no significant difference between PIP-2 treated and PIP-2 untreated pigs.
• the lungs were not inflation-fixed, which limited the utility of the morphologic examination.
• Examination of heart, liver and kidneys likewise showed relatively minor inflammation and hemorrhage that was not significantly different between the PIP-2 treated and untreated animals.
• the relatively minor changes at post-mortem examination presumably reflect the relatively short duration ( ⁇ 8 hours) of LPS exposure.
• ROS reactive oxygen species
• NOX NADPH oxidase
• MJ33 is a lipid molecule that inhibits various PLA2 enzymes such as pancreatic PLA2 as well as the aiPLA2 activity of Prdx6, inhibition of N0X2 activation was demonstrated to be associated with inhibition of aiPLA2 (the PLA2 activity of Prdx6).
• the surfactant-associated protein SP-A binds to Prdx6 and inhibits its aiPLA2 activity.
• Protein truncation was used to determine the minimal effective sequence of the 246 amino acids of human SP-A to inhibit aiPLA2.
• a 9 amino acid sequence, called phospholipase A2 inhibitory peptide (PIP) was determined to have full inhibitory activity.
• the PIP sequence for human SP-A was called PIP-2.
• Additional observed beneficial effects of PIP-2 treatment included: a significant reduction in the increase of indicators of injury to heart muscles as indicated by serum troponin 1 and creatine kinase levels; a significant reduction in the pressor requirements for arterial blood pressure control; a reduced alteration of lung gas exchange as indicated by a decreased abnormal alveolar to arterial (A-a) PO2 gradient; a protection of the lung alveolar-capillary barrier as indicated by a significant reduction in the leakage of protein into the lung alveolar space; a lesser decrease in blood WBC compatible with a decrease in the manifestations of sepsis; a significant reduction in the elevated blood C-reactive protein also compatible with lessened sepsis; and decreased lung inflammation as indicated by a decrease in lung lavage WBC (estimated by MPO content).
• PIP-2 As potential agent for the treatment of clinical ALI and/or sepsis is based on the important role for NOX- generated ROS in the pathophysiology of lung injury. Excessive ROS generation is considered to be of major importance in the pathophysiology of acute lung injury and inhibition of NOXl/2-generated ROS is expected to significantly reduce the oxidative stress associated with this syndrome. Thus, PIP-2 as a nontoxic inhibitor with a relatively favorable tissue half-life can be considered as a strong candidate for testing as adjunctive therapy for prevention or treatment of sepsis and/or ALI. [00103] In sum, PIP-2 reduce mortality in animals in septic shock by 50%, a result which has not been observed in other studies in this model.
• biomarkers for PIP-2 showed near normal levels in the treated animals, while the placebo animals showed significant injury. Without wishing to be bound by theory, due to the nature of sepsis, the results suggest that the placebo animals would have additional mortality due to the poor biomarkers observed. However, some of the mild and modestly affected animals in the PIP-2 treatment group may fully recover, as their biomarkers showed near normal levels after PIP-2 treatment.
• vasopressors are standard of care in septic shock injury.
• One of the impact in septic shock is the 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 injuries.
• the PIP-2 treated animals required a third (1/3) less total mg of vasopressor than the placebo animals, while also achieving greater mean arterial pressure. Achieving this type of mean arterial pressure in a septic shock injury with a novel mechanism of was also unexpected and can have a beneficial effect in patients, , since the use of vasopressors have many negative side effects that PIP-2 did not demonstrate. Therefore, the use of PIP-2 and the related peptides provide significant advantages over the current standard of care for septic shock.

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